Melatonin role in the mitochondrial function

Abstract

Melatonin is an ancient molecule present in unicellular organisms at the very early moment of life. Initially identified as a secretory product of the pineal gland in mammals and in other species, it was considered a hormone related to reproduction. The evidence that melatonin is produced in many organs and tissues of the body, reaching concentrations higher than in the blood, support the multiplicity of the melatonin actions. The best-known actions of melatonin, currently supported by experimental and clinical data, include antioxidant and anti-inflammatory abilities, some of them involving genomic regulation of a series of enzymes. Besides, melatonin displays anticonvulsant and antiexcitotoxic properties. Most of the beneficial consequences resulting from melatonin administration may depend on its effects on mitochondrial physiology. The physiological effects of melatonin on normal mitochondria, its role to prevent mitochondrial impairment, energy failure, and apoptosis in oxidatively-damaged mitochondria, and the beneficial effects of the administration of melatonin in experimental and clinical diseases involving mitochondrial dysfunction and cell death, are revised.

Full text

[Frontiers in Bioscience 12, 947-963, January 1, 2007]
947
Melatonin role in the mitochondrial function
Dario Acuna-Castroviejo, Germaine Escames, Maria I. Rodriguez and Luis C. Lopez
Departamento de Fisiologia, Instituto de Biotecnologia, Universidad de Granada, Avenida de Madrid 11, E-18012 Granada,
Spain
TABLE OF CONTENTS
1. Abstract
2. Introduction
3. Melatonin
3.1. Melatonin production
3.2. Melatonin functions
3.3. Melatonin mechanisms of action
3.3.1. Antioxidant and free radical scavenger properties of melatonin
3.3.1.1. Melatonin and reactive oxygen species
3.3.1.2. Melatonin and reactive nitrogen species
4. Mitochondria
4.1. Mitochondria and energy conservation
4.2. Mitochondria production of ROS and RNS
5. Melatonin and mitochondria in health and disease
5.1. Melatonin actions on mitochondria
5.2. Melatonin, inflammation, and mitochondria
5.3. Melatonin, neurodegenerative diseases, and mitochondria
6. Perspective
7. Acknowledgments
8. References
1. ABSTRACT
Melatonin is an ancient molecule present in
unicellular organisms at the very early moment of life.
Initially identified as a secretory product of the pineal gland
in mammals and in other species, it was considered a
hormone related to reproduction. The evidence that
melatonin is produced in many organs and tissues of the
body, reaching concentrations higher than in the blood,
support the multiplicity of the melatonin actions. The best-
known actions of melatonin, currently supported by
experimental and clinical data, include antioxidant and anti-
inflammatory abilities, some of them involving genomic
regulation of a series of enzymes. Besides, melatonin
displays anticonvulsant and antiexcitotoxic properties.
Most of the beneficial consequences resulting from
melatonin administration may depend on its effects on
mitochondrial physiology. The physiological effects of
melatonin on normal mitochondria, its role to prevent
mitochondrial impairment, energy failure, and apoptosis in
oxidatively-damaged mitochondria, and the beneficial
effects of the administration of melatonin in experimental
and clinical diseases involving mitochondrial dysfunction
and cell death, are revised.
2. INTRODUCTION
Increasing evidence supports the antioxidant and
free radical scavenging properties of melatonin and its
metabolites (1, 2). The relationship between oxidative
stress and mitochondria, the existence of circadian and
seasonal variations in mitochondria, and the presence of
high amounts of melatonin into these organelles, suggest a
physiological role of melatonin on mitochondrial function
(3-7). Subsequent studies concerning the role of melatonin
in health and disease have provided a lot of information
regarding its physiological role and therapeutic applications
due to its antioxidant and anti-inflammatory properties (4-
9). It is now presumed that mitochondrial dysfunction is the
main cause of cell death in most of neurodegenerative
diseases (10, 11). Initially, reactive oxygen species (ROS)
were considered the most aggressive molecules against
mitochondrial function. The recent discovery of the
presence of a nitric oxide synthase (mtNOS) in the
mitochondria points that the NO produced in these
organelles may be related to the impairment of
mitochondrial function in situations such as inflammation
(12, 13). Although it is difficult to know what is the initial
event in the diseases coursing with mitochondrial

Melatonin and mitochondria
948
pathologies, there is agreement that those therapies directed
to maintain a good mitochondrial function are of great
importance to prevent cell death. In this chapter we present
evidences regarding the role of melatonin in mitochondrial
homeostasis that supports the therapeutic utility of the
indoleamine in multiple diseases.
3. MELATONIN
3.1. Melatonin production
Melatonin, a product of tryptophan metabolism,
was primarily isolated in 1956 from the pineal gland and its
structure identified soon after (14). Two main enzymes
control the last steps in the melatonin synthesis, N-
acetyltransferase (NAT) and hydroxyindole-O-
methyltransferase (HIOMT). Although NAT was
considered by years the rate limiting enzyme in melatonin
production (15), there are now evidences suggesting that
this role correspond to HIOMT (16). Melatonin production
by the pineal gland exhibits a circadian rhythm increasing
at night, when the activity of these enzymes is higher (15).
The pattern of the circadian production of melatonin by the
pineal is under the control of the photoperiod. Whereas in
total darkness the rhythm of melatonin production runs
with a period greater than 24 hour, continuous light blunts
this rhythm. This photoneuroendocrine pathway induces
the production of melatonin at night, whereas prevents its
production during the day. Depending on the species, the
nighttime rise in pineal (and blood) melatonin levels are
usually on the order of 2- to 12 folds (from 10 pg/ml during
the day to 120 pg/ml at night) (15). Melatonin produced by
the pineal gland is released to blood and the cerebrospinal
(CSF) fluid and thus, melatonin reaches to every cellular
compartment in the body. Because the pineal gland is
outside the blood-brain barrier, it is accessible to any
molecule in the blood that may modify pineal activity.
Besides, melatonin displays a half-life in the blood of 20-40
min (15), with 90% cleared during a single passage through
the liver. Liver transforms most of the uptake melatonin
into 6-hydroxymelatonin that is mainly conjugated to 6-
sulfatoxymelatonin that is the main metabolite of melatonin
in urine (15).
Melatonin, identified initially as a secretory
product of the pineal gland (17), was considered for several
decades as a regulator of reproduction, mainly in seasonal-
breeding animals (15). But it is now evident that melatonin
is also synthesized in many organs and tissues of the body
(18). Several extrapineal tissues, including retina,
cerebellum, harderian gland, gut, skin, ovary, testes,
lymphocytes, and platelets, airway epithelium, liver,
pancreas, adrenals, kidney, thymus, thyroid, carotid body,
placenta, endometrium, bone marrow, mast cells, natural
killer cells, eosinophilic leukocytes, platelets and
endothelial cells contain melatonin, and some of them are
able to produce the hormone (18-23). These tissues express
the enzymes necessary for melatonin synthesis (24). The
concentration of melatonin in these tissues is higher than in
blood and in some of them, such as bone marrow, bile and
CSF, exceeds 2-3 orders of magnitude its blood levels (18,
25). A common characteristic of extrapineal melatonin is
that it does not leave the organ. The broad extracellular and
intracellular distribution of melatonin, in addition to its
extrapineal production, may explain the role of this
hormone to modulate a number physiological process
through a variety of mechanisms.
3.2. Melatonin functions
The pineal-dependent rhythm in melatonin is
clearly driven by the photoperiodic environment under
which animals are living. Due to the precisely regulated
melatonin circadian rhythm, which it is synchronized to 24
h, it provides information concerning the day- and night-
times, used for synchronization of many other rhythms in
the body (3). It was proposed also that seasonal variations
in the melatonin rhythm may serve as a calendar for
regulatory purposes (26). Brain electrical activity,
neurotransmitter production, secretion, and their receptors,
display circadian rhythms controlled by melatonin. (27-29).
Alterations in melatonin production, either in blind people,
by its reduction with age, or by some pathology, disrupt the
melatonin-dependent rhythms. Thus, alterations in the
behavior, intellectual and cognitive abilities may also
reflect an abnormal pineal melatonin production. This is
not surprising taking in mind that melatonin controls the
rhythm of the neurotransmitters involved in the superior
functions of the brain (30).
Because extrapineal melatonin does not leave the
organ where it is produced, extrapineal melatonin is
considered as a paracrine hormone or signaling molecule.
Three are the main aspects of the extrapineal melatonin
functions studied to date. First, the antioxidant activity of
the hormone, initially reported in 1993 (31), is now
supported by a number of experiments in vitro and in vivo
(1-3, 8, 9). The existence of an inverse relationship between
melatonin content in different tissues and their degree of
oxidative damage has been recently reported (32). Second,
the immunoenhancing properties of melatonin and the
production of the hormone by immune cells are now
accepted (23). Third, an increasing body of evidence
suggests that melatonin exerts homeostatic roles on the
mitochondrion. Melatonin effects on mitochondria, that
have been reported in vitro and in vivo, include increased
membrane fluidity, increased activity of the electron
transfer chain (ETC) complexes and ATP production,
increased mitochondrial membrane potential, reduced
oxidative stress, and closing of the mitochondrial
permeability pore (PTP) (3, 4, 6, 7, 33). Since mitochondria
produce both ROS and reactive nitrogen species (RNS)
including the highly toxic peroxynitrite (ONOO
-
), the
presence of melatonin, a scavenger of both ROS and RNS
in mitochondria, guarantees their protection against
oxidative damage by these reactive molecules, maintaining
at the same time a good mitochondrial function.
After in vivo administration to rats, melatonin
reaches every organ and tissue of the body, but its
concentration varies among subcellular compartments, such
as nucleus and mitochondria (5, 34). Experiments with rat
liver and brain mitochondria were conducted in vitro to
assess their ability to concentrate melatonin. Mitochondria
take up melatonin in a time- and concentration-dependent
manner, with a first order kinetics and a K
M
= 32 nM,

Melatonin and mitochondria
949
Figure 1. Mechanisms of action of melatonin. Melatonin, a hormone derived from serotonin, exerts its effects through both
membrane and nuclear receptors. Cooperation between these receptors has been proposed. Besides, melatonin exerts non-
receptor-mediated effects through its interaction with cytosolic proteins such as Ca
2+
-calmodulin complex and calreticulin.
Finally, melatonin can directly scavenge free radicals.
reaching its maximal concentration 60-90 min after
addition. At this time, the concentration of melatonin in
mitochondria was in the range of 100-200 nM. These data
indicate the existence of an active transport system and
suggest a role for melatonin in mitochondrial homeostasis.
3.3. Melatonin mechanisms of action
Due to the multiplicity of effects of melatonin, it
is not surprising that the hormone may exert its actions by
multiple mechanisms. Several examples of energy
economy during phylogeny leading to the use of one
molecule for multiple purposes include thyroid hormones,
steroid hormones, and peptidic hormones such as
prolactins. The mechanisms of action of melatonin may be
classified into three main groups (Figure 1): a) those
actions related to either membrane or nuclear receptors; b)
the actions linked to cytosolic proteins, and c) the
antioxidant and free radical scavenger activities.
Nevertheless, some of the antioxidant properties of
melatonin are related to genomic, nuclear receptor-related
events. Although there is not full confirmation to date,
recent experiments suggest that melatonin also exerts some
of its effects in the mitochondria through the activation of
mtDNA transcriptional activity, although whether this
effect is related to a mitochondrial receptor of melatonin
remain unclear. Since receptor-mediated actions of
melatonin are out the scope of this chapter, we will not
discuss this topic here.
3.3.1. Antioxidant and free radical scavenger properties
of melatonin
The importance of melatonin as antioxidant
depends on several characteristics: its lipophilic and
hydrophilic nature, its ability to pass all bio-barriers with
ease, and its availability to all tissues and cells. Melatonin
distributes in all cell compartments, being especially high
in the nucleus and mitochondria (5, 34). Melatonin
maintains membrane function and permeability by
preventing lipid peroxidation (LPO) (35) and increasing its
fluidity (33) and maintains mitochondrial function by
reducing hydroperoxide levels and maintaining GSH
homeostasis in both normal conditions and under oxidative
stress (3, 6, 7, 36). This means that melatonin is available
in the sites in which free radicals are forming, thus
decreasing their toxicity (1, 2). Moreover, extrapineal
melatonin is produced for in situ protection against
oxidative damage. This suggests that each organ may
produce the amount of melatonin that it needs
independently of the circulating fluctuations.
Melatonin is a powerful antioxidant and free
radical scavenger and directly scavenges both ROS and

Melatonin and mitochondria
950
RNS (1, 2, 9). Several experiments have been done to
compare the antioxidant activity of melatonin with other
known antioxidants. In vitro, the efficacy of melatonin to
prevent DA autoxidation was significantly higher than
other antioxidants including vitamin E and C (37). In
isolated mitochondria, 100 nM melatonin counteracted t-
butyl hydroperoxide (BPH)-induced GSH depletion,
whereas vitamins C and E and N-acetylcysteine (NAC)
were unable to counteract BPH-induced oxidative stress at
doses of 1 mM (38). In other studies it was reported that
melatonin prevented the oxidation of the radical-trapping
agent 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid
more effectively that did ascorbate, GSH, or trolox (water-
soluble vitamin E) (1). Melatonin reacts with HO
.
at near-
diffusion-controlled rate (1). The calculated second order
rate constant k for the scavenging of the HO
.
by melatonin
was 2.7 x 10
10
M/s (1). This value is similar or even higher
than other well-known HO
.
scavengers (1). Melatonin also
scavenges H
2
O
2
, precursor of HO
.
, with a k of 2.3 x 10
6
M/s. Melatonin also reacts with both NO and ONOO
-
in
vitro (1), whereas in vivo markedly reduced nitrite levels
and nitrotyrosine, reflecting NO levels and tyrosine
nitration by ONOO
-
, respectively (1). Besides, several
metabolites formed when melatonin functions as a free
radical scavenger, i.e. N1-acetyl-N2-formyl-5-
methoxykynuramine (AFMK) and N1-acetyl-5-
methoxykynuramine (AMK) also possess significant
antioxidative and anti-inflammatory activity (1, 39, 40).
Thus not only the parent molecule, melatonin, but its
metabolites as well are protective against oxidative stress
(41). The levels of melatonin and its metabolites determine
the redox status in human serum. Changes in the cellular
redox state mediate the binding activities of some critical
transcription factors such as AP-1 and NF-kappaB and
regulate the gene expression of antioxidant enzymes (42).
Thus, melatonin and its oxidative metabolites may play a
role in modifying the signal transduction pathways and
gene regulation to protect organisms from oxidative stress
3.3.1.1. Melatonin and reactive oxygen species
Melatonin is an efficient scavenger of hydroxyl
radical (HO
.
), and as a consequence of this interaction 3-
hydroxymelatonin is produced (40). Interestingly, this
metabolite is excreted in the urine and it was proposed as a
biological marker of oxidative stress. As it is well known,
HO
.
is a highly reactive compound that rapidly reacts with
proteins, lipids, and DNA inducing oxidative damage. By
scavenging HO
.
, melatonin provides a good mechanism for
protecting against HO
.
-induced oxidative damage in cells
(43). It should be noted here that the effects of melatonin
result more effective than those due to the antioxidant
activity of vitamins E and C (37, 38). In contrast to
vitamins E and C, melatonin not only does not deplete the
cell from GSH, but prevents or even increases its content
(36, 38). The solubility of melatonin, which is not restricted
to a specific cellular compartment as vitamin E, and the
antioxidant cascade of the indoleamine, account for the
antioxidant efficiency of melatonin in comparison with
other antioxidants (40). Melatonin regulates the expression
and activity of several antioxidant enzymes, including
glutathione peroxidase (GPx) and reductase (GRd),
superoxide dismutase (SOD), catalase (CAT), and glucose-
6-phosphate dehydrogenase (G-6-PDH) (40, 44, 45).
Melatonin also increases the cellular content of GSH
through the activation of the gammaglutamylcysteine
synthase, the rate limiting enzyme in GSH synthesis (46).
Melatonin and some of its metabolites comprise the so-
called melatonin family of antioxidants (41). The melatonin
family includes melatonin and the metabolites which are
generated by the interaction of melatonin with ROS and
RNS (1, 2). Similar to other antioxidants, melatonin
possesses a specific electron reduction potential of 0.73 V
(38). Thus, melatonin donates one electron transforming
into a melatonyl cation radical. Another possibility also
proposed is that melatonin might donate a hydrogen atom
from the NH group of the pyrrole ring to generate a neutral
melatonin radical, which in turn could scavenge one O
2
-
to form the final product N
1
-acetyl-N
2
-formyl-5-
methoxykynurenamine (AFMK), just as does the
melatonyl cation radical (2). The other metabolite, N
1
-
acetyl-5-methoxykynurenamine (AMK), is a deformyl
product of AFMK. In the presence of high levels of
ROS, cyclic 3-hydroxymelatonin interacts with them to
form AFMK. Alternatively, both, AFMK and AMK can
be formed through the action of the indoleamine-2,3-
dioxygenase (47). AFMK and melatonin protect against
HO
.
-induced DNA damage in a similar extend, whereas
the ability of the former to protect against lipid
peroxidation is lesser than that of melatonin (48).
AFMK also significantly reduces neuronal cell death
induced by H
2
O
2
, glutamate or beta-amyloid peptide
(48). AMK also prevents HO
.
-induced DNA damage
even more effectively than did melatonin. Thus,
melatonin, AFMK and AMK constitute an antioxidant
cascade produced during the interaction of melatonin
with ROS (41). Together with cyclic 3-
hydroxymelatonin, it was calculated that one melatonin
molecule, via this cascade scavenges possibly up to four
reactive species (48).
3.3.1.2. Melatonin and reactive nitrogen species
One of the most quantitative important ROS in
the body is O
2
-
that it is mainly produced by one-electron
reduction of O
2
in the mitochondria; O
2
-
is not a highly
toxic radical, but it easily reacts with NO forming ONOO
-
(49). The toxicity of ONOO
-
is similar to the one of HO
.
,
and both compounds damage lipids, proteins and DNA at
similar extend. In vitro and in vivo studies reported that
melatonin reduces the production of ONOO
-
(1, 50). It was
shown that ONOO
-
reacts with the nitrogen of the pyrrole
ring of melatonin yielding 1-nitrosomelatonin and 1-
hydroxymelatonin (50). Neutralization of ONOO
-
by
melatonin protects against protein oxidation more
efficiently than GSH, vitamin E or mannitol (41). Other
authors also showed that melatonin reacts with
peroxynitrous acid, yielding 6-hydroxymelatonin that is
excreted normally in the urine, and could serve as marker
of nitrosative stress (41). In vitro and in vivo studies have
shown that 6-hydroxymelatonin protects against oxidative
tissue damage (51). The scavenging mechanism of 6-
hydroxymelatonin may be similar to melatonin although its
antioxidant capacity is even more potent than that of the
later (50). However, under in vivo conditions, the tissue
protective effect of melatonin is always better than that of

Melatonin and mitochondria
951
6-hydroxymelatonin, probably due to the formation of a
series of metabolites with antioxidant properties.
Besides scavenging RNS, melatonin also inhibits
the expression and activity of the iNOS (52). At
physiological concentrations, melatonin administration also
reduces nNOS activity in the brain of different animal
species (53). In vitro, melatonin inhibits nNOS in a dose-
dependent manner, with 1 nM inhibiting 20% of the
activity, and reaching a 40% inhibition at 3 mM (53). In
vivo, melatonin administration at doses of 20-30 mg/kg
inhibits 100% of the activity of iNOS but also a 25%
activity of nNOS. The inhibition of iNOS activity depends
on the reduction of the gene expression, because in vitro,
melatonin has not effect on iNOS activity (54). These
results agree with the different mechanisms of inhibition of
melatonin on nNOS/iNOS activities. Pharmacological
experiments with purified nNOS revealed that melatonin
behaves as a noncompetitive antagonist of nNOS, binding
to Ca
2+
-calmodulin (CaCaM) and impeding the CaCaM-
dependent activation of nNOS (53). Because iNOS is
synthesized with the CaCaM subunit bound to its molecule,
melatonin does not further interact with CaCaM, and
melatonin does not inhibit iNOS activity directly. The
inhibition of iNOS activity by melatonin depends on the
inhibition of the iNOS gene expression (52).
The ability of AFMK and AMK to interact with
nNOS and iNOS was recently tested. Interestingly, AMK,
but not AFMK, inhibits nNOS activity in vitro by the same
mechanism that melatonin, i.e. through its binding to
CaCaM. The difference is that AMK has an IC
50
of 70
microM, whereas the IC
50
for melatonin was > 3 mM (53).
In vivo administration of AMK to normal rats also inhibits
nNOS activity more efficiently than melatonin and so, 10
mg/kg b.w. AMK inhibits 25% the activity of rat brain
nNOS, a similar percentage than 20 mg/kg melatonin.
Interestingly, when the enzyme indoleamine 2,3-
dioxygenase was inhibited with norharmane or 1-
methyltryptophan , the inhibitory activity of melatonin on
nNOS activity both in vitro and in vivo disappeared. It was
then proposed that the inhibitory activity of melatonin on
nNOS activity does not depend on the melatonin directly
but through its transformation to AMK (54). AMK was also
more potent than melatonin to inhibit the iNOS activity in
vivo. In the model of Parkinson=s disease induced by 1-
methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP)
administration to mice, the induction of iNOS in the
substantia nigra caused by the neurotoxin was reduced at
the same extend after the administration of 30 mg/kg
melatonin or 15 mg/kg AMK (unpublished data from this
lab). The data suggest that besides their antioxidant
activity, melatonin metabolites also exert protective
activities through the regulation of the NO/NOS system.
Under oxidative stress, the inflammatory reaction that
follows to oxidative damage induces iNOS expression and
NO production. The parallel transformation of melatonin to
AFMK and AMK yield an additional mechanism of
defense against oxidative stress and inflammation,
producing a cascade of antioxidant and anti-inflammatory
molecules.
4. MITOCHONDRIA
4.1. Mitochondria and energy conservation
Mitochondria possesses an electron transfer chain
coupled to a phosphorylation system that enables the cell to
obtain most of its energy requirements (55). In aerobic
cells, oxidative phosphorylation produces 90-95% of the
total amount of ATP, and more than 90% of the respiratory
phosphorylation is catalyzed by ATP synthase. The
chemiosmotic hypothesis (56) indicates that electron
transfer runs parallel to the proton pumping by the
complexes I, III and IV, yielding a proton gradient (proton-
motive force) across the mitochondrial inner membrane.
Whereas the full four proton reduction transforms O
2
to
water at complex IV, the dissipation of the protonic
gradient through complex V (ATP synthase) yields the
energy enough to phosphorylate ADP and to form ATP.
Regulation of respiration includes ADP (respiratory
control), Ca
2+
and proton leak (57). Recently, the regulation
of mitochondrial respiration by NO has been reported (58).
Mitochondria also posses a constitutive proton leak across
the inner membrane that limits the basal metabolic rate and
the production of potentially dangerous ROS. Besides,
dissipation of energy as heat instead of ATP formation,
permits mitochondria to maintain body temperature at a
level higher than in the environment. This mechanism,
named thermoregulatory uncoupling, causes dissipation of
the mitochondrial membrane potential by an increased
proton conductance of the inner membrane. Non-esterified
fatty acids operate as protonophorous uncouplers with the
help of the special uncoupling proteins (UCPs) (59).
4.2. Mitochondrial production of ROS and RNS
Most of the O
2
taken up by mammalian cells is
processed in the mitochondria and reduced to water via
mitochondrial complex IV. The complete elimination of
every O
2
molecule requires its reduction by 4 electrons.
However, O
2
can be partially reduced by one, two, or three
electrons, yielding O
2
-
, H
2
O
2
and HO
.
, respectively.
Superoxide (O
2
-
) is primarily produced at the level of
ubisemiquinone both at complex I and III (99). To prevent
O
2
-
-dependent damage, this radical is dismutated by
mitochondrial Mn-SOD to H
2
O
2
. In turn, H
2
O
2
has to be
removed to prevent its transformation to the highly toxic
HO
.
. Reduced glutathione (GSH) is of main importance to
maintain the redox status of the mitochondria. Due to the
significance of GSH in mitochondrial physiology, the redox
cycling of GSH in this organelle is normally very active to
avoid any significant lost of GSH (60).
Mitochondria also contain RNS, including NO
and ONOO
-
. It is now recognized that mitochondrial NO
acts as a reversible antagonist of complex IV competing
with O
2
for its binding site. Normal tissue levels of NO and
O
2
are in the ranges of 100-500 nM and 10-30 microM,
respectively. At these concentrations, the NO/O
2
ratio
causes approximately half-maximal inhibition of
mitochondrial respiration rate, suggesting that NO serves to
the physiological regulation of respiration and of the rate of
energy supply to the cell (57). High concentrations of NO
may inhibit not only complex IV but also complexes III and
I, reducing the electron transfer rates through the

Melatonin and mitochondria
952
mitochondrial electron transfer chain 58) and favoring
electron leakage and production of O
2
-
, with a simultaneous
activation of the mitochondrial antioxidant defense.
However, NO reacts very quickly with O
2
-
yielding ONOO
-
that is able to irreversibly damage the respiratory
complexes (61). The effects of ONOO
-
are to be considered
as potentially highly toxic for mitochondria, leading to
mitochondrial dysfunction and cell death (57). The
existence of a specific mitochondrial isoform of NOS
(mtNOS) has been reported, and its role as the enzyme
responsible for the intramitochondrial production of NO is
under active consideration and debate (12, 62, 63). It was
suggested that this constitutively expressed mtNOS isoform
presumably derives from a nNOS isoform (12). Most
recently, we were able to show that mitochondria also
express constitutively another NOS isoform (i-mtNOS) that
is induced in inflammation (13, 64, 65). It is likely that the
NO produced by these isoforms in normal conditions may
serve for regulatory purposes, and that the excess of NO
produced by i-mtNOS in inflammation may deeply inhibit
respiration, in a way that O
2
is redistributed to other
mitochondria or cells where it can be used for energy
production. Mitochondria seem to contain some
mechanisms to reduce RNS. Among them, the reaction
between NO and cytochrome c may serve to remove an
excess of NO (66). Also, the O
2
redistribution due to NO-
dependent electron transfer chain impairment may
constitute an adaptive mechanism to prevent O
2
consumption by these mitochondria and to reduce ROS
production.
5. MELATONIN AND MITOCHONDRIA IN
HEALTH AND DISEASE
Mitochondrial diseases started to be described 30
years ago, and they are nowadays mainly related to
myopathies (67). Soon after, however, a relationship
between mitochondrial alterations and neurological
symptoms led to group these pathologies in mitochondrial
neuromyopathies. Biochemical analysis of the presence of
mitochondrial alterations have revealed the existence of
electron transfer chain defects, impairment of ATP
production, oxidative stress, and/or high sensitivity to PTP
in different degree. Since the recent studies with melatonin
have been mainly related to its antioxidant and anti-
inflammatory properties, we will describe here some of
those mitochondrial alterations in which ROS/RNS are
present.
5.1. Melatonin actions on mitochondria
The relationships between melatonin and
mitochondria are known from several years, and increasing
evidence supports now a physiological role for the hormone
on the organelles. During the last years, experimental and
clinical studies have proved the protective role of melatonin
against mitochondrial failure not only in pathologies
coursing with oxidative stress but also in those
neurodegenerative diseases in which electron transfer
dysfunction is present (68). Experiments in vivo have
shown that intraperitoneal administration of melatonin
significantly enhanced the activity of complexes I and IV in
brain and liver mitochondria of normal rats (69).
Interestingly, the activity of these electron transfer
complexes was maximal after 30 min in liver mitochondria
and after 60 min in brain mitochondria. This difference
depends on the faster availability of melatonin to liver than
to brain after its intraperitoneal administration. After
systemic injection, melatonin appears in cells in the central
nervous system within 30 min and at higher concentrations
than plasma levels (34). The time-dependence of melatonin
also correlates with its half-life in blood. The
administration of melatonin protects mitochondria against
ruthenium red-induced inhibition of the complexes I and IV
(69). To further assess the mechanism of action of
melatonin on mitochondrial bioenergetics, in vitro
experiments were conducted. Melatonin counteracted butyl
hydroperoxide -induced mitochondrial damage, recovering
the GSH pool and the activities of the complexes I and IV
(38). In dose-dependent experiments, it was found that
melatonin significantly increased the activity of these
complexes I and IV at concentrations starting from 1 nM in
rat brain and liver mitochondria (70). These effects of
melatonin may be related, at least in part, to the increase of
the mitochondrial membrane fluidity (33). Thus, in vivo
and in vitro, in normal mitochondria and in oxidatively-
damaged mitochondria, melatonin positively affects the
mitochondrial bioenergetic functions, a finding of great
importance for the extrapolation to pathological situations
(3, 4, 6, 7). The data provide a new homeostatic mechanism
regulating mitochondrial function. First, melatonin
scavenges H
2
O
2
(1, 41), the most important ROS produced
into the mitochondria from O
2
-
, avoiding the waste of the
intramitochondrial GSH pool and subsequent mitochondrial
damage (3, 6, 7). Second, improving mitochondrial
function and ATP synthesis increases the rate of electron
transport and reduces ROS production. These actions
reflect an effect of melatonin to avoid a harmful decrease in
mitochondrial membrane potential that may trigger the
PTP. In some circumstances, melatonin reduces O
2
consumption by mitochondria, an effect that may protect
this organelle from excessive oxidative damage (71). Other
important consequence of the effects of melatonin on
mitochondria is its role in thermogenesis. Since from these
data it becomes apparent that melatonin exerts an opposite
effect to UCPs, melatonin supplementation may reduce
heat production and produce a more efficient use of
substrates in terms of ATP production.
In view of the multiple actions of melatonin on
mitochondria, it cannot be discarded an effect of the
hormone on mtDNA. In preliminary experiments, it was
found that melatonin increases the activity of the complex I
isolated by blue native polyacrylamide gel electrophoresis
(70). In other set of experiments, it was shown that
melatonin administration prevents oxidative degradation of
mtDNA and reduction of mtDNA transcripts in several
tissues including liver, heart, skeletal muscle and brain
(72). Besides, a direct effect of melatonin on mitochondrial
genome expression in brown adipocytes of the Syberian
hamster was reported (73). With these antecedents, we
analyzed the possible effect of melatonin of the expression
of the mtDNA-coded polypeptide subunits I, II, and III of
the complex IV in both in vivo and in vitro, by reverse
transcription polymerase chain reaction. Rats were

Melatonin and mitochondria
953
Figure 2. A diagram showing the relationship between inflammatory reaction, mitochondrial impairment, and cell death.
intraperitoneally injected with melatonin (10 mg/kg body
weight) or vehicle and sacrificed at different times after
treatment to obtain the liver mitochondria used for the
determinations. The results showed a significant increase in
the expression of the mRNAs for the three subunits tested
(5). These results were confirmed by real-time quantitative
PCR, demonstrating that the maximal effect of melatonin to
increase the mRNA content of the three subunits of the
complex IV coded by mtRNA peaked at 8 h after melatonin
injection (unpublished data from this lab).
5.2. Melatonin, inflammation and mitochondria
Sepsis and the consequences of the response to
sepsis are the leading cause of death in medical intensive
care units (74). Common causes of sepsis include bacterial
pathogens, fungi, viruses and parasites. Lipopolysaccharide
(LPS), a component of the cell walls of Gram-negative
bacteria, is the main responsible for the initiation of sepsis
(75). Among other actions, LPS activates a number of
intracellular signaling pathways, including nuclear factor
kappaB (NFkappaB), thereby allowing rapid gene
induction and the expression of inflammatory mediators
which include, besides cytokines, chemokines, lipid
mediators, inducible nitric oxide synthase (iNOS), enzyme
activities, adhesion molecules, myocardial depressant
substances and heat shock proteins (76). The progression of
the inflammatory response leads to multiple organ
dysfunction syndrome (MODS). Mitochondria are primary
targets of injury in systemic organs during the first stage of
sepsis, leading to MODS (13, 52, 77). Septic patients
exhibit an impaired capacity to increase tissue O
2
consumption in response to O
2
delivery. There are a
number of possible mechanisms through which cellular O
2
utilization may be impaired during sepsis, although it is
now considered that NO and RNS play a central role in this
process (Figure 2). In sepsis, the induction of i-mtNOS and
subsequent RNS production were also accompanied by
ROS increase and oxidative damage, with a significant
depletion of GSH (64, 65). Experimental data have probed
the beneficial effects of melatonin in restoring
mitochondrial homeostasis in sepsis; melatonin
administration produced a dose-dependent inhibition of the
activity of iNOS, and a parallel reduction of NO and lipid
peroxidation (52) and a reduction in the expression of
iNOS protein and mRNA, suggesting a genomic effect of
the hormone (Figure 3). Melatonin also recovers the
mitochondrial GSH pool in different organs in
experimental sepsis (64, 65) and prevented mitochondrial
oxidative-nitrosative damage with inhibition of the i-
mtNOS expression and activity induced by LPS in rat lung
and liver mitochondria (13) (Figure 4). The reduction of
NO and ONOO
-
also depends on the ability of melatonin to
scavenge both NO and ONOO
-
(41). Together, these effects
of melatonin explain many of its protective actions against
endotoxemia, including the normalization of the electron
transfer activity and ATP production. (64, 65, 72).

Melatonin and mitochondria
954
Figure 3. Effects of melatonin on the expression of the iNOS protein (left) and mRNA (right) in liver and lungs of rats.
Melatonin (aMT) administration reduced significantly the expression of both protein and mRNA of iNOS induced by
administration of LPS.
Figure 4. Western blot analysis of the effect of melatonin (aMT) on inducible mitochondrial NOS (i-mtNOS) protein expression
in the mitochondria from rat lungs treated with LPS. Melatonin (aMT) administration prevents most of the i-mtNOS induction
produced by LPS.

Melatonin and mitochondria
955
Severe septic patients show a significant
alteration of the circadian melatonin secretion (76).
Cytokines such as IL-1beta and TNF-alpha drastically
reduce pineal serotonin content in pineal explants or cell
cultures from neonate animals (78). Because serotonin is a
precursor for melatonin synthesis, a reduction of the former
in the pineal would likely limit melatonin production and
alters its circadian rhythm, two findings related to the
pathophysiology of septic shock (79). Based on these data
and the multiple protective roles of melatonin in sepsis,
melatonin was used to treat septic human newborns (80).
This study, which constitutes the first report on septic
patients, yielded important data that confirm the beneficial
effects of melatonin against sepsis in humans. This study
demonstrated that melatonin reduced several parameters of
sepsis such as lipid peroxidation. The mortality of
newborns with sepsis is high, usually between 30 and 50%.
In this report (80), three of 10 septic children who were not
treated with melatonin died within 72 h after diagnosis of
sepsis, and more important, none of the 10 septic newborns
treated with melatonin died. The dose of melatonin used in
these treatments was a total of 20 mg orally distributed in
two doses of 10 mg each, with a 1 hour interval, within the
first 12 hours after diagnosis. The comparison of serum
parameters between melatonin-treated and untreated septic
newborns indeed confirmed the antioxidant (reduction in
serum lipid peroxidation products) and the anti-
inflammatory (reduction in C reactive protein) effects of
melatonin. Other parameters including the white blood cell
count and the absolute neutrophil count also significantly
decreased with melatonin treatment, and they remained
elevated in the untreated newborns. In view of the excellent
results with melatonin, the hormone was also used to treat
newborn humans suffering from hypoxia or respiratory
distress. In these studies as well, melatonin improved the
clinical outcome (81).
5.3. Melatonin, neurodegenerative diseases and
mitochondria
The brain is especially sensitive to free radical
damage due to the high utilization of O
2
, its relatively
poorly developed antioxidant defense, and the fact that it
contains large amounts of easily oxidizable fatty acids.
Brain oxidative damage has been considered a common
link in the pathogenesis of a variety of neurodegenerative
disorders (68, 82). Although it is yet unclear whether
oxidative damage is the primary event in the
pathophysiology of neurodegenerative disease, an
increasing body of evidence implicates both ROS and RNS
in the neuronal injury and cell death that occurs in these
pathologies. Moreover, it is now clear that most of the
neurodegenerative diseases course with an increased
activity of iNOS, reflecting the participation of an
inflammatory reaction in these diseases (10). On the other
hand, excitotoxicity is also involved in neuronal death of a
number of neurodegenerative diseases. Excitotoxicity is
associated with mitochondrial dysfunction, loss of Ca
2+
homeostasis, and enhanced oxidative stress (83). The
mechanisms of overstimulation of the NMDA receptors,
which increases Ca
2+
influx into the cell triggering the
excitotoxicity events and apoptosis have been previously
commented. Thus, the sequence of events leading to cell
death in neurodegerative diseases may involve NMDA-
dependent excitotoxicity, and cytosolic and mitochondrial
Ca
2+
overload (most of the mitochondria are located closed
to the NMDA receptors). Moreover, mitochondrial Ca
2+
initiates oxidative stress and can inhibit respiration (11).
Microglia, activated in response to neuronal death, initiates
an inflammatory reaction, with iNOS and i-mtNOS
induction and NO production, which contributes to the
excitotoxic events and to mitochondrial dysfunction, thus
establishing a vicious cycle concluding in cell death. It is
then likely that the elevated mitochondrial NO produced by
i-mtNOS, together with the product of reaction with O
2
-
,
ONOO
-
, could be the major responsible for the
mitochondrial impairment, ATP depletion, and subsequent
cell death in neurodegenerative diseases. In fact,
neurodegenerative diseases of different etiology share the
common feature of oxidative stress, chronic inflammation,
and mitochondrial dysfunction. Thus, mitochondrial
oxidative/nitrosative damage probably constitutes the core
of neurodegeneration. Anti-inflammatory drugs, including
corticosteroids and immunodepressive agents, are used for
the treatment of neurodegenerative diseases, but the
efficacy of these treatments is unclear. The depression that
follows to the anti-inflammatory therapy may increase the
vulnerability of brain tissues to attack by virus such as
adenovirus or herpes virus. In turn, tissue may generate
autoantigens and create autoantibodies.
Biochemical findings revealed the existence of
mitochondrial dysfunction in neurodegenerative diseases
and a protective effect of melatonin administration. In the
case of Parkinson=s disease (PD), there is a deficiency of
the complex I in substantia nigra, and sometimes a
reduction in the activity of the four respiratory complexes
in platelets (84). Inhibition of complex I activity is
accompanied by a reduction of GSH levels, suggesting the
existence of oxidative stress in these mitochondria. The
defect in complex I is accompanied by a defect, with a
lower degree, in complex IV activity and by a decreased
mitochondrial membrane potential. Besides genetic
predisposition to PD, epidemiological studies have
indicated that environmental factors, including exposure to
pesticides, are associated with an increased risk of PD. In
this sense, MPTP, rotenone and paraquat, inhibit
mitochondrial complex I and reproduce most of the features
of PD including mitochondrial oxidative stress and energy
loss, apoptosis initiation and selective dopaminergic
neurodegeneration in the nigrostriatal pathway. The
increase in O
2
-
production, probably dependent of the
complex I inhibition, and in H
2
O
2
concentration in
mitochondria from substantia nigra, are consistently
reported.
Several observations account for the
neuroprotective properties of melatonin in PD. Melatonin
regulates the circadian rhythm of DA and DOPAC, its main
metabolite (85). Melatonin also protects against
excitotoxicity avoiding the autoxidation of dopamine (DA)
that occurs in PD (86). The efficacy of melatonin to prevent
DA autoxidation in vitro was significantly higher than other
antioxidants including vitamin E and C, and that of L-
deprenyl, a MAO B inhibitor that also has antioxidant

Melatonin and mitochondria
956
properties (37). Melatonin administration ameliorated the
reduction of tyrosine hydroxylase-positive fibers, reduced
lipid peroxidation in the striatum (86, 87), and prevented
the MPTP-induced inhibition of the complex I in striatum
and substantia nigra of mice. There was a synergism
between melatonin plus L-deprenyl to increase the
locomotor activity of the mice treated with MPTP,
restoring their normal activity (87). After MPTP
administration, there is an induction of iNOS in substantia
nigra but not in the striatum, whereas the activity of nNOS
increased in the later (88). We found that MPTP
administration induced i-mtNOS without changes in
mtNOS activity in the substantia nigra of MPTP-treated
mice. These changes were accompanied by increased
mitochondrial lipid peroxidation and NO levels
(unpublished data from this lab). Melatonin administration,
besides normalizing complex I activity, lipid peroxidation
and NO levels in mitochondria from substantia nigra of
MPTP-treated mice, counteracted both i-mtNOS and
cytosolic iNOS activity and expression. These effects of
melatonin were accompanied by a total normalization of
the locomotor activity of the mice (87). In parallel to these
studies, we tested a series of synthetic compounds
structurally related to AMK, in search of new
neuroprotective agents. Some of these compounds showed
more potency than AMK to neutralize MPTP toxicity,
although their utility for the treatment of PD in humans
require further studies (89).
To characterize the sequence of
pathophysiological events in PD, a series of experiments
were done in cultured PC12 cells, differentiated with NGF
and treated with rotenone, a selective inhibitor of complex
I. Under these circumstances, PC12 cells show depletion of
DA, increased oxidative stress and reduction of TH
activity. The mitochondria of these cells show an inhibition
of complex I activity, a depletion of the GSH pool and a
reduction in the GRd activity. These mitochondria also
show a loss of mitochondrial membrane potential, ATP
depletion and PTP opening, and most of them die by
apoptosis. At concentrations of 10-100 nM, melatonin
counteracted in a dose-dependent manner the alterations
induced by rotenone: melatonin normalized complex I
activity, counteracted the oxidative stress recovering the
GSH pool and the activity of GRd, and normalized the
mitochondrial membrane potential. As a consequence of
melatonin actions, the ATP production increased and PTP
remained closed, preventing apoptosis almost completely.
Melatonin was much more effective than cyclosporin A to
close the PTP, since 100 nM melatonin displayed the same
effect than 3 microM cyclosporin A (unpublished data from
this lab). The results suggest that the chronology of events
are the following: a) the primary event seems to be the
inhibition of complex I, independently of the cause of PD;
b) as a consequence, an increases in O
2
-
and ROS
production; c) then, the mitochondrial membrane potential
decreases and ATP depletion occurrs; d) PTP opening
induces the first events of apoptosis; e) ROS and cell death
induce microglial reaction and an inflammatory response
increasing the activity of iNOS and i-mtNOS; f) i-mtNOS
(and perhaps iNOS) increases mitochondrial levels of NO
and ONOO
-
, and ROS/RNS induce mitochondrial injury
and electron transfer dysfunction; g) consequently, a further
reduction in mitochondrial membrane potential and ATP
levels with increased levels of ROS/RNS multiply the
apoptotic events, and h) the process enters in a vicious
cycle, increasing neuronal death. Because the significant
consequences of melatonin administration acts at several
steps of these events including electron transfer activity
activity, ROS/RNS production, mitochondrial membrane
potential, and ATP production, clinical studies, aimed to
improve mitochondrial function in PD, should be
conducted to test the beneficial role of melatonin in PD
patients.
Alzheimer=s disease (AD) is another common
neurodegenerative disease coursing with decreased mRNA
expression of mtDNA encoding cytochrome oxidase
subunit II, although it has been proposed that other nDNA-
encoded cytochrome oxidase subunits may be also altered
(90). In AD the beta-amyloid peptide accumulates
extracellular, forming typical insoluble structures or senile
plaques. Beta-amyloid neurotoxicity is associated with
increased oxidative stress, mitochondrial complex IV
inhibition, energy impairment, disruption of Ca
2+
homeostasis, excitotoxicity, apoptosis and necrosis. Beta-
amyloid induces ROS in a metal-catalyzed reaction which
damage neuronal membrane lipids, proteins and nucleic
acids. These process leads to microglia proliferation and
activation, inflammatory reaction and NO/iNOS
system.
The subsequent events triggered by NO are similar to those
in other neurodegenerative diseases. Again, the
participation of i-mtNOS induction in the mitochondrial
dysfunction in AD is yet unknown.
In experimental models of Alzheimer=s disease,
melatonin prevented neurodegenerative changes (91-93),
whereas in humans melatonin administration significantly
slowed the progression of the disease (94, 95). In vitro
experiments were performed in murine neuroblastoma cells
incubated with the beta-amyloid peptide. In these
conditions, oxidative damage to lipids and DNA, and
mitochondrial damage were found. Besides, more than 80%
of the neurons died by apoptosis. The presence of
melatonin in the incubation medium counteracted the beta-
amyloid-induced oxidative stress. The reduction of ROS
levels and the melatonin effect seem associated with an
inhibition of the amyloid molecule capacity to organize
itself into sheets, a process that is required to favor an
enhanced free radical generation. Melatonin was also able
to reduce in parallel cellular death and DNA damage in a
dose-related manner (96). In human platelets melatonin
protected against beta-amyloid-induced damage.
Clinical studies were conducted with melatonin in AD
patients. Melatonin administration at dose of 6 mg daily,
delayed significantly the development of the signs of
AD after 3 years of treatment. In other study, at dose of
9 mg daily, melatonin delayed the signs of AD and
prevented cognitive and behavioral deterioration (95).
Although no studies on mitochondrial function were
performed in these studies, the improvement of
mitochondrial function was one of the main mechanisms
supposedly involved in the beneficial effects of
melatonin.

Melatonin and mitochondria
957
Figure 5. EEG recordings (left) and plasma levels of melatonin (right) before and after melatonin treastment in a 29-months-old
patient with myoclonic epilepsy. Melatonin treatment induced a phase-advance of its rhythm, normlaizing the circadian rhythm
of the hormone in blood. The pharmacological treatment with melatonin yielded a good control of the brain excitability,
normalizing the EEG recordings.
Epilepsy may involve mitochondrial dysfunction
through the excitotoxic pathway that may contribute to
neuronal damage during seizures, as in the case of myoclonic
epilepsy and generalized tonic-clonic seizures. Oxidative
phosphorylation defects, reduced ATP production, ROS/RNS
production, and altered Ca
2+
handling may all contribute to
neuronal damage and epileptogenesis (97). The acute
neurodegenerative process that occurs during seizures
dramatically induces neuronal death and microglia activation,
reflecting the presence of an inflammatory reaction and NO
production. Both, experimental and clinical anticonvulsant
activities of melatonin were reported (97, 98). The
anticonvulsant activity of melatonin was initially related to its
effects on both brain GABA-benzodiazepine receptor complex
and Na
+
, K
+
-ATPase (27-29). Reduced melatonin levels were
related to increased brain damage after stroke or excitotoxic
seizures in rats (177), whereas anticonvulsant activity of
melatonin against seizures induced by a series of drugs in mice
was reported (99, 100). However, due to the inhibitory effect
of melatonin on the NO/NOS system, an effect of the
indoleamine on glutamate-induced excitotoxicity was soon
proposed. Electrophysiological experiments further showed the
antagonism of melatonin against NMDA-induced
excitotoxicity, an effect involving the inhibition of nNOS (101,
102). The effect of melatonin was specific, dose-dependent
and was independent of melatonin receptors (101, 102). An
intracellular action of melatonin in inhibiting the NMDA-
dependent excitotoxic events was further reported with
synthetic kynurenamines, supporting an inhibition of the
NO/NOS system (103). In pentylenetetrazole-induced
generalized convulsions, melatonin administration
significantly increased the latency of the first seizure, and
reduced the number and intensity of the seizures in vivo. The
effects of melatonin were related to increased GABA and
reduced glutamate activity, and to a reduction of brain NO
levels in a dose-dependent manner (103). Some of synthetic
kynurenines, structurally related to melatonin, were also
effective to counteract pentylenetetrazole-induced seizures in
vivo (100). The effects of melatonin against brain
excitotoxicity were the basis for the clinical use of melatonin in
infantile seizures. A 29-months old child having severe
myoclonic epilepsy without response to conventional
anticonvulsant was treated with melatonin (98). Severe
neurological and psychomotor deterioration combined with
increased seizure activity showed a lack of response to the
treatment. Imaging studies including computerized
tomography (CT), single-photon emission computed
tomography (SPECT), and magnetic nuclear resonance
(MNR), electroencephalography (EEG) recordings, blood
biochemistry, and hematological analyses, including measures
of the circadian rhythm of melatonin, were done. At the
moment of the treatment, the patient was in a pre-comatose
stage. After 1 month of melatonin (125 mg daily) plus
phenobarbital therapy for a year, the child seizures were under
control (Figure 5). All analyses, including EEG recordings and
SPECT, were normal. As far as the results of neurological
examination are concerned, only mild hypotony without
focalization remained. Seizures returned

Melatonin and mitochondria
958
Figure 6. Effects of melatonin in the mitochondria. Melatonin displays a series of actions into the mitochondria that reflect its
protective role. Melatonin counteracts excitotoxic events reducing nNOS and iNOS activities; increases electron transfer activity
and AT production, leading to mitochondrial membrane potential increase and thus, ROS/RNS reduction . Besides, melatonin
inhibits the activity of i-mtNOS decreasing RNS production. These actions of melatonin recover GSH pool and close PTP,
preventing apoptosis.
when melatonin was removed from the treatment, but seizures
resumed and the patient=s condition was re-stabilized after
restoring melatonin. During the second year of melatonin
treatment the child progressively became satisfactorily
controlled. The results suggest that neuroprotection by
melatonin also includes excitotoxic events from epileptic
seizures. Further studies with more patients and placebo-
treatment would be beneficial in understanding the potential
use of melatonin as a co-therapy in some cases of seizures
(98).
6. PERSPECTIVE
From the data reviewed here it can be summarized
that mitochondrial dysfunction is a common feature of most of
the diseases related to ROS/RNS excess, independently of the
cause of the disease. Pharmacological intervention with
antioxidants and anti-inflammatory agents may ameliorate the
severity of these diseases. But classical antioxidants, although
may improve some signs of neurodegeneration, have limited
efficacy by several causes. Antioxidants such as vitamin E are
limited by the blood-brain barrier in terms of their ability to
enter the central nervous system. Vitamin C may be toxic in
some circumstances, specially when Fe
2+
levels are increased,
a frequent finding in neurodegenerative diseases. In any case,
high doses of these vitamins would be necessary for
therapeutic effects, and these doses have been reported as pro-
oxidant and/or genotoxic (104). Mitochondrial studies have
shown that these vitamins and other antioxidants such as N-
acetylcysteine frequently lack significant effects in the
recovery of mitochondria from ROS/RNS-induced damage
(38). Experimental and clinical data have shown that melatonin
has low toxicity (98, 105, 106) and the studies conducted
under the guidelines of U.S. National Toxicity Program found
little evidence of melatonin toxicity in rats treated throughout
pregnancy with massive doses (10 to 200 mg/kg daily) (107).
In addition to maternal health, prenatal survival, fetal body
weight, and incidence of fetal malformations were recorded.
None of these indices indicated that melatonin had any
significant toxicity. Thus, melatonin is a secure
pharmacological agent that may be safety used in human
therapy. The activity of melatonin metabolites and synthetic
related compounds against mitochondrial impairment is now
being tested. These studies yield a considerable information
regarding the pharmacophore of melatonin related to it
interaction with nNOS (53, 54, 89). Thus, a promising future
research in this field is the design of new drugs that, acting
through the melatonin pharmacophore, may selectively
antagonize the familyof nNOS, iNOS and i-mtNOS with even
higher potency than melatonin. Such new compounds could
have high therapeutic efficacy in mitochondrial diseases with
reduced side effects resulting from their lack of receptor-
mediated actions. Taking in mind that mitochondria are
now considered a target for drug development, and that the
drugs used for this purpose in neurological diseases should
cross the blood-brain barrier and reach brain mitochondria
(108), melatonin, its metabolites and synthetic analogs, may be
the drugs of election (Figure 6).

Melatonin and mitochondria
959
7. ACKNOWLEDGMENTS
This work was partially supported by grants
PI02/1447, PI02/0817, and G03/137 from the Instituto de
Salud Carlos III, Spain, and SAF01-3191 (Ministerio de
Educación y Ciencia, Spain). Maria I. Rodriguez and Luis
C. Lopez are fellows from the Instituto de Salud Carlos III
(Spain)
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Abbreviations: AD, Alzhemier=s disease; AFMK, N1-
acetyl-N2-formyl-5-methoxykynuramine; AMK, N1-
acetyl-5-methoxykynuramine; CaCaM, calcium-
calmodulin complex; CAT, catalase, CLP, cecal ligation
and puncture; CSF, cephalospinal fluid; DA, dopamine;
DOPAC, 3,4-dihydroxyphenylacetic acid ; GABA,
gamma-aminobutyric acid; GPx, glutathione peroxidase;
GRd, glutathione reductase; GSH, glutathione; GSSG,
disulfide glutathione; H
2
O
2
, hydrogen peroxide; HD,
Huntington=s disease; HIOMT, hidroxyindole-O-
methyltransferase; HO
.
, hydroxyl radical; iNOS,
nitric oxide synthase II, inducible; LPS,
lipopolysaccharide; MODS, multiple organ
dysfunction syndrome; MPTP, 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine; mtNOS, mitochondrial
nitric oxide synthase; NAC, N-acetylcysteine; NAT,
N-acetyltransferase; NFkB, nuclear factor kappa beta;
NK, natural killer; NMDA, N-methyl-D-aspartate;
nNOS, nitric oxide synthase I, constitutive; NO,
nitric oxide; O
2
-
, superoxide anion; ONOO
-
,
peroxynitrite; PD, Parkinson=s disease; PTP,
permeability transition pore; QA, quinolinic acid;
RNS, reactive nitrogen species; ROS, reactive oxygen
species; SOD, superoxide dismutase; TNFalpha,
tumor necrosis factor alpha; UCPs, uncoupling
proteins

Melatonin and mitochondria
963
Key Words: Melatonin, Melatonin Metabolites
Mitochondria, Antioxidant, Oxidative Stress, Reactive
Oxygen Species, Reactive Nitrogen Species, Excitotoxicity,
Mitochondrial Dysfunction, Mitochondrial Diseases,
Neurodegeneration, Review
Send correspondence to: Dario Acuna-Castroviejo,
Ph.D., Departamento de Fisiologia, Facultad de Medicina,
Avenida de Madrid 11, E-18012 Granada, Spain, Tel: 34-
958-246631, Fax: 34-958-246295, E-mail: dacuna@ugr.es
http://www.bioscience.org/current/vol12.htm