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Review
Melatonin as an antioxidant: biochemical mechanisms and
pathophysiological implications in humans
Russel J. Reiter
1½
, Dun-xian Tan
1
, Juan C. Mayo
1
, Rosa M. Sainz, Josefa Leon
1
and Zbigniew Czarnocki
2
1
Department of Cellular and Structural Biology, University of Texas Health Science Center,
San Antonio, Texas, U.S.A.;
2
Department of Chemistry, Warsaw University, Warszawa,
Poland
Received: 17 September, 2003; revised: 17 October, 2003; accepted: 21 October, 2003
Key words: antioxidant, antioxidant enzymes, free radicals, melatonin, neurodegeneration, respiratory
distress, sepsis
This brief resume enumerates the multiple actions of melatonin as an antioxidant.
This indoleamine is produced in the vertebrate pineal gland, the retina and possibly
some other organs. Additionally, however, it is found in invertebrates, bacteria, uni
-
cellular organisms as well as in plants, all of which do not have a pineal gland.
Melatonin’s functions as an antioxidant include: a), direct free radical scavenging,
b), stimulation of antioxidative enzymes, c), increasing the efficiency of mitochon
-
drial oxidative phosphorylation and reducing electron leakage (thereby lowering
free radical generation), and 3), augmenting the efficiency of other antioxidants.
There may be other functions of melatonin, yet undiscovered, which enhance its abil
-
ity to protect against molecular damage by oxygen and nitrogen-based toxic reac
-
tants. Numerous in vitro and in vivo studies have documented the ability of both
physiological and pharmacological concentrations to melatonin to protect against
free radical destruction. Furthermore, clinical tests utilizing melatonin have proven
highly successful; because of the positive outcomes of these studies, melatonin’s use
in disease states and processes where free radical damage is involved should be in
-
creased.
Vol. 50 No. 4/2003
1129–1146
QUARTERLY
½
Corresponding author: Russel J. Reiter, Ph.D., Department of Cellular and Structural Biology, Mail
Code 7762, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX
78229-3900 U.S.A.; phone: 210/567-3859; fax: 210/567-6948; e-mail: Reiter@UTHSCSA.EDU
Abbreviations: AD, Alzheimer's disease; AFMK, N
1
-acetyl-N
2
-formyl-5-methoxykynuramine; CAT,
catalase; DMPO, 5,5-dimethylpyrolidine oxide; ETC, electron transport chain; G6PD, glucose-6-phos
-
phate dehydrogenase; GPx, glutathione peroxidase; GRd, glutathione reductase; LOO, peroxyl radical;
MPTP, 1-methyl-4-phenylpyridinium; RDS, respiratory distress syndrome; RNS, reactive nitrogen spe
-
cies; ROS, recative oxygen species; SOD, superoxide dismutase; t-BHP, t-butyl-hydroperoxide.
N-Acetyl-5-methoxytryptamine, commonly
known as melatonin (Fig. 1), is a synthetic
product of the vertebrate pineal gland as well
as of other select organs. The biochemical
pathway concerned with the synthesis of
melatonin has been well described as have
been the neural mechanisms governing pi
-
neal melatonin production (Reiter, 1991).
The indoleamine was initially found to func
-
tion as a mediator of circannual reproductive
rhythms (Reiter, 1980) as well as of circadian
cycles (Kennaway & Wright, 2002). Subse
-
quently, however, melatonin was shown to
have significantly broader actions including
oncostatic effects (Blask et al., 2002), immune
system stimulation (Guerrero & Reiter, 2002)
and anti-inflammatory functions (Cuzzocrea
& Reiter, 2002).
Even more recently, and somewhat unex
-
pectedly, melatonin was identified as a power
-
ful direct free radical scavenger (Tan et al.,
2002) and indirect antioxidant (Reiter et al.,
2000c; Rodriquez et al., 2004). What seems
particularly unusual is the high efficacy of
melatonin as a protector against reactive oxy
-
gen (ROS) and reactive nitrogen species
(RNS). This field of research has witnessed
an explosive expansion in the last decade and
whereas all of the mechanisms of melatonin’s
effects as an executioner of free radicals and
related products have not yet been identified,
there is no doubt concerning its ability to re
-
strain the molecular damage resulting from
toxic oxygen and nitrogen-based reactants
(AcuZa-Castroviejo et al., 2002; Reiter et al.,
2002b). This review summarizes some of the
mechanisms of melatonin’s protective actions
as well as documents that it significantly re
-
duces oxidative stress at many levels. It is
noted, however, that this brief resume cannot
do justice to the massive number of reports
that have been published on these subjects
and the reader is urged to consult other re
-
views for additional details and information
(Hardeland et al., 1995; Reiter et al., 2000b,
2001; Tan et al., 2002; 2003b).
DIRECT ANTIOXIDANT ACTIONS OF
MELATONIN
Melatonin seems to function via a number
of means to reduce oxidative stress. Thus,
the experimental evidence supports its ac-
tions as a direct free radical scavenger (Har-
deland et al., 1993; 1995; Allegra et al., 2003),
as an indirect antioxidant when stimulating
antioxidant enzymes (Reiter et al., 2000c;
Rodriquez et al., 2004), its stimulation of the
synthesis of glutathione (an essential intra
-
cellular antioxidant) (Urata et al., 1999), its
ability to augment the activities of other anti
-
oxidants (or vice versa) (Gitto et al., 2001a),
its protection of antioxidative enzymes from
oxidative damage (Mayo et al., 2002; 2003),
and its ability to increase the efficiency of mi
-
tochondrial electron transport chain (ETC)
thereby lowering electron leakage and reduc
-
ing free radical generation (AcuZa-Castro
-
viejo et al., 2002; Okatani et al., 2003a).
While melatonin has proven highly effective
in lowering molecular damage under condi
-
tions of elevated oxidative stress (Reiter,
1998; Reiter & Tan, 2003), the contribution of
each of the above-mentioned processes to the
ability of this indole to restrain the resulting
molecular mutilation that accompanies exag
-
1130 R.J. Reiter and others 2003
Figure 1. Molecular structure of the antioxidant
melatonin.
This molecule was discovered to be a direct free radical
scavenger roughly a decade ago and subsequently
there has been a vast amount of research documenting
its potent and diverse antioxidant capabilities.
-
gerated free radical generation remains un
-
known.
Melatonin as a direct scavenger of oxygen-
based free radicals and related species
There is now a vast literature documenting
melatonin’s interaction with both ROS and
RNS (Reiter et al., 2001; Poeggeler et al.,
2002; Tan et al., 2002; Allegra et al., 2003).
The initial evidence illustrating melatonin’s
ability to neutralize the highly toxic hydroxyl
radical (×OH) (Fig. 2) appeared roughly 10
years ago (Tan et al., 1993). Since then, nu
-
merous reports have appeared which confirm
this action of melatonin (Poeggeler et al.,
1994; Hardeland et al., 1995; Matuszak et al.,
1997; Bandyopadhyay et al., 2002; Brömme et
al., 2002; Li et al., 2002) and, furthermore, a
potential product of that interaction has been
identified to be cyclic 3-hydroxymelatonin
(Tan et al., 1998). In the proposed scheme,
each molecule of melatonin scavenges two
×OH; this study also showed that cyclic
3-hydroxymelatonin is excreted in the urine
(human and rat) and the quantity of this by
-
product is proportional to the amount of
melatonin administered to an animal and to
the degree of oxidative stress the animal has
experienced. The findings also indicate that
cyclic 3-hydroxymelatonin is a footprint mole
-
cule that appears in the urine and that it is an
index of in vivo ×OH scavenging by mela
-
tonin. Finally, cyclic 3-hydroxymelatonin it
-
Vol. 50 Melatonin as an antioxidant 1131
Figure 2. Oxygen and nitrogen-based free radicals and associated reactants that are generated in cells
by various processes.
Free radicals are defined as molecules that have an unpaired electron in their valence orbital. Free radicals and the
related reactants are not equally toxic. It is generally conceded that the most reactive, and therefore damaging,
products are the oxygen-based hydroxyl radical (×OH) and the nitrogen-based peroxynitrite anion (ONOO
–
). Arg,
L-arginine; BH
4
, 5,6,7,8-tetrahydro-L-biopterin; Cit, L-citruline; ETC, electron transport chain; FAD, flavin adenine
dinucleotide (oxidized); FADH
2
, flavin adenine dinucleotide (reduced); Gly, glycine; MOP, myloperoxidase; NAD
+
,
nicotinamide adenine dinucleotide (oxidized); NADH, nicotinamide adenine dinucleotide (reduced); P-450,
cytochrome P-450.
self has free radical scavenging activity (Tan
et al., 2003b; Lopez-Burillo et al., 2003).
Stasica and co-workers (2000), using a com
-
putational approach, determined the most
likely probable site on the indole ring of
melatonin that may bind a ×OH; the C2 car
-
bon was proposed as the likely site of attack.
Additional details concerning the structural
properties of melatonin that make it an effi
-
cient scavenger of the ×OH as well as the po
-
tential reactions of the indole as a radical
scavenger are reviewed by Tan and colleagues
(2002).
Hydrogen peroxide (H
2
O
2
), a non-radical
ROS, is generated in vivo by several enzyme
systems and, additionally, it is produced
intracellularly by the dismutation of the
superoxide anion radical (O
2
×–
) (Fig. 2). In
vivo,H
2
O
2
is a weak oxidizing and reducing
agent. Also, no electric charge allows H
2
O
2
to
traverse cell membranes and is therefore ac-
cessible to sites significantly removed from
its point of generation. Although H
2
O
2
is
weakly reactive, its major toxicity derives
from its conversion to the highly toxic ×OH
via the Fenton or Haber-Weiss reactions.
Melatonin as a scavenger of H
2
O
2
in a pure
chemical system was initially documented by
Tan et al. (2000). A mechanism of the oxida
-
tion of melatonin by H
2
O
2
was suggested on
the basis of the major resulting metabolite,
i.e., N
1
-acetyl-N
2
-formyl-5-methoxykynurami
-
ne (AFMK). AFMK was confirmed using elec
-
tron ionization mass spectrometry and pro
-
ton and carbon nuclear magnetic resonance.
Whether intracellular melatonin neutralizes
H
2
O
2
in a manner as described by Tan et al.
(2000) is unknown. If it does, it would act like
the H
2
O
2
-metabolizing enzymes, i.e., gluta
-
thione peroxidase (GSH-Px) and catalase
(CAT), in the removal of this oxidizing agent
and, importantly, reduce the generation of
the ×OH. That AFMK is a byproduct of the in
-
teraction of melatonin with H
2
O
2
has been re
-
ported by others as well including under in
vivo conditions (Burkhardt et al., 2001;
Carampin et al., 2003; Rozov et al., 2003).
One recent report questioned whether
melatonin interacts directly with H
2
O
2
(Fowler et al., 2003). Why this group failed to
document what other reports found is not ap
-
parent but could be related to the fact they
tested a single dose of melatonin.
Recently, significant attention has been fo
-
cused on AFMK as a scavenger of oxy
-
gen-based reactants as well. Cyclic voltametry
has shown that AFMK is capable of donating
two electrons; furthermore, the kynuramine
reduces damage to DNA and lipids in a high
free radical environment and lowers neuronal
death when these cells are exposed to either
H
2
O
2
, glutamate or amyloid b
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 the
resulting products, i.e., cyclic 3-hydroxyme-
latonin and AFMK, may also function as scav-
engers of toxic reactants. This cascade of
scavenging actions may be one reason ac-
counting for the unexpectedly high efficacy of
melatonin in reducing free radical damage in
vivo. Finally, another product, N-acetyl-5-me-
thoxykynuramine (AMK), is likewise capable
of neutralizing some oxygen-based reactants
(Tan et al., 2002; Ressmeyer et al., 2003) as is
the chief hepatic enzymatic metabolite of
melatonin, 6-hydroxymelatonin (Qi et al.,
2000; Hara et al., 2001).
O
2
×–
(Fig. 2) is generated during respiration
in mitochondria when electrons leak from the
ETC and during the respiratory burst of
phagocytic cells. Relative to the ×OH, O
2
×–
has low toxicity but it rapidly couples with ni
-
tric oxide (NO×) to produce a non-radical ni
-
trogen-based reactant, the peroxynitrite an
-
ion (ONOO
–
) (Fig. 2); this product is consid
-
ered to be almost as damaging as is the ×OH.
In addition to its inherent toxicity, ONOO
–
via peroxynitrous acid (ONOOH) may be me
-
tabolized to the ×OH in vivo.
The efficacy of melatonin in neutralizing the
O
2
×–
is only poorly defined. Melatonin re
-
portedly scavenges this reactant in a pure
chemical system where a hypoxanthine/ xan
-
1132 R.J. Reiter and others 2003
thine system was used to generate the O
2
×–
(Marshall et al., 1996); the ability of
melatonin to quench the O
2
×–
is also sup
-
ported by evidence that melatonin modestly
diminished the electron spin resonance (ESR)
signal produced by the adduct, 5,5-dime
-
thylpyrroline oxide (DMPO)-O
2
×–
(Zang et al.,
1998). However, the role, if any, of melatonin
in neutralizing the O
2
×–
is unclear, particu
-
larly in vivo.
Macromolecular damage in vivo is also a
consequence of singlet oxygen (
1
O
2
), an en
-
ergy-rich form of oxygen;
1
O
2
is usually pro
-
duced in photosensitizing reactions of a vari
-
ety of substrates including dyes and biological
pigments. Poeggeler and co-workers (1996)
were the first to show that melatonin neutral
-
ized
1
O
2
, during which AFMK was generated.
This quenching ability of melatonin was con-
firmed by Zang et al. (1998) and by Roberts
and colleagues (2000). That AFMK is the
product formed when melatonin is oxidized
by
1
O
2
has been confirmed (De Almeida et al.,
2003). In light of these findings, it appears
that AFMK is a product common to several
interactions of melatonin with oxygen-based
reactants.
The evidence that melatonin functions as a
chain breaking antioxidant by scavenging the
peroxyl radical (LOO×) remains problematic.
The first reports on this subject placed
melatonin among the very best scavengers in
terms of its ability to neutralize the LOO×
(Fig. 2); thus, the claim was made that mela
-
tonin is twice as effective as vitamin E, the
primier chain breaking antioxidant, in inter
-
fering with the propagation of lipid pero
-
xidation (Pieri et al., 1994; 1995). These re
-
ports, however, have not been universally
confirmed. Using a markedly different sys
-
tem to perform their tests, Livrea and
co-workers (1997) also analyzed the ability of
melatonin in terms of its chain breaking activ
-
ity. Using either non-peroxidable unilamellar
dimirystoyl phosphatidycholine liposomes or
peroxidable soybean phosphatidylcholine
liposomes, this group reported that mela
-
tonin was not a particularly effective chain
breaking antioxidant. The data accumulated
by Livrea et al. (1997) are consistent with
those of Antunes and colleagues (1999) who
thoroughly evaluated the lipoperoxyl-trap
-
ping efficiency of melatonin and concluded
that it had limited ability to neutralize di
-
rectly the LOO×. Given that melatonin is a
heterocyclic aromatic amine, this group noted
that molecules containing an NH group in a
5-membered pyrrole and carbazole ring do
not typically function as highly effective chain
breaking antioxidants.
Despite the controversy regarding the abil
-
ity of melatonin to interact with the LOO×, in
vivo melatonin has consistently been found to
be highly efficient in limiting the peroxi
-
dation of lipids (Reiter et al., 1998). Curtail
-
ing the progress of self-propagating lipid
breakdown by melatonin may be a result of its
ability to scavenge the initiating agents, e.g.,
×OH, ONOO
–
, etc., rather than being due to
its apparent limited capability as a direct
LOO× scavenger, i.e., to function as a chain
breaking antioxidant.
Due to the electron-deficient nature of ha-
lide ions, haloperoxyl radicals are signifi-
cantly more reactive than the alkylperoxyl
radical; accordingly, the trichloromethyl
-
peroxyl radical (CCl
3
OO×) was found to be po
-
tently trapped by melatonin (Marshall et al.,
1996). In a pulse radiolysis study, this find
-
ing was also reported by Mahal and
co-workers (1999). This latter group also
showed, like Scaiano (1995), that melatonin
traps the tert-butoxyl radical.
Hypochlorous acid (HOCl) formation is cata
-
lyzed by myloperoxidase (MPO) in activated
neutrophils (Fig. 2). HOCl is a powerful oxi
-
dizing agent and it damages a wide variety of
biomolecules. Presumably by electron dona
-
tion, Dellegar et al. (1999) reported that mela
-
tonin detoxifies HOCl. In the process,
melatonin is theoretically converted to the
melatoninyl cation radical which could scav
-
enge another radical. This melatonin radical
intermediate has also been proposed in other
Vol. 50 Melatonin as an antioxidant 1133
scavenging actions of the indoleamine (Tan et
al., 1993; 1998).
Melatonin as a direct scavenger of nitro
-
gen-based free radicals and related species
NO× (nitrogen monoxide), a molecule widely
produced in mammals where it has a variety
of beneficial functions is a rather weak free
radical. NO× is involved in a number of in
-
flammatory processes that can lead to exten
-
sive tissue injury. Additionally, much of the
toxicity of NO× may be a consequence of its
coupling with O
2
×–
which results in the for
-
mation of the highly reactive ONOO
–
(Fig. 2).
That melatonin detoxifies NO× has been re
-
ported by several groups (Mahal et al., 1999;
Noda et al., 1999; Blanchard et al., 2000). The
latter group, however, showed that melatonin
interacts with NO× only in the presence of
molecular oxygen, a finding suggesting that
melatonin may in fact react with a molecule
derived from nitric oxide, possibly ONOO
–
.
The requirement for O
2
in the reaction of
melatonin with NO× was confirmed by
Turjanski et al. (2000a; 2000b). The chief
product of the melatonin/NO× reaction is re-
portedly N-nitrosomelatonin. Semiempirical
AM1 computations are consistent with the
nitrosation of melatonin by NO×/O
2
(Turjanski et al., 2000c).
The coupling of two relatively unreactive
species, i.e., NO× and O
2
×–
, generates the po
-
tently oxidizing ONOO
–
(Fig. 2); this combi
-
nation reaction has a very high rate constant
(5.0 ´ 10
9
M
–1
s
–1
). The toxicity of peroxy
-
nitrite could relate to any of three reactive
species, i.e., ground state peroxynitrous acid
(ONOOH), the activated form of the acid
(ONOOH*) as well as to its conjugated base
(ONOO
–
). Zhang and colleagues (1998; 1999)
documented that melatonin is a substrate for
peroxynitrite. Melatonin was shown to react
with ONOO
–
with first-order kinetics; how
-
ever, the rate constant for the reaction of
melatonin with ONOOH was considerably
higher (a reaction unimportant at physiologi
-
cal pH). Blanchard et al. (2000) also found
that melatonin interacted with ONOO
–
but
with the formation of different products than
those reported by Zhang et al. (1998; 1999).
These differences cannot currently be ex
-
plained.
INDIRECT ANTIOXIDANT ACTIONS
OF MELATONIN
Besides its ability to directly scavenge oxy
-
gen and nitrogen-based reactants, melatonin
has a number of indirect means by which it
may reduce oxidative stress. The relative im
-
portance of the direct and indirect anti
-
oxidative processes of melatonin in vivo re
-
main unknown.
Melatonin stimulation of antioxidative
enzymes
Antioxidative enzymes provide a major de-
fense mechanism against free radical damage
either by metabolizing them to less reactive
species or to non-toxic byproducts (Fig. 3).
The important antioxidative enzymes that
have been investigated relative to melatonin
are the superoxide dismutases (SOD), both
MnSOD and CuZnSOD, catalase (CAT),
glutathione peroxidase (GPx), glutathione
reductase (GRd) and glucose-6-phosphate
dehydrogenase (G6PD) (Reiter et al., 2000c;
Mayo et al., 2002; Rodriquez et al., 2004).
The initial reports documenting melatonin’s
stimulatory effect on GPx appeared almost 10
years ago when it was shown that pharmaco
-
logical doses of melatonin given to rats
(Barlow-Walden et al., 1995) and chicks
(Pablos et al., 1995) in vivo resulted in a
marked augmentation in the activity of this
enzyme. GPx reduces free radical damage be
-
cause it metabolizes H
2
O
2
(and other perox
-
ides) to water; in the process, however,
glutathione (GSH) is oxidized to its disulfide,
GSSG (Fig. 3). GSSG is then quickly reduced
back to GSH by GRd, an enzyme which has
1134 R.J. Reiter and others 2003
also been shown to be stimulated by
melatonin (Pablos et al., 1998). The recycling
of GSH may well be a major action of mela
-
tonin in curtailing oxidative stress. The abil
-
ity of melatonin to regulate the GSH/GSSG
balance by modulating enzyme activities ap
-
pears to involve an action of melatonin at a
nuclear binding site (Pablos et al., 1997). The
other GSH metabolizing enzyme, i.e., CAT,
also increases its activity in response to
melatonin (Naidu et al., 2003).
Subsequent studies showed that the stimula
-
tion of the activities of GPx and GRd is
achieved with physiological levels of mela
-
tonin. Thus, Pablos et al. (1998) observed
that the normal nighttime rise in the activi
-
ties of these enzymes in a number of tissues is
extinguished if chicks are kept under con
-
stant light; light exposure at night, of course,
prevents the nocturnal rise in physiological
melatonin concentrations which, because of
the loss of this stimulatory signal, prevents
the activities of GPx and GRd from rising.
GRd requires the co-factor NADPH which is
generated by the antioxidative enzyme G6PD
(Fig. 3). Although the amount of data is lim
-
ited, there is one report claiming that
melatonin also stimulates G6PD (Pierrefiche
& Laborit, 1995). This would be important in
GSH recycling since NADPH is a necessary
cofactor for G6PD.
Vol. 50 Melatonin as an antioxidant 1135
Figure 3. Free radicals and other reactants are enzymatically removed from cells by a series of
antioxidative enzymes.
Melatonin stimulates the activity of several of these as summarized in the text. Melatonin also promotes the pro
-
duction of glutathione, an important intracellular antioxidant. Arg,
L-arginine; GH
4
, 5,6,7,8-tetrahydro-biopterin;
Cat, catalase; Cyt c, cytochrome c; GSSG, glutathione (oxidized; glutathione disulfide); GSH, glutathione (reduced);
GPx, glutathione peroxidase; GRd, glutathione reductase; GSH-T, glutathione S-transferase; GSNO, nitrosylated
glutathione; HbO
2
, oxyhemoglobin; PDG, phosphate-dependent glutaminase; SOD, superoxide dismutase.
Subsequent to this early series of studies,
numerous reports have confirmed and ex
-
tended the evidence concerning the promo
-
tional effects of melatonin on the antioxi
-
dative enzymes (Rodriquez et al., 2004) in
-
cluding, not only GPx and GRd but SOD as
well. Furthermore, due to the ease with
which it crosses the placenta, melatonin,
when administered to pregnant rats, results
in a rise in GPx and SOD activities in the
brain of the fetuses (Okatani et al., 2000).
Also, in the human chorion melatonin has
been found to increase GPx activity (Okatani
et al., 2001).
In addition to estimating enzyme activities,
gene expression for antioxidative enzymes
have been studied following melatonin admin
-
istration. For example, Mayo et al. (2002)
found that the depressions in gene expression
for neural GPx, CuZnSOD and MnSOD that
occurred after treatment of rats with the
neurotoxin 6-hydroxydopamine were prevent-
ed by melatonin. Similarly, others (Kotler et
al., 1998; Antolin et al., 2002) have also ob-
served that melatonin enhances gene expres-
sion for antioxidative enzymes either under
basal conditions or after their inhibition by
neurotoxic agents. While the direct free radi-
cal scavenging properties of melatonin are in
-
dependent of any receptor for the indole, its
ability to alter the activity of antioxidative en
-
zymes likely requires an interaction of mela
-
tonin with either membrane or nuclear recep
-
tors.
While melatonin clearly functions as a di
-
rect free radical scavenger and indirectly re
-
duces oxidative stress via the stimulation of
antioxidative enzymes, the relative impor
-
tance of each of these processes in reducing
tissue damage due to free radicals in an unre
-
solved issue. The high efficacy of melatonin
in preventing oxidative mutilation of essen
-
tial biomolecules suggests that both mecha
-
nisms must be important and, in fact, other
processes, as summarized below, may like
-
wise contribute to melatonin’s unexpectedly
strong antioxidative capabilities.
Melatonin stimulation of glutathione
synthesis
GSH is very abundant intracellular free radi
-
cal scavenger and antioxidant (Fig. 3). A single
report has shown that melatonin stimulates its
rate limiting enzyme, g -glutamylcysteine
synthase, thereby increasing intracellular GSH
concentrations (Urata et al., 1999). This action
of melatonin, unlike the direct free radical scav
-
enging function of the indoleamine, is likely me
-
diated by specific receptors. The stimulation of
GSH synthesis by melatonin could be a major
antioxidative action of melatonin. Considering
the potential importance of the findings of
Urata and co-workers (1999), it is in need of
confirmation particularly in vivo and in a vari
-
ety of cell types.
Synergistic actions of melatonin with clas-
sic antioxidants
According to Gitto and co-workers (2001a),
under in vitro conditions and using end prod-
ucts of lipid peroxidation as an indices of free
radical damage, melatonin augments the pro-
tective actions of vitamin E, vitamin C and
GSH against free radical-mediated oxidation
of polyunsaturated fatty fats. The clear impli-
cation, and the conclusion reached by the au
-
thors, is that combinations of melatonin with
other antioxidants clearly increase their effi
-
cacy. The mechanism of the synergy remains
unknown and confirmation of these findings,
particularly in vivo, is important. When com
-
pared under conditions of high oxidative
stress in vivo, melatonin has proven superior
to vitamins C and E in reducing oxidative
damage (Tan et al., 2002).
Actions of melatonin at the level
of the mitochondria
Mitochondria are a major source of free rad
-
icals and as a consequence these subcellular
organelles are exposed to extensive oxidative
abuse. The inner mitochondrial membrane is
the site of the ETC (Fig. 4), a system of
1136 R.J. Reiter and others 2003
oxido-reductant protein complexes (com
-
plexes I, II, III and IV). In aerobic cells, mito
-
chondrial oxidative phosphorylation
(OXPHOS) is responsible for an estimated
90–95% of the total ATP generated by cells.
Deficiencies in the ETC can lead to the leak
-
age of electrons which thereafter form free
radicals and other toxic reactants which re
-
sults in molecular damage in mitochondria;
this damage culminates in and contributes to
what are referred to as mitochondria-related
diseases (AcuZa-Castroviejo et al., 2002).
That melatonin has important actions at the
level of mitochondria is suggested by a num
-
ber of observations: a), melatonin is an effi
-
cient scavenger of ROS/RNS which are abun
-
dantly produced in mitochondria; b), al
-
though mitochondria are incapable of GSH
synthesis (they take it up from the cytosol),
they do possess GPx and GRd for GSH cy
-
cling, both enzymes of which are stimulated
by melatonin; c), melatonin has been shown
to have antiapoptotic effects, with the
apoptotic signals originating in mitochon
-
dria; d), melatonin may be in higher concen
-
trations in mitochondria than elsewhere in
the cell and higher than serum concentra
-
tions of melatonin (AcuZa-Castroviejo et al.,
2002).
Long term melatonin administration has
been reported to increase the number of mito
-
chondria in cells (Decker & Quay, 1982) while
experiments with radioactive melatonin sug
-
gests mitochondrial binding sites for the
indole (Poon & Pang, 1992). Additionally,
melatonin was shown to inhibit NADPH-de
-
pendent lipid peroxidation in human placen
-
tal mitochondria (Milczarek et al., 2000), to
protect the fetal rat brain against oxidant-me
-
diated mitochondrial damage (Wakatsuki et
al., 2001) and to stimulate mitochondrial res
-
piration in the brain and liver of senes
-
cence-accelerated mice (Okatani et al., 2002a;
2002b; 2003a; 2003b).
The first tests of melatonin in reference to
mitochondrial physiology were performed in
vivo. In these studies, melatonin was shown
to significantly increase hepatic and brain
complex I and complex IV activities of the mi
-
tochondrial ETC (Martin et al., 2000a). Fur
-
Vol. 50 Melatonin as an antioxidant 1137
Figure 4. Actions of melatonin at the mitochondrial level increase its efficiency as an antioxidant by re
-
ducing free radical generation and increasing ATP production.
These actions of melatonin are summarized in the text. Cyt c, cytochrome c; RNS, reactive nitrogen species; ROS,
reactive oxygen species; ETC, electron transport chain.
thermore, it was shown that melatonin re
-
verses the inhibitory effect of 1-methyl-4-
phenylpyridinium (MPTP) (Absi et al., 2000)
and ruthenium red (Martin et al., 2000a) on
the activities of these complexes.
In in vitro experiments, Martin et al. (2000b)
documented that when oxidative damage was
induced in mitochondria by incubating them
with t-butyl hydroperoxide (t-BHP), the effect
was prevented with a 100 nM concentration
of melatonin; conversely, neither the addition
of N-acetylcysteine, vitamin E nor vitamin C
protected mitochondria against t-BHP toxic
-
ity. Melatonin’s stimulation of complexes I
and IV activities were dose-dependent.
Finally, melatonin reduces cyanide toxicity at
the level of mitochondria as well (Yamamoto
& Yang, 1996) These actions of melatonin
would be very important physiologically con-
sidering ETC efficiency is coupled to
OXPHOS. Subsequent studies, in fact, re-
ported that ATP production is also elevated
when mitochondria are treated with mela-
tonin (Martin et al., 2001). As an energy
source, ATP is critical to the cell for virtually
all functions including the repair of oxida-
tively-damaged molecules. Thus, besides pro-
tecting molecules from damage due to ROS
and RNS, once molecules are damaged
melatonin may indirectly hasten their repair.
This is an area of melatonin research that
awaits further experimentation. Also, in
-
creasing the efficiency of the ETC theoreti
-
cally, at least, may reduce electron leakage
and free radical production. The actions of
melatonin at the mitochondrial are summa
-
rized in Fig. 4.
PATHOPHYSIOLOGICAL EVIDENCE
OF THE ANTIOXIDANT FUNCTIONS
OF MELATONIN IN HUMANS
Antioxidants have attracted a great deal of
attention as potential agents for forestalling
age-related free radical-based diseases
(Halliwell, 2001; Harman, 2002; Reiter et al.,
2003). The information on the scavenging ac
-
tions of antioxidants may be particularly im
-
portant for the aged where free radical-medi
-
ated diseases are numerous and, further
-
more, the aging process itself is believed, in
part, to be a result of the persistent accumula
-
tion of molecular debris resulting from the
unending mutilation by free radicals (Har
-
man, 1998; 1999; Fossel, 2002).
In the last 10 years, a vast amount of pub
-
lished literature has been amassed that pro
-
vides unequivocal documentation that, in
vivo, melatonin has the capability of diminish
-
ing destruction of DNA, proteins and lipids
that are a result of their reactions with ROS
and RNS. The number of publications regard
-
ing these actions in non-human mammals is
so massive that it is not possible to discuss
these findings in the current report and the
reader is referred to other sources for this in-
formation (Reiter et al., 2000a; 2002a; 2003;
Cardinali et al., 2003; Cheung, 2003). Rather,
summarized below are some of the studies in
which melatonin has been used to combat
free radical damage in humans.
Based on a substantial amount of data docu-
menting melatonin’s ability to reduce neural
damage in models of Alzheimer’s disease
(AD) (Pappolla et al., 2000; 2003), several
groups have administered melatonin to indi
-
viduals diagnosed with this neuro
-
degenerative condition in attempt to amelio
-
rate disease symptoms. The first of these re
-
ports appeared in 1998 when Brusco et al.
(1998a) showed that giving one of a pair of
monozygotic twins (both with AD) 6 mg
melatonin daily for 36 months significantly
delayed the progression of the disease and re
-
duced the degree of brain atrophy (as esti
-
mated by nuclear magnetic resonance imag
-
ing of the CNS). Melatonin is known to
readily cross the blood-brain barrier and
neuronal loss in AD is believed to be a conse
-
quence of free radical-mediated apoptosis.
Thus, in this study melatonin’s antioxidant
functions presumably helped preserve neu
-
rons from mutilation and death. Using be
-
1138 R.J. Reiter and others 2003
havioral endpoints, several studies have also
shown that melatonin may also improve loco
-
motor activity and affect in AD patients
(Brusco et al., 1998b; Cohen-Mansfield et al.,
2000). In addition to the report that recently
appeared (Asayama et al., 2003), more exten
-
sive investigations that are double-blind and
placebo-controlled are needed. There is cer
-
tainly ample experimental evidence to justify
treating AD patients and elderly with related
degenerative diseases of the CNS with
melatonin (Reiter et al., 2000a; Cheung,
2003). Also, there is virtually no acute or
chronic toxicity of melatonin (Jahnke et al.,
1999; Jan et al., 2000; Seabra et al., 2000)
which should encourage its long-term use in
individuals with age-related neurodegenera
-
tive diseases.
Melatonin has proven useful as a treatment
for septic shock in premature newborns as
well (Gitto et al., 2001b). Sepsis is a serious
condition and occurs in 1–10 cases per 1000
births with even higher rates than this in
low-birth-weight neonates. The mortality
rates in newborns who become septic can be
as high as 50%. Sepsis culminates in multiple
organ failure and death with the tissue dam-
aged generally believed to be due, at least in
part, to excessive free radical generation.
Since melatonin had been shown to be an ef
-
fective treatment for bacterial lipopolysaccha
-
ride (Sewerynek et al., 1995), which causes
multiple organ failure in rats, Gitto and
co-workers (2001b) tested its efficacy as a
treatment for sepsis in human premature
newborns.
Twenty newborns judged to be septic were
randomly divided into 2 groups of 10 infants
each; all were given conventional antibiotic
therapy but 10 were also supplemented with 2
doses of 10 mg oral melatonin (separated by a
1 h interval). Within 1 h and also at 4 h after
melatonin administration, the levels of lipid
peroxidation products in the serum were al
-
ready depressed relative to those in non-mela
-
tonin treated children. Additionally, over the
next 72 h all clinical parameters improved sig
-
nificantly more quickly in the children given
melatonin. As is usual, 3 or 10 neonates not
treated with melatonin died; conversely, due
to their more rapid recovery all of the melato
-
nin treated children survived. Gitto et al.
(2001b) attributed the beneficial actions of
melatonin in this study to the antioxidant
properties of the indoleamine although there
may have been other yet to be defined actions
which permitted the children to exhibit a
more rapid recovery from sepsis. Regardless
of the mechanisms of protection by melato
-
nin, the outcome of this study seemingly justi
-
fies the use of melatonin in the treatment of
this serious condition not only in children but
in adults as well.
A variety of conditions in neonates is associ
-
ated with elevated oxidative stress (Gitto et
al., 2002). Considering the high efficacy and
low toxicity of melatonin in these conditions
and stimulated by the successful use of mela-
tonin as described above (Gitto et al., 2001b),
Fulia and co-workers (2001) used the indo-
leamine to treat newborns who were suffering
from transient asphyxia; free radical damage
has been implicated in the pathophysiology of
neonatal asphyxia. Twenty newborns with
perinatal asphyxia diagnosed within the first
6 h of life were studied, 10 of who were given
80 mg melatonin orally. In the asphyxiated
neonates given melatonin, serum levels of
malondialdehyde (a lipid peroxidation prod
-
uct) and nitrite/nitrate concentrations were
significantly reduced relative to those in the
non-melatonin treated, asphyxiated children.
Likewise, the clinical improvement was faster
in the neonates given melatonin and all of
these individuals survived; conversely, 3 of 10
non-melatonin-treated, asphyxiated children
died (Fulia et al., 2001).
Reactive oxygen species have also been im
-
plicated in the pathogenesis of respiratory
distress syndrome (RDS) and its complica
-
tions (Gitto et al., 2001c). Given this, the ra
-
tionale for treating children with RDS (grade
III or IV) with melatonin is clear. In this case,
40 RDS newborns were given melatonin (100
Vol. 50 Melatonin as an antioxidant 1139
mg given intravenously over a 2 day period)
and 34 were provided conventional therapy
only. At 24 and 72 h and at 7 days after
melatonin administration, serum interleukin
(IL)-6, IL-8, tumor necrosis factor alpha
(TNF-a) and nitrite/nitrate levels were signif
-
icantly lower in the melatonin-treated RDS
neonates relative to the newborns suffering
with RDS but given conventional treatment
(Gitto et al., 2004). Clearly, melatonin im
-
proved the outcome of the RDS afflicted new
-
borns by reducing oxidative and inflamma
-
tory parameters associated with this condi
-
tion.
Melatonin has also been tested as an agent
to reduce oxidative stress in adult humans
subjected to cardiopulmonary bypass surgery
(CPB) (Ochoa et al., 2003). Melatonin when
given in advance of surgery onset reduced the
degree of lipid peroxidation products in eryth-
rocyte membranes of blood collected at vari-
ous intervals after the onset of the operation.
Another index of the breakdown of mem-
brane lipids also documented the protective
effect of melatonin. Thus, the increase in red
blood cell membrane rigidity (decreased
membrane fluidity) was also attenuated in the
CPB individuals treated with melatonin. An
increased membrane rigidity correlates posi
-
tively with augmented levels of products of
lipid peroxidation (Garcia et al., 1999).
CONCLUDING REMARKS
Since the discovery of melatonin as an anti
-
oxidant in 1993 (Tan et al., 1993), there has
been an burgeoning number of reports docu
-
menting this action under an almost unlim
-
ited number of conditions, many of which
have direct clinical relevance. Thus, mela
-
tonin has been shown to reduce the toxicity of
drugs and in some cases improve their effi
-
cacy (Reiter et al., 2002c), to reduce the sever
-
ity and degree of tissue damage following
ischemia/reperfusion in the brain (Cheung,
2003) and other organs, to prevent degenera
-
tive changes in the CNS in models of Alzhei
-
mer’s (Pappolla et al., 2000) and Parkinson’s
disease (Antolin et al., 2002), to reduce free
radical damage to DNA which may lead to
cancer (Reiter et al., 1998), and many other
situations too numerous to mention in this
brief report.
A major unresolved issue, as already men
-
tioned above, relates to the significance of the
various actions of melatonin that function in
reduction of oxidative stress. At this point, it
is unknown which of the multiple actions of
melatonin, i.e., whether free radical scaveng-
ing, stimulation of antioxidative enzymes, in-
creasing the efficacy of mitochondrial ETC
and reducing electron leakage, improving the
efficiency of other antioxidants, etc., are most
important in contributing to its high efficacy.
It is also likely that both receptor-independ
-
ent and receptor-dependent actions of mela
-
tonin participate in its function as an antioxi
-
dant (Tan et al., 2003a). Its successful use in
human conditions where excessive free radi
-
cal generation occurs, however, should en
-
courage its continued use in the treatment of
other disease processes, and there seem to be
many, where oxidative stress is a component.
1140 R.J. Reiter and others 2003
REFERENCES
Absi E, Ayala A, Machado A, Prado J. (2000) Protective effect of melatonin against the 1-methyl-4-phenylpyridinium-
induced inhibition of complex I of the mitochondrial respiratory chain. J Pineal Res.; 29: 40–7. MEDLINE
Acuna-Castroviejo D, Escames G, Carozo A, Leon J, Khaldy H, Reiter RJ. (2002) Melatonin, mitochondrial homeostasis
and mitochondrial-related diseases. Curr Top Med Chem.; 2: 133–52.
MEDLINE
Allegra M, Reiter RJ, Tan DX, Gentile C, Tesoriere L, Livrea MA. (2003) The chemistry of melatonin’s interaction with
reactive species. J Pineal Res.; 34: 1–10.
MEDLINE
Antolin I, Mayo JC, Sainz RM, del Brio ML, Herrera F, Martin V, Rodriquez C. (2002) Protective effect of melatonin in a
chronic experimental model of Parkinson’s disease. Brain Res.; 943: 163–73.
MEDLINE
Antunes F, Barclay LRC, Ingold KU, King M, Norris JO, Sciano JL, Xi F. (1999) On the antioxidant activity of melatonin.
Free Radic Biol Med.; 26: 117–28.
MEDLINE
Asayama K, Yamadera H, Ito T, Suzuki H, Kudo Y, Endo S. (2003) Double blind study of melatonin effects on the sleep-
wake rhythm, cognitive and non-cognitive functions in Alzheimer type dementia. J Nippon Med Sch.; 70: 334–41.
MEDLINE
Bandyopadhyay D, Bandyopadhyay A, Das PK, Reiter RJ. (2002) Melatonin protects against gastric ulceration and
increases the efficacy of ranitidine and omeprazole in reducing gastric damage. J Pineal Res.; 33: 1–7.
MEDLINE
Barlow-Walden LR, Reiter RJ, Abe M, Pablos M, Menendez-Pelaez A, Chen LD, Poeggeler B. (1995) Melatonin
stimulates brain glutathione peroxidase activity. Neurochem Int.; 26: 497–52.
MEDLINE
Blanchard B, Pompon D, Ducroq C. (2000) Nitrosation of melatonin by nitric oxide and peroxynitrite. J Pineal Res.; 29:
184–92.
MEDLINE
Blask DE, Sauer LA, Dauchy RT. (2002) Melatonin as a chronobiotic/anticancer agent: Cellular, biochemical, and
molecular mechanisms of action and their implications for circadian-based therapy. Curr Top Med Chem.; 2: 113–32.
MEDLINE
Bromme HJ, Morke W, Peschke E. (2002) Transformation of barbituric acid into alloxan by hydroxyl radicals: interaction
with melatonin and other hydroxyl radical scavengers. J Pineal Res.; 33: 239–47.
MEDLINE
Brusco LI, Marquez M, Cardinali DP. (1998a) Monozygotic twins with Alzheimer’s disease treated with melatonin: Case
report. J Pineal Res.; 25: 260–3.
MEDLINE
Brusco LI, Marquez M, Cardinali DP. (2000) Melatonin treatment stabilizes chronobiological and cognitive symptoms in
Alzheimer’s disease. Neuroendocrinol Lett.; 21: 39–42.
MEDLINE
Burkhardt S, Poeggeler B, Tan DX, Rosner C, Gruetzner T, Nitzki F, Schoenke M, Thuermann S, Reiter RJ, Hardeland R.
(2001) Oxidation products formed from melatonin in various radical-generating systems. In Actions and redox properties
of melatonin in various radical-generating systems. Hardeland R, ed, pp 9–22. Cuvillier, Gottingen.
Carampin P, Rosan S, Dalzoppo D, Zagotto G, Zatla P. (2003) Some biochemical properties of melatonin and the
characterization of a relevant metabolite arising from its interaction with H
2
O
2
. J Pineal Res.; 34: 134–42. MEDLINE
Cardinali DP, Ladizesky MG, Boggio V, Cutrera RA, Mautalen C. (2003) Melatonin effects on bone: Experimental facts
and clinical perspectives. J Pineal Res.; 34: 81–7. MEDLINE
Cheung RTF. (2003) The utility of melatonin in reducing cerebral damage resulting from ischemia and reperfusion. J
Pineal Res.; 34: 153–60.
MEDLINE
Cohen-Mansfield JS, Garfinkel D, Lipton S. (2000) Melatonin for treatment of sun downing in elderly persons with
dementia – a preliminary study. Arch Gerontol Geriatrics.; 35: 65–71.
Cuzzocrea S, Reiter RJ. (2002) Pharmacological actions of melatonin in acute and chronic inflammation. Curr Top Med
Chem.; 2: 153–66.
MEDLINE
De Almeida EA, Martinez GR, Klitzke CF, de Medeiros MHG, Di Mascio P. (2003) Oxidation of melatonin by singlet
molecular oxygen (O
2
(
1
Dg)) produces N
1
-acetyl-N
2
-formyl- 5-methoxykynuramine. J Pineal Res.; 35: 131–7. MEDLINE
Decker JF, Quay WB. (1982) Stimulatory effects of melatonin on ependymal epithelium of choroid plexuses in golden
hamsters. J Neural Transm.; 55: 53–67.
MEDLINE
Dellegar SM, Murphy SA, Boune AE. (1999) Identification of factors affecting the rate of deactivation of hypochlorous
acid by melatonin. Biochem Biophys Res Commun.; 257: 431–39.
MEDLINE
Fossel M. (2002) Cell senescence in human aging and disease. Ann NY Acad Sci.; 959: 14–23.
MEDLINE
Fowler G, Daroszewska M, Ingold KU. (2003) Melatonin does not directly scavenge hydrogen peroxide. Free Radic Biol
Med.; 34: 77–83.
MEDLINE
Fulia F, Gitto E, Cuzzocrea S, Reiter RJ, Dugo L, Gitto P, Barberi S, Cordaro S, Barber I. (2001) Increased levels of
malondialdehyde and nitrite/nitrate in the blood of asphyxiated newborns: Reduction by melatonin. J Pineal Res.; 31:
343–9.
MEDLINE
Garcia JJ, Reiter RJ, Pie J, Ortiz GG, Cabrera J, Sainz RM, Acuna-Castroviejo D. (1999) Role of pinoline and melatonin
in stabilizing hepatic microsomal membranes against oxidative stress. J Bioenerg Biomembr.; 31: 609–16.
MEDLINE
Gitto E, Tan DX, Reiter RJ, Karbownik M, Manchester LC, Cuzzocrea S, Fulia F, Barberi I. (2001a) Individual and
synergistic actions of melatonin: Studies with vitamin E, vitamin C, glutathione and desferoxamine in liver homogenates. J
Pharm Pharmacol.; 53: 1393–401.
MEDLINE
Gitto E, Karbownik M, Reiter RJ, Tan DX, Cuzzocrea S, Chiurazzi P, Cordaro S, Corona G, Trimarchi G, Barberi I.
(2001b) Effects of melatonin treatment in septic newborns. Pediat Res.; 50: 756–60.
MEDLINE
Gitto E, Reiter RJ, Karbownik M, Tan DX, Barberi I. (2001c) Respiratory distress syndrome in the newborn: Role of
oxidative stress. Intensive Care Med.; 27: 1116–23.
MEDLINE
Gitto E, Reiter RJ, Karbownik M, Tan DX, Gitto P, Barberi S, Barberi I. (2002) Causes of oxidative stress in the pre- and
postnatal period. Biol Neonate.; 81: 146–57.
MEDLINE
Gitto E, Reiter RJ, Cordaro SP, La Rosa M, Chiurazzi P, Trimarchi G, Gitto P, Calabro MP, Barberi I. (2004) Oxidative
and respiratory parameters in respiratory distress syndrome of preterm newborns: Beneficial effects of melatonin. Am J
Perinatol.; in press.
Guerrero JM, Reiter RJ. (2002) Melatonin-immune system relationships. Curr Top Med Chem.; 2: 167–80.
MEDLINE
Halliwell B. (2001) Free radical reactions in human disease. In Environmental stressors in health and disease. Fuchs J,
Packer L, eds, pp 1–16. Marcel Dekker, New York.
Hara M, Yoshida M, Nishijima H, Yokosuka M, Iigo M, Ohtani-Kaneko R, Shimada A, Hasegawa T, Akama Y, Hirata K.
(2001) Melatonin, a pineal secretory product with antioxidant properties, protects against cisplatin-induced nephrotoxicity
in rats. J Pineal Res.; 30: 129–38. MEDLINE
Hardeland R, Reiter RJ, Poeggeler B, Tan DX. (1993) The significance of the metabolism of the neurohormone melatonin:
Antioxidative protection and formation of bioactive substances. Neurosci Biobehav Rev.; 17: 347–57.
MEDLINE
Hardeland R, Balzer I, Poeggeler B, Fuhrberg B, Uria H, Bahrmann G, Wolf R, Meyer TJ, Reiter RJ. (1995) On the
primary functions of melatonin in evolution: mediation of photoperiodic signals in unicell, photoxidation, and scavenging
of free radicals. J Pineal Res.; 18: 104–11.
MEDLINE
Harman D. (1998) Aging: Phenomenon and theories. Ann NY Acad Sci.; 854: 1–7.
MEDLINE
Harman D. (1999) Free radical theory of aging: Increasing the average life expectancy at birth and the maximum life span.
J Anti-Aging Med.; 2: 199–208.
Harman D. (2002) Alzheimer’s disease: Role of aging in pathogenesis. Ann NY Acad Sci.; 959: 384–95.
MEDLINE
Jahnke G, Marr M, Myers C. (1999) Maternal and developmental toxicity evaluation of melatonin administration orally to
pregnant Sprague-Dawley rats. Toxicol Sci.; 50: 271–9.
MEDLINE
Jan JE, Hamilton D, Seward N, Fast DK, Freeman RD, Landon M. (2000) Clinical trials of controlled-release melatonin in
children with sleep-wake disorders. J Pineal Res.; 29: 34–9.
MEDLINE
Kennaway DJ, Wright H. (2002) Melatonin and circadian rhythms. Curr Top Med Chem.; 2: 199–209
MEDLINE
Kotler ML, Rodriquez C, Sainz RM, Antolin I, Menendez-Pelaez A. (1998) Melatonin increases gene expression for
antioxidant enzymes in rat brain cortex. J Pineal Res.; 24: 83–9.
MEDLINE
Li XJ, Gu J, Lu SD, Sun FY. (2002) Melatonin attenuates MPTP-induced dopaminergic neuronal injury associated with
scavenging hydroxyl radical. J Pineal Res.; 32: 47–52.
MEDLINE
Livrea MA, Tesoriere L, D’Arpa D, Morreale M. (1997) Reaction of melatonin with lipoperoxyl radicals in phospholipid
bilayers. Free Radic Biol Med.; 23: 706–11.
MEDLINE
Lopez-Burillo S, Tan DX, Rodriquez-Gallego V, Manchester LC, Mayo JC, Sainz RM, Reiter RJ. (2003) Melatonin and
its derivatives cyclic 3-hydroxymelatonin, N
1
-acetyl-N
2
-formyl-5-methoxykynuramine and 6-hydroxymelatonin reduce
oxidative damage induced by Fenton reagents. J Pineal Res.; 34: 178–84.
Mahal HS, Sharma HS, Mukherjee T. (1999) Antioxidant properties of melatonin: A pulse radiolysis study. Free Radic
Biol Med.; 26: 557–65. MEDLINE
Marshall KA, Reiter RJ, Poeggeler B, Aruoma OI, Halliwell B. (1996) Evaluation of the antioxidant activity of melatonin
in vitro. Free Radic Biol Med.; 21: 307–15.
MEDLINE
Martin M, Macias M, Escames G, Reiter RJ, Agapito MT, Ortiz GG, Acuna-Castroviejo D. (2000a) Melatonin-induced
increased activity of the respiratory chain complexes I and IV can prevent mitochondrial damage induced by ruthenium
red in vitro. J Pineal Res.; 28: 242–8. MEDLINE
Martin M, Macias M, Escames G, Leon J, Acuna-Castroviejo D. (2000b) Melatonin but not vitamins C and E maintains
glutathione homeostasis in t-butylhydroperoxide induced mitochondrial oxidative stress. FASEB J.; 14: 1677–9.
MEDLINE
Martin M, Macias M, Leon J, Escames G, Khaldy H, Acuna-Castroviejo D. (2002) Melatonin increases the activity of
oxidative phosphorylation enzymes and the production of ATP in rat brain and liver mitochondria. Int J Biochem Cell
Biol.; 34: 348–57.
MEDLINE
Matuszak A, Reszka KJ, Chignell CF. (1997) Reaction of melatonin and related indoles with hydroxyl radicals: EPR and
spin trapping investigations. Free Radic Biol Med.; 23: 367–72.
MEDLINE
Mayo JC, Sainz RM, Antolin I, Herrera F, Martin V, Rodriquez C. (2002) Melatonin regulation of antioxidant enzyme
gene expression. Cell Mol Life Sci.; 59: 1706–13.
MEDLINE
Mayo JC, Tan DX, Sainz RM, Lopez-Burillo S, Reiter RJ. (2003) Oxidative damage to catalase induced by peroxyl
radicals: Functional protection by melatonin and other antioxidants. Free Radic Res.; 37: 543–53.
MEDLINE
Milczarek R, Klimek J, Zelewski L. (2000) Melatonin inhibits NADPH-dependent lipid peroxidation in human placental
mitochondria. Horm Metab Res.; 32: 84–5.
MEDLINE
Naidu PS, Singh A, Kaur P, Sandhir R, Kulkawi SK. (2003) Possible mechanism of action of melatonin attenuation of
haloperidol-induced orofacial dyskinesia. Pharmacol Biochem Behav.; 74: 641–8.
MEDLINE
Noda Y, Mori A, Liburty R, Packer L. (1999) Melatonin and its precursors scavenge nitric oxide. J Pineal Res.; 27:
159–64.
MEDLINE
Ochoa JJ, Vilchez MJ, Palacios MA, Garcia JJ, Reiter RJ, Munoz-Hoyos A. (2003) Melatonin protects against lipid
peroxidation and membrane rigidity in erythrocytes from patients undergoing cardiopulmonary bypass surgery. J Pineal
Res.; 35: 104–8.
MEDLINE
Okatani Y, Wakatsuki A, Kaneda C. (2000) Melatonin increases activities of glutathione peroxidase and superoxide
dismutase in fetal rat brain. J Pineal Res.; 28: 89–96.
MEDLINE
Okatani Y, Wakatsuki A, Shinohara K, Kaneda C, Fukaya T. (2001) Melatonin stimulates glutathione peroxidase activity
in human chorion. J Pineal Res.; 30: 199–205.
MEDLINE
Okatani Y, Wakatsuki A, Reiter RJ. (2002a) Melatonin protects hepatic mitochondrial chain activity in senescence-
accelerated mice. J Pineal Res.; 32: 143–8.
MEDLINE
Okatani Y, Wakatsuki A, Reiter RJ, Miyahara Y. (2002b) Hepatic mitochondrial dysfunction in senescence-accelerated
mice: Correction by long-term, orally administered physiological levels of melatonin. J Pineal Res.; 33: 127–33.
MEDLINE
Okatani Y, Wakatsuki A, Reiter RJ, Miyahara Y. (2003a) Acutely administered melatonin restores hepatic mitochondrial
physiology in old mice. Int J Biochem Cell Biol.; 35: 367–75.
MEDLINE
Okatani Y, Wakatsuki A, Reiter RJ, Enzan H, Miyahara Y. (2003b) Protective effective of melatonin against
mitochondrial injury induced by ischemia and reperfusion of rat liver. Eur J Pharmacol.; 469: 145–52. MEDLINE
Pablos MI, Agapito MT, Gutierrez R, Recio JM, Reiter RJ, Barlow-Walden LF, Acuna-Castroviejo D, Menendez-Pelaez
A. (1995) Melatonin stimulates the activity of the detoxifying enzyme glutathione peroxidase in several tissues of chicks. J
Pineal Res.; 19: 111–5.
MEDLINE
Pablos MI, Guerrero JM, Ortiz GG, Agapito MT, Reiter RJ. (1997) Both melatonin and a putative receptor agonist CGP
52608 stimulate glutathione peroxidase and glutathione reductase activities in mouse brain in vivo. Neuroendocrinol Lett.;
18: 49–58.
Pablos MI, Reiter RJ, Ortiz GG, Guerrero JM, Agapito MT, Chuang JI, Sewerynek E. (1998) Rhythms of glutathione
peroxidase and glutathione reductase in brain of chick and their inhibition by light. Neurochem Int.; 32: 69–75.
MEDLINE
Pappolla MA, Chyan YJ, Poeggeler B, Frangione B, Wilson G, Chiso J, Reiter RJ. (2000) An assessment of the
antioxidant and antiamyloidogenic properties of melatonin: Implications for Alzheimer’s disease. J Neural Transm.; 107:
203–31.
MEDLINE
Pappolla MA, Reiter RJ, Bryant-Thomas TK, Poeggeler B. (2003) Oxidative mediated neurodegeneration in Alzheimer’s
disease: Melatonin and related antioxidants as neuroprotective agents. Curr Med Chem.; 3: 233–43.
Pieri C, Marra M, Moroni F, Recchioni R, Marcheselli F. (1994) Melatonin: A peroxyl radical scavenger more effective
than vitamin E. Life Sci.; 55: 271–76.
MEDLINE
Pieri C, Moroni M, Marcheselli F, Recchioni R. (1995) Melatonin is an efficient antioxidant. Arch Gerontol Geriatr.; 20:
159–65.
Pierrefiche G, Laborit H. (1995) Oxygen radicals, melatonin and aging. Exp Gerontol.; 30: 213–27.
MEDLINE
Poeggeler B, Saarela S, Reiter RJ, Tan DX, Chen LD, Manchester LC, Barlow-Walden L. (1994) Melatonin — a highly
potent endogenous scavenger and electron donor: New aspects of the oxidation chemistry of this indole assessed in vitro.
Ann NY Acad Sci.; 738: 419–20.
MEDLINE
Poeggeler B, Reiter RJ, Hardeland R, Tan DX, Barlow-Walden LR. (1996) Melatonin and structurally related endogenous
indoles act as potent electron donors and radical scavengers in vitro. Redox Rep.; 2: 179–84.
MEDLINE
Poeggeler B, Thuermann S, Dose A, Schoenke M, Burkhardt S, Hardeland R. (2002) Melatonin’s unique scavenging
properties – roles of its functional substituents as revealed by a comparison with its structural analogues. J Pineal Res.; 33:
20–30.
Poon AMS, Pang SF. (1992) 2[
125
I]iodomelatonin binding sites in spleens of guinea pigs. Life Sci.; 50: 1719–26.
MEDLINE
Qi W, Reiter RJ, Tan DX, Manchester LC, Siu AW, Garcia JJ. (2000) Increased levels of oxidatively damaged DNA
induced by chromium(III) and H
2
O
2
: Protection by melatonin and related indoles. J Pineal Res.; 29: 54–61. MEDLINE
Reiter RJ. (1980) The pineal and its hormones in the control of reproduction in mammals. Endocr Rev.; 1: 109–31.
MEDLINE
Reiter RJ. (1991) Pineal melatonin: Cell biology of its synthesis and of its physiological interactions. Endocr Rev.; 12:
151–80.
MEDLINE
Reiter RJ. (1998) Oxidative damage in the central nervous system: Protection by melatonin. Prog Neurobiol.; 56: 359–84.
MEDLINE
Reiter RJ, Tan DX. (2003) Melatonin: A novel protective agent against oxidative injury of the ischemic/reperfused heart.
Cardiovasc Res.; 58: 10–9.
MEDLINE
Reiter RJ, Tan DX, Kim SJ, Qi W. (1998). Melatonin as a pharmacological agent against damage to lipids and DNA. Proc
West Pharmacol Soc.; 41: 229–36.
MEDLINE
Reiter RJ, Cabrera J, Sainz RM, Mayo JC, Manchester LC, Tan DX. (2000a) Melatonin as a pharmacological agent against
neuronal loss in experimental models of Huntington’s disease, Alzheimer’s disease and Parkinsonism. Ann NY Acad Sci.;
890: 471–85.
MEDLINE
Reiter RJ, Tan DX, Acuna-Castroviejo D, Burkhardt S, Karbownik M. (2000b) Melatonin: Mechanisms and actions as an
antioxidant. Curr Top Biophys.; 24: 171–83.
Reiter RJ, Tan DX, Osuna C, Gitto E. (2000c) Actions of melatonin in the reduction of oxidative stress: A review. J
Biomed Sci.; 7: 444–58.
MEDLINE
Reiter RJ, Tan DX, Manchester LC, Qi W. (2001) Biochemical reactivity of melatonin with reactive oxygen and nitrogen
species: A review of the evidence. Cell Biochem Biophys.; 34: 237–56.
MEDLINE
Reiter RJ, Tan DX, Allegra M. (2002a) Melatonin: Reducing molecular pathology and dysfunction due to free radicals and
associated reactants. Neuroendocrinol Lett.; 23: 3–8.
MEDLINE
Reiter RJ, Tan DX, Manchester LC, Calvo JR. (2002b) Antioxidative capacity of melatonin. In Handbook of antioxidants,
2nd edn. Cadenas E, Packer L, ed, pp 565–613. Marcel Dekker, New York.
Reiter RJ, Tan DX, Sainz RM, Mayo JC. (2002c) Melatonin: Reducing the toxicity and increasing the efficacy of drugs. J
Pharm Pharmacol.; 54: 1299–321.
MEDLINE
Reiter RJ, Sainz RM, Lopez-Burillo S, Mayo JC, Manchester LC, Tan DX. (2003) Melatonin ameliorates neurological
damage and neurophysiological deficits in experimental models of stroke. Ann NY Acad Sci.; 993: 35–47.
MEDLINE
Ressmeyer AR, Mayo JC, Zelosko V, Sainz RM, Tan DX, Poeggeler B, Antolin I, Reiter RJ, Hardeland R. (2003)
Antioxidant properties of the melatonin metabolite N-acetyl-5-methoxykynuramine (AMK): Scavenging of free radicals
and prevention of protein destruction. Redox Rep.; 8: 205–13.
MEDLINE
Roberts JE, Hu DN, Martinez L. (2000) Photophysical studies on melatonin and its receptor agonists. J Pineal Res.; 29:
94–9.
MEDLINE
Rodriquez C, Mayo JC, Sainz RM, Antolin I, Herrera F, Martin V, Reiter RJ. (2004) Regulation of antioxidant enzymes: A
significant role for melatonin. J Pineal Res.; in press.
Rozov SV, Filatova EV, Orlov AA, Volkova AV, Zhloba AA, Blashko EL, Pozdeyev NV. (2003) N
1
-Acetyl-N
2
-formyl-5-
methoxykynuramine is a product of melatonin oxidation in rats. J Pineal Res.; 35: 245–50. MEDLINE
Scaiano JC. (1995) Exploratory laser flash photolysis study of free radical reactions and magnetic field effects in
melatonin chemistry. J Pineal Res.; 19: 189–95.
MEDLINE
Seabra M de LV, Bignotto M, Pinto LR Jr, Tufik S. (2000) Randomized double blind clinical trial, controlled with
placebo, of the toxicology of chronic melatonin treatment. J Pineal Res.; 29: 193–200. MEDLINE
Sewerynek E, Melchiorri D, Reiter RJ, Ortiz GG, Lewinski A. (1995) Lipopolysaccharide-induced hepatotoxicity is
inhibited by the antioxidant melatonin. Eur J Pharmacol.; 293: 327–34.
MEDLINE
Stasica P, Paneth P, Rosiak JM. (2000) Hydroxyl radical reaction with melatonin molecule: A computational study. J
Pineal Res.; 29: 125–7.
MEDLINE
Tan DX, Chen LD, Poeggeler B, Manchester LC, Reiter RJ. (1993) Melatonin: A potent, endogenous hydroxyl radical
scavenger. Endocrine J.; 1: 57–60.
Tan DX, Manchester LC, Reiter RJ, Plummer BF, Hardies LJ, Weintraub ST, Vijayalaxmi, Shepard AMM. (1998) A
novel melatonin metabolite, cyclic 3-hydroxymelatonin: A biomarker of in vivo hydroxyl radical generation. Biochem
Biophys Res Commun.; 253: 614–20.
MEDLINE
Tan DX, Manchester LC, Reiter RJ, Plummer BL, Limson J, Weintraub ST, Qi W. (2000) Melatonin directly scavenges
hydrogen peroxide: A potentially new metabolic pathway of melatonin biotransformation. Free Radic Biol Med.; 29:
1177–85.
MEDLINE
Tan DX, Manchester LC, Burkhardt S, Sainz RM, Mayo JC, Kohen R, Shohami E, Huo YS, Hardeland R, Reiter RJ.
(2001) N
1
-Acetyl-N
2
-formyl-5-methoxykynuramine, a biogenic amine and melatonin metabolite, functions as a potent
antioxidant. FASEB J.; 15: 2294–6. MEDLINE
Tan DX, Reiter RJ, Manchester LC, Yan MT, El-Sawi M, Sainz RM, Mayo JC, Kohen R, Allegra M, Hardeland R. (2002)
Chemical and physical properties and potential mechanisms: Melatonin as a broad-spectrum antioxidant and free radical
scavenger. Curr Top Med Chem.; 2: 181–98.
MEDLINE
Tan DX, Manchester LC, Hardeland R, Lopez-Burillo S, Mayo JC, Sainz RM, Reiter RJ. (2003a) Melatonin: A hormone,
a tissue factor, an autocoid, a paracoid and an antioxidant vitamin. J Pineal Res.; 34: 75–8.
MEDLINE
Tan DX, Hardeland R, Manchester LC, Poeggeler B, Lopez-Burillo S, Mayo JC, Sainz RM, Reiter RJ. (2003b)
Mechanistic and comparative studies of melatonin and classic antioxidants in terms of their interactions with the ABTS
cation radical. J Pineal Res.; 34: 249–59.
MEDLINE
Turjanski AG, Chaia Z, Rosenstein RE, Estrin DA, Doctorovich F, Piro O. (2000a) N-Nitrosomelatonin. Acta Crystallogr
C.; 56: 682–3.
MEDLINE
Turjanski AG, Leonik F, Rosenstein RE, Estrin DA, Doctorovich F. (2000b) Scavenging of NO by melatonin. J Am Chem
Soc.; 122: 10468–9.
Turjanski AG, Saenz DA, Doctorovich F, Estrin DA, Rosenstein RE. (2000c) Nitrosation of melatonin by nitric oxide: A
computational study. J Pineal Res.; 31: 97–101.
MEDLINE
Urata Y, Honma S, Goto S, Todoroki S, Ueda T, Cho S, Honma K, Kondo T. (1999) Melatonin induces gamma-
glutamylcysteine synthetase mediated by activator protein-1 in human vascular endothelial cells. Free Radic Biol Med.;
27: 838–47.
MEDLINE
Wakatsuki A, Okatani Y, Shivohara K, Ikenoue N, Fukaya T. (2001) Melatonin protects against ischemia/reperfusion-
induced oxidative damage to mitochondria in fetal rat brain. J Pineal Res.; 31: 167–72.
MEDLINE
Yamamoto HA, Yang HW. (1996) Preventive effect of melatonin against cyanide-induced seizures and lipid peroxidation
in mice. Neurosci Lett.; 207: 89–92. MEDLINE
Zang LY, Cosma G, Gardner H, Vallynathan V. (1998) Scavenging of reactive oxygen species by melatonin. Biochim
Biophys Acta.; 1425: 469–77.
MEDLINE
Zhang H, Squadrito GI, Pryor WA. (1998) The reaction of melatonin with peroxynitrite: Formation of melatonin radical
cation and absence of stable nitrated products. Biochem Biophys Res Commun.; 25: 83–7.
Zhang H, Squadrito GL, Uppu R, Pryor WA. (1999) Reaction of peroxynitrite with melatonin: A mechanistic study. Chem
Res Toxicol.; 12: 526–34.
MEDLINE
