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)).

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.

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.

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.

Melatonin and cell death: differential actions on apoptosis in normal and cancer cells.

Melatonin is a natural compound synthesized by a variety of organs. It has been shown to function as a cell-protective agent. Since 1994, when the first paper was published documenting the role of melatonin in apoptosis, the number of reports in this area has increased rapidly. Much of the research conducted falls into three major categories: first, the role of melatonin in inhibiting apoptosis in immune cells; second, the role of melatonin in preventing neuronal apoptosis and finally, the role of melatonin in increasing apoptotic cell death in cancer cells. The mechanisms whereby melatonin influences apoptosis have not clarified, although a number of mechanistic options have been suggested. Apoptotic cell death is a physiological phenomenon related to homeostasis and proper functioning of tissues and organs; however, a failure in the apoptotic program is related to a number of diseases. The participation of melatonin in apoptosis in numerous cell types and its potential importance in a variety of diseases such as immunodeficiency, neurodegeneration and cancer is summarized in this review.

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.

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.

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.

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.

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.

Changes in lipid peroxidation during pregnancy and after delivery in rats: effect of pinealectomy.

Pregnancy is a physiological state accompanied by a high energy demand of many bodily functions and an increased oxygen requirement. Because of the increased intake and utilization of oxygen, increased levels of oxidative stress would be expected. In the present study, the degree of lipid peroxidation was examined in different tissues from non-pregnant and pregnant rats after the delivery of their young. Melatonin and other indole metabolites are known to be direct free radical scavengers and indirect antioxidants. Thus the effect of pinealectomy at 1 month before pregnancy on the accumulation of lipid damage was investigated in non-pregnant and pregnant rats after the delivery of their young. Malonaldehyde and 4-hydroxyalkenal concentrations were measured in the lung, uterus, liver, brain, kidney, thymus and spleen from intact and pinealectomized pregnant rats soon after birth of their young and at 14 and 21 days after delivery. The same parameters were also evaluated in intact and pinealectomized non-pregnant rats. Shortly after delivery, lipid oxidative damage was increased in lung, uterus, brain, kidney and thymus of the mothers. No differences were detected in liver and spleen. Pinealectomy enhanced this effect in the uterus and lung. It is concluded that during pregnancy high levels of oxidative stress induce an increase in oxidative damage to lipids, which in some cases is inhibited by the antioxidative actions of pineal indoles.

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.

5-methoxytryptophol preserves hepatic microsomal membrane fluidity during oxidative stress.

Lipid peroxidation is a degenerative chain reaction in biological membranes that may be initiated by exposure to free radicals. This process is associated with changes in the membrane fluidity and loss of several cell membrane-dependent functions. 5-methoxytryptophol (ML) is an indole isolated from the mammalian pineal gland. The purpose of this study was to investigate the effects of ML (0. 01mM-10mM) on membrane fluidity modulated by lipid peroxidation. Hepatic microsomes obtained from rats were incubated with or without ML (0.01-10 mM). Then lipid peroxidation was induced by FeCl(3), ADP, and NADPH. Membrane fluidity was determined using fluorescence spectroscopy. Malonaldehyde (MDA) +4-hydroxyalkenals (4-HDA) concentrations were estimated as an indicator of the degree of lipid peroxidation. With oxidative stress, membrane fluidity decreased and MDA+4-HDA levels increased. ML (0.01-3 mM) reduced membrane rigidity and the rise in MDA+4-HDA formation in a concentration-dependent manner. 10 mM ML protected against lipid peroxidation but failed to prevent the membrane rigidity. In the absence of oxidative reagents, ML (0.3-10 mM) decreased membrane fluidity whereas MDA+4-HDA levels remained unchanged. This indicates that ML may interact with membrane lipids. The results presented here suggest that ML may be another pineal indoleamine (in addition to melatonin) that resists membrane rigidity due to lipid peroxidation.

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.

Melatonin reduces lipid peroxidation and tissue edema in cerulein-induced acute pancreatitis in rats.

Since oxygen free radicals and lipid peroxidation have been implicated in the pathogenesis of an early stage of acute pancreatitis, we examined whether melatonin, a recently discovered free-radical scavenger, could attenuate pancreatic injury in Sprague-Dawley rats with cerulein-induced pancreatitis. Acute pancreatitis was induced by four intraperitoneal injections of cerulein (50 microg/kg body wt) given at 1-hr intervals. Thirty minutes after the last cerulein injection, the rats were killed and the degree of pancreatic edema, the level of lipid peroxidation in the pancreas, and serum amylase activity were increased significantly. Pretreatment with melatonin (10 or 50 mg/kg body wt) 30 min before each cerulein injection resulted in a significant reduction in pancreatic edema and the levels of lipid peroxidation. Serum amylase activity, however, was not significantly influenced by either dose of melatonin. Moreover, we found that cerulein administration was associated with stomach edema as well as high levels of lipid peroxidation in the stomach and small intestine, which were also reduced by melatonin. Melatonin's protective effects in cerulein-treated rats presumably relate to its radical scavenging ability and to other antioxidative processes induced by melatonin.

Melatonin regulates glucocorticoid receptor: an answer to its antiapoptotic action in thymus.

We have previouslyreported that low doses of melatonin inhibit apoptosis in both dexamethasone-treated cultured thymocytes (standard model for the study of apoptosis) and the intact thymus. Here we elucidate the mechanism by which this agent protects thymocytes from cell death induced by glucocorticoids. Our results demonstrate an effect of melatonin on the mRNA for antioxidant enzymes in thymocytes, also showing an unexpected regulation by dexamethasone of these mRNA. Both an effect of melatonin on the general machinery of apoptosis and a possible regulation of the expression of the cell death related genes bcl-2 and p53 are shown not to be involved. We found melatonin to down-regulate the mRNA for the glucocorticoid receptor in thymocytes (glucocorticoids up-regulate their own receptor). The decrease by melatonin of mRNA levels for this receptor in IM-9 cells (where glucocorticoids down-regulate it) demonstrates that melatonin actually down-regulates glucocorticoid receptor. These findings allow us to propose the effects of melatonin on this receptor as the likely mediator of its thymocyte protection against dexamethasone-induced cell death. This effect of melatonin, given the oxidant properties of glucocorticoids, adds another mechanism to explain its antioxidant effects.

The oxidant/antioxidant network: role of melatonin.

Melatonin is now known to be a multifaceted free radical scavenger and antioxidant. It detoxifies a variety of free radicals and reactive oxygen intermediates including the hydroxyl radical, peroxynitrite anion, singlet oxygen and nitric oxide. Additionally, it reportedly stimulates several antioxidative enzymes including glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and superoxide dismutase; conversely, it inhibits a prooxidative enzyme, nitric oxide synthase. Melatonin also crosses all morphophysiological barriers, e.g., the blood-brain barrier, placenta, and distributes throughout the cell; these features increase the efficacy of melatonin as an antioxidant. Melatonin has been shown to markedly protect both membrane lipids and nuclear DNA from oxidative damage. In every experimental model in which melatonin has been tested, it has been found to resist macromolecular damage and the associated dysfunction associated with free radicals.

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.

Role of pinoline and melatonin in stabilizing hepatic microsomal membranes against oxidative stress.

We investigated the influence of pinoline (0.01-1.5 mM) on microsomal membrane fluidity before and after rigidity was induced by oxidative stress. In addition, we tested the effect of pinoline in the presence of 1 mM melatonin. The fluidity in rat hepatic microsomes was monitored using fluorescence spectroscopy and it was compared to the inhibition of malonaldehyde (MDA) plus 4-hydroxyalkenals (4-HDA) production as a reflection of lipid peroxidation. Below 0.6 mM, pinoline inhibited membrane rigidity in a manner parallel to its inhibitory effect on MDA + 4-HDA formation. At concentrations between 1-1.5 mM, pinoline was less effective in stabilizing microsomal membranes than was predicted from its inhibition of lipid peroxidation. The addition of 1 mM melatonin enhanced the membrane-stabilizing activity of pinoline (0.01-0.6 mM). This cooperative effect was not observed for concentrations of pinoline between 1-1.5 mM. When pinoline was tested without induced oxidative damage, 1-1.5 mM pinoline maintained membrane fluidity at the same level as that recorded after induced lipid peroxidation. The results suggest that pinoline may be another pineal molecule that prevents membrane rigidity mediated by lipid peroxidation and this ability is enhanced by melatonin.

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.