Melatonin mitigates mitochondrial malfunction
Apoptosis is a form of programmed cell death that
physiologically plays a role in embryogenesis, metamor-
phosis, diﬀerentiation, proliferation/homeostasis, and as a
defensive mechanism to remove infected, mutated, or
damaged cells . Under normal conditions, a balance
between apoptosis and cell survival is important in the
development of multicellular organisms and in the regula-
tion and maintenance of cell populations in tissues. In fact,
dysfunction of the apoptotic program is implicated in a
variety of pathological conditions. Thus, defects in apop-
tosis can result in cancer, autoimmune diseases and the
spread of viral infections, while neurodegenerative disor-
ders, AIDS and ischemic diseases are caused or enhanced
by excessive apoptosis . As a consequence, modulation of
the diﬀerent molecular pathways of the apoptotic process
has emerged as an attractive therapeutic strategy for these
diseases . In particular, recent studies have focused on the
extrinsic or mitochondrial pathway of apoptosis which
leads to mitochondrial membrane permeabilization (MMP)
and translocation of a number of soluble proteins localized
in the matrix and in the intermembrane space to the cytosol
. The cause of MMP is the opening of a nonspeciﬁc pore
in the inner mitochondrial membrane, known as mitoch-
ondrial transition pore (MTP), as a consequence of a rise in
matrix calcium levels . Several factors are known to
greatly enhance the sensitivity of the MTP to calcium, of
which the most potent and relevant are oxidative stress,
ATP depletion, mitochondrial depolarization, among oth-
Melatonin is a highly conservative molecule found in
organism from unicells to vertebrates . First discovered
as the main secretory product of the pineal gland, it is
known to be present in the blood, where its concentrations
exhibit a circadian rhythm; it is also found in high
concentrations in others body ﬂuids and tissues and is
diﬀerentially distributed in subcellular organelles as well
[8–10]. Its wide extracellular and intracellular distribution
may explain the complexity of melatonin’s role in modu-
lating a diverse number of physiological processes through
diﬀerent mechanisms of action. Classically, the eﬀects of
melatonin were considered to be receptor mediated; more
recently, nonreceptor mediated actions, including its free
radical scavenging activities, have been uncovered [11–13].
Although two distinct receptors/binding sites have been
identiﬁed, i.e. membrane  and nuclear [15, 16], they may
not act separately . New recent studies suggest that
Abstract: Melatonin, or N-acetyl-5-methoxytryptamine, is a compound
derived from tryptophan that is found in all organisms from unicells to
vertebrates. This indoleamine may act as a protective agent in disease
conditions such as Parkinson’s, Alzheimer’s, aging, sepsis and other
disorders including ischemia/reperfusion. In addition, melatonin has been
proposed as a drug for the treatment of cancer. These disorders have in
common a dysfunction of the apoptotic program. Thus, while defects which
reduce apoptotic processes can exaggerate cancer, neurodegenerative
disorders and ischemic conditions are made worse by enhanced apoptosis.
The mechanism by which melatonin controls cell death is not entirely known.
Recently, mitochondria, which are implicated in the intrinsic pathway of
apoptosis, have been identiﬁed as a target for melatonin actions. It is known
that melatonin scavenges oxygen and nitrogen-based reactants generated in
mitochondria. This limits the loss of the intramitochondrial glutathione and
lowers mitochondrial protein damage, improving electron transport chain
(ETC) activity and reducing mtDNA damage. Melatonin also increases the
activity of the complex I and complex IV of the ETC, thereby improving
mitochondrial respiration and increasing ATP synthesis under normal and
stressful conditions. These eﬀects reﬂect the ability of melatonin to reduce
the harmful reduction in the mitochondrial membrane potential that may
trigger mitochondrial transition pore (MTP) opening and the apoptotic
cascade. In addition, a reported direct action of melatonin in the control of
currents through the MTP opens a new perspective in the understanding of
the regulation of apoptotic cell death by the indoleamine.
, Darı´o Acun˜a-
, Germane Escames
Department of Cellular and Structural Biology,
University of Texas Health Science Center,
San Antonio, TX;
Fisiologı´a, Facultad de Medicina, Instituto de
Biotecnologı´a, Universidad de Granada,
Key words: apoptosis, melatonin,
mitochondria, mitochondrial transition pore,
Address reprint requests to Russel J. Reiter,
Department of Cellular and Structural Biology,
Mail Code 7762, University of Texas Health
Science Center, San Antonio, 7703 Floyd Curl
Drive, San Antonio, TX 78229-3900, USA.
Received July 1, 2004;
accepted August 30, 2004.
J. Pineal Res. 2005; 38:1–9
Blackwell Munksgaard, 2004
Journal of Pineal Research
calreticulin may represent a new class of high-aﬃnity
melatonin-binding sites involved in some functions of the
indoleamine including genomic regulation . For exam-
ple, some of the antioxidant properties of melatonin are
because of a genomic eﬀect in regulating protein expression
and activities of antioxidant enzymes  as well as the
inducible (iNOS) and mitochondrial (mtNOS) isoforms of
nitric oxide synthase [20, 21]. Melatonin inhibits nNOS
activity due its binding to the calcium–calmodulin complex
. Some compounds structurally related to melatonin
including its neural metabolite, N-acetyl-5-methoxykynu-
renamine (AMK), also inhibit nNOS activity in rat striatum
in a dose-dependent manner. This suggests that the eﬀect of
melatonin on cerebral nNOS may be mediated, at least in
part, through its metabolites [22, 23]. AMK and N
-formyl-5-methoxykinuramine (AFMK) are formed dur-
ing the enzymatic metabolism of melatonin in the brain
, but also as secondary products when melatonin acts as
free radical scavenger of reactive oxygen (ROS) and
reactive nitrogen species (RNS). Interestingly, these metab-
olites are also eﬃcient antioxidants [25, 26].
The recent discovery that mitochondria are a target for
melatonin opened a new perspective to understand the
mechanism of action of this indoleamine . Melatonin
has a direct role in mitochondrial homeostasis [9, 28, 29],
which may explain the protective eﬀect of this molecule in
diseases such as Parkinson’s disease, Alzheimer’s disease,
epilepsy, aging, ischemia–reperfusion and sepsis, all of
which have mitochondrial dysfunction as a primary or
secondary cause of the condition . As apoptosis is a
mechanism involved in the cell death described in these
diseases, it was expected that melatonin may exhibit
antiapoptotic eﬀects . In fact, several ﬁndings document
a role for melatonin in modulating experimentally induced
apoptosis by a variety of agents. The indoleamine inhibits
apoptosis in immune cells [31, 32], peripheral tissues [33, 34]
and prevents neuronal cell death in models of Parkinsonism
[35–37], Alzheimer’s disease [38–41] and ischemia–reper-
fusion injury [42–44]. The mechanism by which melatonin
reduces apoptosis seems to be related to its antioxidant and
free radical scavenging properties. However, recently, a new
mechanism has revealed that the antiapoptotic eﬀects of
melatonin may be explained by a direct interaction with the
MTP . Interestingly, melatonin acts as a proapoptotic
agent in cancer models , and, therefore, it appears to
have diﬀerential actions in regulating the apoptotic process
in normal and cancer cells .
Mitochondria and cell death
Apoptosis and necrosis are two forms of cell death, with
clearly distinguishable morphological and biochemical
features . Apoptosis is morphologically characterized
by cytoplasmic contraction, chromatin condensation, nuc-
lear fragmentation, internucleosomal DNA fragmentation,
plasma membrane bleb formation, apoptotic body forma-
tion and retention of organelle integrity . Many of these
changes are activated speciﬁcally by a set of cysteine
proteases called caspases. They possess an active site,
cysteine, and cleave substrates after aspartic acid residues
. Apoptotic cells are rapidly sequestered by phagocytes or
by neighboring cells before they can lyse, spill their contents
and cause an inﬂammatory reaction .
In contrast to apoptosis, necrosis does not involve any
regular DNA or protein degradation pattern and is
accompanied by swelling of the entire cytoplasm (oncosis)
and of the mitochondrial matrix, both of which occur
shortly before the cell membrane ruptures .
These two types of cellular demise can occur concurrently
in tissues or cell cultures exposed to the same stimulus 
and, often, the intensity of the same initial insult dictates
the prevalence of either apoptosis or necrosis and it can also
vary among experiments . This suggests that while some
early events may be common to both types of cell death, a
downstream controller may be required to direct cells
toward the organized execution of apoptosis . Thus, the
early phase of both modes of cell death may involve a
similar change in MMP .
The cause of the MMP is the opening of a nonspeciﬁc
pore in the inner mitochondrial membrane, known as the
MTP. Opening of the MTP allows the passage of any
molecule of >1500 Da across the inner mitochondrial
membrane; it can be rapidly closed by chelation of calcium.
Because the MTP also allows rapid passage of protons, its
opening is accompanied by depolarization of the mito-
chondria and uncoupling of oxidative phosphorylation. In
addition, the equilibration of small solutes across the inner
mitochondrial membrane leaves behind high concentrations
of proteins in the matrix and these exert a colloidal osmotic
pressure that is responsible for the extensive swelling of
mitochondria associated with MTP opening .
If the MTP remains open, ATP levels can be totally
depleted leading to cell necrosis. On the contrary, transient
opening of the MTP may be involved in the intrinsic
pathway or mitochondrial-mediated apoptosis through the
release of proteins usually conﬁned to the mitochondrial
compartment. Known as apoptogenic proteins, these
released molecules include cytochrome c , AIF ,
HtrA2/Omi , SMAC/Diablo  and EndoG  of
which cytochrome c has been the most intensively studied.
Upon intrinsic apoptotic stimulation, cytochrome c is
released into the cytosol where it triggers the formation of
the apoptosome, a multimeric molecule composed of
apoptotic protease activating factor-1 (Apaf-1), dATP
and cytochrome c . At present, the only known function
of the apoptosome is the recruitment and activation of
caspase 9 . The caspase 9/apoptosome complex targets
and activates caspase 3. This is considered the point of no
return in the apoptotic signaling cascade . However,
mitochondria play an important role in apoptosis even in
the absence of the MTP opening as release of proapoptotic
factors from the intermembrane space of mitochondria may
occur through changes in the outer membrane permeability.
These are induced by proapoptotic proteins such as Bax
and Bid, two members of the Bcl-2 protein family .
The exact composition of the MTP is not known; it is
currently believed to involve cytosolic proteins (hexoquin-
ase), outer membrane proteins (peripheral benzodiazepine
receptor, voltage-dependent anion channel or VDAC),
intermembrane proteins (creatine kinase), inner membrane
proteins (adenine nucleotide translocator or ANT); and
also matrix proteins (cyclophilin D) .
Leo´n et al.
It appears that any major change in energy balance
(absence of oxygen, depletion of ATP, depletion of NADH/
NADPH, disruption of the DW
) or changes in the redox
balance (oxidation/depletion of reduced gluthatione, exces-
sive production of ROS/RNS) may induce MTP opening.
In addition, determined signal transduction pathways
triggered via intracellular or cell surface receptors can
result in MTP opening. Thus, second messengers such as
increases in cytosolic calcium concentration, ceramide and
caspase 1-like enzymes facilitate MTP .
The Bcl-2 family of proteins are potent regulators of
apoptosis. This family is divided into three groups, based
on structural similarities and functional criteria. Members
of group I (Bcl-2 and Bcl-x
) possess antiapoptotic activity,
whereas members of groups II (Bax and Bak) and group III
(Bid) promote cell death . One hypothesis proposes that
permeabilization of the mitochondrial outer membrane to
small proteins occurs through interaction of a Bcl-2 family
member with the MTP. Bax has been shown to induce the
MTP in cells upon induction of apoptosis via an interaction
with VDAC. However, other experiments suggested the
involvement of ANT in Bax-mediated apoptosis .
Apoptotic cell death can also be triggered when death
signals, i.e. tumor necrosis factor (TNF) or Fas ligand,
interact with the death receptors at the plasma membrane,
resulting in the recruitment of adaptor molecules such as
the Fas-associated protein with the death domain, which is
responsible for activating caspase 8. Activated caspase 8
can directly activate caspase 3 and caspase 7, but it can also
cleave Bid. The cleaved C-terminal Bid (truncated Bid or
tBid) translocates to the mitochondria and induces the
release of cytochrome c, linking the death receptor pathway
with the mitochondrial pathway . Interaction of tBid
with the mitochondria does not seem to require the
activation of the MTP or Bax, although tBid and Bax can
function synergistically . In addition, Bid-induced
cytochrome c release can be antagonized by Bcl-2 death
repressor protein .
Mitochondria, free radicals and cell death
Cells possess multiple sites for ROS/RNS production and a
number of mechanisms for their detoxiﬁcation . Small
ﬂuctuations in the steady-state concentrations of ROS/RNS
may play a role in intracellular signaling ; however,
uncontrolled increases in these metabolites lead to free
radical-mediated chain reactions which indiscriminately
target proteins, lipids and DNA resulting in cell death .
Mitochondria are considered the main source of free
radicals in the cell and oxidants produced by the electron
transport chain (ETC) have been implicated in cell death
. Most available data indicate that the origin of
excessive ROS generation is a consequence of an impair-
ment of the ETC .
The major consequence of an increased ROS production
is the subsequent decreased availability of intracellular
antioxidants such as NAD(P)H or GSH, leading to an
imbalance in the redox status. This, in turn, results in
damage to the mitochondrial respiratory chain and a
further elevation of free radical generation . Other
major consequence of a reduction in the mitochondrial
GSH content is the opening of the MTP because of the
oxidation of critical sulfhydryl groups present in the
The ROS produced by mitochondria can be discharged
into the cytoplasm where they induce calcium release from
the endoplasmic reticulum, which leads to mitochondrial
calcium loading. The increase in the concentration of
mitochondrial calcium can induce opening of the MTP .
Other consequence due to the accumulation of calcium in
the mitochondria include the induction of mtNOS causing a
rise in nitric oxide (NO
) and peroxynitrite (ONOO
production which induce (cyclosporine-insensitive) cyto-
chrome c release associated with peroxidation of mitoch-
ondrial lipids .
Melatonin and mitochondria
In vitro and in vivo experiments have shown that melatonin
can inﬂuence mitochondrial homeostasis. Thus, melatonin
increases the activities of the brain and liver mitochondrial
respiratory complexes I and IV in a time-dependent manner
after its administration to rats . Melatonin also coun-
teracts ruthenium red-induced inhibition of complexes I
and IV in brain and liver mitochondria .
Further experiments indicate that the indoleamine, but
not other endogenous antioxidants such as vitamins C and
E, regulates the glutathione redox status in isolated brain
and hepatic mitochondria, correcting it when it is disrupted
by oxidative stress . Under normal conditions, melatonin
reduces mitochondrial hydroperoxide levels and stimulates
the activity of the two enzymes involved in the GSH-GSSG
balance, i.e. glutathione peroxidase (GPx) and glutathione
reductase (GRd) . Melatonin is also able to counteract
the oxidative damage induced by high doses of t-butyl
hydroperoxide (t-BHP), restoring GSH levels and GPx and
GRd activities and scavenging hydroperoxides. However,
vitamins C and E have no such eﬀect under these conditions
. These results are in agreement with other data showing
the eﬀects of melatonin on GSH homeostasis in brain tissue
 and in gastric mucosa and testis . As a result of the
interaction of melatonin with complexes I and IV and the
subsequent promotion of electron ﬂux through the ETC,
melatonin increases ATP production under basal condi-
tions and counteracts cyanide-induced depletion of ATP
associated with complex IV inhibition . Although the
indoleamine also reportedly stimulates metabolism of
isolated mitochondria from frog oocytes , other experi-
ments have shown that melatonin reduces the oxygen
consumption of liver mitochondria , an eﬀect that
may protect this organelle from excessive oxidative damage
The antioxidant and free radical scavenging capacity of
melatonin protects proteins of the ETC and mtDNA from
the ROS/RNS-induced oxidative damage . Melatonin
also interacts with lipid bilayers, reducing lipid peroxida-
tion and stabilizing mitochondrial inner membranes , an
eﬀect that may improve ETC activity . In a model of
sepsis induced by the administration of lipopolysacharide in
rats, melatonin prevented functional deterioration which
occurs as a result of mtNOS-induced mitochondrial failure.
In this situation, melatonin administration also reduced
Melatonin, mitochondria and apoptosis
both mtNOS activity and NO
production and also
counteracted the inhibition of complexes I and IV .
Other studies have described one possible mechanism by
which melatonin increases the activity of the complex IV;
this protective action may be due, at least in part, to an
eﬀect on the expression of mtDNA. Melatonin increases the
expression of mtDNA encoded polypeptide subunits I, II
and III of complex IV in mitochondria from rat liver in a
time-dependent manner which correlates with the increase
in complex IV activity . In experiments with fresh
mitochondria prepared from rats treated for 10 days with
melatonin, the indoleamine reduced the levels of mRNA in
these animals, compared with non-melatonin-treated con-
trols . These eﬀects were also produced by AMK and
this compound was more potent than melatonin itself .
Interestingly, AMK is a metabolite of melatonin and its
in vivo production could be responsible for some of the
apparent actions of melatonin .
Collectively, these results may help to explain the
protective eﬀects of melatonin in neurodegenerative dis-
eases and other disorders which involve mitochondrial
dysfunction. Thus, melatonin prevents the inhibition of
mitochondrial complex I activity induced by MPTP 
and limits dopamine autooxidation . Melatonin is also
neuroprotective in in vitro models of Alzheimer’s disease
through its stimulatory eﬀects on complex V activity [9, 28,
38]. Furthermore, the antiepileptic properties of melatonin
may be due to the regulation of the central GABA-
benzodiazepine receptor complex and inhibition of the
glutamate-mediated response . However, other studies
reveal that melatonin acts by inhibiting ROS-induced
mitochondrial dysfunction in vivo [78, 87] as well as in
cultured cells . In the senescence accelerated mouse,
either chronic or acute melatonin administration restores
the activity of the mitochondrial complexes [89–91]. Treat-
ment with melatonin before injury protects against mitoch-
ondrial dysfunction induced by ischemia–reperfusion of rat
liver  and restores hepatic energetic status by inhibiting
both activation of iNOS and the production of TNFa .
As mitochondrial dysfunction can lead to ATP depletion,
depolarization and initiation of apoptotic processes, it is
possible that the antiapoptotic eﬀects of melatonin in the
situations described above may be a result of its protective
actions [27, 30, 82]. However, recent ﬁndings have shown
that the interaction of melatonin with mitochondria in
terms of antiapoptotic agent is more complex than
Melatonin, mitochondria and apoptosis
Mitochondrial dysfunction associated with the loss of
calcium homeostasis and enhanced cellular oxidative stress
have long been recognized to play a major role in cell death
associated with excitotoxicity , a well-known process
that has been implicated in neurodegeneration in Hunting-
ton’s disease, Alzheimer’s disease, Parkinsonism, epilepsy
and disorders such as ischemia–reperfusion . Excitotox-
icity results from the over-stimulation of ionotropic glu-
tamate receptors, in particular, the N-methyl-d-aspartate
(NMDA) and the a-amino-hydroxy-5-methyl-4-isoxazole-
propionate (AMPA) receptors which lacks the GluR2
subunit [96, 97]. Over-stimulation can occur as a result of
an increase in the liberation of excitatory aminoacids from
the presynaptic neuron. However, energy depletion caused
by mitochondrial dysfunction can result in neuronal depo-
larization, opening of NMDA receptors and the inﬂux of
calcium then activates several intracellular enzymes, inclu-
ding phospholipase A2, NOS, xanthine dehydrogenase,
calcineurin and endonucleases, many of which elicit the
generation of endogenous ROS. Additionally, when taken
up by mitochondria, calcium can induce MTP opening and
cell death .
Melatonin is a potent antiexcitotoxic agent which has
been documented in both in vivo and in vitro experiments
. Electrophysiological experiments demonstrate the
antagonism of melatonin on the NMDA receptor [98–
101]. This eﬀect is dose-dependent and, as a consequence of
the treatment, the NMDA receptor channel pore remains
closed, thereby preventing the opening of L-type calcium
channels and calcium inﬂux . Other experiments have
shown that melatonin also inhibits activation of nNOS
through its binding to the calcium–calmodulin complex,
reducing the production of both NO
as the presynaptic release of additional glutamate .
Some synthetic melatonin-related kynurenines also reduce
striatal NMDA excitability in a dose-dependent manner;
some of these kynurenines were 100 times more potent than
melatonin in this action. The eﬀects of these drugs were
linked to their inhibition of nNOS activity and a reduction
production and were not because of an interaction
with melatonin membrane receptors [22, 23]. Further
experiments demonstrate that melatonin is able to diminish
the rises in cytosolic calcium induced by NMDA in cultured
mouse striatal neurons . Taken together, these results
show that melatonin limits cytosolic calcium rises and, as a
consequence, the concomitant production of free radicals;
additionally, melatonin reduces the associated mitochond-
rial membrane depolarization . Other experiments
carried out using rat brain astrocytes  and cultured
PC12 cells  show that melatonin prevents ROS-
induced calcium overload and mitochondrial membrane
depolarization. In these two reports, melatonin indirectly
inhibited the opening of the MTP and blocked MTP-
dependent cytochrome c release, the downstream activation
of caspase 3 and the cell death by apoptosis . In a
recent in vivo experiment as well, melatonin was reported to
inhibit caspase 3 activation in the mouse brain damaged by
ischemia–reperfusion . However, recordings have been
obtained from the inner mitochondrial membrane of rat
liver mitoplasts using the patch-clamp approach and have
demonstrated a direct eﬀect of melatonin on the MTP
activity at the single channel level. These results showed
that melatonin strongly inhibits MTP currents in a dose-
dependent manner with an IC
of 0.8 m .
Studies in peripheral tissues have suggested that melato-
nin inhibits apoptotic processes via its antioxidant proper-
ties. For example, melatonin protects against cyclosporin
A-induced hemolysis in human erythrocytes because of
depuration resulting from O
produced by mitochondria
. Melatonin is also highly protective against mitoch-
ondrial ROS-induced cardiotoxicity resulting from doxo-
rubicin treatment. In this study, pretreatment with
Leo´n et al.
melatonin prevented the release of lactate dehydrogenase
and restored membrane potential .
Many lines of evidence indicate an antiapoptotic eﬀect of
melatonin on thymic cells. The methoxyindole reduces
DNA fragmentation induced by glucocorticoids in cultured
thymocytes . A reduction in glucocorticoid-receptor
mRNA levels in the intact thymus as well as in cultured
thymocytes that were treated with melatonin seem to be the
most likely mechanism whereby melatonin inhibits gluco-
corticoid-induced cell death . Other studies reported
that melatonin inhibits DNA fragmentation and the release
of cytochrome c from mitochondria of mouse thymocytes
treated with dexamethasone. Melatonin may act by inhib-
iting the mitochondrial pathway, presumably through the
regulation of Bax protein levels , although melatonin
was ineﬀective per se on this parameter.
Interestingly, proapoptotic eﬀects of melatonin have
been noted in a number of tumor cell lines . In MCF-
7 breast tumor cell studies conducted in the absence of
exogenous steroid hormones, treatment with melatonin
produced a 64% reduction in the cellular ATP levels
through a membrane receptor-modulated pathway .
These ﬁndings in tumor cells are in contrast to the described
actions of melatonin in normal cells and suggest melato-
nin’s potential use in killing cancer cells while preserving
the function of normal cells.
The actions of melatonin on mitochondria may be mediated
via at least three mechanisms (Fig. 1). First, antioxidant and
free radical scavenging properties of the indoleamine protect
the organelle from oxidative damage. Secondly, its actions at
the mtDNA level increase the expression of complex IV.
Thirdly, a direct interaction of melatonin with the MTP was
found recently. These eﬀects suggest that melatonin, because
of these direct and indirect mitochondrial actions, may have
utility as an antiapoptotic agent for normal cells.
In addition, some of the products that are produced
when melatonin detoxiﬁes reactive species [25, 111–114],
especially AMK and AFMK, are also both eﬃcient
antioxidants [25, 83, 115] that may be found in mitochon-
dria; these metabolites can also act at the mitochondrial
genomic level, resulting in a cascade of protective reactions.
Given that these compounds exert the same actions as
melatonin, they also could act as antiapoptotic drugs in
normal cells and as proapoptotic agents in cancer models.
In all the studies where comparisons were made, melato-
nin’s metabolites AMK and AFMK were more potent than
melatonin itself . Therefore, these compounds may exert
the same regulatory eﬀects on apoptotic processes in a more
eﬃcient manner than melatonin.
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