ORI GI N A L P A PE R

Melatonin: New Places in Therapy

Deepa S. Maharaj Æ Beverley D. Glass Æ Santy Daya

Published online: 7 September 2007� The Biochemical Society 2007

Abstract The fact that the full extent of the function of the pineal gland has not yetbeen elucidated, has stimulated melatonin research worldwide. This review introducesmelatonin’s mechanism of action, direct and indirect antioxidant actions as well as theantioxidant properties of its metabolites, 6-hydroxymelatonin (6-OHM) and N-acetyl-N-formyl-5-methoxykynurenamine (AFMK). At present the mechanism of action isproposed to be receptor-, protein- and nonprotein-mediated. From its popular role inthe 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 inrespect 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 itsmetabolites, AFMK and 6-OHM will be presented. It is these free radical scavengingand antioxidant properties of melatonin that has shifted the focus from that of merelystrengthening circadian rhythms to that of neuroprotectant: a new place in therapy.

Keywords Melatonin � Circadian rhythm � Jetlag �Free radical scavenger � Antioxidant � Neuroprotection

D. S. Maharaj � S. Daya (&)Faculty of Pharmacy, Rhodes University, P.O. Box 94, Grahamstown 6140, South Africae-mail: s.daya@ru.ac.za

B. D. GlassSchool of Pharmacy and Molecular Sciences, James Cook University, Townsville,QLD 4811, Australia

Biosci Rep (2007) 27:299–320DOI 10.1007/s10540-007-9052-1

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Abbreviation

6-OHM 6-hydroxymelatoninAFMK N-acetyl-N-formyl-5-methoxykynurenamineGPx Glutathione peroxidaseGRd Glutathione reductaseSOD Superoxide dismutaseNOS Nitic oxide synthaseCaCaM Ca2+-calmodulinNO• Nitric oxideiNOS inducible Nitric oxide synthetase•OH Hydroxyl radicalO��2 Superoxide anions

H2O2 Hydrogen peroxideCAT CatalaseTHA Terephthalic acidDHBA Dihydroxybenzoic acid1O2 Singlet oxygenLOO• Peroxyl radicalsONOO– Nitric oxide/peroxynitrite anionsG6PD Glucose-6-phosphate dehydrogenaseGSH Reduced glutathioneAD Alzheimers diseasePD Parkinsons disease

Introduction

Most scientists believe the pineal gland to be a relic of the early days of humandevelopment, eventually becoming obsolete due to evolution. After centuries ofdisregard, the pineal gland has in the last decade been acknowledged as an importantfunctional neuroendocrine gland. The pineal gland was considered to be the regulator ofthe flow of the spirit, and thus the philosopher, Rene Descartes, proclaimed that thepineal gland was the seat of the soul. The full extent of its function is however as yetunknown, resulting in worldwide research in an attempt to elucidate the role of thepineal gland in the mammalian body with melatonin, the focus of the larger part of thispineal research. The present review introduces the neuroprotective properties ofmelatonin and its metabolites, 6-hydroxymelatonin (6-OHM) and N-acetyl-N-formyl-5-methoxykynurenamine (AFMK) and draws attention to its use in clinical practice.Although melatonin, because of being identified in bovine pineal tissue has previouslybeen classified as a hormone, recent studies by Tan et al. (2003) include classificationsboth as a vitamin and an antioxidant.

From a phylogenetic point of view, melatonin is a molecule present in organismsfrom unicells to mammals. Since tryptophan and serotonin, both precursors ofmelatonin, are present at the early stages of cell phylogeny, the presence of melatoninis also suggested. Tryptophan metabolites including melatonin are antioxidants, and

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from a hypothetical point of view, the first function of melatonin may have been as anantioxidant (Acuna-Castroviejo et al. 2001). Melatonin has thus evolved as anantioxidant and vitamin and where it is produced in humans for example has inaddition acquired autocoid, paracoid and hormonal properties (Tan et al. 2003).

The discovery of different targets in the cell suggests the mechanism of action tooccur by (Acuna-Castroviejo et al. 2001):

(a) receptor-mediated,(b) protein-mediated(c) and non protein-mediated effects

Receptor-mediated melatonin events involve both membrane and nuclear receptors.Although membrane melatonin receptors have been identified and are well character-ized in humans (Conway et al. 2000), some of the receptor-related antioxidant effects ofmelatonin are also seen to be related to its nuclear receptors (Becker-Andre et al. 1994;Garcia-Maurino et al. 2000).

The expression of some enzymes, mainly related to the endogenous antioxidantsystem of the cell, such as glutathione peroxidase (GPx), glutathione reductase (GRd),superoxide dismutase (SOD) and nitic oxide synthase (NOS) (Antolin et al. 1996;Crespo et al. 1999), are under genomic regulation by melatonin. Melatonin has beenreported to influence both antioxidant enzyme activity and cellular mRNA levels forthese enzymes both under physiological conditions and those conditions of elevatedoxidative stress (Rodriguez et al. 2004). An interaction between membrane and nuclearmelatonin signaling has been thus proposed (Carlberg and Wiesenberg 1995).

Experimental evidence has clearly demonstrated the interaction of melatonin withCa2+-calmodulin (CaCaM), a ubiquitous protein in the cell with high affinity binding ofmelatonin to CaCaM characterised (Romero et al. 1998). Melatonin is thought tomodulate the CaCaM signaling pathway either by changing the intracellular Ca2+

concentration via activation of its G-protein-coupled membrane receptors, or through adirect interaction with CaCaM (Turjanski et al. 2004). The significance of the melatonin-CaCaM interaction was emphasized in a series of experiments showing changes in thecytoskeletal rearrangements due to this interaction (Benitez-King 2000). Also the bindingof melatonin to CaCaM inhibits intracellular CaCaM-dependent enzymes such as NOS(Leon et al. 2000). Hence, melatonin inhibits nitric oxide (NO•) production. In a recentreport, authors have shown that melatonin decreased NO• concentration due to thesuppression of inducible nitric oxide synthetase (iNOS) expression (Zhang et al. 2004).

Melatonin and Oxidation Stress

Introduction

Of significance is that melatonin levels decrease with increasing age such that, in theelderly, melatonin concentrations in the blood are only a fraction of those in the young(Reiter 1992). Undoubtedly the aging process is multi-factorial, and no single factor hasbeen identified, which satisfactorily explains this phenomenon. Although many theoriesrelating the pineal gland and its secretory product melatonin to aging, have beenproposed, the role of this agent in the aging process is still unclear (Karasek 2004).However, for several reasons it seems reasonable to postulate a role for melatonin inthis process. Melatonin levels decline gradually over the life-span and may be related to

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lowered sleep efficacy, very often associated with advancing age, as well as todeterioration of many circadian rhythms. Melatonin exhibits immunomodulatoryproperties, and a remodeling of the immune system function is an integral part ofaging. Finally, because melatonin is a potent free radical scavenger, its deficiency mayresult in reduced antioxidant protection in the elderly which may have significance notonly for aging per se but also may contribute to the incidence or severity of some age-related diseases (Karasek 2004). Functionally, melatonin has been linked to theregulation of seasonal reproduction (Reiter 1980; Pang et al. 1998), strengthening ofcircadian rhythms (Ardelt 1989), stimulation of the immune system (Guerrero andReiter 2002; Maestroni 2001), inhibition of cancer initiation (Karbownik and Reiter2002), tumour growth (Sauer et al. 2001; Vladimir et al. 2003), sleep processes (Dijk andCajochen 1997), and the potential for use in photodynamic therapy in the destruction oftumors (Perotti et al. 2002; Maharaj et al. 2005a). That melatonin functions as apowerful free radical scavenger and antioxidant was only uncovered in the last decade(Tan et al. 1993a; Hardeland et al. 1995; Reiter et al. 2002). Considering the diminishedmelatonin production in aged organisms (Reiter 1992), the functions of melatonin arelikewise attenuated in the elderly.

Melatonin and Free Radicals

In the last 10–12 years, many reports have documented melatonin’s ability to directlyneutralize free radicals and related toxicants. When injected into animals or given orally,melatonin levels quickly rise in the blood and, this is followed shortly thereafter, by itsuptake into tissue (Menendez-Pelaez et al. 1993; Menendez-Pelaez and Reiter 1993). Thereare no morphophysiological barriers to melatonin; this is apparent in reference to the brainwhere melatonin concentrations increase soon after peripheral administration of theindoleamine (Menendez-Pelaez et al. 1993). The first indication that melatonin may be adirect free radical scavenger actually appeared in 1991, but the details relating to what wasdone and the specific findings are difficult to unravel because of incomplete methodologicaldetails (Ianas et al. 1991). However, the authors did conclude, that melatonin possesses bothantioxidant and pro-oxidant activity, a feature common to a number of so-calledantioxidants. Two years later, Tan et al. (1993a, b) provided strong evidence that melatoninwas highly effective in detoxifying the highly reactive hydroxyl radical (•OH).

Melatonin: Direct Scavenging Actions

Melatonin and Superoxide Anions ( O��2 )

The efficacy of melatonin in neutralising O��2 is only poorly defined. Melatonin hasbeen shown to be minimally reactive with O��2 (Chan and Tang 1996; Marshall et al.1996), although a study in which electron spin resonance was used to identifyDMPO(5,5–dimethylpyrroline-N-oxide)- O��2 adducts, melatonin was reported tomodestly interact with O��2 (Zang et al. 1998). However, the role, if any, of melatoninin neutralising the O��2 is unclear, particularly in vivo.

Melatonin and Hydrogen Peroxide (H2O2)

Melatonin has been shown to remove H2O2 in at least three different ways; it stimulatesthe activities of the two H2O2-metabolizing enzymes, GPx and catalase (CAT) (Pablos

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et al. 1995; Montilla et al. 1997; Reiter et al. 2000b, c) and it directly interacts with H2O2

to remove it from the cell (Zang et al. 1998). Recent evidence, however has uncovered apathway whereby melatonin directly interacts with H2O2 to diminish its levels in a purechemical system (Tan et al. 2000a, b). The product that results from the melatonin-H2O2 interaction is AFMK (Tan et al. 2001). Additionally, AFMK was shown to becapable of donating two electrons and, therefore, being a direct free radical scavenger inits own right (Reiter et al. 2002). In specific experiments, AFMK reduced DNA damageinduced by a combination of H2O2 and the transition metal Cr3+, and also limited thedestruction of lipids resulting from their exposure to H2O2 and other transition metals,such as Fe2+ (Tan et al. 2001). Hence, not only melatonin but also at least one of itsmetabolites is highly efficient in reducing free radical damage. This is referred to as theantioxidant cascade of melatonin; a process that greatly increases the efficiency of thisubiquitously acting free radical scavenger and antioxidant.

Melatonin and •OH

Definitive evidence that melatonin is a direct scavenger of the toxic •OH was providedby Tan et al. (1993a) and Poeggeler et al. (1994). For these studies H2O2 was exposed to254 nm ultraviolet light to generate •OH which was captured with the spin-trappingagent, DMPO using a well defined cell free system. The resulting adducts (•OH-DMPO)were identified and quantified using electron spin resonance spectroscopy, widelyaccepted as the most definitive method for identifying such adducts. When melatoninwas added to the mixture in increasing concentrations, it progressively reduced •OH-DMPO, proving it had scavenged •OH rendering it no longer available for adductformation. Tan et al. (1993a) compared melatonin to two well-known •OH scavengers,glutathione and mannitol, and found that melatonin was significantly better than thesetwo agents. Structure activity relationship studies revealed that both the acetyl group onthe side chain and the methoxy group at position five of the indole nucleus wereimportant for melatonin’s •OH scavenging ability (Fig. 1). Thus, melatonin scavenges•OH by contributing an electron, thereby rendering the radical non-reactive, butbecoming itself a radical, the indolyl cation radical (Reiter et al. 1996). This product isnot very reactive and is therefore non-toxic to the cell (Lewis et al. 1990). It is believedthat this indolyl cation radical then scavengers the O��2 , forming AFMK which isexcreted in the urine.

In 1998, it was shown that each melatonin molecule actually scavenged two •OHs andgenerated the product, cyclic-3-hydroxymelatonin (Tan et al. 1998). These authors alsoindicated that cyclic 3-hydroxymelatonin is a footprint molecule that appears in theurine and an index of in vivo scavenging by melatonin. Many other studies haveconfirmed melatonin’s ability to detoxify •OH (Matuszak et al. 1997; Poeggeler et al.2002). The calculated biomolecular rate constant for the melatonin/•OH reaction is0.6 · 1011 M–1 S–1 (Poeggeler et al. 1996).

N

CH3O CH2CH2NHCOCH35

HFig. 1 Chemical structure of melatonin showing the structure activity relationship studies

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The •OH scavenging ability of melatonin in vitro has also been confirmed by Pahklaet al. (1998) who used terephthalic acid (THA) as a chemical dosimeter of •OH, since itforms the adduct, THA-•OH (Barreto et al. 1994). In this system, the reduction of theformation of this adduct by melatonin was concentration dependent.

Poeggeler et al. (1994, 1995, 1996) further showed that melatonin is also an efficientradical scavenger in other in vitro systems. By measuring changes in indole fluores-cence, they showed that melatonin was rapidly oxidized by •OH generated with Fentonreagents but not by iron itself. Likewise, they found that melatonin was oxidised in thepresence of H2O2 only and found that melatonin was synergistic with other antioxidantsfor example, vitamins C and E, in the scavenging of radicals. This is an importantobservation, because it suggests that in vivo, particularly in the presence of other freeradical scavengers, melatonin could have a role as a physiologically relevant antioxidant.

Evidence that exogenously administered melatonin acts in vivo to scavenge •OH hasbeen provided by Li et al. (1997). This group used the salicylate trapping method toshow that melatonin administration to rats undergoing ischemia-reperfusion of the brainreduced dihydroxybenzoic acid (DHBA) in the microdialysate retrieved from theischemic brain. DHBA is a specific product formed by the interaction of the •OH andsalicylate and its reduction indicates that melatonin scavenged •OH thereby leading to areduced DHBA formation. This was the first evidence showing that melatonin functionsas a •OH quencher in vivo.

The significance of melatonin as an •OH scavenger relates to the fact that thisreactant is generally considered the most damaging of all endogenously generatedreactive agents. Once produced it plunders any molecule it encounters in its immediatevicinity (Reiter et al. 2002). Indeed, its high reactivity prevents it from moving morethan a few molecular diameters from where it was produced before it biochemicallyalters a neighbouring molecule. Thus, for any scavenger to combat •OH-mediateddamage, it must be at the site where the radical is produced to prevent its destructiveactions. Unlike some other well known antioxidants that are exclusively lipid (e.g.vitamin E) or water (e.g. vitamin C) soluble and, therefore, exhibit a limitedintracellular distribution, melatonin is amphiphilic allowing it to reduce •OH-mediateddamage both in lipid and aqueous subcellular compartments (Reiter et al. 2002).Evidence has shown that melatonin is clearly highly soluble in a lipid-based medium(Costa et al. 1997) and it has been shown to dissolve to some extent in an aqueousmedium (Shida et al. 1994).

Stasica et al. (2000), using a computational approach, determined the most likelyprobable site on the indole ring of melatonin that may bind a •OH; the C2 carbon wasproposed as the likely site of attack. Previous studies have established that •OH reactsvery rapidly with melatonin in aqueous solution (Matuszak et al. 1997) to form anumber of metabolites. The secondary and tertiary metabolites formed in vitro andin vivo, for example, 6-OHM, N-acetyl-5-methoxykynuramine and AFMK (Horstmanet al. 2002) believed to be generated when melatonin interacts with free radicals, arealso regarded as effective free radical scavengers (Tan et al. 2000b, Maharaj et al. 2002,2003a, b, 2005b). Melatonin metabolites are able to neutralize O2 by-products like theirparent compound, melatonin (Reiter et al. 2002). This effect of melatonin and itsmetabolites has been referred to as the antioxidant cascade and allows melatonin and itsmetabolites to scavenge additional radicals beyond what the parent compound,melatonin, is capable of doing (Tan et al. 2000b; Maharaj et al. 2002). This metaboliccascade permits melatonin to directly or indirectly scavenge a number of radicals unlikethe classic antioxidants where the ratio of scavenger to radicals neutralized is 1:1.

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Melatonin and Singlet Oxygen (1O2)

Indirect evidence suggesting that melatonin neutralizes 1O2 was first provided byCagnoli et al. (1995), when they showed that the brain of photosensitized rose bengaltreated rats was protected by melatonin administration. Poeggeler et al. (1996) showedthat melatonin neutralized 1O2 during which time AFMK was synthesized. Thisquenching ability of melatonin was confirmed by Zang et al. (1998) and Roberts et al.(2000). Melatonin was also shown to prevent both neuronal apoptosis and inhibition ofcreatine kinase activity. Since melatonin in pharmacological concentrations overcomesthe photodynamic injury to neurons, the authors assumed that melatonin directlyneutralised 1O2. The formation of AFMK when melatonin is oxidized by 1O2 has beenconfirmed by de Almeida et al. (2003). In light of these findings, it appears that AFMKis a product common to several interactions of melatonin with oxygen-based reactants.In a recent study conducted by Maharaj et al. (2005a), the authors showed thatmelatonin produces radicals upon laser irradiation, while lamp photolysis in the samestudy showed that melatonin is able to scavenge 1O2 produced by naphthalene. Theauthors concluded that while melatonin is a free radical scavenger under biologicalconditions, 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 inphotodynamic therapy in the destruction of tumors thus providing a new paradigm inthe therapeutic potential for melatonin.

Melatonin and Peroxyl Radicals (LOO•)

One of the most extensively studied processes in free radical biology is lipidperoxidation, wherein the LOO• radical is generated; this radical then oxidizes anotheradjacent lipid molecule to maintain the chain reaction of lipid peroxidation. However,the evidence that melatonin functions as a chain breaking antioxidant by scavengingLOO• remains problematic. Earlier studies by Pieri et al. (1994, 1995) placed melatoninamong the very best scavengers in terms of its abiltiy to neutralize the LOO•, claimingthat melatonin was a more efficient LOO• scavenger than is vitamin E, which isconsidered to be the premier chain-breaking antioxidant. This claim however, has notbeen verified in subsequent studies (Livera et al. 1997). Despite the controversyregarding the ability of melatonin to interact with the LOO•, in vivo melatonin hasconsistently been found to be highly efficient in limiting the peroxidation of lipids(Reiter et al. 1998a, b). Thus, it appears that melatonin’s ability to reduce lipidperoxidation in vivo is probably not related to its function as a chain-breakingantioxidant but could be associated with melatonin’s ability to scavenge the initiatingradicals (Reiter et al. 2000a) and to other actions within the molecular lipid bilayer(Garcia et al. 1998, Tesoriere et al. 1999). While melatonin appears not to have anyparticular ability to scavenge the LOO•, it does neutralize the trichloromethylperoxylradical, an interaction that has a rate constant of 2.7 · 108 M–1 S–1 (Marshall et al.1996). This was confirmed by Mahal et al. (1999) in a pulse radiolysis study.

Melatonin and Nitric Oxide/Peroxynitrite Anions (ONOO–)

In a test of melatonin’s proficiency to scavenge ONOO–, it met the challenge andneutralized this reactant, and its metabolites (Gilad et al. 1997; Zang et al. 1998;

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Blanchard et al. 2000). Likewise, in many situations where ONOO– was induced in vivo,exogenously administered melatonin curtailed the molecular and physiological damagethat normally accompanies ONOO– exposure (Cuzzocrea and Reiter 2001). Cuzzocreaet al. (1997) showed that melatonin in vivo reduced the inflammatory response inducedby carageenan where both NO• and ONOO– are believed to mediate the inflammation(Beckman and Koppenol 1996). In a comprehensive study by Cuzzocrea et al. (1997),these authors demonstrated, using several different endpoints, melatonin’s anti-inflammatory ability and theorized that this is related to the ability of the indole toinhibit NOS activity and to scavenge ONOO– and the •OH. These findings wereextended by the same group (Cuzzocrea et al. 1998) where melatonin was found to be apotent inhibitor of the severe inflammatory response in rats following the injection of anon-bacterial proinflammatory moleucle, zymosan; this moleucle, in addition toinitiating a marked inflammatory response, causes multiple organ failure (Mainouset al. 1995). Again, Cuzzocrea et al. (1998) speculated that melatonin’s protectiveeffects are a consequence of its ability to reduce NO• formation and scavenge ONOO–

and associated oxidants.In addition, melatonin has been shown to incapacitate NO• (Mahal et al. 1999; Noda

et al. 1999; Blanchard et al. 2000). Although, inherently relatively unreactive, NO•

quickly couples with O��2 to form ONOO– (Beckman et al. 1990) that is capable ofmeting out significant molecular destruction (Phelps et al. 1995). Thus, by scavengingNO•, melatonin indirectly limits oxidative stress (Reiter et al. 2002). Besides directlyscavenging NO•, melatonin reduces its generation under some circumstances byinhibiting the activity of its rate-limiting enzyme, NOS (Pozo et al. 1997; Crespo et al.1999).

Melatonin: Indirect Antioxidant Actions

Whereas the role of melatonin as a direct free radical (and associated reactants)scavenger is obviously extensive, precisely how these reactions relate to the indole’sability to protect against such a wide variety of toxicants in vivo remains to be resolved(Reiter et al. 2002). This relates to the fact that melatonin, in addition to its ability todirectly neutralize reactive species, also limits their generation or metabolizesintermediates to innocuous products. As noted earlier melatonin inhibits NOS (Pozoet al. 1997; Crespo et al. 1999) under some circumstances which lowers tissue damagethat is a consequence of either NO• itself or of the product formed (i.e. ONOO–), whenit couples with O��2 . At physiological concentrations, melatonin has been shown toinhibit NOS activity in rat cerebellar (Pozo et al. 1994) and hypothalamus (Bettahi et al.1996). The melatonin-induced suppression of NOS acitivity is believed to be aconsequence of the binding of calmodulin by melatonin (Pozo et al. 1997). Nitic oxidesynthase is a calmodulin-activated enzyme (Bredth and Snyder 1990) and by bindingcalmodulin, melatonin may limit its availability for this function. With a drop in NO•

synthesis, the formation of ONOO– is curtailed, and the potential oxidative damage isaverted (Pryor and Squadrito 1995). Whether melatonin reduces NOS activity in alltissues that contain this enzyme is unknown.

Furthermore, melatonin stimulates several other important antioxidative enzymesincluding SOD, both MnSOD and CuZnSOD (Antolin et al. 1996; Kotler et al. 1998;Albarran et al. 2001), CAT, (Reiter et al. 2000d), GPx (Barlow-Walden et al. 1995;Pablos et al. 1997), GRd (Pablos et al. 1997) and glucose-6-phosphate dehydrogenase(G6PD) (Reiter et al. 2000d; Rodriquez et al. 2004). SOD is a member of the family of

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enzymes that play a role in the dismutation of O��2 from cells thereby lowering theformation of the highly reactive and damaging ONOO– (Beckman et al. 1990).

Usually when one considers the removal of H2O2 from cells, it is via its enzymaticconversion to innocuous agents. The two major enzymes involved in these conversionsare GPx and catalase. The GPx and catalase enzymes are involved in converting H2O2

to non-toxic products in the body (Chance et al. 1979). The initial reports documentingmelatonin’s stimulatory effect on GPx appeared almost 10 years ago when it was shownthat pharmacological doses of melatonin given to rats (Barlow-Walden et al. 1995) andchicks (Pablos et al. 1997) in vivo resulted in a marked augmentation in the activity ofthis enzyme. GPx reduces free radical damage because it metabolizes H2O2 (and otherperoxides) to water; in the process, however, reduced glutathione (GSH) is oxidized toits disulfide, GSSG. GSSG which is rapidly reduced back to GSH by GRd. In addition,melatonin has been reported to increase the activity of GRd, another importantantioxidant enzyme (Pablos et al. 1998). Studies by Montilla et al. (1997) and Reiteret al. (2000a, b) have further confirmed melatonin’s ability to stimulate catalase, GPxand GRd activity. The enzyme GPx utilizes GSH, an intracellular thiol that is typicallyin mM concentrations, as a substrate. Maintaining high intracellular concentrations ofGSH seems to be a function of melatonin since this indole stimulates the activity of itsrate-limiting enzyme, gama-glutamylcysteine synthase (Urata et al. 1999). When GSH ismetabolised by GPx, a reaction that also requires H2O2 or other hydroperoxides, it isconverted to GSSG. Within cells the GSH:GSSG ratio is normally greatly in favour ofthe former, and to maintain this ratio GSSG is rapidly metabolized back to GSH byGRd. As noted above experimental evidence has shown that melatonin also promotesthe activity of GRd thereby helping to maintain high levels of reduced glutathione(Hara et al. 2001).

Furthermore, GRd requires the co-factor NADPH which is generated by theantioxidative enzyme G6PD. Although the amount of data is limited, there is one reportclaiming that melatonin also stimulates G6PD (Pierrefiche and Laborit 1995). Thiswould be important in GSH recycling since NADPH is a necessary cofactor for G6PD.GSH is a very abundant intracellular free radical scavenger and antioxidant. Therecycling of GSH may well be a major action of melatonin in curtailing oxidative stress.The ability of melatonin to regulate the GSH/GSSG balance by modulating enzymeactivities appears to involve an action of melatonin at a nuclear binding site (Pabloset al. 1997). The other GSH metabolizing enzyme, i.e., CAT, also increases its activity inresponse to melatonin (Naidu et al. 2003). A single report has shown that melatoninstimulates the rate limiting enzyme of GSH, c-glutamylcysteine synthase, therebyincreasing intracellular GSH concentrations (Urata et al. 1999). This action ofmelatonin, unlike the direct free radical scavenging function of the indoleamine, islikely to be mediated by specific receptors. The stimulation of GSH synthesis bymelatonin could contribute to major antioxidative activity of melatonin. Considering thepotential importance of the findings of Urata et al. (1999), it is in need of confirmationparticularly in vivo and in a variety of cell types.

Although membrane receptors for melatonin have been identified in many cells(Dubocovich et al. 1999), nuclear binding sites for the indole have also beendocumented (Garcia-Maurino et al. 1998; Guerrero et al. 2000). Either or both ofthese may be involved in the mechanisms by which melatonin promotes the activity ofSOD, GPx and GRd. Indeed, in one case, Pablos et al. (1997) showed that a nuclearmelatonin receptor agonist, like melatonin itself, stimulated both GPx and GRd in themouse brain. Whether this is a common mechanism by which melatonin increases the

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activity of these enzymes (or prevents their decrease in high oxidative stress conditions)remains to be investigated. With reference to the stimulation of GSH synthesis asreported by Urata et al. (1999), it is assumed that this action of melatonin similarlyinvolves a receptor-mediated process, although the location of the receptor remainsunresolved.

Melatonin as a Metal Chelator

The depletion of melatonin with age has been associated with a number ofneurodegenerative diseases such as Alzheimers (AD) and Parkinsons disease (PD).Coincidental with a number of neurodegenerative diseases is the accumulation of metalssuch as aluminuim in AD (Limson et al. 1998), iron in PD and copper in Wilson’sdisease and AD (Parmar et al. 2002). Furthermore, both lead and cadmium are highlytoxic to living systems, mostly because they easily displace softer metals such as zinc atthe active sites of proteins and enzymes, thereby inactivating them (Limson et al. 1998).Thus the role of melatonin as a metal chelator has been investigated by a number ofresearchers.

Limson et al. (1998) investigated the possible role of melatonin in metal regulation inthe central nervous system by using an electrochemical technique, adsorptive strippingvoltammetry. The authors showed that melatonin formed complexes with aluminium(III), cadmium (II), copper (II), iron (III), lead and zinc thus confering a role of metaldetoxification on melatonin. The metal chelating abiltiy of melatonin has been shown toincrease with increasing concentrations of melatonin (Limson et al. 1998; Gulcin et al.2003). In addition, the authors stated that the fact that melatonin binds to iron (III) notiron (II) suggests that melatonin removes iron (III) unbound to a protein, thuspreventing it from reducing back to iron (II), the form in which it generates a freeradical via the Fenton reaction. Factors favouring the metal binding role of melatonininclude (1) its hydrophilic, lipophilic nature, which allows it to freely move across allcellular barriers facilitating the removal of toxic metals from the CNS, and (2) the factthat it counteracts free radical damage. Gulcin et al. (2003) confirmed that melatonin isan effective metal chelating agent and showed that a 60 mg/ml concentration ofmelatonin exhibited a 95% chelating effect on ferrous ions. Parmar et al. (2002) alsoshowed that melatonin binds effectively to both copper (II) and copper (I) ions.Furthermore, melatonin has also been shown to form complexes with lithium,potassium, sodium and calcium (Lack et al. 2001). The ability of melatonin to serveas a metal chelator provides further evidence supporting its role as a neuroprotectiveagent.

Beyond these actions, melatonin has additional means whereby it reduces theoxidative mutilation of essential macromolecules. Melatonin maintains the optimalfluidity of cellular membranes (Garcia et al. 1998). This is accomplished by reducing theperoxidation of inherent polyunsaturated fatty acids and indirectly reducing increasedmembrane rigidity (Garcia et al. 1998) by positioning itself within cellular membranesto restrict damage to polyunsaturated fatty acids by toxic reactants (Tesoriere et al.1999). Even more important than reducing oxidative damage may be melatonin’s actionat the level of the electron transport chain in mitochondria (Acuna-Castroviejo et al.2001). In addition to the indirect evidence suggesting an action of melatonin onmitochondrial electron transfer (Gilad et al. 1997), several studies have shown that theindole directly stimulates mitochondrial enzyme activity associated with oxidativephosphorylation; these include NADH-coenzyme Q reductase (complex I) and

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cytochrome C oxidase (complex IV) (Martin et al. 2000a, b. Additionally, besidesstimulating oxidative phosphorylation that could reduce electron leakage and freeradical generation (Acuna-Castroviejo et al. 2001), melatonin treatment of rat brain andliver mitochondria in vitro increases ATP production (Martin et al. 2002). This action ofmelatonin may be highly significant in reducing accumulated oxidative damage byproviding energy for molecular repair processs. How melatonin influences themitochondrial enzymes and processes remains enigmatic (Reiter et al. 2002).

Clearly, the number of mechanisms at melatonin’s disposal to reduce moleculardestruction and cellular dysfunction due to oxygen and nitrogen based reactants isextensive. These actions have been shown, for the most part, to be operative in vitro andin vivo situations and are summarized in Fig. 2.

Melatonin and NeurodeGeneration

Undoubtedly, one of the major challenges for contempory neurology is the defferal andprevention of age-associated neurodegenerative conditions that are commonplace in apopulation whose lifespan has been shown to increase in recent decades. Thedebilitating consequences of brain deterioration and malfunction obviously compromisethe quality of life and longevity and, additionally, they are financially taxing to society.The scientific quest to identify the causes and effective treatments for these devastatingconditions is diverse and intensive. While oxidative stress may be one feature that linksmany neurological deficits, it is also obvious that these diseases have extremely complexetiopathologies and it is unlikely that a single agent will totally combat theirdevelopment (Reiter 1998a).

Melatonin, is of interest in the context of neurodegenerative diseases for severalreasons:

1. The endogenous production of this molecule falls dramatically with age (Djeridaneet al. 2005) coincidental with the onset of many of the age-associated neurodegen-erative conditions (Hurn et al. 1996; Karasek 2004);

2. Melatonin readily crosses the blood brain barrier and after its exogenousadministration it is found in high concentrations in the brain, sometimes exceedingthose in the blood manifold (Menendez-Pelaez and Reiter 1993; Menendez-Pelaezet al. 1993; Finnochiaro and Glikin 1998);

Stimulates antioxidative enzymes Detoxifies oxygen-basedradicals/reactive species

Detoxifies nitrogen-basedradicals/reactive species

Stimulates singlet oxygenformation – role inphotodynamic therapy

Increases efficiency of oxidative phosphorylation

Stabilizes cellularmembranes

Inhibits pro-oxidant enzyme

Forms oxidative products that are antioxidanti.e. AFMK, AMK ,cyclic 3-hydroxymelatoninand 6-hydroxymelatonin

Reduces pro-inflammatorycytokines and reducesadhesion molecules

Reduces NF- B bindingto DNA

NH

CH3O CH2CH2NHCOCH3

Melatonin Lipophilic – crosses all morphophysiological barriers

Serves as a metalchelator

Fig. 2 Diagramatic representation of the multiple melantonin actions which protect against toxicity

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3. Melatonin is a ubiquitously acting free radical scavenger and antioxidant (Harde-land et al. 1993; Reiter et al. 2004) which in models of neurological diseases hasproven effectivity in reducing oxidative damage and preserving neurologicalfunction (Reiter et al. 1998b, 2004);

4. The only procedure, that is, food restriction, in animal models of aging thatsignificantly delays senescence also retards the age-associated loss of melatonin(Stokkan et al. 1991) suggesting a potential association between the loss ofmelatonin and the signs of aging.

AFMK as a Free Radical Scavenger

Recently, significant attention has been focused on AFMK as a scavenger of oxygen basedreactants. Cyclic voltametry has shown that AFMK is capable of donating two electrons;furthermore, the kynurenamine reduces damage to DNA and lipids in a high free radicalenvironment and lowers neuronal death when these cells are exposed to either H2O2,glutamate or amyloid 25–35. Each of these is known to generate free radicals (Tan et al.2001). This indicates that not only the parent molecule, i.e., melatonin, but also theresulting products, i.e., cyclic 3-hydroxymelatonin and AFMK, may also function asscavengers of toxic reactants. A recent review by Tan et al. (2007) further highlights thefact that melatonin via the AFMK pathway is highly efficient, scavenging up to 10 ROS/reactive nitrogen species (RNS) for a single melatonin molecule. During times of oxidativestress, melatonin levels decline due to their being metabolized by interaction with thesereactive species. Because more AFMK is produced during high oxidative stress, thismetabolite and cyclic 3-hydroxymelatonin may be indicative of the level of oxidative stress(Tan et al. 2007). This cascade of scavenging actions may be one reason accounting for theunexpectedly high efficacy of melatonin in reducing free radical damage in vivo. Silva et al.(2004) showed that AFMK inhibits lipopolysaccharide-mediated production of tumornecrosis factor-alpha and interleukin-8. Moreover, these authors showed that theinhibitory activity of AFMK is stronger than that of melatonin and interestingly,monocytes efficiently oxidize melatonin to AFMK. AFMK and AMK (N-acetyl-N2-formyl-5-methoxykynuramine) have also been studied for involvement in the inhibition ofneuronal nitric oxide synthase (nNOS) activity in vitro and in the rat striatum in vivo, withauthors discovering the fact that AMK and not AFMK was responsible for the inhibitionof nNOS both in vitro and in vivo. Studies revealed that AMK was a more potent inhibitorthan melatonin by 25% (Tan et al. 2007). Therefore for the inhibition of nNOS, melatoninneeds to be converted to AMK and the resulting ratio is thus an important consideration.This also raises the question of the clinical use of AMK in excito-toxicity, for example,where it is important that reductions of nNOS levels be achieved (Leon et al. 2006)Furthermore, a recent publication by Onuki et al. (2005) showed that AFMK is able toinhibit 5-aminolevulinic acid-induced DNA damage. Finally, another product, N-acetyl-5-methoxykynuramine, is likewise capable of neutralizing some oxygen-based reactants(Tan et al. 2002; Ressmeyer et al. 2003) as is the chief hepatic enzymatic metabolite ofmelatonin, 6-OHM (Hara et al. 2001; Maharaj et al. 2003a, b, 2005b).

Hydroxymelatonin (6-OHM) as a Free Radical Scavenger

The chief, hepatic metabolite, 6-OHM is also reportedly an effective free radicalscavenger (Maharaj et al. 2002). Thus, even when melatonin, itself is metabolically

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converted to 6-OHM before it functions in the direct detoxification of radical(s), 6-OHM is capable of similar radical scavenging ability (Reiter and Tan 2003). In addition,6-OHM is formed when melatonin is reacted with peroxynitrite in the absence ofbicarbonate (Zang et al. 1998). The 6-OHM, has been shown to have some physiologicalactivities similar to those of melatonin (Vaughan et al. 1976). It has been claimed thisdegradation product may be more potent than melatonin in inhibiting lipid peroxidation(Pierrefiche et al. 1993; Hara et al. 1997). Pierrefiche et al. (1993), where it has beenreported that 6-OHM is 30-fold more potent than melatonin in reducing lipidperoxidation. Furthermore, Hara et al. (1997) and Lui et al. (2002) confirmed theseresults and showed that 6-OHM was as potent as melatonin in regard to its antioxidantactivity. In addition, Hara et al. (2001) demonstrated that 6-OHM treatment preventsthe cisplatin-induced increase in GSSG in renal tissue. Maharaj et al. (2003a) reportedthat 6-OHM is capable of reducing cyanide induced oxidative stress and in anotherstudy showed 6-OHM capable of binding iron (III) and converting it to iron (II) which isa more biologically usable form of iron (Maharaj et al. 2003b). Furthermore, the authorsshowed that 6-OHM reduces iron (II)-induced lipid peroxidation and thus does notdrive the Fenton reaction. Yoshida et al. (2003) showed that 6-OHM effectivelyscavengers superoxide anions and this was recently confirmed by Maharaj et al. (2005b)who in addition showed 6-OHM to scavenge 1O2.

However, Matuszak et al. (1997), reported that this hydroxylated indole, in contrastto non-hydroxylated melatonin, functions as both an •OH promoter and •OH scavenger.The pro-oxidant ability of 6-OHM was further supported by Sakano et al. (2004) whoshowed 6-OHM to induce site-specific DNA damage in the presence of copper (II). Theauthors concluded that melatonin may exhibit carcinogenic potential through oxidativeDNA damage by its metabolite. As evident from these numerous studies there is muchcontrovesy surrounding whether 6-OHM serves as a more potent or equipotentantioxidant as compared to melatonin and whether it possess pro-oxidant properties.

Melatonin in Clinical Use

Melatonin: Epilepsy, Neuroprotection, Parkinson’s amd Alzheimers Disease andCancer

Melatonin and the actions of other antioxidants are of interest in the clinical arena(Reiter 1998c). The fact that melatonin inhibits brain glutamate receptors and nitricoxide production has suggested that it may in addition to acting as a neuroprotectiveagent also exert an antiexcitotoxic effect and thus may have a role to play in decreasingepileptic manifestations in humans (Molina-Carballo et al. 1997). The addition ofmelatonin to the phenobarbital therapy in a child with severe myoclonic epilepsyresulted in a seizure-free year. As proof of these findings, reduction of the melatonindose resulted in the resumation of of the seizures and subsequent restoration re-stabilized the patient’s condition, confirming melatonin’s useful role in mechanisms ofneuroprotection. In a study by Gupta et al. (2004), melatonin’s effect on the blood levelsof antioxidant enzymes in children with epilepsy receiving carbamazepine was assessedThe effect of the add-on melatonin as compared to a placebo on the antioxidantenzymes, GPx and GRd in children receiving carbamazepine was determined. The finalconclusions drawn were that melatonin exerts antioxidant activity in patients receivingcarbamazepine therapy due to the fact that an increase in the GRd activity in the

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melatonin group was noted. The potential of melatonin in reducing morbidity-mortalityafter cranio-cerebral trauma (CCT) has been reviewed by Maldonado et al. (2007),where melatonin is described as a protective agent against damage after CCT. Thisprotective action may be attributed to the following: role as a scavenger of ROS andRNS, stimulation of antioxidative enzymes and inhibition of pro-inflammatorycytokines. Melatonin is also reported to reduce the toxicity of drugs such as non-steroidal antiinflammatories, haloperidol, phenobarbital and many others used in thetreatment of this condition. Melatonin’s ability to cross the blood brain barrier and itseffect amongst others on the contusion volume and cellular membranes highlights itsrole in the treatment of CCT (Maldonado et al. 2007).

As early as 1973, a study in the Lancet (Shaw et al. 1973) reported that no alterationin parkinsonian disabilities was observed in pateints treated with melatonin. Morerecently however (Bruguerolle et al. 2002) there has been interest in the existence ofcircadian rhythms which have been shown to be altered, resulting in sleep disturbancesin Parkinson’s disease patients. In these patients, as well as a loss of circadian rhythm ofblood pressure, biologic indices such as cortisol, catecholamines and melatonin arealtered. In Parkinson’s patients (Zisaprel et al. 2001) melatonin may on the one handexacerbate symptoms (interference with dopamine release) but on the other handprotect against neurodegeneration due to its antioxidant properties.

Due to melatonin’s role as an antioxidant and neuroprotectant and its importance inaging, there is a potential role in AD (Wu and Swaab 2005). The neuroprotective effectsin AD have been further demonstrated by Zhou et al., who reported in 2003 for the firsttime, decreased cerebrospinal fluid levels of melatonin in subjects with earlyneuropathological changes in the temporal cortex where the AD process commences.These decreased levels are seen as an early event in the development of AD. Note hasalso been taken of the fact that circadian disorders such as sleep-wake cycledisturbances associated with aging are more pronounced in AD. Some positive resultshave been achieved by reactivation of the circadian system using light therapy andmelatonin supplements (Bliwise 2004). In patients with AD, in order to treat disruptedsleep some non-pharmacological interventions such as exercise and illumination haveproved successful with the usefullness of melatonin as an hypnotic in this populationproving to be questionable (Skene and Swaab, 2003). In a double blind study (9subjects—placebo; 11 melatonin 3 mg) to examine the effects of melatonin on sleep-time and activity and cognitive and non-cognitive functions, melatonin significantlyprolonged sleep time and decreased activity in the night with no significant difference indaytime sleeptime or activity between the two groups. Evaluation of the cognitive andnon-cognitive functions with the Alzheimers Disease Assessment Scale, showedsignificant differences between the placebo and melatonin groups both in terms ofcognition and non-cognition (Asayaam et al. 2003). These results are consistent withfindings which reported that AD patients receiving 9 mg of melatonin presented with asignificant improvement in sleep quality with stabilization of behavioural and cognitiveparameters (Cardinali et al. 2002). Because of an association between melatonin andcancer, a group of researchers have conducted a review of randomised controlled trials(RCTs) of melatonin either as a sole treatment or as an adjunct in the treatment of solidtumour cancers. Results (643 patients, unblinded) indicated that there is a reduction inthe risk of death after a year with a relative risk of 0.66 reported. Thus the use ofmelatonin may be considered beneficial especially because of the low cost and incidenceof side effects (Mills et al. 2005).

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These studies although by no means exhaustive hightlight the role of melatonin in themanagement of epilepsy, AD and PD and cancer, providing evidence of the fact thatmelatonin has indeed a new place in therapy.

Melatonin: The Skin

An appropriately named article (One Molecule, many derivatives: A never-endinginteraction of melatonin with reactive oxygen and nitrogen species) by Tan et al. (2007)provides an excellent summary for this review, in addition to highlighting its protectivefunction in tissues exposed to hostile environments such as the skin. Skin aging has beenattributed to ROS. In addition to melatonin’s roles as an antioxidant and free radicalscavenger, its role in skin architecture was investigated (Esregoglu et al. 2005).Methodology involving long-term pinealectomy and subsequent treatment with mela-tonin clearly indicates that melatonin in increasing thickness of the epidermis anddermis, and levels of the antioxidant enzymes, catalase and glutathione peroxidase is aneffective anti-aging factor. Thus taking into account the decreasing melatonin levelswith age, exogenous administration of melatonin should reduce age-related skindiseases. Similarly melatonin administration to pinealectomised rats increased the levelsof not only glutathione peroxidase but also glutathione and superoxide dismutase,reducing skin flap necrosis and thus improving the outcome for skin flaps still widelyused in plastic surgery (Gurlek et al. 2004). Because of melatonin’s antioxidant anddirect scavenging ability, it became the focus of a study to determine its protective effectagainst UV-induced cell damage in HaCaT keratinocytes. Findings contrasting with theprevious two papers indicated that preincubation with melatonin showed protectiveeffects, whilst postirradiation treatments showed no effects. With the mechanismattributed to inhibition of apoptosis, conclusions were drawn that pretreatment withmelatonin led to protection of the keratinocytes against UVB-irradiation (Fischer et al.2006).

Conclusion

While melatonin’s physicochemical properties allow it to diffuse into most tissues andcells in the body, it has a relatively short half-life in the blood, being excreted as 6-OHMand/or AFMK depending on whether the catabolic pathway is in the CNS or liver. Maet al. (2006) have reported on the use of LC-MS/MS and GC-MS in the determinationof melatonin and six of its metabolites, including 6-OHM and AFMK. The advantage ofthis method is not only related to its sensitivity in the detection of these urinarymetabolites, but also its confirmation of the antioxidant activity of melatonin. This wasachieved by measuring the amount of AFMK in the urine with reduced levels attributedto the lack of survival of AFMK to be excreted in the urine due to its antioxidantactivity. AFMK is thus proposed as one of the biomarkers of in vivo exposure to ROSand thus its investigation in chronic inflammatory disease would obviously be of interest.Therefore, other than its proven antioxidant activity, melatonin may have someapplication as an antiinflammatory agent (Ma et al. 2006).

The role of melatonin as a powerful free radical scavenger and antioxidant has cometo the forefront in the last decade with reports on its ability to interact with bothsuperoxide anions and hydrogen peroxide. However, it is of significance that melatonin

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appears to be successful in scavenging hydroxyl radicals as they are considered to be themost damaging of all endogenously generated reactive agents. There is also indirectevidence that melatonin neutralises singlet oxygen and is effective in reducing lipidperoxidation by not only scavenging initiating radicals, but also due to other actions inthe molecular lipid bilayer. Melatonin has also been reported to be efficient inscavenging peroxy nitrite anions, reducing nitric oxide formation and possessing indirectantioxidant activity.

Melatonin’s hydrophilic/lipophilic nature, its free radical and antioxidant activity,together with declining endogenous production has shifted the focus from merelystrenghthening circadian rhythms to that of neuroprotectant, with the fact that themajor metabolites and photodegradants, 6-OHM and AFMK possess similar activity,strengthening the argument. The fact that melatonin levels decline with age associatedwith deterioration in circadian rhythms has raised the question of its role in age-relateddiseases. The wealth of research reviewed in this article, including melatonin in clinicaluse, offers conclusive proof that there are indeed new places in therapy for melatonin.

Acknowledgements The authors thank NRF for funding. DSM thanks the MRC for the postdoctoralfellowship. The authors would also like to thank Rhodes University in Grahamstown, South Africa andJames Cook University in Townsville, Australia for financial assistance.

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