Protective effects of melatonin in reducing oxidative stress and in preserving the fluidity of biological membranes: a review

REVIEW ARTICLE

Protective effects of melatonin in reducing oxidative stress and in

preserving the fluidity of biological membranes a review

Abstract Free radicals generated within subcellular compartments damage

macromolecules which lead to severe structural changes and functional

alterations of cellular organelles A manifestation of free radical injury to

biological membranes is the process of lipid peroxidation an autooxidative

chain reaction in which polyunsaturated fatty acids in the membrane are

the substrate There is considerable evidence that damage to

polyunsaturated fatty acids tends to reduce membrane fluidity However

adequate levels of fluidity are essential for the proper functioning of

biological membranes Thus there is considerable interest in antioxidant

molecules which are able to stabilize membranes because of their protective

effects against lipid peroxidation Melatonin is an indoleamine that

modulates a wide variety of endocrine neural and immune functions Over

the last two decades intensive research has proven this molecule as well as

its metabolites to possess substantial antioxidant activity In addition to

their ability to scavenge several reactive oxygen and nitrogen species

melatonin increases the activity of the glutathione redox enzymes that is

glutathione peroxidase and reductase as well as other antioxidant enzymes

These beneficial effects of melatonin are more significant because of its

small molecular size and its amphipathic behaviour which facilitates ease of

melatonin penetration into every subcellular compartment In the present

work we review the current information related to the beneficial effects of

melatonin in maintaining the fluidity of biological membranes against free

radical attack and further we discuss its implications for ageing and

disease

Joaquın J Garcıa1Laura Lopez-Pingarron2Priscilla Almeida-Souza1Alejandro Tres2 Pilar Escudero2Francisco A Garcıa-Gil3Dun-Xian Tan4 Russel J Reiter4Jose M Ramırez3 and MilagrosBernal-Perez5

1Department of Pharmacology and Physiology

University of Zaragoza Zaragoza Spain2Department of Medicine Psychiatry and

Dermatology University of Zaragoza Zaragoza

Spain 3Department of Surgery Gynaecology

and Obstetrics University of Zaragoza

Zaragoza Spain 4Department of Cellular and

Structural Biology University of Texas Health

Science Center at San Antonio San Antonio

TX USA 5Department of Preventive Medicine

and Public Health University of Zaragoza

Zaragoza Spain

Key words lipid bilayer lipid peroxidation

melatonin membrane fluidity oxidative stress

Address reprint requests to Joaquin J Garcıa

Department of Pharmacology and Physiology

University of Zaragoza cDomingo Miral sn

50009 Zaragoza Spain

E-mail jjgarciaunizares

Received February 19 2014

Accepted February 20 2014

Introduction

Melatonin (N-acetyl-5-methoxytryptamine) is synthesizedfrom the essential amino acid L-tryptophan by a consecu-tive action of four enzymes tryptophan hydroxylase

L-aromatic amino acid decarboxylase N-acetyltransferaseand hydroxyindole-O-methyltransferase (Fig 1) Althoughmelatonin is an indole produced and secreted into theblood by the pineal gland in mammals both melatonin

and the enzymes involved in its production have beenshown to be present in numerous other tissues includingthe ovary testes bone marrow gut placenta liver [1ndash6]Melatonin has a broad spectrum of physiological effects

in the animal kingdom from unicellular organisms tohigher vertebrates [7ndash9] and also in plants [10ndash14] The

main regulator of melatonin secretion by the vertebratepineal gland is the light-dark cycle acting via the suprach-iasmatic nucleus which through a multisynaptic sympa-thetic pathway promotes the production and release of

melatonin during darkness [15] Thus during the dayhuman serum melatonin concentrations are low (5ndash20pgmL) while at night its synthesis and release are stimu-

lated and blood levels reach peak values (80ndash150 pgmL)

[16] Consequently this temporal information is used as acircadian-based means of inducing photoperiodic and cir-

cadian responses and it serves as both a daily and seasonaltimer [17] The chronobiotic properties of melatonin aremajor physiological functions of the indoleamine produced

by the pineal gland Melatonin also exhibits remarkablefunctional versatility due to its actions on the neuralimmune and endocrine systems as well as its effects onmetabolism [18ndash22] In contrast the physiological signifi-

cance of melatonin produced in peripheral tissues seems tohave intracrine autocrine and paracrine roles as this mela-tonin does not enter the circulation [23ndash25] Studies duringthe last decade document that melatonin has both recep-tor-mediated and receptor-independent actions Theindoleamine acts by binding to membrane and nuclear

receptors [26 27] by interacting with cytosolic proteinsfor example calmodulin [28] and as a powerful direct freeradical scavenger [29]

As the in vitro scavenging properties of melatonin werediscovered numerous reports have accumulated showingthat melatonin also limits in vivo oxidative destruction oflipids proteins and both nuclear and mitochondrial DNA

[25 29] Melatonin is especially effective as an antioxidant

225

J Pineal Res 2014 56225ndash237Doi101111jpi12128

copy 2014 John Wiley amp Sons AS

Published by John Wiley amp Sons Ltd

Journal of Pineal Research

Mo

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Bio

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Mel

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because it utilizes a wide variety of means to reduce oxida-tive stress Firstly melatonin scavenges several toxicreactants including the highly toxic hydroxyl radical (˙OH)[30ndash32] and what may be even more important it takes

advantage of its derivatives which likewise are highly effi-cient radical scavengers When melatonin interacts withOH it initiates a scavenging cascade reaction (Fig 2) in

which the metabolites produced cyclic 3-hydroxymelato-nin N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK)and N1-acetyl-5-methoxykynuramine (AMK) also func-

tion in the neutralizing of free radicals [33ndash36] Thus onemolecule of melatonin eventually may scavenge up to eightor more radicals Additionally melatonin also detoxifies

the peroxynitrite anion (ONOO) hydrogen peroxide thesuperoxide anion radical (O

2 ) singlet oxygen as well asother toxic reactants [37ndash44] These actions of melatoninin scavenging free radicals throughout the cell depend on

its chemical characteristics that is melatonin is lipophilicand hydrophilic and thus it crosses all cell barriers withease and is available to all tissues cells and subcellular

organelles Therefore the melatoninrsquos beneficial actionsextend to every organism and every organIn addition to its direct scavenging actions melatonin

functions as an indirect antioxidant as well It does so bymeans of its ability to stimulate the expression and activityof antioxidant enzymes which remove free radicals andtheir precursors [45ndash50] Also melatonin or its metabolite

AMK inhibits one pro-oxidative enzyme that is induc-ible nitric oxide synthase (iNOS) [51] NO˙ has the capa-bility of coupling with the O

2 to form the ONOOHence melatonin limits the formation of ONOO a non-radical reactant which is equally toxic as ˙OH by reducingthe activity of iNOS

One additional important functional feature of melato-ninrsquos ability to reduce oxidative stress has been uncoveredin recent years Mitochondria are mainly responsible for

energy generation through oxidative phosphorylationyielding ATP which is required for every cell functionUnfortunately large numbers of free radicals are gener-ated within mitochondria when electrons escape during

their transfer through the respiratory chain By stimulatingcomplex I and complex IV of the mitochondrial respira-tory chain melatonin reduces electron leakage and free

radical generation that is a consequence of the respiratoryprocess [52ndash57] This is referred to as radical avoidance[38] Finally melatonin also contributes to the mainte-nance of the production of the ATP by the mitochondria

via maintaining high levels of mitochondrial glutathione(GSH) This reduces the necessity of the mitochondria tak-ing up extra GSH from the cytosol [23]

The combination of these beneficial properties of mela-tonin in the mitochondria and its ability to directly scav-enge free radicals and to stimulate the antioxidant

enzymes in all cell compartments makes this indoleamineunique in terms of its antioxidant capabilities In a recentstudy where melatonin was compared with synthetically

produced mitochondrially targeted antioxidants melato-nin was found to be superior in protecting mitochondriafrom free-radical-mediated damage [58]

Fluidity of biological membranes and lipidperoxidation

Every eukaryotic cell is surrounded by a plasma mem-brane that separates it from the extracellular environmentMoreover membranes also enclose the organelles which

divide the cell into discrete compartments wherein specificbiochemical processes take placeThe most abundant constituents of cellular membranes

are phospholipids From a chemical structure point of

view all membranes contain the following phospholipidsphosphatidylcholine phosphatidylserine phosphatidyleth-anolamine sphingomyelin and phosphatidylinositol These

molecules are amphipathic structures composed of polar(or hydrophilic) and nonpolar (or hydrophobic) moietiesIn an aqueous environment phospholipids tend to orient

with their polar heads facing the surrounding aqueous sur-faces while the hydrophobic fatty acyl chains project awayfrom contact with water Thereby when phospholipids are

dispersed in water they spontaneously form lipid bilayersEach phospholipid layer of a cell membrane is referred toas a leafletPhospholipids are not distributed uniformly across the

bilayer rather there is an asymmetrical distributionbetween the inner and outer leaflets of membranes Besidesphospholipids animal membranes also contain cholesterol

5-MethoxytryptopholTryptophanHydroxyindole-O-Tryptophanmethyltransferasehydroxylase

- -- 5 Hydroxytryptophan 5 Hydroxytryptophol5 Methoxytryptophan- -Hydroxyindole O

- AldehydeL aromatic amino acidmethyltransferase reductasedecarboxylasey

5-Hydroxyindol-5 Hydroxytryptamine5-Methoxytryptamine acetaldehydeHydroxyindole-O-

Monoamine methyltransferase AldehydeN-acetyltransferaseyfoxidase dehydrogenase

N-Acetyl-5- 5-Hydroxyindol5-Methoxyindolhydroxytryptamine acetic acidacetic acid

Hydroxyindole-O-Hydroxyindole-O-methyltransferasemethyltransferase

-- - - 5 MethoxyindolN Acetyl 5 methoxytryptamineti idacetic acidmelatonin( )

Fig 1 Pathway for tryptophan meta-bolism and synthesis of melatonin inmammals Indoleamines that have adocumented ability to reduce membranerigidity and lipid peroxidation aredepicted in red

226

Garcıa et al

Its steroid nucleus lies parallel to the fatty acyl chains ofmembrane phospholipids The lipid bilayer serves as amatrix for embedded proteins which function as trans-porters ion channels receptor-effector-coupled systems

for hormones and neurotransmitters etc The ratio of pro-teins to lipids varies widely among membranes from differ-ent structures for example the inner mitochondrial

membrane is roughly 75 protein while the myelin mem-brane is composed of only 18 protein [29] Additionallysome membrane lipids and proteins are glycolipids and

glycoproteins respectively with covalently bound carbo-hydrate side chains that protrude from the external surfaceof the membrane

Singer and Nicolson [59] proposed the term fluid mosaicmodel which suggests that biological membranes are fluidstructures Fluidity is defined as the quality of ease ofmovement and represents the reciprocal value of mem-

brane viscosity [60 61] In general the term means a com-bination of the mobilities of different membranecomponents Many of the constituent molecules of cellular

membranes are free to diffuse in the plane of the mem-brane and even lsquoflip-floprsquo from one phospholipid leaflet tothe other at slow rates (Fig 3) However some compo-

nents are not free to diffuse or to flip-flop in the mem-brane For example large hydrophilic membranemolecules are unlikely to flip-flop because they must bedragged through the nonpolar interior of the lipid bilayer

Several chemical and physical agents modulate lipid flu-idity of biological membranes such as (i) the length anddegree of saturation of the fatty acid chains (ii) the nature

of polar head groups which influences the mobility of thehydrocarbon chains (iii) the concentration of cholesterolin the lipid bilayer because its steroid nucleus lies parallel

to the fatty acyl chains of membrane phospholipids (iv)the protein density in the membrane (v) the temperatureof the membrane and (vi) the presence of natural or syn-

thetic amphipathic substances in the bilayer for examplesteroids vitamins anaesthetics and barbiturics [62 63]Changes in membrane fluidity are critically important to

the homeostasis of various cell functions including

survival differentiation and the activity of cell death

pathways [64] Thus lipid bilayer fluidity modulates theactivity andor efficiency of membrane proteins such asion channels and transporters as well as receptors [65ndash70]Even slight changes in membrane fluidity may cause aber-

rant cellular function and induce pathological processes[71ndash74] Accordingly several methods have been proposedto evaluate membrane fluidity in a wide variety of biologi-

cal membranes The most extensively used of these hasbeen the fluorescence polarization anisotropy of diphenyl-hexatriene (DPH) derivatives and electron paramagnetic

resonance using fatty acid spin-label agents [61]A free radical is any species capable of independent exis-

tence that contains one or more unpaired electrons that

is those that occupy an atomic or molecular orbital byitself [75] Over the last four decades peroxidation of fattyacids induced by free radicals has been studied in greatdetail as a deleterious process that occurs in plasma as well

as in intracellular membranes The lipid peroxidation reac-tion is divided into three successive phases initiationpropagation and termination (Fig 4) Initiation takes

place through an abstraction of a hydrogen atom from afatty acid containing two or more separated doubledbounds leading to a carbon-centred alkyl radical with a

simultaneous rearrangement of the double bounds tobecome conjugated Thereafter the alkyl radical formedreacts with oxygen which is nonpolar and thereby solu-ble in the hydrocarbon core of lipid bilayers giving rise to

a peroxyl radical Propagation which involves the abstrac-tion of hydrogen from a neighbouring fatty acid by per-oxyl radicals results in the formation of a lipid

hydroperoxide and a new alkyl radical Finally termina-tion of the lipid peroxidation process is generally believedto take place by an interaction between two free radicals

resulting in the termination of a nonradical product [75]Lipid hydroperoxides can also undergo degradation into

hydrocarbons alcohols ethers epoxides and aldehydes

Among the latter malondialdehyde (MDA) and4-hydroxy-alkenals (4-HDA) are of special importancebecause they can cross-link phospholipids proteins andDNA [76] Because of this many of the assay methods

to establish free-radical-induced injury to biological

OHCH -CH -NH-CO-CHCH O 2 2 3 CH O3 3

NCO-CH3Reactive oxygen speciesN N

H H

-MELATONIN CYCLIC 3 HYDROXYMELATONIN

CH O- - - - - CO-CH2-CH2-NH-CO-CH33CO CH2 CH2 NH CO CH3CH3O

-NH CHONH2

i sup1- -Nsup2- -Nsup1-ACETYL- Reactive oxygen species N ACETYL FORMYLC5-METHOXYKYNURAMINE5-METHOXYKYNURAMINE Enzyme

Fig 2 Antioxidative cascade of mela-tonin Besides the antioxidant activity ofmelatonin the derivatives generated fromthe interaction of the indoleamine withfree radicals also are potent scavengers Ithas been estimated that a single moleculeof melatonin may neutralize up to eighttoxic reactive oxygen species

227

Melatonin effects on membrane fluidity

membranes have measured MDA concentrations Lipid-free radicals can abstract hydrogen atoms from adjacentproteins leaving the protein with an unpaired electron

thereby modifying the structure and function of membraneproteinsLoss of freedom of motion in biological membranes

after oxidative stress is well documented [77ndash79] Twostructural reasons have been proposed as a causal relation-ship for the loss of membrane fluidity during oxidative

stress First there may be a reduction in the polyunsatu-ratedsaturated fatty acid ratio in membrane composition[78] because free radicals have a particular affinity for

electron-rich unsaturated covalent bounds which arefound in polyunsaturated fatty acids [80] Second the for-mation of cross-linking among the membrane lipid moie-

ties may limit motion within the membrane contributingto rigidity [77]

Melatonin and order in the lipid bilayer

In the published literature there are a limited number of

studies related to the interaction of melatonin in mem-branes at the molecular level All have used lipid vesiclesbased on synthetic membranes built with one or a fewphospholipid types Lipid vesicles are useful tools to exam-

ine a variety of physicochemical properties in the analysisof proteinndashlipid or drugndashmembrane interactions Howevera criticism of these models is that they reproduce few

experimental paradigms which may limit the conclusionsbased on them compared with those obtained using genu-ine biological membranes

The effect of melatonin in these model membranes hasbeen mainly to reduce order in other words increasedfluidity The presence of melatonin in dipalmitoyl phos-phatidylcholine dimyristoyl phosphatidylcholine and di-

palmitoyl phosphatidylglycerol multilamellar liposomesincreased lipid dynamics [81ndash83] The ability of melatoninto elevate fluidity in these vesicles should be considered as

another cooperative mechanism by which melatonin pro-tects biological membranes against lipid peroxidationwhich causes a marked reduction in lipid dynamics leading

to membrane rigidityThe precise mechanism underlying the effect of melato-

nin in the lipid vesicles is not well established A report

using electron spin resonance spectra of labels placed onthe surface and at different depths of dimyristoylglycerolphosphatidylcholine large vesicles showed an average

(A)

(B)

(C)

(D)

Fig 3 The types of possible movementfor phospholipids in a lipid bilayerinclude A) lateral diffusion in the planeof the membrane B) flip-flop ormigration from the monolayer on oneside to the other C) rotation of the lipidabout its long axis D) flexion-extensionof the hydrocarbon chains

XInitiation

Initiation

RH + X R

Propagation R + O ROO2

ROO + RH ROOH + R

Termination

N2 ROO No polymersPropagation

Fig 4 Process of lipid peroxidation in biological membranes Afree radical (X˙) removes a hydrogen atom from a polyunsaturedfatty acid (RH) resulting in a conjugated diene that quickly reactswith an oxygen molecule forming a lipid peroxyl radical (ROO˙)ROO˙ removes one hydrogen atom from a second RH forming alipid hydroperoxide (ROOH) and a second conjugated diene Sev-eral measurable end-products are produced during this autooxida-tive process for example malondialdehyde 4-hydroxy-alkenalsand isoprostanes

228

Garcıa et al

shallow position for melatonin in the membrane althoughnitroxides placed deep in the bilayer were also able toquench melatonin fluorescence this finding suggests thatthe indoleamine is also in the hydrophobic core [84] In

dry cholesterol-lecithin-mixed reversed micelles dispersedin carbon tetrachloride (CCl4) melatonin is mainly locatedin and oriented in the nanodomain constituted by the

hydrophilic groups of cholesterol and lecithin [85] This isin agreement with the hypothesis that melatonin positionsitself preferentially in a superficial location in lipid bilayers

near the polar head group of phospholipids [86ndash88] It hasbeen proposed that this positioning of melatonin in the bi-layers might be responsible for the observed disordering in

the tails of the phospholipids [86] and this may be benefi-cial in some physiopathological processes as it is generallyaccepted that a higher disorder in the membrane phospho-lipids makes the interactions of antioxidants with lipid

radicals more efficient and thus it may reduce the delete-rious effects of lipid peroxidation

Melatonin protects against membranerigidity due to lipid peroxidation

Among a wide variety of actions of melatonin in protect-ing organ and tissues against oxidative injury mediated byfree radicals the curtailment of lipid peroxidation hasbeen repeatedly reported and has aroused special interest

because of its therapeutic potential [29 89ndash94]Quantification of lipid peroxidation due to oxidative

stress has been pursued by measuring several products

generated as a consequence of the interaction of free radi-cals with the membrane phospholipids Assays as those forthiobarbituric- or thiobarbituric-like reactive substances

have been extensively used as indices of lipid peroxidationMultiple recent studies have documented the utility andefficacy of melatonin in preventing elevation of MDA+4-HDA concentrations caused by numerous diseases and bytoxicological experimental models to generate in vivo andin vitro lipid peroxidation [95ndash102]The measurements of isoprostanes in the biological

membranes are considered a more sensitive method toevaluate lipid peroxidation than are MDA concentrations[103] These compounds are chemically stable products

of free-radical-mediated damage to arachidonic acid Sev-eral reports that have used the isoprostane levels to eval-uate melatonin behaviour have employed models of

brain injury Melatonin attenuated the production of 8-isoprostanes following experimental umbilical occlusionof mid-gestation foetal sheep [104] Also melatonin treat-ment of animal models of cerebral hypoxiandashischaemia

produced a significant drop in isoprostane concentrationsin the cerebral cortex as well as a reduction in theencephalopathy mediated by reduction in the inflamma-

tory cell recruitment and glial cell activation in theseareas when compared to nontreated animals [105 106]Moreover it has been shown that the indoleamine

reduced oxidative stress secondary to traumatic braininjury and even melatonin levels in the cerebrospinalfluid increased after traumatic brain injury which is

believed to be an adaptive response to oxidative stressandor inflammation [107]

In addition to these reports that have documented theneuroprotective effects of the indoleamine melatonin alsoreduced isoprostane generation in the liver kidney andplasma of rats following treatment with the bipyridyl her-

bicide diquat [108 109] Finally free radical overproduc-tion and lipid peroxidation that occurs during intenseexercise contribute significantly to induce muscle damage

Melatonin administration reduced urinary isoprostane lev-els in men who participated in a run of 50 km when com-pared to placebo-treated individuals [110]

Given that melatonin reduces lipid peroxidation in everycell and tissue it was assumed that in doing so the indolewould also maintain cell membranes in a state of optimal

fluidity The initial study which noted that melatoninreduced membrane rigidity due to lipid peroxidation wasperformed by our group with the aid of a well known in vi-tro model frequently used to induce lipid peroxidation in

hepatic microsomes [111] The incubation of microsomalmembranes with FeCl3 adenosine-5-diphosphate and nico-tinamide adenine dinucleotide phosphate was followed by

MDA accumulation and a loss in membrane fluidity Theaddition of melatonin prevented both the rise in MDA andin membrane rigidity these actions were concentration

dependent (Fig 5) [111] In this study microsomal mem-brane fluidity was assessed by fluorescence spectroscopy amethod based on the intercalation into the membrane of afluorescent molecule which when illuminated by polarized

light emits a fluorescent signal The degree of polarizationof this signal depends on the state of mobility of the probereflecting motion in the membrane lipid environment

These results were soon confirmed in another studydesigned to test the cooperative effects of melatonin withtamoxifen an anti-oestrogenic drug currently used for the

treatment of breast cancer In this investigation melatonin

100

80

60

40

In

hibi

tion

20

0

ndash4 ndash2ndash6 ndash5 ndash3

Melatonin (Log[])

Fig 5 Ability of melatonin to reduce membrane rigidity (yellow)and lipid peroxidation (red) in microsomes obtained from the liverof Sprague-Dawley rats Oxidative stress was induced by additionof FeCl3 NADPH and ADP Percentage inhibitions are expressedas means SE Obtained from four independent experiments[Redrawn from 100]

229


enhanced the ability of tamoxifen to limit the reduction inmicrosomal membrane fluidity that occurred as a conse-quence of lipid peroxidation [111] Whether the change inmembrane fluidity induced by melatonin relates to its on-

costatic role in breast cancer and other tumors is yetunknown [16 112ndash115]Melatonin is the major indoleamine synthesized from

tryptophan in organisms and is an important moleculethat provides cellular protection and antioxidant activitySeveral structurally related indoles including 5-hydroxy-

tryptophan [116] 5-methoxytryptophol [117] N-acetylse-rotonin [118] and indole-3-propionic acid [119 120] aswell as pinoline a szlig-carboline formed by condensation

between indoleamines and aldehydes may also act as pow-erful radical scavengers while stabilizing membranes [121]While melatonin clearly is capable of significantly reduc-

ing lipid peroxidation it is not particularly effective as a

direct peroxyl radical scavenger [31 122] that is it doesnot have great efficiency as a chain-breaking antioxidantBecause of this it has been assumed that melatoninrsquos pro-

tective actions on lipids stems from its ability to neutralizethe toxic reactants that initiate the chain of events thatlead to massive lipid peroxidation Indeed this is the case

as melatonin has been shown to detoxify the two mayorinitiators of the peroxidation of fatty acids namely the˙OH [30 123ndash125] and the ONOO [37 126]There may however be additional means that are oper-

ative which are involved when melatonin stymies lipid per-oxidation It has recently become apparent that derivativesof melatonin that are formed when the indoleamine func-

tions as a scavenger may actually be more effective thanmelatonin itself in neutralizing the peroxyl radical Thusthe experimental data indicate the cyclic 3-hydroxymelato-

nin and AMK are capable of scavenging the peroxyl radi-cal with greater efficacy than melatonin itself [33] Thissuggests melatonin via these metabolites may be an indi-

rect chain-breaking antioxidant in addition to its directscavenging of the toxic reactants that initiate this devastat-ing processAgeing is a characteristic of all organisms and is defined

as a progressive endogenous and irreversible physiologicaldecline that increases vulnerability to disease and finally todeath [127] The nature of the ageing process has been

subject of numerous hypotheses Among other experimen-tal proposals a prominent theory to explain the incessantdeterioration with age is the accumulation of oxidatively

damaged molecules which are the result of free radicalsproduced during aerobic respiration [128ndash131] Severalstudies in experimental animals and humans show age-related changes in the rhythm of melatonin secretion As

pineal melatonin production is diminished during ageing[132ndash135] it has been speculated that the loss of this anti-oxidant may contribute to the accumulation of free radical

damage that occurs in the later stages of life [136ndash138] Inold rats (25-month-old) we showed that membrane fluid-ity of microsomes isolated from the liver where markedly

lower than hepatic microsomes isolated from 2-month-oldrats Likewise pinealectomy induces a life-long reductionin endogenous melatonin levels and the outcome was an

exaggerated membrane rigidity arising as a result of physi-ological ageing [139]

One particular experimental animal model in which tostudy disorders that are manifest late in life is the senes-cence-accelerated mouse It involves two strains a senes-cence-accelerated prone mouse (SAMP) and a senescence-

accelerated resistant mouse (SAMR) Following a normalprocess of development the SAMP strain exhibits a shortlife span with signs of senile ageing including loss of skin

glossiness increased hair coarseness hair loss perioph-thalmic injuries and lordokyphosis of the spine [140] TheSAMR strain displays normal ageing characteristics [140

141] Both a higher oxidative stress and a reduced mito-chondrial function have been reported in various organsof SAMP8 compared with normal ageing SAMR1 used as

a control [142 143] Oral melatonin administration toSAMP animals protects against age-related oxidative dam-age of lipids proteins and DNA in the brain and liver[144ndash146] and moreover protects the mitochondrial respi-

ratory chain activity from accelerated ageing [57 146]In a recent study we evaluated membrane fluidity in

central nervous system neurons and mitochondrial mem-

branes obtained from SAMP8 and SAMR1 mice at 5 and10 months of age additionally we evaluated the effect ofchronic treatment of these mice with melatonin from age 1

to 10 months on these parameters As anticipated ageingpromoted rigidity in synaptosomal and mitochondrialmembranes in untreated SAMP mice Melatonin adminis-tration reduced the rigidity (Fig 6) especially in the mito-

chondrial membranes [101] Beyond the structural changesthat are proposed as the basic mechanisms for membranerigidity due to the accumulative lipid damage mediated by

free radicals it is interesting that ageing also increasescholesterol content in the membranes [147] Cholesterolusually orients itself parallel to the phospholipids bilayer

and negatively influences permeability fluidity and themechanical properties of lipid bilayers [148ndash150] whichleads to the development of more rigid membranes Bongi-

orno et al [85] have shown that melatonin competes with

38

37

36

35

34

Mem

bran

e flu

idit

y (1

Pol

ariz

atio

n)

33

SAMR1SAMR1SAMR1 SAMP8SAMP8SAMP8

5 months 10 months 10 months + aMT

Fig 6 Effects of age and chronic melatonin (aMT) treatment onmitochondrial membrane fluidity Mitochondria were isolatedfrom the senescence-accelerated resistant mouse (SAMR1)(n = 49) and the senescence-accelerated prone mouse (SAMP8)(n = 47) Values are mean SE P le 005 versus SAMR1

(5 months) P le 005 versus SAMP8 (10 months) [Redrawnfrom 101]

230

Garcıa et al

cholesterol for binding to lecithin and that the indole-amine may even displace cholesterol from the bilayer Ifthis can be documented in vivo melatonin could reducethe increased rigidity of cell membranes during ageing

Thus besides its antioxidant activity the biophysicaleffects of melatonin on the lipid membrane dynamics mayalso contribute to its cellular protection in ageing What is

of additional interest is that endogenous melatonin pro-duction falls during ageing [132 133 135 151] whichcould be a factor in age-associated membrane rigidity

Currently there is a great deal of effort to identify effec-tive antioxidant drugs to prevent or to treat free-radical-mediated tissue damage Melatonin has recently proven to

protect human tissues from free-radical-induced mutila-tion for example the indoleamine efficiently protectedagainst lipid peroxidation and membrane rigidity in ery-throcytes of patients undergoing cardiopulmonary bypass

surgery [152] a therapeutic technique with a high degreeof surgical risk [153 154]d-Aminolevulinic acid (ALA) is a precursor of haeme

synthesis Under physiological conditions serum ALAconcentrations in humans are practically undetectable Anincrease in ALA is typically related to acute intermittent

porphyria hereditary tyrosinemia or to lead poisoningThe administration of ALA to rats caused both a signifi-cant increase in hepatic lipid peroxidation in homogenatesand rigidity in the microsomal membranes when compared

to these parameters in control animals Melatonin com-pletely counteracted the effects of ALA [155]Radiation injury of living cells is to a large extent due

to free radical generation The most commonly damagedbiomolecule due to ionizing radiation is DNA conse-quently ionizing radiation exposure is justifiably consid-

ered a carcinogenic agent [156 157] In addition to DNAhowever lipids and proteins the major macromoleculespresent in biological membranes are also attacked by free

radicals induced by ionizing radiation [158] Microsomalmembranes isolated from the liver of rats subjected towhole body ionizing radiation exhibited a significantreduction in membrane fluidity when compared to those

of nonirradiated rats Moreover DNA from the hepato-cytes had elevated concentrations of 8-hydroxy-2-deoxygu-anosine a DNA base adduct that is considered a key

biomarker related to carcinogenesis [159] When melatoninwas administered in advance of ionizing radiation itcompletely prevented both the rigidity in the hepatic

microsomal membranes and the rise in 8-hydroxy-2-deoxyguanosine levels [160]Recent investigations have also shown melatoninrsquos bene-

ficial actions against chemical agents a-Naphtylisothiocy-

anate (ANIT) is a well-known toxic substance thatproduces a cholangiolitic hepatitis characterized by intra-hepatic cholestasis hepatocellular and biliary epithelial cell

necrosis and bile duct obstruction [161 162] Rats treatedwith ANIT-developed cholestasis within 24 hr as indi-cated by both serum levels of alanine aminotransferase

and aspartic acid aminotransferase activities and serumtotal bilirubin concentration Moreover lipid peroxidationand rigidity in homogenates and microsomal membranes

obtained from the liver were observed to be higher in theANIT-treated rats than in control animals Melatonin or

6-hydroxy-melatonin treatments completely reversedcholestasis lipid peroxidation and hepatic microsomalmembrane rigidity [163]Hydrazines are extensively used in laboratory industrial

and therapeutic fields A wide variety of toxic effects ofthe hydrazines have been described including autoimmunedisturbances [164 165] leukemogenesis [166] haemolytic

anaemia [167 168] and cancer [169ndash171] We comparedthe effect of melatonin or ascorbic acid against experimen-tally induced damage to cellular membranes caused by

phenylhydrazine (PHZ) Melatonin treatment in rats givenprior to PHZ administration prevented the decrease inmembrane fluidity Unlike with melatonin treatment the

rigidity of hepatic microsomes from animals treated withPHZ plus ascorbic acid was even greater than in rats givenPHZ only [172]CCl4 is a toxin that produces hepatocyte fatty degenera-

tion cellular necrosis fibrosis cirrhosis and cancer in ratsand other animal species [173] The administration of mel-atonin or pinoline fully prevented cell membrane rigidity

in the liver due to CCl4 in rats In this study treatmentwith melatonin was more effective than pinoline in reduc-ing lipid peroxidation [95]

A last point of consideration should be the safety ofmelatonin in clinical use Firstly in experimental studiesmelatonin doses up to 800 mgkg failed to cause death inmice A lethal dose in 50 of mice that is LD50 has not

been determined despite attempts to do so [174] Secondlywhen very high doses of melatonin were administered(200 mgkg) to pregnant rats no toxicity was observed

[175] Finally many studies including those in childrenand adult humans have shown that melatonin has verylow toxicity [176] although there may occasionally be

apparent aberrant isolated reactions to melatonin [177178] Melatonin has been used regularly by numerous indi-viduals for many years in different countries with few side

effects being reported

Conclusions

Peroxidation of membrane lipids has traditionally beenthought to be a major consequence of rampaging free rad-icals The end result of lipid peroxidation is the chemical

alteration of polyunsaturated fatty acids with the disrup-tion of the integrity of cellular and subcellular membranesThe dynamic properties of the lipid bilayer play a central

role in the regulation of many important physiologicalevents in the cell Therefore disruption of the structuralproperties due to oxidative stress has serious consequencesfor cellular function

Melatonin exhibits remarkable functional versatility topreserve the morphological and functional aspects of thecell membrane from free radical attack These include its

ability to scavenge free radicals to enhance of the activ-ity of the antioxidant enzymes and to optimize the trans-fer of electrons through the electron transport chain in

the inner mitochondrial membrane Importantly melato-nin has no reproducible adverse effects in humans or ani-mals and crosses all physiological barriers easily for

example bloodndashbrain barrier and membranes of cells andorganelles

231


As a result of its cytoprotective effects many recentreviews summarize the numerous beneficial actions of mel-atonin in several clinical models of diseases [16 179ndash185]Accordingly experimental controlled trials are being con-

ducted to clarify the therapeutic role of this clinicallyattractive antioxidant and nontoxic moleculeThe evidence summarized in this review strongly sug-

gests the ability of melatonin to preserve optimal levels offluidity in biological membranes and to resist the rigidityinduced by free radical attack this must be considered

another important mechanism by which melatonin plays abeneficial role in protecting against free-radical-relateddiseases

Acknowledgements

This work was supported by grants from the lsquoGobierno de

Aragonrsquo (Aging and Oxidative Stress Physiology GrantNo B40) and from the lsquoInstituto de Salud Carlos IIIrsquo(RD1200430035)

Conflict of interest

The authors have no conflict of interest to declare

References

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molecular details in the human pineal gland in the light of

phylogeny structure function and chronobiological dis-

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adjuvant therapy of malignant tumors Med Sci Monit

2008 14RA64ndashRA70

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endar Experientia 1993 49654ndash664

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actions recent insights and new perspectives J Pineal Res

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nin mitochondria and neuroprotection In Melatonin

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sites in purified cell nuclei of rat liver J Pineal Res 1994

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MT2 melatonin receptors in mammals Endocrine 2005

27101ndash110

28 BENITEZ-KING G Melatonin as a cytoskeletal modulator

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Pharm J 2010 141ndash9

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Garcıa et al

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32 GALANO A On the direct scavenging activity of melatonin

towards hydroxyl and a series of peroxyl radicals Phys

Chem Chem Phys 2011 137178ndash718833 GALANO A TAN DX REITER RJ On the free radical scav-

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AMK J Pineal Res 2013 54245ndash257

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2003 341ndash10

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directly scavenges free radicals generated in red blood cells

and a cell-free system chemiluminescence measurements

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the production of ATP in rat brain and liver mitochondria

Int J Biochem Cell Biol 2002 34348ndash35757 OKATANI Y WAKATSUKI A REITER RJ et al Hepatic mito-

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that protect mitochondria reduce interleukin-6 and oxida-

tive stress improve mitochondrial function and reduce bio-

chemical markers of organ dysfunction in a rat model of

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interacts with lipid bilayers a study by fluorescence and

ESR spectroscopies FEBS Lett 1997 416103ndash106

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mixed reversed micelles used as cell membrane models


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1999 26108ndash112

87 De LIMA VR CARO MS MUNFORD ML et al Influence of

melatonin on the order of phosphatidylcholine-based mem-

branes J Pineal Res 2010 49169ndash17588 DROLLE E KUCERKA N HOOPES MI et al Effect of melato-

nin and cholesterol on the structure of DOPC and DPPC

membranes Biochim Biophys Acta 2013 18282247ndash2254

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reverses lipopolysaccharide-induced gastro-intestinal motil-

ity disturbances through the inhibition of oxidative stress

J Pineal Res 2008 4445ndash5191 GITTO E PELLEGRINO S GITTO P et al Oxidative stress of

the newborn in the pre- and postnatal period and the clini-

cal utility of melatonin J Pineal Res 2009 46128ndash139

92 K euroUC euroUKAKIN B LYKKESFELDT J NIELSEN HJ et al Utility of

melatonin to treat surgical stress after major vascular sur-

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93 REITER RJ PAREDES SD KORKMAZ A et al Melatonin com-

bats molecular terrorism at the mitochondrial level Inter-

discip Toxicol 2008 1137ndash14994 TAMURA H TAKASAKI A MIWA I et al Oxidative stress

impairs oocyte quality and melatonin protects oocytes from

free radical damage and improves fertilization rate J Pineal

Res 2008 44280ndash28795 ARANDA M ALBENDEA CD LOSTALE F et al In vivo hepatic

oxidative stress because of carbon tetrachloride toxicity

protection by melatonin and pinoline J Pineal Res 2010

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sis of the protective effects of melatonin and vitamin E on

streptozocin-induced diabetes mellitus J Pineal Res 2002

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reduces dinitrobenzene sulfonic acid-induced colitis


98 DZIEGIEL P SUDER E SUROWIAK P et al Role of exogenous

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malondialdehyde and nitritenitrate in the blood of asphyxi-

ated newborns reduction by melatonin J Pineal Res 2001

31343ndash349

100 GARCIA JJ REITER RJ GUERRERO JM et al Melatonin pre-

vents changes in microsomal membrane fluidity during

induced lipid peroxidation FEBS Lett 1997 408297ndash300101 GARCIA JJ PI ~NOL-RIPOLL G MARTINEZ-BALLARIN E et al

Melatonin reduces membrane rigidity and oxidative dam-

age in the brain of SAMP8 mice Neurobiol Aging 2011

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234

Garcıa et al

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tive products of lipid peroxidation Prog Lipid Res 1997

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tation fetal sheep following umbilical cord occlusion Pedi-

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melatonin in hypoxic-ischemic brain damage an experi-

mental study J Matern Fetal Neonatal Med 2012

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F-isoprostanes and total F-neuroprostanes in a model of

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after severe traumatic brain injury and correlates with oxi-

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on diquat-induced lipid peroxidation in vivo as assessed by

the measurement of F2- isoprostanes J Pineal Res 2006

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tion J Membr Biol 1998 16259ndash65

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peutic role of melatonin in oncology J Pineal Res 1995

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pathological growth Front Neuroendocrinol 2000 21133ndash170

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application to oral cavity tumours J Oral Pathol Med

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stress J Membr Biol 2009 23193ndash99117 GARCIA JJ REITER RJ CABRERA JJ et al 5-methoxytrypto-

phol preserves hepatic microsomal membrane fluidity dur-

ing oxidative stress J Cell Biochem 2000 76651ndash657

118 GARCIA JJ REITER RJ KARBOWNIK M et al N-acetylseroto-

nin suppresses hepatic microsomal membrane rigidity asso-

ciated with lipid peroxidation Eur J Pharmacol 2001

428169ndash175

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induced free radical- mediated reduction in microsomal

membrane fluidity reversal by indole-3-propionic acid J

Bioenerg Biomembr 2001 3373ndash78

120 KARBOWNIK M REITER RJ GARCIA JJ et al Indole-3-propi-

onic acid a melatonin-related molecule protects hepatic

microsomal membranes from iron-induced oxidative dam-

age relevance to cancer reduction J Cell Biochem 2001

81507ndash513121 GARCIA JJ REITER RJ PIE J et al Role of pinoline and

melatonin in stabilizing hepatic microsomal membranes

against oxidative stress J Bioenerg Biomembr 1999

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the antioxidant activity of melatonin in vitro Free Radic

Biol Med 1996 21307ndash315

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of melatonin on hydroxyl radicals generated by alloxan

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tonin and related indoles with hydroxyl radicals EPR and

spin trapping investigations Free Radic Biol Med 1997

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effect of melatonin in carrageenan-induced models of local

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actions of melatonin in oxygen radical pathophysiology

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Chem B 2004 10819083ndash19085150 KUCERKA N PENCER J NIEH MP et al Influence of choles-

terol on the bilayer properties of monounsaturated phos-

phatidylcholine unilamellar vesicles Eur Phys J E Soft

Matter 2007 23247ndash254151 SANCHEZ-HIDALGO M De la LASTRA CA CARRASCOSA-SAL-

MORAL MP et al Age-related changes in melatonin synthesis

in rat extrapineal tissues Exp Gerontol 2009 44328ndash334

152 OCHOA JJ VILCHEZ MJ PALACIOS MA et al Melatonin pro-

tects against lipid peroxidation and membrane rigidity in

erythrocytes from patients undergoing cardiopulmonary

bypass surgery J Pineal Res 2003 35104ndash108

153 ROMANOFF ME KINGSLEY CP Anesthetic management in

the precardiopulmonary by-pass period In A Practical

Approach to Cardiac Anaesthesia HENSLEY FA MARTIN

DE eds Little Brown Boston 1995 pp 219ndash230

154 STARKOPF J TAMME K ZILMER M et al The evidence of

oxidative stress in cardiac surgery and septic patients a

comparative study Clin Chim Acta 1997 26277ndash88155 KARBOWNIK M REITER RJ GARCIA JJ et al Melatonin

reduces rat hepatic macromolecular damage due to oxida-

tive stress caused by delta-aminolevulinic acid Biochim

Biophys Acta 2000 1523140ndash146156 TEOULE R Radiation-induced DNA damage and its repair

Int J Radiat Biol Relat Stud Phys Chem Med 1987

51573ndash589

157 AMES BN GOLD LS Endogenous mutagens and the causes

of aging and cancer Mutat Res 1991 2503ndash16

158 MARTINEZ-CAYUELA M Oxygen free radicals and human

disease Biochimie 1995 77147ndash161

159 FLOYD RA The role of 8-hydroxydeoxyguanosine in carci-

nogenesis Carcinogenesis 1990 111447ndash1450

160 KARBOWNIK M REITER RJ QI W et al Protective effects of

melatonin against oxidation of guanine bases in DNA and

decreased microsomal membrane fluidity in rat liver

induced by whole body ionizing radiation Mol Cell Bio-

chem 2000 211137ndash144161 PLAA GL PRIESTLY BG Intrahepatic cholestasis induced

by drugs and chemicals Pharmacol Rev 1976 28207ndash273

162 ROTH RA DAHM LJ Neutrophil- and glutathione-mediated

hepatotoxicity of alpha- naphthylisothiocyanate Drug

Metab Rev 1997 29153ndash165163 CALVO JR REITER RJ GARCIA JJ et al Characterization of

the protective effects of melatonin and related indoles

against alpha naphthylisothiocyanate-induced liver injury

in rats J Cell Biochem 2001 80461ndash470164 NeuroASSBERGER L JOHANSSON AC BJeuroORCK S Antibodies to

neutrophil granulocyte myeloperoxidase and elastase auto-

immune responses in glomerulonephritis due to hydralazine

treatment J Intern Med 1991 229261ndash265165 SPEIRS C FIELDER AH CHAPEL H et al Complement sys-

tem protein C4 and susceptibility to hydralazine-induced

systemic lupus erythematosus Lancet 1989 1922ndash924

166 LEVINE EG BLOOMFIELD CD Leukemias and myelodysplas-

tic syndromes secondary to drug radiation and environ-

mental exposure Semin Oncol 1992 1947ndash84167 BRUGNARA C de FRANCESCHI L Effect of cell age and phen-

ylhydrazine on the cation transport properties of rabbit ery-

throcytes J Cell Physiol 1993 154271ndash280168 GOLDBERG B STERN A The mechanism of oxidative hemo-

lysis produced by phenylhydrazine Mol Pharmacol 1977

13832ndash839

169 BOSAN WS LAMBERT CE SHANK RC The role of formalde-

hyde in hydrazine-induced methylation of liver DNA guan-

ine Carcinogenesis 1986 7413ndash418170 PARODI S De FLORA S CAVANNA M et al DNA-damaging

activity in vivo and bacterial mutagenicity of sixteen hydra-

zine derivatives as related quantitatively to their carcinoge-

nicity Cancer Res 1981 411469ndash1482171 VIDAL-VANACLOCHA F ALONSO-VARONA A AYALA R et al

Coincident implantation growth and interaction sites

within the liver of cancer and reactive hematopoietic cells

Int J Cancer 1990 46267ndash271172 KARBOWNIK M REITER RJ GARCIA JJ et al Melatonin

reduces phenylhydrazine-induced oxidative damage to cel-

lular membranes evidence for the involvement of iron Int

J Biochem Cell Biol 2000 321045ndash1054173 MANIBUSAN MK ODIN M EASTMOND DA Postulated car-

bon tetrachloride mode of action a review J Environ Sci

Health C Environ Carcinog Ecotoxicol Rev 2007 25185ndash

209

174 BARCHAS J DACOSTA F SPECTOR S Acute pharmacology of

melatonin Nature 1967 214919ndash920175 JAHNKE G MARR M MYERS C et al Maternal and devel-

opmental toxicity evaluation of melatonin administered

orally to pregnant Sprague-Dawley rats Toxicol Sci 1999

50271ndash279176 MOLINA-CARBALLO A MU ~NOZ-HOYOS A REITER RJ et al

Utility of high doses of melatonin as adjunctive anticonvul-

sant therapy in a child with severe myoclonic epilepsy two

yearsrsquo experience J Pineal Res 1997 2397ndash105177 De BLEECKER JL LAMONT BH VERSTRAETE AG et al Mela-

tonin and painful gynecomastia Neurology 1999 53435ndash

436

236

Garcıa et al

178 CALVO JR GUERRERO JM OSUNA C et al Melatonin trig-

gers Crohnrsquos disease symptoms J Pineal Res 2002 32277ndash

278

179 LISSONI P Is there a role for melatonin in supportive care

Support Care Cancer 2002 10110ndash116180 MONTILLA P T UNEZ I Melatonin Present and Future 1st

edn Nova Science Publishers Inc New York 2006

181 PANDI-PERUMAL SR CARDINALI DP Melatonin From Mol-

ecules to Therapy 1st edn Nova Science Publishers Inc

New York 2007

182 ROSALES-CORRAL SA ACU ~NA-CASTROVIEJO D COTO-MONTES

A et al Alzheimerrsquos disease pathological mechanisms and

the beneficial role of melatonin J Pineal Res 2012 52167ndash202

183 SANCHEZ-BARCELO EJ MEDIAVILLA MD TAN DX et al

Clinical uses of melatonin evaluation of human trials Curr

Med Chem 2010 172070ndash2095184 OKATANI Y WAKATSUKI A REITER RJ et al Melatonin

reduces oxidative damage of neural lipids and proteins in

senescence-accelerated mouse Neurobiol Aging 2002

23639ndash644185 KORKMAZ A REITER RJ TOPAL T et al Melatonin an

established antioxidant worthy of use in clinical trials Mol

Med 2009 1543ndash50

237


because it utilizes a wide variety of means to reduce oxida-tive stress Firstly melatonin scavenges several toxicreactants including the highly toxic hydroxyl radical (˙OH)[30ndash32] and what may be even more important it takes

advantage of its derivatives which likewise are highly effi-cient radical scavengers When melatonin interacts withOH it initiates a scavenging cascade reaction (Fig 2) in

which the metabolites produced cyclic 3-hydroxymelato-nin N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK)and N1-acetyl-5-methoxykynuramine (AMK) also func-

tion in the neutralizing of free radicals [33ndash36] Thus onemolecule of melatonin eventually may scavenge up to eightor more radicals Additionally melatonin also detoxifies

the peroxynitrite anion (ONOO) hydrogen peroxide thesuperoxide anion radical (O

2 ) singlet oxygen as well asother toxic reactants [37ndash44] These actions of melatoninin scavenging free radicals throughout the cell depend on

its chemical characteristics that is melatonin is lipophilicand hydrophilic and thus it crosses all cell barriers withease and is available to all tissues cells and subcellular

organelles Therefore the melatoninrsquos beneficial actionsextend to every organism and every organIn addition to its direct scavenging actions melatonin

functions as an indirect antioxidant as well It does so bymeans of its ability to stimulate the expression and activityof antioxidant enzymes which remove free radicals andtheir precursors [45ndash50] Also melatonin or its metabolite

AMK inhibits one pro-oxidative enzyme that is induc-ible nitric oxide synthase (iNOS) [51] NO˙ has the capa-bility of coupling with the O

2 to form the ONOOHence melatonin limits the formation of ONOO a non-radical reactant which is equally toxic as ˙OH by reducingthe activity of iNOS

One additional important functional feature of melato-ninrsquos ability to reduce oxidative stress has been uncoveredin recent years Mitochondria are mainly responsible for

energy generation through oxidative phosphorylationyielding ATP which is required for every cell functionUnfortunately large numbers of free radicals are gener-ated within mitochondria when electrons escape during

their transfer through the respiratory chain By stimulatingcomplex I and complex IV of the mitochondrial respira-tory chain melatonin reduces electron leakage and free

radical generation that is a consequence of the respiratoryprocess [52ndash57] This is referred to as radical avoidance[38] Finally melatonin also contributes to the mainte-nance of the production of the ATP by the mitochondria

via maintaining high levels of mitochondrial glutathione(GSH) This reduces the necessity of the mitochondria tak-ing up extra GSH from the cytosol [23]

The combination of these beneficial properties of mela-tonin in the mitochondria and its ability to directly scav-enge free radicals and to stimulate the antioxidant

enzymes in all cell compartments makes this indoleamineunique in terms of its antioxidant capabilities In a recentstudy where melatonin was compared with synthetically

produced mitochondrially targeted antioxidants melato-nin was found to be superior in protecting mitochondriafrom free-radical-mediated damage [58]

Fluidity of biological membranes and lipidperoxidation

Every eukaryotic cell is surrounded by a plasma mem-brane that separates it from the extracellular environmentMoreover membranes also enclose the organelles which

divide the cell into discrete compartments wherein specificbiochemical processes take placeThe most abundant constituents of cellular membranes

are phospholipids From a chemical structure point of

view all membranes contain the following phospholipidsphosphatidylcholine phosphatidylserine phosphatidyleth-anolamine sphingomyelin and phosphatidylinositol These

molecules are amphipathic structures composed of polar(or hydrophilic) and nonpolar (or hydrophobic) moietiesIn an aqueous environment phospholipids tend to orient

with their polar heads facing the surrounding aqueous sur-faces while the hydrophobic fatty acyl chains project awayfrom contact with water Thereby when phospholipids are

dispersed in water they spontaneously form lipid bilayersEach phospholipid layer of a cell membrane is referred toas a leafletPhospholipids are not distributed uniformly across the

bilayer rather there is an asymmetrical distributionbetween the inner and outer leaflets of membranes Besidesphospholipids animal membranes also contain cholesterol

5-MethoxytryptopholTryptophanHydroxyindole-O-Tryptophanmethyltransferasehydroxylase

- -- 5 Hydroxytryptophan 5 Hydroxytryptophol5 Methoxytryptophan- -Hydroxyindole O

- AldehydeL aromatic amino acidmethyltransferase reductasedecarboxylasey

5-Hydroxyindol-5 Hydroxytryptamine5-Methoxytryptamine acetaldehydeHydroxyindole-O-

Monoamine methyltransferase AldehydeN-acetyltransferaseyfoxidase dehydrogenase

N-Acetyl-5- 5-Hydroxyindol5-Methoxyindolhydroxytryptamine acetic acidacetic acid

Hydroxyindole-O-Hydroxyindole-O-methyltransferasemethyltransferase

-- - - 5 MethoxyindolN Acetyl 5 methoxytryptamineti idacetic acidmelatonin( )

Fig 1 Pathway for tryptophan meta-bolism and synthesis of melatonin inmammals Indoleamines that have adocumented ability to reduce membranerigidity and lipid peroxidation aredepicted in red

226

Garcıa et al
































H H



-NH CHONH2



227



















(A)

(B)

(C)

(D)


XInitiation

Initiation

RH + X R


ROO + RH ROOH + R

Termination



228

Garcıa et al































100

80

60

40

In

hibi

tion

20

0


Melatonin (Log[])


229



































38

37

36

35

34

Mem

bran

e flu

idit

y (1

Pol

ariz

atio

n)

33





230

Garcıa et al

































Conclusions








231






Acknowledgements





References




















1472206ndash214



































Res 2012 52365ndash375

















18537ndash539








27101ndash110






232

Garcıa et al


















2001 34237ndash256





2003 341ndash10











45235ndash246














Int 1995 26497ndash502




1995 19111ndash115




3269ndash75


















381ndash9























207ndash213





738251ndash261



206







233







E782
























246ndash350





Med 1994 17411ndash418








1828







2002 53209ndash215















1999 26108ndash112





































31343ndash349









234

Garcıa et al






















40326ndash331
















2011 40593ndash597












428169ndash175






















































235




















35367ndash375































51573ndash589






























13832ndash839















209










436

236

Garcıa et al



278






New York 2007












237

































H H



-NH CHONH2



227



















(A)

(B)

(C)

(D)


XInitiation

Initiation

RH + X R


ROO + RH ROOH + R

Termination



228

Garcıa et al































100

80

60

40

In

hibi

tion

20

0


Melatonin (Log[])


229



































38

37

36

35

34

Mem

bran

e flu

idit

y (1

Pol

ariz

atio

n)

33





230

Garcıa et al

































Conclusions








231






Acknowledgements





References




















1472206ndash214



































Res 2012 52365ndash375

















18537ndash539








27101ndash110






232

Garcıa et al


















2001 34237ndash256





2003 341ndash10











45235ndash246














Int 1995 26497ndash502




1995 19111ndash115




3269ndash75


















381ndash9























207ndash213





738251ndash261



206







233







E782
























246ndash350





Med 1994 17411ndash418








1828







2002 53209ndash215















1999 26108ndash112





































31343ndash349









234

Garcıa et al






















40326ndash331
















2011 40593ndash597












428169ndash175






















































235




















35367ndash375































51573ndash589






























13832ndash839















209










436

236

Garcıa et al



278






New York 2007












237



















(A)

(B)

(C)

(D)


XInitiation

Initiation

RH + X R


ROO + RH ROOH + R

Termination



228

Garcıa et al































100

80

60

40

In

hibi

tion

20

0


Melatonin (Log[])


229



































38

37

36

35

34

Mem

bran

e flu

idit

y (1

Pol

ariz

atio

n)

33





230

Garcıa et al

































Conclusions








231






Acknowledgements





References




















1472206ndash214



































Res 2012 52365ndash375

















18537ndash539








27101ndash110






232

Garcıa et al


















2001 34237ndash256





2003 341ndash10











45235ndash246














Int 1995 26497ndash502




1995 19111ndash115




3269ndash75


















381ndash9























207ndash213





738251ndash261



206







233







E782
























246ndash350





Med 1994 17411ndash418








1828







2002 53209ndash215















1999 26108ndash112





































31343ndash349









234

Garcıa et al






















40326ndash331
















2011 40593ndash597












428169ndash175






















































235




















35367ndash375































51573ndash589






























13832ndash839















209










436

236

Garcıa et al



278






New York 2007












237
































100

80

60

40

In

hibi

tion

20

0


Melatonin (Log[])


229



































38

37

36

35

34

Mem

bran

e flu

idit

y (1

Pol

ariz

atio

n)

33





230

Garcıa et al

































Conclusions








231






Acknowledgements





References




















1472206ndash214



































Res 2012 52365ndash375

















18537ndash539








27101ndash110






232

Garcıa et al


















2001 34237ndash256





2003 341ndash10











45235ndash246














Int 1995 26497ndash502




1995 19111ndash115




3269ndash75


















381ndash9























207ndash213





738251ndash261



206







233







E782
























246ndash350





Med 1994 17411ndash418








1828







2002 53209ndash215















1999 26108ndash112





































31343ndash349









234

Garcıa et al






















40326ndash331
















2011 40593ndash597












428169ndash175






















































235




















35367ndash375































51573ndash589






























13832ndash839















209










436

236

Garcıa et al



278






New York 2007












237



































38

37

36

35

34

Mem

bran

e flu

idit

y (1

Pol

ariz

atio

n)

33





230

Garcıa et al

































Conclusions








231






Acknowledgements





References




















1472206ndash214



































Res 2012 52365ndash375

















18537ndash539








27101ndash110






232

Garcıa et al


















2001 34237ndash256





2003 341ndash10











45235ndash246














Int 1995 26497ndash502




1995 19111ndash115




3269ndash75


















381ndash9























207ndash213





738251ndash261



206







233







E782
























246ndash350





Med 1994 17411ndash418








1828







2002 53209ndash215















1999 26108ndash112





































31343ndash349









234

Garcıa et al






















40326ndash331
















2011 40593ndash597












428169ndash175






















































235




















35367ndash375































51573ndash589






























13832ndash839















209










436

236

Garcıa et al



278






New York 2007












237


































Conclusions








231






Acknowledgements





References




















1472206ndash214



































Res 2012 52365ndash375

















18537ndash539








27101ndash110






232

Garcıa et al


















2001 34237ndash256





2003 341ndash10











45235ndash246














Int 1995 26497ndash502




1995 19111ndash115




3269ndash75


















381ndash9























207ndash213





738251ndash261



206







233







E782
























246ndash350





Med 1994 17411ndash418








1828







2002 53209ndash215















1999 26108ndash112





































31343ndash349









234

Garcıa et al






















40326ndash331
















2011 40593ndash597












428169ndash175






















































235




















35367ndash375































51573ndash589






























13832ndash839















209










436

236

Garcıa et al



278






New York 2007












237






Acknowledgements





References




















1472206ndash214



































Res 2012 52365ndash375

















18537ndash539








27101ndash110






232

Garcıa et al


















2001 34237ndash256





2003 341ndash10











45235ndash246














Int 1995 26497ndash502




1995 19111ndash115




3269ndash75


















381ndash9























207ndash213





738251ndash261



206







233







E782
























246ndash350





Med 1994 17411ndash418








1828







2002 53209ndash215















1999 26108ndash112





































31343ndash349









234

Garcıa et al






















40326ndash331
















2011 40593ndash597












428169ndash175






















































235




















35367ndash375































51573ndash589






























13832ndash839















209










436

236

Garcıa et al



278






New York 2007












237



















2001 34237ndash256





2003 341ndash10











45235ndash246














Int 1995 26497ndash502




1995 19111ndash115




3269ndash75


















381ndash9























207ndash213





738251ndash261



206







233







E782
























246ndash350





Med 1994 17411ndash418








1828







2002 53209ndash215















1999 26108ndash112





































31343ndash349









234

Garcıa et al






















40326ndash331
















2011 40593ndash597












428169ndash175






















































235




















35367ndash375































51573ndash589






























13832ndash839















209










436

236

Garcıa et al



278






New York 2007












237







E782
























246ndash350





Med 1994 17411ndash418








1828







2002 53209ndash215















1999 26108ndash112





































31343ndash349









234

Garcıa et al






















40326ndash331
















2011 40593ndash597












428169ndash175






















































235




















35367ndash375































51573ndash589






























13832ndash839















209










436

236

Garcıa et al



278






New York 2007












237























40326ndash331
















2011 40593ndash597












428169ndash175






















































235




















35367ndash375































51573ndash589






























13832ndash839















209










436

236

Garcıa et al



278






New York 2007












237




















35367ndash375































51573ndash589






























13832ndash839















209










436

236

Garcıa et al



278






New York 2007












237




278






New York 2007












237


Documents

Protective effects of melatonin in reducing oxidative stress and in preserving the fluidity of biological membranes: a review