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Nitrones have been extensively used for the detection of transient free radicals using electron paramagnetic resonance. Since the mid-80's, nitrones have also been widely used as protective agents against oxidative stress in several biological models. Due to the high potency of nitrones, there has been extensive research on the development of derivatives with improved biological and spin trapping properties as well as enhanced intra-cellular compartmentalization. The chemical and pharmacological properties of nitrones depend mainly on the connectivity as well as on the nature and the position of the substituents on the nitrone group. Therefore, novel bioactive molecules have been designed and the development of specific nitrone derivatives is aimed at providing new therapeutic approaches and perspectives in prevention, treatment and rehabilitation. This review focuses on the effects that are exerted by the most promising nitrone antioxidants that are available. A comprehensive description of the unique molecular mechanism and mediators that are targeted by these compounds is given to guide and enable novel and successful approaches to the treatment of a broad spectrum of diseases associated with stress and aging. New promising nitrone compounds are now available for further development by translational medicine that exert superior bioactivity and efficacy.

The mineralocorticoid receptor (MR) and its ligand aldosterone regulate renal sodium reabsorption and blood pressure and much knowledge has been accumulated in MR physiopathology, cellular and molecular targets. In contrast, our understanding of this hormonal system in non-classical targets (heart, blood vessels, neurons, keratinocytes...) is limited, particularly in the mammalian skin. We review here the few available data that point on MR in the skin and that document cutaneous MR expression and function, based on mouse models and very limited observations in humans. Mice that overexpress the MR in the basal epidermal keratinocytes display developmental and post-natal abnormalities of the epidermis and hair follicle, raising exciting new questions regarding skin biology. The MR as a transcription factor may be an unexpected novel player in regulating keratinocyte and hair physiology and pathology. Because its activating ligand also includes glucocorticoids, that are widely used in dermatology, we propose that the MR may be also involved in the side-effects of corticoids, opening novel options for therapeutical intervention.

Here, we examine the currently available information which supports that the adipokine, leptin, is a major player in the biology and pathology of mammalian skin and its appendages. Specifically, the potent metabolic effects of leptin and its mimetics may be utilized to improve, preserve and restore skin regeneration and hair cycle progression, and may halt or even partially reverse some aspects of skin ageing. Since leptin can enhance mitochondrial activity and biogenesis, this may contribute to the wound healing-promoting and hair growth-modulatory effects of leptin. Leptin dependent intracellular signalling by the Janus kinase 2 dependent signal transducer and activator of transcription 3, adenosine monophosphate kinase, and peroxisome proliferator-activated receptor (PPAR) gamma coactivator/PPAR converges to mediate mitochondrial metabolic activation and enhanced cell proliferation which may orchestrate the potent developmental, trophic and protective effects of leptin. Since leptin and leptin mimetics have already been clinically tested, investigative dermatology is well-advised to place greater emphasis on the systematic exploration of the cutaneous dimensions and dermatological potential of this pleiotropic hormone.

The redox-active quinalphos main metabolite, 2-hydroxyquinoxaline, is particularly effective under excitation by light. We have studied the photocatalytic destruction of melatonin and its precursors, because the cytoprotective indoleamine has been detected in high quantities in mammalian skin. In photooxidation reactions, in which melatonin, N-acetylserotonin and serotonin are destroyed by 2-hydroxyquinoxaline, the photocatalyst is virtually not consumed. Rates of melatonin and serotonin destruction are not changed by the singlet oxygen quencher 1,4-diazabicyclo-(2,2,2)-octane, indicating that this oxygen species is not involved in the primary reactions, so that the persistence of 2-hydroxyquinoxaline has to be explained by redox cycling. This should imply formation of an organic radical, presumably the quinoxaline-2-oxyl radical, from which 2-hydroxyquinoxaline is regenerated by electron abstraction from indolic radical scavengers. Electron donation by 2-hydroxyquinoxaline is demonstrated by reduction of the 2,2'-azino-bis-(3-ethylbenzthiazolinyl-6-sulfonic acid) cation radical under ultrasound excitation. The compound 2-hydroxyquinoxaline interacts with the specific superoxide anion scavenger Tiron. Formation of oligomeric products from melatonin and serotonin is strongly inhibited by sodium dithionite. Products from photocatalytic indolamine conversion are predominantly dimers and oligomers. No kynuramines were detected in the case of serotonin oxidation, and melatonin's otherwise prevailing oxidation product N(1)-acetyl-N(2)-formyl-5-methoxykynuramine, another cytoprotective metabolite, is only formed in relatively small quantities. The proportion between products from melatonin is changed by 1,4-diazabicyclo-(2,2,2)-octane: singlet oxygen, also formed under the influence of excited 2-hydroxyquinoxaline, only affects secondary reactions.

The N-methyl-d-aspartate (NMDA) subtype of glutamate receptors plays an important role in brain physiology, but excessive receptor stimulation results in seizures and excitotoxic nerve cell death. NMDA receptor-mediated neuronal excitation and injury can be prevented by high, non-physiological concentrations of the neuroinhibitory tryptophan metabolite kynurenic acid (KYNA). Here we report that endogenous KYNA, which is formed in and released from astrocytes, controls NMDA receptors in vivo. This was revealed with the aid of the dopaminergic drugs d-amphetamine and apomorphine, which cause rapid, transient decreases in striatal KYNA levels in rats. Intrastriatal injections of the excitotoxins NMDA or quinolinate (but not the non-NMDA receptor agonist kainate) at the time of maximal KYNA reduction resulted in two- to threefold increases in excitotoxic lesion size. Pre-treatment with a kynurenine 3-hydroxylase inhibitor or with dopamine receptor antagonists, i.e., two classes of pharmacological agents that prevented the reduction in brain KYNA caused by dopaminergic stimulation, abolished the potentiation of neurotoxicity. Thus, the present study identifies a previously unappreciated role of KYNA as a functional link between dopamine receptor stimulation and NMDA neurotoxicity in the striatum.

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

A new flavone sulfonic acid 1 named niruriflavone was isolated from the 70% ethanolic extract of the whole plant of Phyllanthus niruri (Euphorbiaceae), together with 6,10,14-trimethyl-2- pentadecanone, hypophyllanthin, gallic acid, brevifolin carboxylic acid, methyl brevifolin carboxylate, isoquercetin, quercetin-3-O-β -D-glucopyranosyl(1→ 4)-α-rhamnopyranoside, corilagin, and isocorilagin, whose structures were determined by spectroscopic methods and comparison with published data. In an ABTS cation radical reduction assay, niruriflavone (1) exhibited potent radical scavenging properties. A biological test system based on bioluminescence of the dinoflagellate Lingulodinium polyedrum did not reveal any prooxidant properties of 1 at 50 μM.

The search for effective treatments that prevent oxidative stress associated with premature ageing and neurodegenerative diseases is an important area of neurochemical research. As age- and disease-related oxidative stress is frequently associated with mitochondrial dysfunction, amphiphilic antioxidant agents of high stability and selectivity that target these organelles can provide on-site protection. Such an amphiphilic nitrone protected human neuroblastoma cells at low micromolar concentrations against oxidative damage and death induced by exposure to the β-amyloid peptide, hydrogen peroxide and 3-hydroxykynurenine. Daily administration of the antioxidant at a concentration of only 5 μm significantly increased the lifespan of the individually cultured rotifer Philodina acuticornis odiosa Milne. This compound is unique in its exceptional anti-ageing efficacy, being one order of magnitude more potent than any other compound previously tested on rotifers. The nitrone protected these aquatic animals against the lethal toxicity of hydrogen peroxide and doxorubicin and greatly enhanced their survival when co-administered with these oxidotoxins. These findings indicate that amphiphilic antioxidants have a great potential as neuroprotective agents in preventing the death of cells and organisms exposed to enhanced oxidative stress and damage.

The melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK) was found to be unstable in air when adsorbed on a thin-layer silica gel chromatography plate, a result that is in good agreement with the relatively high reactivity of this compound. Three novel main products were separated from the reaction mixture and identified by mass spectrometry and nuclear magnetic resonance data as: (i) 3-acetamidomethyl-6-methoxycinnolinone (AMMC), (ii) 3-nitro-AMK (AMNK, N1-acetyl-5-methoxy-3-nitrokynuramine), and (iii) N-[2-(6-methoxyquinazolin-4-yl)-ethyl]-acetamide (MQA). AMMC and AMNK are shown to be nonenzymatically formed also in solution, by nitric oxide (NO) in the first case, and by a mixture of peroxynitrite and hydrogen carbonate, in the second one. The use of three different NO donors, PAPA-NONOate, S-nitroso-N-acetylpenicillamine and sodium nitroprussiate led to essentially the same results, with regard to a highly preferential formation of AMMC; AMNK was not detected in these reaction systems. Competition experiments with the NO scavenger N-acetylcysteine indicate a somewhat lower reactivity compared with the competitor. Peroxynitrite led to AMNK formation in the presence of physiological concentrations of hydrogen carbonate at pH 7.4, but not in its absence, indicating that nitration involves a mixture of carbonate radicals and NO2, formed from the peroxynitrite-CO2 adduct. No AMMC was detected after AMK exposure to peroxynitrite. Both AMNK and AMMC exhibited a much lower reactivity toward 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) cation radicals than did AMK. In a competition assay for hydroxyl radicals, AMMC showed prooxidant properties, whereas AMNK was a moderate antioxidant. AMMC and AMNK should represent relatively stable physiological products, although their rates of synthesis are still unknown and may be low. Formation of these compounds may contribute to the disappearance of AMK from tissues and body fluids.

Quinolinic acid is a well-known excitotoxin that induces oxidative stress and damage. In the present study, oxidative damage to biomolecules was followed by measuring lipid peroxidation and protein carbonyl formation in rat brain tissue culture over a period of 24 hr of exposure to this prooxidant agent at a concentration of 0.5 mm. Quinolinic acid enhanced lipid peroxidation in an early stage of tissue culture, and protein carbonyl at a later stage. These data confirm and extend previous studies demonstrating that quinolinic acid can induce significant oxidative damage. Melatonin, an antioxidant and neuroprotective agent with multiple actions as a radical scavenger and signaling molecule, completely prevented these prooxidant actions of quinolinic acid at a concentration of 1 mm. Morphological lesions and neurotoxicity induced by quinolinic acid were evaluated by light microscopy. Quinolinic acid produced extensive apoptosis/necrosis which was significantly attenuated by melatonin. Cotreatment with melatonin exerted a profound protective effect antagonizing the neurotoxicity induced by quinolinic acid. Glutathione reductase and catalase activities were increased by quinolinic acid and these effects were antagonized by melatonin. Furthermore, melatonin induced superoxide dismutase activity. Quinolinic acid and melatonin acted independently and by different mechanisms in modulating antioxidant enzyme activities. Our findings using quinolinic acid and melatonin clearly demonstrate that such changes should always be seen in the context of oxidative neurotoxicity and antioxidant neuroprotection.

Interactions of melatonin and nicotinamide adenine dinucleotide (NADH) have been studied in different experimental models including NADH-promoted oxyhemoglobin oxidation, vanadate-induced NADH oxidation and paraquat-induced NADH depletion in cultured PC12 cells. Our findings indicate that melatonin preserves NADH levels under oxidative stress both in cell-free systems and in cultured PC12 cells. These interactions likely involve electron donation by melatonin and reduction of the NAD radical. As a result, the NAD radical is recycled to NADH and melatonin is oxidized to N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK). NADH is a central molecule at the crossroads between energy metabolism and the antioxidant defense system in organisms. Recycling of NADH by melatonin might improve the efficiency of NADH as an energy carrier and as an antioxidant. Interactions between melatonin and NADH may be implicated in mitochondrial metabolism.

In numerous experimental systems, the neurohormone melatonin has been shown to protect against oxidative stress, an effect which appears to be the result of a combination of different actions. In this study, we have investigated the possible contribution to radical scavenging by substituted kynuramines formed from melatonin via pyrrole ring cleavage. N1-Acetyl-5-methoxykynuramine (AMK), a metabolite deriving from melatonin by mechanisms involving free radicals, exhibits potent antioxidant properties exceeding those of its direct precursor N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and its analog N1-acetylkynuramine (AK). Scavenging of hydroxyl radicals was demonstrated by competition with ABTS in a Fenton reaction system at pH 5 and by competition with DMSO in a hemin-catalyzed H2O2 system at pH 8. Under catalysis by hemin, oxidation of AMK was accompanied by the emission of chemiluminescence. AMK was a potent reductant of ABTS cation radicals, but, in the absence of catalysts, a poor scavenger of superoxide anions. In accordance with the latter observation, AMK was fairly stable in a pH 8 H2O2 system devoid of hemin. Contrary to AFMK, AMK was easily oxidized in a reaction mixture generating carbonate radicals. In an oxidative protein destruction assay based on peroxyl radical formation, AMK proved to be highly protective. No prooxidant properties of AMK were detected in a sensitive biological test system based on light emission by the bioluminescent dinoflagellate Lingulodinium polyedrum. AMK may contribute to the antioxidant properties of the indolic precursor melatonin.

Melatonin and classic antioxidants possess the capacity to scavenge ABTSb+ with IC50s of 4, 11, 15.5, 15.5, 17 and 21 microm for melatonin, glutathione, vitamin C, trolox, NADH and NADPH, respectively. In terms of scavenging ABTSb+, melatonin exhibits a different profile than that of the classic antioxidants. Classic antioxidants scavenge one or less ABTSb+, while each melatonin molecule can scavenge more than one ABTSb+, probably with a maximum of four. Classic antioxidants do not synergize when combined in terms of scavenging ABTSb+. However, a synergistic action is observed when melatonin is combined with any of the classic antioxidants. Cyclic voltammetry indicates that melatonin donates an electron at the potential of 715 mV. The scavenging mechanism of melatonin on ABTSb+ may involve multiple-electron donations via intermediates through a stepwise process. Intermediates including the melatoninyl cation radical, the melatoninyl neutral radical and cyclic 3-hydroxymelatonin (cyclic 3-OHM) and N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) seem to participate in these reactions. More interestingly, the pH of the solution dramatically modifies the ABTSb+ scavenging capacity of melatonin while pH changes have no measurable influence on the scavenging activity of classic antioxidants. An acidic pH markedly reduces the ABTSb+ scavenging capacity of melatonin while an increased pH promotes the interaction of melatonin and ABTSb+. The major melatonin metabolites that develop when melatonin interacts with ABTSb+ are cyclic 3-OHM and AFMK. Cyclic 3-OHM is the intermediate between melatonin and AFMK, and cyclic 3-OHM also has the ability to scavenge ABTSb+. Melatonin and the metabolites which are generated via the interaction of melatonin with ABTSb+, i.e. the melatoninyl cation radical, melatoninyl neutral radical and cyclic 3-OHM, all scavenge ABTSb+. This unique cascade action of melatonin, in terms of scavenging, increases its efficiency to neutralized ABTSb+; this contrasts with the effects of the classic antioxidants.

Oxidation of melatonin was followed by measuring chemiluminescence emitted during pyrrole ring cleavage, a process leading to the main oxidation product of this indoleamine, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK). Radical reactions of melatonin were studied in two variants of a moderately alkaline (pH 8) H2O2 system, one of which contained hemin as a catalyst. In both systems, light emission from melatonin oxidation lasted several hours. Time courses and turnover rates depended on the presence or absence of hemin; the catalyst enhanced light emission many-fold. In the two reaction systems, the presence of hydrogen carbonate (HCO)(3)(-) enhanced chemiluminescence by more than 10-fold, indicating scavenging of carbonate radicals. In the presence of 10% dimethylsulfoxide (DMSO) or 1 m mannitol, HCO(3)(-)-dependent as well as independent light emissions were only partially inhibited. With regard to the stimulatory effect of HCO(3)(-), this implies a formation of carbonate radicals (CO)(3)(-) independent of hydroxyl (OH) radicals, presumably involving superoxide anions abundantly present in the system. Tiron, a scavenger of superoxide anions, strongly and almost instantaneously inhibited chemiluminescence, in accordance to the requirement of this reactive oxygen species for AFMK formation and its involvement in -radical formation. Melatonin's capability of scavenging CO(3)(-) may contribute to its protective potency.

Melatonin's O-methyl and N-acetyl residues are not only the basis of its amphilicity enabling the molecule to enter all organs and all subcellular compartments, but are also decisive for its antioxidant properties. We have compared melatonin's redox chemistry with that of several structural analogs: tryptamine, N-acetyltryptamine, serotonin, N-acetylserotonin, 5-methoxytryptamine, 6-chloromelatonin and 2-iodomelatonin. Scavenging of hydroxyl radicals (*OH) was measured in a scavenger competition assay based on ABTS cation radical (ABTS(*)+) formation. The capability of undergoing single-electron transfer reactions was studied using an ABTS(*)+ reduction assay, reflecting the more general property of scavenging organic cation radicals. Direct scavenging of superoxide anions (O2(*)-), under non-catalyzed conditions, was investigated in a hematoxylin autoxidation assay. Measurements of chemiluminescence were used for studying scavenging of O2(*)- under catalyzed conditions, either by hemin-mediated interaction or by combination with the respective indolyl cation radicals. Light emission was determined in the absence or presence of the *OH scavenger dimethylsulfoxide and the O2(*)- scavenger Tiron. Products formed by oxidation of the respective indoles in a moderately alkaline, hemin-catalyzed H2O2 system were analyzed by thin-layer chromatography and fluorometry. Absence of either the O-methyl or the N-acetyl residue causes marked diminutions in the capacities of scavenging *OH and ABTS(*)+ as well as in chemiluminescence emitted during oxidation. The importance of the N-acetyl group is insofar remarkable as it seems, at first glance, to be isolated from the indolic moiety; interactions between side chain and indolic moiety are therefore decisive for melatonin's redox properties. The 5-hydroxylated compounds are not generally more efficient scavengers, but particularly better reducers of ABTS(*)+; in the alkaline H2O2 system generating *OH and O2(*)-, melatonin was much more rapidly oxidized than the 5-hydroxylated and non-substituted analogs. Oxidative products formed from any of the compounds studied contained much less of substituted kynuramines as in the case of melatonin, indicating that radical chain termination by O2(*)- is considerably more efficient with melatonin. These findings are supported by measurements of chemiluminescence, which largely reflects pyrrole ring cleavage as a result of combination with superoxide anions. In this regard, only 6-chloromelatonin equalled melatonin, whereas the efficiency of 2-iodomelatonin was much lower, another indication for the importance of 2,3-dioxygenation.

Exposure of neuronal cells to the Alzheimer's amyloid beta protein (Abeta) results in extensive oxidative damage of bio-molecules that are profoundly harmful to neuronal homeostasis. It has been demonstrated that melatonin protects neurons against Abeta-mediated neurotoxicity, including cell death and a spectrum of oxidative lesions. We undertook the current study to determine whether melatonin membrane receptors are involved in the mechanism of neuroprotection against Abeta neurotoxicity. For this purpose, we characterized the free-radical scavenging potency of several compounds exhibiting various affinities for melatonin membrane receptors (MLT 1a and 1b). Abeta-mediated neurotoxicity was assessed in human neuroblastoma cells and in primary hippocampal neurons. In sharp contrast with melatonin, no neuroprotection against Abeta toxicity was observed when we used melatonin membrane receptor agonists that were devoid of antioxidant activity. In contrast, the cells were fully protected in parallel control experiments when either melatonin, or the structurally unrelated free-radical scavenger phenyl-N-t-butyl nitrone (PBN), were added to Abeta-containing culture media. This study demonstrates that the neuroprotective properties of melatonin against Abeta-mediated toxicity does not require binding of melatonin to a membrane receptor and is likely the result of the antioxidant and antiamyloidogenic features of the agent.

Inheritance of apoE4 is a strong risk factor for the development of late-onset sporadic Alzheimer's disease (AD). Several lines of evidence suggest that apoE4 binds to the Alzheimer Abeta protein and, under certain experimental conditions, promotes formation of beta-sheet structures and amyloid fibrils. Deposition of amyloid fibrils is a critical step in the development of AD. We report here that addition of melatonin to Abeta in the presence of apoE resulted in a potent isoform-specific inhibition of fibril formation, the extent of which was far greater than that of the inhibition produced by melatonin alone. This effect was structure-dependent and unrelated to the antioxidant properties of melatonin, since it could be reproduced neither with the structurally related indole N-acetyl-5-hydroxytryptamine nor with the antioxidants ascorbate, alpha-tocophenol, and PBN. The enhanced inhibitory effects of melatonin and apoE were lost when bovine serum albumin was substituted for apoE. In addition, Abeta in combination with apoE was highly neurotoxic (apoE4 > apoE3) to neuronal cells in culture, and this activity was also prevented by melatonin. These findings suggest that reductions in brain melatonin, which occur during aging, may contribute to a proamyloidogenic microenvironment in the aging brain.

Summary. This review summarizes recent advancements in our understanding of the potential role of the amyloid ‚ protein in Alzheimer’s disease. It also discusses the significance of amyloid ‚ in initiating the generation of partially reduced oxygen species and points out their role in damaging essential macromolecules in the CNS which leads to neuronal dysfunction and loss. Recently acquired experimental data links these destructive oxidative processes with some neurodegenerative aspects of Alzheimer’s disease. The experimental findings related to the free radical scavenging and antioxidative properties of melatonin are tabulated and its efficacy and the likely mechanisms involved in its ability to reduce neuronal damage mediated by oxygen-based reactive species in experimental models of Alzheimer’s disease are summarized. Besides the direct scavenging properties and indirect antioxidant actions of melatonin, its ability to protect neurons probably also stems from its antiamyloidogenic properties. Melatonin is also unique because of the ease with which it passes through the blood-brain barrier.

Widespread cerebral deposition of a 40-43-amino acid peptide called the amyloid beta-protein (Abeta) in the form of amyloid fibrils is one of the most prominent neuropathologic features of Alzheimer's disease. Numerous studies suggest that Abeta is toxic to neurons by free radical-mediated mechanisms. We have previously reported that melatonin prevents oxidative stress and death of neurons exposed to Abeta. In the process of screening indole compounds for neuroprotection against Abeta, potent neuroprotective properties were uncovered for an endogenous related species, indole-3-propionic acid (IPA). This compound has previously been identified in the plasma and cerebrospinal fluid of humans, but its functions are not known. IPA completely protected primary neurons and neuroblastoma cells against oxidative damage and death caused by exposure to Abeta, by inhibition of superoxide dismutase, or by treatment with hydrogen peroxide. In kinetic competition experiments using free radical-trapping agents, the capacity of IPA to scavenge hydroxyl radicals exceeded that of melatonin, an indoleamine considered to be the most potent naturally occurring scavenger of free radicals. In contrast with other antioxidants, IPA was not converted to reactive intermediates with pro-oxidant activity. These findings may have therapeutic applications in a broad range of clinical situations.

Tissue levels of the endogenous excitatory amino acid receptor antagonist kynurenic acid (KYNA) and of its bioprecursor L-kynurenine were measured in rats of different ages after d-amphetamine administration. In adult animals, extracellular KYNA concentrations were also determined in vivo by hippocampal microdialysis. In the adult brain, d-amphetamine caused a transient, dose-dependent decrease in tissue content and extracellular levels of KYNA, reaching a nadir of approximately 70% of control values after 1 h at 5 mg/kg. Quantitatively similar decrements were observed in four different brain regions. Seven, 14 and 28-day-old pups were particularly sensitive to the drug, showing a reduction in forebrain KYNA levels to 25%, 40% and 35% of control values, respectively, 1 h after the administration of 5 mg/kg d-amphetamine. Notably, no changes in brain L-kynurenine levels and in liver L-kynurenine and KYNA concentrations were found after d-amphetamine administration. Thus, endogenous monoamines released by d-amphetamine may interfere with the transamination of L-kynurenine to KYNA specifically in the brain. These results suggest that d-amphetamine increases excitatory amino acid receptor function temporarily by reducing the levels of endogenous KYNA.

Several brain-specific mechanisms control the formation of the endogenous excitatory amino acid receptor antagonist kynurenic acid (KYNA) in the adult rat brain. Two of these, dopaminergic neurotransmission and cellular energy metabolism, were examined in the brain of immature (postnatal day 7) rats. The results indicate that during the early postnatal period cerebral KYNA synthesis is exceptionally amenable to modulation by dopaminergic mechanisms but rather insensitive to fluctuations in cellular energy status. These findings may be of relevance for the role of KYNA in the function and dysfunction of the developing brain.

This study was designed to examine the effects of d-amphetamine (D-AMPH) and D1- and D2-selective dopaminergic drugs on the concentration of the broad-spectrum excitatory amino acid receptor antagonist kynurenic acid (KYNA) in the striatum of developing and adult rats. At all ages, KYNA levels were significantly reduced 1 h after the systemic administration of D-AMPH (5 mg/kg). SKF 38393 (5 mg/kg) and quinpirole (2 mg/kg) also caused a rapid decrease in striatal KYNA, but only in postnatal day (PND) 7 and 14 rats. All these effects were readily prevented by specific dopamine receptor antagonists. The possible functional significance of the reduction in KYNA levels was tested in PND 14 animals. When pretreated with D-AMPH (5 mg/kg), these rats showed markedly increased vulnerability to an intrastriatal injection of the excitotoxin NMDA. These data suggest that KYNA plays a role as a mediator of dopamine-glutamate interactions in the rat striatum.

Melatonin protection against ethanol-induced gastroduodenal injury was investigated in duodenum-ligated rats. Melatonin, injected i.p. 30 min before administration of 1 ml of absolute ethanol, given by gavage, significantly decreased ethanol-induced macroscopic, histological and biochemical changes in the gastroduodenal mucosa. Ethanol-induced lesions were detectable as haemorrhagic streaks. Ethanol administration damaged 36% and 25% of the total gastric and duodenal surface, respectively. Melatonin treatment reduced ethanol-induced gastric and duodenal damage to 14% and 8%, respectively. When indomethacin was given together with ethanol, the gastric damaged area was 44% of the total surface, while the duodenal damaged area was 35%; melatonin administration reduced the damage to only 13% of the total gastric surface and to 12% of total duodenal surface. Both stomach and duodenum of ethanol-treated animals showed polymorphonuclear leukocyte (PMN) infiltration. The number of PMN increased more than 600 and 200 times in stomach and duodenum, respectively, following ethanol administration. Melatonin treatment reduced ethanol-induced PMN infiltration by 38% in the stomach and 20% in the duodenum. In indomethacin-ethanol-treated rats, the number of PMN increased by 875% compared to control group in the stomach and by 264% in duodenum. Melatonin administration reduced the indomethacin-ethanol-induced PMN rise by 57% in the stomach and 40% in the duodenum. Gastroduodenal total glutathione (tGSH) concentration and glutathione reductase (GSSG-Rd) activity were significantly reduced following ethanol and indomethacin-ethanol administration. Melatonin ameliorated both the decrease in tGSH concentration as well as the reduction of GSSG-Rd activity elicited by ethanol both in the stomach and duodenum; melatonin was effective against indomethacin-ethanol-induced damage only in the stomach. Ethanol-induced gastroduodenal damage is believed to be mediated by the generation of free radicals. Recently, a number of in vivo and in vitro experiments have shown melatonin to be an effective antioxidant and free radical scavenger; thus, we conclude that the protection by melatonin against ethanol-induced gastroduodenal injury is due, at least in part, to its radical scavenging activity. British Journal of Pharmacology (1997) 121, 264–270; doi:10.1038/sj.bjp.0701104

We evaluated the inhibitory effect of melatonin, a recently discovered scavenger of free radicals, on cataract formation in the newborn rat. The glutathione synthesis inhibitor, buthionine sulfoximine (BSO) (3 mmol/kg), was intraperitoneally injected into newborn rats for 3 consecutive days starting on day 2 after birth. These glutathione depleted rats develop cataracts. Melatonin (4 mg/kg) was injected intraperitoneally into half of the rats once a day beginning at day 2 after birth; the other half of the animals received solvent daily. The incidence of cataract was observed on day 16, after the eyes of the newborn animals had opened. Both reduced glutathione (GSH) and oxidized glutathione (GSSG) levels were measured. Cataracts were observed in all animals (18/18) treated with BSO plus solvent. The incidence of the cataract in the animals cotreated with melatonin was only 6.2% (1/15). Total lenticular glutathione (GSH + GSSG) levels in BSO only treated rats were reduced by 97%. The total glutathione in the lens of the BSO plus melatonin group was significantly higher (by 3%) than that of the BSO only group. The percentage of the total glutathione as GSSG for the BSO plus solvent group was higher than the control value. Cotreatment of BSO injected rats with melatonin (4 mg/kg/day) clearly reduced cataract formation proving that it is directly or indirectly protective against oxidative stress which accompanies glutathione deficiency. The inhibitory effects of melatonin on cataract formation in this study could be due to melatonin's free radical scavenging activity or due to its stimulatory effect on glutathione production.

Recent findings suggest that the ability of melatonin to enter all body tissues and to be metabolized, enzymatically or nonenzymatically, in any of them results in a spectrum of effects, which exceed substantially those transduced by membrane receptors. These actions comprise the formation of various bioactive compounds such as N-acetylserotonin, 5-methoxytryptamine, N,N-dimethyl-5-methoxytryptamine, 5-methoxytryptophol, cyclic 2-hydroxymelatonin, pinoline, and 5-methoxylated kynuramines. Apart from enzymatic metabolism, nonenzymatic reactions with free radicals, in particular the superoxide anion and the hydroxyl radical, represent a new and significant aspect of melatonin's biological role. Melatonin represents the most potent physiological scavenger of hydroxyl radicals found to date, and recent findings suggest an essential role of this indoleamine for protection from hydroxyl radical-induced carcinogenesis and neurodegeneration.

Melatonin inhibits DNA-adduct formation induced by the chemical carcinogen safrole in a dose-dependent manner. Total DNA-adduct formation after in vivo administration of 300 mg/kg safrole measured by 32P-postlabeling analysis of carcinogen-modified DNA in rat liver was 36,751 +/- 2290 counts/min/10 micrograms DNA. Coadministration of 300 mg/kg safrole with either 0.2 mg/kg (low dose) or 0.4 mg/kg (high dose) melatonin reduced DNA-adduct formation induced by safrole to 22,182 +/- 987 counts/min/10 micrograms DNA and 462 +/- 283 counts/min/10 micrograms DNA, respectively. Circulating melatonin concentrations at the termination of the study in safrole, low melatonin and high melatonin groups were 50 +/- 8, 3140 +/- 430 and 10,040 +/- 2610 pg/ml serum, respectively. The results suggest that melatonin protects against safrole associated DNA damage.

Melatonin is a very potent and efficient endogenous radical scavenger. The pineal indolamine reacts with the highly toxic hydroxyl radical and provides on-site protection against oxidative damage to biomolecules within every cellular compartment. Melatonin acts as a primary non-enzymatic antioxidative defense against the devastating actions of the extremely reactive hydroxyl radical. Melatonin and structurally related tryptophan metabolites are evolutionary conservative molecules principally involved in the prevention of oxidative stress in organisms as different as algae and rats. The rate of aging and the time of onset of age-related diseases in rodents can be retarded by the administration of melatonin or treatments that preserve the endogenous rhythm of melatonin formation. The release of excitatory amino acids such as glutamate enhances endogenous hydroxyl radical formation. The activation of central excitatory amino acid receptors suppress melatonin synthesis and is therefore accompanied by a reduced detoxification rate of hydroxyl radicals. Aged animals and humans are melatonin-deficient and more sensitive to oxidative stress. Experiments investigating the effects of endogenous excitatory amino acid antagonists and stimulants of melatonin biosynthesis such as magnesium may finally lead to novel therapeutic approaches for the prevention of degeneration and dysdifferentiation associated with diseases related to premature aging.

Our group has demonstrated that the amphiphilic character of alpha-phenyl-N-tert-butyl nitrone based agents is a key feature in determining their bioactivity and protection against oxidative toxicity. In this work, we report the synthesis of a new class of amphiphilic amide nitrones. Their hydroxyl radical scavenging activity and radical reducing potency were shown using ABTS competition and ABTS(+) reduction assays, respectively. Cyclic voltammetry was used to investigate their redox behavior, and the effects of the substitution of the PBN on the charge density of the nitronyl atoms, the electron affinity, and the ionization potential were computationally rationalized. The protective effects of amphiphilic amide nitrones in cell cultures exposed to oxidotoxins greatly exceeded those exerted by the parent compound PBN. They decreased electron and proton leakage as well as hydrogen peroxide formation in isolated rat brain mitochondria at nanomolar concentration. They also significantly enhanced mitochondrial membrane potential. Finally, dopamine-induced inhibition of complex I activity was antagonized by pretreatment with these agents. These findings indicate that amphiphilic amide nitrones are much more than just radical scavenging antioxidants but may act as a new class of bioenergetic agents directly on mitochondrial electron and proton transport.

Aging is a multi-factorial process, however, it is generally accepted that reactive oxygen species (ROS) are significant contributors. Mitochondria are important players in the aging process because they produce most of the cellular ROS. Despite the strength of the free-radical hypothesis, the use of free radical scavengers to delay aging has generated mixed results in vertebrate models, and clinical evidence of efficacy is lacking. This is in part due to the production of pro-oxidant metabolites by many antioxidants while scavenging ROS, which counteract their potentially beneficial effects. As such, a more effective approach is to enhance mitochondrial metabolism by reducing electron leakage with attendant reduction of ROS generation. Here, we report on the actions of a novel endogenous indole derivative, indolepropionamide (IPAM), which is similar in structure to melatonin. Our results suggest that IPAM binds to the rate-limiting component of oxidative phosphorylation in complex I of the respiratory chain and acts as a stabilizer of energy metabolism, thereby reducing ROS production. IPAM reversed the age-dependent decline of mitochondrial energetic capacity and increased rotifer lifespan, and it may, in fact, constitute a novel endogenous anti-aging substance of physiological importance.

Background The noncompetitive NMDA antagonists phencyclidine (PCP) and dizocilpine (MK-801) have been considered for use as neuroprotective therapeutic agents, although both produce injury in neurons of cingulate and retrosplenial cortices in rodents. The low-affinity, noncompetitive NMDA antagonist dextrorphan has been considered for use as a neuroprotective therapeutic drug. The aim of the present work was to evaluate the neurotoxicity of dextrorphan. Methods Sprague-Dawley male rats were used and injected with either saline or dextrorphan (30 mg/kg i.p.). The animals were sacrificed 30 min later, and the brain was examined for histopathological changes. Results After systemic administration of the drug, hyperchromatic and shrunken nuclei with chromatin condensation and disruption were observed. Also, granular and vacuolated cytoplasm was apparent in pyramidal neurons in the retrosplenial (posterior cingulate) cortex. Status spongiosus (spongy degeneration) of the neuropil was also detected. Conclusions Morphological changes are similar to those described previously, which are induced by high-affinity, noncompetitive NMDA antagonists, such as MK-801.

Melatonin (CAS 73-31-4) has both hypnotic and sleep/wake rhythm regulating properties. These sleep promoting actions, which are already demonstrable in healthy humans, have been found useful in subjects suffering from circadian rhythm sleep disorders (CRSD) like delayed sleep phase syndrome (DSPS), jet lag and shift-work sleep disorder. Low nocturnal melatonin production and secretion have been documented in elderly insomniacs, and exogenous melatonin has been shown to be beneficial in treating sleep disturbances of these patients. In comparison to a number of sleep-promoting compounds that are usually prescribed, such as benzodiazepines and z-drugs (zolpidem and zopiclon belonging to the latter ones), melatonin has several advantages of clinical value: it does not cause hangover nor withdrawal effects and is devoid of any addictive potential. However, recent meta-analyses revealed that melatonin is not sufficiently effective in treating most primary sleep disorders. Some of the reasons for a limited efficacy of this natural hormone are related to its extremely short half-life in the circulation, and to the fact that sleep maintenance is also regulated by mechanisms downstream of primary melatonergic actions. Hence, there is an urgent need for the development of melatonin receptor agonists with a longer half-life, which could be suitable for a successful treatment of insomnia. Such requirements are fulfilled by ramelteon (CAS 196597-26-9), which possesses a high affinity for the melatonin receptors MT1 and MT2 present in the circadian pacemaker, the suprachiasmatic nucleus (SCN). Ramelteon also has a substantially longer half-life than melatonin. This new drug has been successfully used in treating elderly insomniacs without any adverse effects reported, and is promising for treating patients with primary insomnia and also those suffering from CRSD. Since sleep disturbances constitute the most prevalent symptoms of various forms of depression, the need for the development of an ideal antidepressant was felt, which would both improve sleep and mitigate depressive symptoms. Since most of the currently used antidepressants, including the selective serotonin re-uptake inhibitors worsen the sleep disturbances of depressive patients, another novel melatonergic drug, agomelatine (CAS 138112-76-2), holds some promise because of its particular combination of actions: it has a high affinity for MT1 and MT2 receptors in the SCN, but it acts additionally as a 5-HT(2C) antagonist [5-hydroxytryptamine (serotonin) receptor 2C antagonist]. The latter property, which is decisive for the antidepressive action, would not favor but potentially antagonize sleep, but this is overcome during night by the melatonergic, sleep-promoting effect. This drug has been found beneficial in treating patients with major depressive and seasonal affective disorders. Unlike the other antidepressants, agomelatine improves both sleep and clinical symptoms of depressive illness and does not have any of the side effects on sleep seen with other compounds in use. This property seems to be of particular value because of the aggravating effects of disturbed sleep in the development of depressive symptoms. Based on these facts, agomelatine seems to be a drug of superior efficacy with a promising future in the treatment of depressive disorders. However, long-term safety studies are required for both ramelteon and agomelatine, with a consideration of the pharmacology of their metabolites, their effects on redox metabolism, and of eventual undesired melatonergic effects, e. g., on reproductive functions. According to current data, both compounds seem to be safe during short-term treatment

A new series of hydrophilic, lipophilic, and amphiphilic alpha-phenyl-N-tert-butylnitrone (PBN) derivatives were synthesized to explore the relationship between their hydrophilic-lipophilic properties and antioxidant potency. Very potent protective effects of amphiphilic lactobionamide and tris(hydroxymethyl)aminomethane PBN derivatives were observed in mitochondrial preparations, in cell cultures, and in rotifers exposed to unspecific and mitochondria targeted oxidotoxins.

The quinalphos metabolite 2-hydroxyquinoxaline (HQO), previously shown to photocatalytically destroy antioxidant vitamins and biogenic amines in vitro, was tested for toxicity in several small aquatic organisms and for mutagenicity in Salmonella typhimurium. In the rotifer Philodina acuticornis, HQO caused the disappearance of large individuals and increased hydroperoxide concentration. The latter effect was not only observed in animals kept in a light/dark cycle, but also in constant darkness, indicating that HQO can assume a reactive state and/or form reactive intermediates under the influence of either light or redox-active metabolites, in particular, free radicals. Cell proliferation was inhibited in the ciliate Paramecium bursaria. In the dinoflagellate Lingulodinium polyedrum, which allows early detection of cellular stress on the basis of bioluminescence measurements, strong rises in light emission became apparent on the 2nd day of exposure to HQO and continued until cells died between 12 and 18 days of treatment. Oxidative damage of protein by HQO was demonstrated by measuring protein carbonyl in L. polyedrumin vivo as well as in light-exposed bovine serum albumin in vitro. In an Ames test of mutagenicity, HQO proved to be genotoxic in both light- and dark-exposed bacteria. HQO appears as a source of secondary quinalphos toxicity, which deserves further attention.

The use of classical antioxidants is limited by their low bioavailabilities, and therefore, high doses are usually required to display significant protective activity. In a recent article (J. Med. Chem. 2003, 46, 5230) we showed that the ability of the alpha-phenyl-N-tert-butylnitrone (PBN) to restore the viability of ATPase-deficient human skin fibroblasts was greatly enhanced by grafting it on a fluorinated amphiphilic carrier. With the aim of extending this concept to other antioxidants, we present here the design, the synthesis, and the physicochemical measurements of a new series of fluorinated amphiphilic antioxidant derivatives. The hydroxyl radical scavenging activity and the radical reducing potency of these newly designed compounds were respectively demonstrated in an ABTS competition and an ABTS(*+) reduction assay. We also showed that the protective effects of amphiphilic antioxidants derived from PBN, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) or lipoic acid (5-[1,2]-dithiolan-3-ylpentanoic acid) in primary cortical mixed cell cultures exposed to oxidotoxins are greatly improved compared to their parent compounds in the following rank-order: (1) PBN, (2) Trolox, and (3) lipoic acid. In contrast, the protective activity of indole-3-propionic acid was slightly decreased by grafting it on the amphiphilic carrier. Similar observations were made in in vivo experiments using aquatic invertebrate microorganisms, called rotifers, which were exposed to lethal concentrations of nonselective (H(2)O(2)) and mitochondria-selective (doxorubicin) oxidotoxins. The conclusion of these studies is that fluorinated amphiphilic PBN, Trolox, and lipoic acid derivatives exhibit very potent protective activities in in vitro and in vivo experiments. The findings demonstrated herein therefore strongly suggest that the amphiphilic character enhances the bioavailability of the antioxidants and allows for a selective targeting of mitochondria.

Toxicity of the pesticide quinalphos may comprise secondary, delayed effects by its main metabolite 2-hydroxyquinoxaline (HQO). We demonstrate that HQO can destroy photocatalytically vitamins C and E, catecholamines, serotonin, melatonin, the melatonin metabolite AMK (N(1)-acetyl-5-methoxykynuramine), and unsubstituted and substituted anthranilic acids when exposed to visible light. In order to avoid HQO-independent ascorbate oxidation by light and to exclude actions by hydroxyl radicals, experiments on this vitamin were carried out in ethanolic solutions. Other substances tested (vitamin E, melatonin, anthranilic acids) were also photocatalytically destroyed by HQO in ethanol. After product analyses had indicated that HQO was not, or only poorly, degraded in the light, despite its catalytic action on other compounds, we followed directly the time course of HQO and ascorbate concentrations in ethanol. While ascorbate was largely destroyed, no change in HQO was demonstrable within 2 h of incubation. Destruction was not prevented by the singlet oxygen quencher DABCO. Obviously, HQO is capable of undergoing a process of organic redox cycling, perhaps via an intermediate quinoxaline-2-oxyl radical. Health problems from HQO intoxication may not only arise from the loss of valuable biomolecules, such as antioxidant vitamins and biogenic amines, but also from the formation of potentially toxic products. Dimerization and oligomerization are involved in several oxidation processes catalyzed by HQO, especially in the indoleamines, in dopamine, and presumably also in vitamin E. Melatonin oxidation by HQO did not only lead to the well-known - and usually protective - metabolite AFMK (N(1)-acetyl-N(2)-formyl-5-methoxykynuramine), but also to a high number of additional products, among them dimers and trimers. DABCO did not prevent melatonin destruction, but changed the spectrum of products. Serotonin was preferentially converted to a dimer, which can further oligomerize. Several indole dimers are known to be highly neurotoxic, as well as oxidation products formed from catecholamines via the adrenochrome/noradrenochrome pathway. Destruction of melatonin may cause deficiencies in circadian physiology, in immune functions and in antioxidative protection.

Melatonin protection against H2O2-induced lipid peroxidation in brain homogenates was measured in vitro. The level of malonaldehyde (MDA) plus 4-hydroxyalkenals (4-HDA) was assayed in brain homogenates as an index of induced membrane oxidative damage. Brain homogenates were co-incubated with H2O2 alone or in combination with melatonin. Brain MDA + 4-HDA increased after H2O2 (0.1-5 mM) with the effect being both concentration- and time-dependent. Melatonin (0.1-5 mM) protected against H2O2-induced lipid peroxidation of brain homogenates in a concentration-dependent manner.

The hydroxyl radical scavenging activity of indole-3-propionate was evaluated by kinetic competition studies with the hydroxyl radical trapping reagent 2,2'-azino-bis-(3-ethyl-benz-thiazoline-6-sulfonic acid) (ABTS) and by measuring hydroxyl radical-initiated lipid peroxidation in the rat striatum. Using ABTS, the indole was shown to act as a potent hydroxyl radical scavenger with a rate constant of 7.8x1010 mol l-1 s-1. Hydroxyl radical-initiated lipid peroxidation, determined by measuring tissue malondialdehyde formation, was inhibited dose-dependently both in vitro and in vivo. Indole-3-propionate reacts with hydroxyl radicals at a diffusion controlled rate and can thereby provide on-site protection against the oxidative damage of biomolecules induced by these highly reactive and toxic oxygen intermediates. While it remains to be established if endogenous brain tissue levels of indole-3-propionate are sufficiently high to have a significant impact on total antioxidative capacity, the compound itself or a structurally related agent may be useful as an antioxidant adjuvant to combat hydroxyl radical-mediated oxidative stress.

Using in vivo and in vitro paradigms, the regulation and function of the brain metabolite kynurenic acid (KYNA) was examined in rats on postnatal days (PND) 7 and 14. As shown previously in adult rats, glucose removal and d-amphetamine (d-Amph) administration caused decreases in KYNA formation, while exposure to pyruvate up-regulated KYNA synthesis. The effect of glucose deprivation was substantially blunted in immature animals. In PND 14 rats, d-Amph pre-treatment exacerbated the excitotoxic effects of an intrastriatal N-methyl-D-aspartate (NMDA) injection. This potentiation was prevented by m-nitrobenzoylalanine, a kynurenine 3-hydroxylase inhibitor that also antagonized the KYNA reduction caused by d-Amph. These and additional experiments with the competitive NMDA receptor antagonist CGP 40116 indicate the existence of a functionally significant, novel high-affinity receptor for KYNA in the brain.

Melatonin is being increasingly promoted as a treatment for "jet lag" and insomnia and has been suggested to act as an antioxidant in vivo. The antioxidant and potential pro-oxidant activities of melatonin were investigated in vitro. Melatonin was able to scavenge hypochlorous acid (HOCl) at a rate sufficient to protect catalase against inactivation by this molecule. Melatonin could also prevent the oxidation of 5-thio-2-nitrobenzoic acid by HOCl. Melatonin decreased the peroxidation of ox-brain phospholipids with a calculated IC50 of (210 +/- 2.3) microM. In contrast, serotonin which also scavenged HOCl, was much more effective in decreasing phospholipid peroxidation (IC50 15 +/- 5 microM). Both compounds reacted with trichloromethylperoxyl radical (CCl3O2) with rate constants of (2.7 +/- 0.2) x 10(8) and (1.2 +/- 0.1) x 10(8)M-1 s- respectively. Melatonin did not scavenge superoxide radical and weakly protected DNA against damage by the ferric bleomycin system. By contrast serotonin was weakly pro-oxidant in the ferric-bleomycin system and strongly pro-oxidant in the Fe(3+)-EDTA/H2O-deoxyribose system. Solubility restrictions precluded examination of melatonin in this system. Our data show that melatonin exerts only limited direct antioxidant activities.

Because of the overexpression of the enzyme superoxide dismutase, individuals with Down syndrome (DS) are believed to suffer from increased oxidative stress as a result of the excessive production of oxygen-based free radicals; their exposure to higher than normal free radical production may account in part for signs of premature aging, early onset of cataracts, and of Alzheimer's disease. Free radicals are normally neutralized by free radical scavengers and other antioxidants. The pineal hormone melatonin is a potent scavenger of both the hydroxyl and peroxyl radicals, both of which are highly toxic, and a stimulator of the antioxidative enzyme glutathione peroxidase. Considering this, we deemed it important to define the day/night rhythm and levels of melatonin production in DS subjects. To do this, we assessed the urinary excretion of the chief melatonin metabolite, 6-hydroxymelatonin sulfate, throughout a 24 hr period in DS subjects; comparisons were made with the metabolite levels in the urine of non-Down siblings and parents of the DS subjects. All 8 non-Down subjects exhibited what was classified as normal urinary excretion of 6-hydroxymelatonin sulfate with the usual low daytime and high night-time levels of the melatonin metabolite. Of 12 DS subjects studied, 10 exhibited the normal day/night rhythm in urinary 6-hydroxymelatonin sulfate levels; 2 subjects were devoid of a rhythm. However, when all the data from each group were averaged, there were no noticeable differences in the absolute levels or 24 hr variations in urinary 6-hydroxymelatonin sulfate excretion between DS and non-Down subjects.

The hormone synthesized by the pineal gland, melatonin, has been shown to be a direct free radical scavenger both in vivo and in vitro. Thus, it potently protects cells from the damage induced by oxidative agents. In this study, we demonstrate that melatonin increases glutathione peroxidase activity in several tissues from chicks. This stimulation is time dependent and maximal increases are seen 90 min after melatonin injection (500 micrograms/kg intraperitoneally), although enzymatic activity is still elevated 135 min after its administration. No significant increases were detected 45 min after the injection. Glutathione peroxidase is generally considered to be an important antioxidative enzyme because it metabolizes hydrogen peroxide and other hydroperoxides. Thus, melatonin not only is a direct scavenger of toxic radicals but in an avian species, as in mammals, it stimulates the antioxidative enzyme glutathione peroxidase. The ability of melatonin to increase glutathione peroxidase activity is consistent with its general role as an antioxidant.

The ability of melatonin to modify H 2 O 2 -induced lipid peroxidation in brain homogenates was determined. The concentrations of brain malonaldehyde (MDA) and 4-hydroxyalkenals (4-HDA) were assayed as an index of induced membrane oxidative damage. Homogenates from five different regions of the brain (cerebral cortex, cerebellum, hippocampus, hypothalamus, and corpus striatum) derived from two different strains of rats, Sprague-Dawley and Wistar, were incubated with either H 2 O 2 (5 mM) alone or H 2 O 2 together with melatonin at increasing concentrations ranging from 0.1 to 4 mM. The basal level of lipid peroxidation was strain-dependent and about 100% higher in homogenates from the brain of Wistar rats than those measured in Sprague-Dawley rats. MDA + 4-HDA levels increased after H 2 O 2 treatment in homogenates obtained from each region of the brain in both rat strains but the sensitivity of the homogenates from Sprague-Dawley rats was greater than that for the homogenates from Wistar rats (increases after H 2 O 2 from 45 to 165% compared 20 to 40% for Sprague-Dawley and Wistar rats, respectively). Melatonin co-treatment reduced H 2 O 2 -induced lipid peroxidation in brain homogenates in a concentration-dependent manner ; the degree of protection against lipid peroxidation was similar in all brain regions.

Exogenously administered melatonin causes a 2-fold rise in glutathione peroxidase activity within 30 min in the brain of the rat. Furthermore, brain glutathione peroxidase activity is higher at night than during the day and is correlated with high night-time tissue melatonin levels. Glutathione peroxidase is thought to be the principal enzyme eliminating peroxides in the brain. This antioxidative enzyme reduces the formation of hydroxyl radicals formed via iron-catalyzed Fenton-type reactions from hydrogen peroxide by reducing this oxidant to water. Since the hydroxyl radical is the most noxious oxygen radical known, induction of brain glutathione peroxidase might be an important mechanism by which melatonin exerts its potent neuroprotective effects.

Melatonin is widely abundant in many eukaryotic taxa, including vari-ous animal phyla, angiosperms, and unicells. In the bioluminescent dinoflagellate Gonyaulax polyedra, melatonin is produced in concentrations sometimes exceed-ing those found in the pineal gland, exhibits a circadian rhythm with a pro-nounced nocturnal maximum, and mimics the short-day response of asexual encystment. Even more efficient as a cyst inducer is 5-methoxytryptamine (5MT), which is also periodically formed in Gonyaulax. In this unicell, the photoperiodic signal-transduction pathway presumably involves melatonin formation, its deace-tylation to 5MT, 5MT-dependent transfer of protons from an acidic vacuole, and cytoplasmic acidification. According to this concept, we observe that cyst forma-tion can be induced by various monoamine oxidase inhibitors and protonophores, that 5MT dramatically stimulates H+-dependent bioluminescence and leads to a decrease of cytoplasmic pH, as shown by measurements of dicyanohydroquinone fluorescence. Cellular components from Gonyaulax catalyze the photooxidation of melatonin. Its property of being easily destroyed by light in the presence of cel-lular catalysts may have been the reason that many organisms have developed mechanisms utilizing this indoleamine as a mediator of darkness. Photooxidative reactions of melatonin, as studied with crude Gonyaulax extracts and, more in de-tail, with protoporphyrin IX as a catalyst, lead to the formation of N1 -acetyl-N -formyl-5-methoxykynuramine (AFMK) as one of the main products. Photochemical mechanisms involve interactions with a photooxidant cation radi-cal leading to the formation of a melatonyl cation radical, which subsequently combines with a superoxide anion. Photooxidation of melatonin represents one of several possibilities of a more general, biologically highly important property of this indoleamine to act as an extremely efficient radical scavenger, including its feature of terminating radical reaction chains by a final combination with the su-peroxide anion. Trapping of free radicals may reflect the primary and evolutionar-ily most ancient role of melatonin in living beings.

The effect of various concentrations of melatonin on the growth of ME-180 human cervical cancer cells in vitro was examined. Melatonin at a concentration of 2 mM inhibited the growth of the cells after 48 h of melatonin treatment. At concentrations of 2 μM or 0.1 mM melatonin had no effect on cell proliferation. To determine whether the inhibitory effect of melatonin on the growth of the cervical cancer cells was linked to intracellular glutathione concentrations, experiments were performed in which intracellular glutathione levels were depressed by the addition of buthionine sulfoximine to the incubation medium 24 h before the addition of melatonin. The results show that 2 mM melatonin treatment still inhibits the growth of cells when glutathione levels are depressed by 95%. Even with depressed glutathione levels, 0.1 mM melatonin still had no effect on cell growth. Thus, melatonin's ability to inhibit ME-180 cervical cell growth in vitro may be independent of intracellular glutathione concentrations. It was also found that during one passage the intracellular glutathione levels of cervical cancer cells gradually decreases. When 4.5-day-old medium was replaced with new medium, intracellular glutathione levels partially recovered within 36 h. This suggests that the observed gradual reduction of cellular glutathione during incubation was a result of a reduction of some constituent in the medium after prolonged culture of the cells.

This survey summarizes the findings, accumulated within the last 2 years, concerning melatonin's role in defending against toxic free radicals. Free radicals are chemical constituents that have an unpaired electron in their outer orbital and, because of this feature, are highly reactive. Inspired oxygen, which sustains life, also is harmful because up to 5% of the oxygen (O2) taken in is converted to oxygen-free radicals. The addition of a single electron to O2 produces the superoxide anion radical (O2-.); O2-. is catalytic-reduced by superoxide dismutase, to hydrogen peroxide (H2O2). Although H2O2 is not itself a free radical, it can be toxic at high concentrations and, more importantly, it can be reduced to the hydroxyl radical (.OH). The .OH is the most toxic of the oxygen-based radicals and it wreaks havoc within cells, particularly with macromolecules. In recent in vitro studies, melatonin was shown to be a very efficient neutralizer of the .OH; indeed, in the system used to test its free radical scavenging ability it was found to be significantly more effective than the well known antioxidant, glutathione (GSH), in doing so. Likewise, melatonin has been shown to stimulate glutathione peroxidase (GSH-Px) activity in neural tissue; GSH-PX metabolizes reduced glutathione to its oxidized form and in doing so it converts H2O2 to H2O, thereby reducing generation of the .OH by eliminating its precursor. More recent studies have shown that melatonin is also a more efficient scavenger of the peroxyl radical than is vitamin E. The peroxyl radical is generated during lipid peroxidation and propagates the chain reaction that leads to massive lipid destruction in cell membranes. In vivo studies have demonstrated that melatonin is remarkably potent in protecting against free radical damage induced by a variety of means. Thus, DNA damage resulting from either the exposure of animals to the chemical carcinogen safrole or to ionizing radiation is markedly reduced when melatonin is co-administered. Likewise, the induction of cataracts, generally accepted as being a consequence of free radical attack on lenticular macromolecules, in newborn rats injected with a GSH-depleting drug are prevented when the animals are given daily melatonin injections. Also, paraquat-induced lipid peroxidation in the lungs of rats is overcome when they also receive melatonin during the exposure period. Paraquat is a highly toxic herbicide that inflicts at least part of its damage by generating free radicals.(ABSTRACT TRUNCATED AT 400 WORDS)