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The fact that the full extent of the function of the pineal gland has not yet been elucidated, has stimulated melatonin research worldwide. This review introduces melatonin's mechanism of action, direct and indirect antioxidant actions as well as the antioxidant properties of its metabolites, 6-hydroxymelatonin (6-OHM) and N-acetyl-N-formyl-5-methoxykynurenamine (AFMK). At present the mechanism of action is proposed to be receptor-, protein- and nonprotein-mediated. From its popular role in the treatment of jetlag, melatonin is now implicated in the reduction of oxidative stess, both as a free radical scavenger and antioxidant. Melatonin's direct scavenging action in respect of the following will be discussed: superoxide anions, hydrogen peroxide, hydroxyl radicals, singlet oxygen, peroxy radicals and nitric oxide/peroxy nitrite anions. In addition melatonin also possesses indirect antioxidant activity and the role of its metabolites, AFMK and 6-OHM will be presented. It is these free radical scavenging and antioxidant properties of melatonin that has shifted the focus from that of merely strengthening circadian rhythms to that of neuroprotectant: a new place in therapy.

Oxidative damage of biological macromolecules is a hallmark of most neurodegenerative disorders such as Alzheimer, Parkinson and diffuse Lewy body diseases. Another important phenomenon involved in these disorders is the alteration of iron homeostasis, with an increase in iron levels. The present study investigated whether 6-hydroxymelatonin (6-OHM) can reduce Fe2+-induced lipid peroxidation and necrotic cell damage in the rat hippocampus in vivo. It was found that 6-OHM administration proved successful in reducing Fe2+-induced neurotoxicity in rat hippocampus. This study provides some evidence of the neuroprotective effects of 6-OHM.

Melatonin, a naturally occurring chemical mediator, although assigned a diverse range of functions, has attracted interest in recent years because of its ability to function as a free radical scavenger. Because of the implications of singlet oxygen in neurotoxicity, the objective of the study was to investigate the ability of melatonin to quench singlet oxygen generated using laser irradiation or lamp photolysis. The results show that melatonin produces radicals upon laser irradiation while the lamp photolysis studies show that melatonin is able to scavenge singlet oxygen produced by naphthalene. While melatonin is a free radical scavenger under biological conditions, it acts as a generator of singlet oxygen and or radicals (as PhiDelta is 1.41) when irradiated with laser light, implying that it has the potential to be used in photodynamic therapy in the destruction of tumors.

Both 6-hydroxymelatonin and N-acetyl-N-formyl-5-methoxykynurenamine are photodegradants and enzymatic metabolites of melatonin and are known to retain equipotent activity against potassium cyanide-induced superoxide generation compared to melatonin. It is not clear whether one or both of these metabolites is responsible for this effect. The present study therefore investigates the possible manner in which 6-hydroxymelatonin protects against oxidative stress induced by cyanide in rat brain homogenates. We examined the ability of 6-hydroxymelatonin to scavenge KCN-induced superoxide anion generation as well as lipid peroxidation. In addition, we also examined the effect of this indole on lactate dehydrogenase activity (LDH) as well as mitochondrial electron transport using dichlorophenol-indophenol as an electron acceptor. The results of this study show that 6-hydroxymelatonin significantly reduces KCN-induced superoxide anion generation, which is accompanied by a commensurate reduction in lipid peroxidation. Partial reversal of the KCN-induced reduction in mitochondrial electron transport is accompanied by a similar reversal of mitochondrial LDH activity blunted by KCN. It can thus be proposed that 6-hydroxymelatonin is potentially neuroprotective against KCN-induced neurotoxicity.

Disorders of iron accumulation are known to produce hepatotoxicity. Agents, which can reduce Fe(3+) to a more usable form Fe(2+) could potentially limit such damage. Since it has been previously demonstrated that the pineal secretory product, melatonin, is able to bind iron, we decided to investigate the potential protective properties of the principal melatonin metabolite and degradant, 6-hydroxymelatonin (6-OHM). Using adsorptive cathode stripping voltammetry (AdCSV) we showed that Fe(3+) in the presence of 6-OHM is converted to Fe(2+). We further demonstrated that 6-OHM reduces the Fe(2+)-induced rise in lipid peroxidation in rat liver homogenates. The results imply that 6-OHM facilitates the conversion of Fe(3+) to Fe(2+) which is a more biologically usable form of iron. While such a conversion could also potentially make more Fe(2+) available for driving the Fenton reaction and the consequent generation of the dangerous hydroxyl radical, 6-OHM is able to quench these radicals, thereby providing tissue protection.

Ultraviolet (UV) light is known to induce the generation of free radicals in biological tissues such as skin. Of these free radicals, the O2-. and particularly the.OH radical can induce cellular damage including lipid peroxidation. Thus, the use of antioxidants to prevent such damage induced by UV irradiation has received much attention recently. One such antioxidant, which has the potential to be incorporated into sunscreens, is the pineal secretory product melatonin. One of the concerns of using melatonin in sunscreens is its photostability. In the present study, we investigated the photostability of melatonin subjected to UV irradiation. In addition, we used liquid chromatography mass spectrometry (LC-MS) to identify the degradants and we also assessed the ability of the degradants to inhibit O2-. generation as well as lipid peroxidation in rat brain homogenate. The results show that UV irradiation of melatonin (0.1 mg/mL) using a 400-W lamp for 2 hr caused a significant decline of melatonin to 18% of its original concentration after 20 min, with the decline continuing until the melatonin concentration reaches zero at 120 min. The LC-MS results show that the degradants of melatonin are 6-hydroxymelatonin and N1-acetyl-N2-formyl-5-methoxykynurenamine (AFMK). These degradants were able to provide equipotent activity against potassium cyanide (KCN)-induced superoxide generation compared to non-irradiated melatonin. Thus, the study shows that although melatonin is rapidly degraded by UV irradiation, the degradants retain antioxidant activity, making melatonin a likely candidate for inclusion in sunscreens.

Copper is an essential trace element which forms an integral component of many enzymes. While trace amounts of copper are needed to sustain life, excess copper is extremely toxic. Copper has been implicated in various neurodegenerative disorders, such as Wilson's and Alzheimer's diseases. Previous studies showed that melatonin, the principle secretory product of the pineal gland, binds Cupric chloride (Cu2+) and that this may have implications in copper-induced neurodegenerative diseases. In the present study, in vitro copper-mediated lipid peroxidation was induced. Melatonin (5 mM) protected against copper-mediated lipid peroxidation in liver homogenates. Electron micrographs of in vivo administered Cu2+ and melatonin show that melatonin affords some protection to rat hepatocytes in the presence of copper. Electrochemical studies performed show that melatonin, in addition to binding Cu2+, may provide protection against copper-mediated free radical damage by binding Cu1+. The findings of these studies provide further evidence for the neuroprotective role of melatonin.

Copper toxicity has been implicated in various neurodegenerative disorders such as Wilson's disease and Alzheimer's disease. Free copper in the brain is toxic and leads to neuronal and cellular damage, through free radical generation. Melatonin has been investigated as a possible copper ion chelator. Melatonin could prevent copper-induced neuronal and cellular damage through binding with copper and preventing copper-induced free radical generation. The effect of copper on pineal indolamine synthesis has not been studied extensively. In the present study, copper (2 mg/kg) and melatonin (12 mg/kg) were administered daily to Wistar rats for a 2-week and 6-week period. Pineal organ culture was utilized to monitor pineal indolamine synthesis. The pineals from the 2-week copper/melatonin-treated group showed a statistically significant decrease in 5-methoxytryptophol synthesis (p < 0.01), compared to the pineals from the copper-treated group. Conversly, in the 6-week experiment, 5-methoxytryptophol synthesis was increased in both the copper- and copper/melatonin-treated groups. There was a statistically significant decrease in the N-acetyl serotonin level in the pineals from the 6-week copper-treated animals, as compared to the control- and copper/melatonin-treated group (p < 0.01). These results imply that copper reduces N-acetyltransferase activity, which results in a decrease in N-acetyl serotonin synthesis. Melatonin when coadministered with copper appears to prevent the N-acetyltransferase inhibition by copper. Copper exerts contradictory effects on 5-methoxytryptophol synthesis. Further investigations need to be carried out to examine the effects of copper on the pineal enzymes.

Melatonin (N-acetyl-5-methoxytryptamine) has a diverse range of functions, including the control of neuroendocrine events. A number of studies have shown that melatonin may be of potential benefit for the treatment of insomnia, as well as neurodegenerative disorders. At present, there are numerous dosage forms of melatonin, with the oral route of administration being most popular. Presently, there is little information on the stability of melatonin over a pH range. With the changes in pH in the gastro-intestinal tract, as well as in different experimental conditions, information on the stability of melatonin would be important. We used a high-performance liquid chromatography method to determine the stability of melatonin solutions over a pH range (1.2-12) at room temperature and at 37 degrees C over a period of 21 days. The results show that no melatonin degradation occurred in the first 2 days. From days 3 to 21, there was a gradual decline in melatonin at all pHs, with the decline not exceeding 30%. No decline in melatonin levels occurred in the first 2 days at 37 degrees C. From days 3 to 21, melatonin levels declined gradually, with the decline not exceeding 29%.

In the present study, we investigated the ability of serotonin and melatonin to bind metals that occur naturally in the brain. An electrochemical technique called adsorptive cathodic stripping voltammetry (AdCSV) was employed to study the metal-serotonin or metal melatonin interactions. The results show that both serotonin and melatonin form stable complexes with lithium and potassium, with serotonin favouring lithium over potassium, and melatonin favouring potassium over lithium. Coordination between either serotonin or melatonin and calcium was not favoured. The stability of the complexes formed between serotonin and the metals decreased with the metals as follows: Li+ > K+ > Al3+ > Na+ > Ca2-. The trend for melatonin-metal complexes was K+ > Li+ > Na+ > Al3+ > Ca2+ The binding and stable complex formation between both ligands, serotonin and melatonin with lithium, potassium and sodium is of biological importance. The binding of serotonin to lithium could provide an explanation for the therapeutic effects of lithium in depression treatment, whereas the binding of aluminium by melatonin could provide insight into the role of this element in the aetiology of Alzheimer's disease.

The protective effects of melatonin against the neurotoxin, quinolinic acid, were investigated in rat brain homogenate using the thiobarbituric acid assay. Quinolinic acid increased lipid peroxidation in a dose dependent manner. When homogenate was cotreated with melatonin there was a significant decrease in lipid peroxidation. The results of the present report show that melatonin may play a protective role in the brain against the neurohormone quinolinic acid, which has been identified as a causative agent in Huntington's Disease.

The neuroprotective effect of melatonin against the quinolinic acid-induced degeneration of rat hippocampal neurons was investigated. Three groups of rats were given intrahippocampal injections of either; saline, quinolinic acid or i.p. injections of melatonin prior to and after being injected with quinolinic acid. On the fifth day after the intrahippocampal injections the brains were removed and the hippocampi either sectioned and stained for microscopic examination or used in glutamate receptor binding studies. The results show that melatonin protects hippocampal neurons from quinolinic acid-induced neurodegeneration and partially prevents the decrease in glutamate receptor numbers caused by quinolinic acid. Thus, melatonin has the potential to reduce hippocampal neuronal damage induced by neurotoxins such as quinolinic acid.

Melatonin, a pineal secretory product, and its precursors, tryptophan and serotonin, were examined for their metal binding affinities for both essential and toxic metals: aluminium, cadmium, copper, iron, lead, and zinc. An electrochemical technique, adsorptive stripping voltammetry, showed the varying abilities of melatonin and its precursors to bind the metals in situ. The results show that the following metal complexes were formed: aluminium with melatonin, tryptophan, and serotonin; cadmium with melatonin and tryptophan; copper with melatonin and serotonin; iron(III) with melatonin and serotonin; lead with melatonin, tryptophan, and serotonin; and zinc with melatonin and tryptophan. Iron(II) showed the formation of an in situ complex with tryptophan only. These studies suggest a further role for melatonin in the reduction of free radical generation and metal detoxification, and they may explain the accumulation of aluminium in Alzheimer's disease.

The effects of desipramine and fluoxetine were examined on rat hepatic tryptophan-2,3-dioxygenase activity in the presence or absence of exogenous melatonin. Male Wistar rats were treated intraperitoneally over 8 days. The antidepressants were also administered individually and their impact on the enzyme noted. Desipramine reduced basal hepatic tryptophan-2,3-dioxygenase activity in the liver while fluoxetine had no observable effect. However, fluoxetine also prevented the hemin induced elevation in total enzyme activity. Exogenous melatonin (0.25mg/kg/day) for 8 days counteracted the inhibitory effects of both desipramine and fluoxetine at concentrations of 2.5mg/kg/day. Total enzyme activity was restored when melatonin was administered in combination with either drug. The experimental findings indicate that there is chemical antagonism at the surface of the enzyme between melatonin and the antidepressants that abolishes the observed inhibitory effects of the administered compounds.

In view of the biogenic amine deficiency hypothesis of depression and the contentious claim that hepatic tryptophan-2,3-dioxygenase (TDO) is the major peripheral determinant of brain tryptophan, brain serotonin (5-HT), and ultimately melatonin, the regulation of TDO by melatonin and 5-HT is investigated and discussed. It is concluded that TDO activity is regulated differentially by melatonin and 5-HT. Melatonin is a competitive inhibitor of TDO and 5-HT is predominantly an allosteric inhibitor. In the presence of 5-HT the effects of melatonin are nullified. However melatonin alone is a more potent inhibitor of TDO. Melatonin is also shown to be a reversible negative effector of TDO.

Follicular fluid samples were obtained from the largest pre-ovulatory follicle of 120 women undergoing in-vitro fertilization and were examined for melatonin by enzyme-linked immunosorbent assay and the steroids oestradiol and progesterone by radioimmunoassay. The concentrations (mean +/- SE) of melatonin (213.4 +/- 18.9 pmol/l) and progesterone (20.1 +/- 1.1 mumol/l) in follicular fluid during the autumn and winter (dark) months were significantly higher than during the spring and summer (light) months, melatonin (138.4 +/- 12.5 pmol/l) and progesterone (11.6 +/- 0.8 mumol/l). By contrast, oestradiol concentrations were significantly lower during the dark months than during the light months (264.7 +/- 44.1 and 661.8 +/- 55.1 nmol/l respectively). There was a positive correlation between follicular fluid melatonin and progesterone concentrations (r = 0.271, P < 0.05, n = 120) and a negative relationship between melatonin and oestradiol (r = -0.254, P < 0.05, n = 120). The effects of melatonin alone and in combination with human chorionic gonadotrophin (HCG) or follicle stimulating hormone (FSH) on steroidogenesis by human granulosa cell culture were also investigated. Melatonin had minimal effects on oestradiol or progesterone production by granulosa cells. Interestingly, the oestradiol response in culture appeared to be different according to the time of the year when harvested. During the light period oestradiol production was enhanced. Melatonin also synergized with HCG in increasing progesterone production on days 6 and 7 after treatment during both light and dark periods. FSH stimulated oestradiol production by the cells on day 2 of culture. Melatonin had no effect on FSH stimulation of oestradiol production. The results of this study suggest that melatonin may be involved in the regulation of steroidogenesis by the human ovaries.

Liver tryptophan 2,3-dioxygenase is a cytosolic enzyme that plays a crucial role in the regulation of circulating levels of tryptophan. Stimulation of the activity of this enzyme by heme enhances the catabolism of tryptophan, making less tryptophan available for uptake into the brain. Melatonin, the major hormone of the pineal gland, is shown to cause competitive inhibition of this enzyme (Ki = 2.70 microM). This structural analog of the substrate L-tryptophan is a negative homotropic cooperative modulator of the enzyme. The enzyme has a Km = 100 microM, and the substrate concentration required for optimum activity was found to be 2.5 mM with substrate inhibition becoming a feature at higher levels of tryptophan.

Melatonin concentrations and aromatase stimulating activity were determined in human seminal plasma and correlated with sperm density and motility. Aromatase stimulating activity was determined with an in vitro rat granulosa cell system and melatonin by radioimmunoassay. Compared to normal semen, aromatase stimulating activity was lower in azoospermic individuals, while melatonin was higher in oligospermic and azoospermic samples. Aromatase stimulating activity correlated positively with sperm concentrations and a negative correlation was found between melatonin and sperm progression. These findings suggest that low sperm production is associated with low aromatase stimulating bioactivity in seminal plasma; and melatonin may have an effect upon both sperm production and motility.

When rats were exposed to extended light into the normal dark phase, the administration of 5-aminolevulinic acid (5-ALA) markedly stimulated the accumulation of tryptophan (TRP) and the synthesis of serotonin (5-HT) in the forebrain. When a single injection of melatonin (25 micrograms) was given near the end of the 5-ALA treatment, the rise in forebrain TRP levels was either totally prevented or reversed; likewise, the increased levels of 5-HT were also suppressed. Melatonin by itself also slightly suppressed forebrain TRP levels in non-5-ALA-treated rats. Whether these are pharmacological or physiological effects of melatonin remains to be determined.

An attempt was made to use the pineal gland as a model for the study of the primary mode of action of alpha-methyldopa, which is still unclear. Organ cultures of pineal glands from rats treated chronically with alpha-methyldopa showed enhanced conversion of radio-active serotonin to melatonin (aMT) as well as its precursor, N-acetyl-serotonin (aHT). This treatment was also found to raise serotonin-N-acetyltransferase (NAT) activity. These increases associated with alpha-methyldopa treatment were further enhanced by the beta-adrenergic agonist, isoproterenol, suggesting a supersensitivity-type effect occurring at the level of the beta-receptor. A subsequent binding study, however, showed a decrease in beta-receptor binding with exposure to alpha-methyldopa, providing mitigating evidence against the occurrence of a supersensitivity phenomenon. It is possible that a metabolite of alpha-methyldopa acts as an alpha 1 and beta-agonist, resulting in greater melatonin (aMT) and N-acetylserotonin (aHT) synthesis than by a beta-agonist, isoproterenol.

Prior melatonin administration (1,5 and 10 mg/kg b. wt) causes a significant reduction in gastric ulceration induced in male rats by restraint immobilization in the presence of low temperatures.

A naturally occurring compound, 6-methoxybenzoxazolinone (6-MBOA), present in grasses, has been shown to induce sexual maturation in a number of rodent species. The structural similarity of 6-MBOA and melatonin has led researchers to suspect that 6-MBOA might induce its progonadal effects by directly altering pineal function. Previous studies have shown that 6-MBOA has the properties of a weak beta-adrenergic agonist capable of stimulating rat pineal N-acetyltransferase (NAT) activity at pharmacological concentrations of 10(-3) M. In the present study we have examined the effect of 6-MBOA on both the pineal melatonin synthesizing enzymes, namely, NAT and hydroxyindole-O-methyltransferase (HIOMT) as well as on melatonin production, in organ cultured rat pineal glands. In addition, we have also examined the ability of 6-MBOA to displace a ligand from rat brain alpha- and beta-adrenoceptors. Our results confirm that 6-MBOA stimulates NAT activity and melatonin production at the high concentration of 10(-3) M. It appears to have no effect on HIOMT activity. A competition study shows that 6-MBOA is able of displacing ligands at the alpha- and beta-adrenoceptors but only at concentrations greater than 10(-4) M. Whether such high concentrations of 6-MBOA reach the pineal of rodent in their natural habitat is unknown. However, if 6-MBOA does mediate progonadal effects by altering pineal function it would be expected that 6-MBOA would ultimately inhibit the effects of melatonin. The possibilities are that the high melatonin levels induced by 6-MBOA cause desensitization of melatonin receptors or that 6-MBOA is an antagonist at the level of the melatonin receptor, thus restricting the inhibitory effects of melatonin on the reproductive system.

The daytime administration of the heme precursor 5-aminolevulinate (5-ALA) has been shown to reduce brain tryptophan and serotonin levels owing to saturation of liver tryptophan pyrrolase. Saturation of this enzyme with heme results in enhanced activity, leading to increased catabolism of tryptophan and thus making less tryptophan available to the brain. Tryptophan is also the precursor of melatonin, a primary secretory product of the pineal gland. Reduced melatonin levels, which could be a consequence of altered tryptophan metabolism, have been associated with depressive disorders in humans. In view of this, in the present study we examined the effects of 5-ALA administration on forebrain tryptophan and serotonin levels as well as pineal serotonin, melatonin, and the pineal melatonin-forming enzymes N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT). 5-ALA induced a reduction in forebrain tryptophan and serotonin levels during the light phase and caused the opposite effect in the dark phase. Allopurinol, an inhibitor of hepatic tryptophan pyrrolase activity, prevented the reduction in the indole levels induced by 5-ALA. Although 5-ALA induced these changes in the forebrain, it did not alter either daytime or nighttime pineal NAT, HIMOT, serotonin, or melatonin levels. It appears that high serotonin levels and melatonin production in the pineal are conserved even in the face of low circulating tryptophan levels, changes which alter brain tryptophan and serotonin concentrations.

Abstract: Hepatic tryptophan 2,3-dioxygenase (TDO) is one of the rate-limiting enzymes in tryptophan catabolism and plays an important role in regulating the physiological flux of tryptophan into relevant metabolic pathways. In this study, we determined the effect of the non-steroidal anti-inflammatory agents, tolmetin and sulindac, on rat liver TDO activity and the subsequent changes in the hippocampal and striatal neurotransmitter levels. The amount of melatonin produced by the pineal gland was also measured using high performance liquid chromatography (HPLC). Treatment of rats with tolmetin or sulindac (5 mg/kg/bd for 5 days) significantly inhibited liver TDO activity. The results show that whilst tolmetin and sulindac increase serotonin levels in the hippocampus, these agents also significantly reduce dopamine levels in the striatum. Tolmetin, but not sulindac, increased the amount of melatonin produced by the pineal gland. The results of this study suggest that whilst tolmetin and sulindac may be beneficial for patients suffering from depression, these agents also have the potential to induce adverse effects in patients suffering with neurological disorders such as Parkinson''s disease. [Copyright &y& Elsevier]