Current Topics in Medicinal Chemistry 2002, 2, 133-151

133

Melatonin, Mitochondrial Homeostasis and Mitochondrial-Related Diseases

Darío Acuña Castroviejo

1

*

, Germaine Escames

1

, Angel Carazo

1

, Josefa León

1

, Huda Khaldy

1

 and

Russel J. Reiter

2

1

Instituto de Biotecnología, Departamento de Fisiología, Universidad de Granada, Granada, Spain

2

Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio,

San Antonio, Texas, USA

Abstract: The recently described ‘hydrogen hypothesis’ invokes metabolic symbiosis as the driving force for
a symbiotic association  between an anaerobic, strictly hydrogen-dependent organism (the host) and an
eubacterium (the symbiont) that is able to respire, but which generates molecular hydrogen as an end product
of anaerobic metabolism. The resulting proto-eukaryotic cell would have acquired the essentials of eukaryotic
energy metabolism, evolving not only aerobic respiration, but also the cost of oxygen consumption, i.e.,
generation of reactive oxygen species (ROS) and oxidative damage. Mitochondria contain their own genome
with a modified genetic code that is highly conserved among mammals. Control of gene expression suggests
that transcription of certain mitochondrial genes may be regulated in response to the redox potential of the
mitochondrial membrane. Mitochondria are involved in energy production and conservation, and they have an
uncoupling mechanism to produce heat instead of ATP. Also, mitochondria are involved in programmed cell
death. Increasing evidence suggests the participation of mitochondria in neurodegenerative and
neuromuscular diseases involving alterations in both nuclear (nDNA) and mitochondrial (mtDNA) DNA.
Melatonin is now known as a powerful antioxidant and increasing experimental evidence shows its beneficial
effects against oxidative stress-induced macromolecular damage and diseases, including those in which
mitochondrial function is affected. This review summarizes the data and mechanisms of action of melatonin in
relation to mitochondrial pathologies.

INTRODUCTION

into ATP [3]. The respiration-produced 

∆µ

H

+

 can be utilized

by mitochondria not only to form ATP but also to support
other energy-consuming processes such as transport of
certain solutes from the cytosol to the matrix. Mitochondria
are also of central importance for physiological Ca

2+

handling, acting as a reservoir for Ca

2+

. Mitochondrial Ca

2+

regulates the activity of mitochondrial dehydrogenases as
well as nucleic acid and protein synthesis [4]. Several factors
have been proposed to regulate respiration including ATP
(respiratory control), Ca

2+

 and proton leak [5].

Mitochondrial Function

Mitochondria are specialized for the rapid oxidation of

NADH and FADH produced during glycolysis, Krebs cycle
and 

β

-oxidation of fatty acids by the transfer electrons from

these precursors to oxygen. The electron transport chain
(ETC) is a system of oxido-reductant protein complexes
(complexes I, II, III and IV) in the inner mitochondrial
membrane. According to the chemiosmotic hypothesis, C-I,
C-III and C-IV pump protons yielding a proton gradient
along the mitochondrial inner membrane, which is a source
of free energy that is dissipated when protons enter the inner
mitochondrial membrane through ATP synthase [1]. During
this process, ADP is phosphorylated to ATP. Mitochondrial
DNA encodes several components of the respiratory
complexes: 7 of C-I; cyt b

560

 corresponding to a cofactor of

C-II; 3 of C-IV and 2 of ATP synthase [2]. In aerobic cells,
oxidative phosphorylation (OXPHOS) is responsible for
production of 90-95% of the total amount of ATP, and more
than 90% of respiratory phosphorylation is catalyzed by
ATP synthase, an enzyme converting the respiratory chain-
produced electrochemical proton potential difference (

∆µ

H

+

)

Dissipation of energy as heat to maintain body

temperature at a level higher than in the environment is
another important function of mitochondria. The mechanism
is referred to as thermoregulatory uncoupling of respiration
and phosphorylation. Uncoupling results in dissipation of
the respiratory chain-produced 

∆µ

H

+

 due to increased proton

conductance of the inner membrane. Thus, energy released
by respiration is dissipated immediately as heat without
formation and hydrolysis of ATP. Non-esterified fatty acids
have been proven as the compounds mediating the
thermoregulatory uncoupling. They operate as
protonophorous uncouplers with the help of special
uncoupling proteins (UCPs) [6]. It is known that T

3

influences in rat the expression of nine nuclear-encoded
respiratory genes, regulates mitochondrial RNA synthesis
through both the activation of a mitochondrial transcription
factor (mtTFA) and the specific mitochondrial T

3

 receptors,

*Address correspondence to this author at the Departamento de Fisiologia,
Facultad de  Medicina, Universidad de Granada, Avenida de Madrid, 11,
E-18012 Granada, Spain; E-mail: dacuna@ugr.es

1568-0266/02 $35.00+.00

© 2002 Bentham Science Publishers Ltd.

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Castroviejo et al.

and stimulates the expression of the mRNA for UCP2 and
UCP3 [7-9].

transcription, mtTFA appears to have a function in
maintenance of mtDNA [2]. Because the mitochondrial
genes encode only a few proteins, almost all of the
mitochondrial proteins must be imported into the
mitochondria after their synthesis by cytoplasmic free
ribosomes as preproteins [14]. These usually have 20 amino
acid N-terminal extensions (presequences), which can direct
the preproteins to the mitochondria [15]. Cytoplasmic
import factors deliver the preproteins to the outer surface of
the mitochondria; then import systems of the outer
membrane (Tom, translocase of the outer membrane) and the
inner  membrane (Tim, translocase of the inner membrane)
transport the preproteins to their final destinations [16, 17].
Fundamental mechanisms of mitochondrial protein import
seem to be conserved from eukaryotes to mammals.

UCPs are elementary proton transporters across the inner

mitochondrial membrane. Proton transport is driven only
exclusively by the membrane potential [10]. Fatty acids
provide one or more carboxyl groups along the translocation
channel, and deliver their protons to an acceptor group
(carboxyl groups of the other fatty acid), which in turn
delivers protons into the matrix. While UCP1 is known to
play an important role in regulating heat production during
cold exposure, possible roles for UCP2 and UCP3 include:
regulation of ATP synthesis; control of reactive oxygen
species (ROS) production by mitochondria; control of
adaptative thermogenesis in response to cold and diet; and
regulation of fatty acid oxidation [11, 12]. A failure to
control ROS damage can cause the collapse of multiple vital
functions, including mitochondrial energy conservation
which culminates in the loss of membrane integrity and cell
death by necrosis and/or apoptosis [6]. UCP2-dependent
uncoupling mitochondrial depolarization reduces ROS
production and thus inhibits the permeability transition pore
(PTP) avoiding the proapoptotic cascade. It was suggested
that superoxide anion (O

2

¯

) production decreases because

uncoupling increases the rate of electron transport,
diminishing the probability that electrons will escape from
the respiratory chain and interact with molecular oxygen.
Also, a direct interaction between UCP2 with the
antiapoptotic bcl-2 family was also proposed [6].

Mitochondria and Apoptosis

Although some aspects remain to be elucidated [18, 19],

the mitochondria exert a central role in eukaryote life and
death [20]. These organelles are involved in apoptosis (and
necrosis) by promoting the release of proapoptotic factors
including cytochrome c and other "death factors" in the
intermembrane space [21]. The activation of the apoptotic
cascade leads to cell death [22]. In some situations, Ca

2+

overload leads to mitochondrial swelling, loss of respiratory
control, collapse of 

∆ψ

m

, and release of matrix Ca

2+

 caused

by a permeabilization of the mitochondrial inner membrane
(PTP) to molecules up to 1.5 kDa. Structurally, the PTP is
formed by the adenine nucleotide translocase (ANT), and
electrophysiological studies also have shown an interaction
of PTP with the membrane porins and with the
mitochondrial benzodiazepine receptor [4, 23].

Mitochondrial DNA

Mitochondrial DNA consists of a closed-circular,

doubled-stranded DNA  molecule of about 16.6 kbp. Most
information is encoded on the heavy (H) strand, with genes
for two rRNAs, 14 tRNAs, and 12 polypeptides. The light
(L) strand codes for 8 tRNA, and a single polypeptide. All
13 polypeptides are constituents of the enzyme complexes of
the ETC. The genes lack introns and, except for one
regulatory region, intergenetic sequences are absent or
limited to a few bases. Replication and transcription in
mitochondria depend upon trans-acting nuclear-encoded
factors [2, 13].

The PTP can switch from low- to high-conductance

states. The conformational switch appears to be dependent
on the saturation of the internal Ca

2+

 binding of the channel.

The low-conductance state of PTP may be responsible for
mitochondrial volume homeostasis and contributes to a
significant part of the final cytosolic Ca

2+

 signalling [23].

Thus, under its low-conductance conformation, the PTP
does not impair mitochondrial function and is operated by
changes in matrix pH accompanying mitochondrial Ca

2+

uptake. The high-conductance state of the PTP is activated
by the cooperative binding of two Ca

2+

  ions to its matrix

domain, with a molecular cutoff of 1.5 kDa. This induces a
complete collapse of the proton gradient, allowing for the
efflux of a variety of other ions and of small molecules such
as pyrimidine and adenine nucleotides, and promotes the
diffusion of components from the incubation medium into
the matrix, such as sucrose. The high-conductance state of
PTP is highly regulated and exhibits the features of a Ca

2+

-,

voltage- and pH-gated channel [23], modulated by the redox
and phosphate potentials. Opening of the PTP appears to be
regulated by direct binding of a mitochondrial cycloplilin
(cyclophilin D) to its matrix domain, accounting for the
inhibitory effect of cyclosphorin A (CsA).

Transcription of mtDNA is controlled by a human

dissociable transcription factor (mtTFA) acting in concert
with the mitochondrial RNA polymerase and a factor
mediating attenuation of transcription (mtTERM). As no
intron sequences are present in vertebrate mtDNA, and
intergenetic sequences are minimal, processing of the long
polycistronic H- and L-strand messengers is thought to be a
relatively simple process requiring only a few enzymes.
Genes for tRNAs flank the two rRNAs genes and nearly
every protein gene, suggesting that the secondary structure of
the tRNA sequences provide the punctuation marks in the
reading of the mtDNA information. One initiation factor
(mtIF-2) and three mitochondrial elongation factors (mtEFs)
have been identified and participate in the polypeptidic chain
elongation. Mammalian mtDNA replication is a slow
process and replication is unidirectionally. DNA polymerase

γ

 is the only DNA polymerase present in mitochondria, and

it is required for mtDNA synthesis. In addition to its role in

The PTP may be also regulated by ROS leaking from the

ETC. The shift from a low to a high-conductance state can
be promoted by the oxidation of NADPH by oxidative
stress. This impairs the antioxidant function of glutathione

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   135

(GSH) [24]. The participation of ROS in opening PTP is
clear, since no PTP opening occurs in the absence of
molecular oxygen [24]. The PTP possesses at least two
redox-sensitive sites and both increase the probability of
opening after oxidation: the S-site, a dithiol in apparent
redox equilibrium with matrix GSH, and the P-site, in
apparent redox equilibrium with the pyridine nucleotides
[25]. Glutathione disulfide (GSSG) is probably the
immediate oxidant of the S-site and many pore inducers
such as hydrogen peroxide (H

2

O

2

) appear to affect the pore

through changes in the level of the GSH rather than direct
oxidation of the S-site. In turn, oxidation of the P-site by
oxidized pyridine nucleotides can induce PTP under
conditions where the GSH pool is maintained in a fully
reduced state. Under conditions of oxidative stress, the
mitochondrial levels of GSH and reduced pyridine
nucleotides are connected through energy-linked
transhydrogenase and glutathione reductase (GRd) and thus
it is difficult for these compounds to independently
modulate the S- and P-site in vivo [25].

mechanisms controlling the mitochondrial genetic system
and intergenomic communication have been recently
summarized [32]. Thus, defects in mitochondrial
metabolism may be associated with mutations of mtDNA or
nDNA. Abnormalities of mitochondrial metabolism that
cause human disease have been recognized for more than 30
years. They encompass defects in fatty acid oxidation, Krebs
cycle enzymes and the OXPHOS system. Some of these
pathologies do not induce, as a primary cause, an alteration
in mitochondrial metabolism, but result from it. That is the
case during energy alterations due to changes in hormones
(hyper and hypothyroidism-induced changes in UCPs
expression altering 

∆ψ

m

) [3, 9, 33], or as a consequence of

ischemia/reperfusion, excitotoxicity, or sepsis. In each of
these, there is an increase in ROS production and an
alteration in mitochondrial function that may lead to cell
death [34, 35]. Moreover, as mtDNA encodes proteins of the
OXPHOS, such mutations frequently result in a deficiency
in one or more constituents of these enzymes.

A recently-described group of alterations involves

mitochondrial transmembrane carrier deficiencies that
constitute the mitochondriopathies [36]. Increasing amounts
of ROS damage DNA and produces an increase in poly-
(ADP-ribose) synthetase (PARS) to repair the damaged
DNA. This enzyme ADP-ribosyates proteins depleting the
intracellular concentration of its substrate, NAD

+

, slowing

the ETC and ATP production [37, 38]. Primary OXPHOS
defects [39] are caused by mutations of mtDNA or nDNA
genes encoding subunits of the ETC complexes, including
mutations affecting mitochondrial targeting of protein, i.e.,
the N-terminus sequence. A defect in the import of the
Rieske iron-sulphur center has been postulated. Other
alterations of mtDNA including tRNA and protein encoding
genes of the ETC complexes, and cyt b. In turn, OXPHOS
deficiencies may result in increased ROS [39]. Secondary,
OXPHOS deficiencies are both due to genetic and
environmental factors. Alterations in mtDNA transcription,
translation and replication are also included [39].
Endogenous and exogenous toxins may impair OXPHOS.
Aging itself may be due to ROS over production and
mitochondrial damage. Toxins such as MPTP simulate
neurodegenerative diseases further demonstrating the
involvement of ROS in this pathology [39, 40].

Apoptosis may be visualized as a process of the

glycolyzing host cell to punish respiring guests if they
formed excessive ROS [3]. In fact, the protomitochondria
brought respiration to the partnership and with it the power
to kill the new cell through the production of ROS [26]. It
is obvious that in modern organisms the functions of
apoptosis (at mitoptosis) are not restricted by the
elimination of ROS-overproducing mitochondria and cells.
However, apoptotic stimuli are processed inside the cell in
such a way that an increase in intramitochondrial
(intracellular) levels of ROS is initiated. The production of
O

2

¯

 and H

2

O

2

 by the ETC is the inevitable side effect of

ETC induced by the one or two electron reduction of O

2

 [3].

In some instances, the production of ROS increases and may
induce PTP opening. The increased PTP mitochondria
cannot survive due to the collapse of 

∆ψ

m

, since PTP

permits the efflux of molecules up to 1.5 kDa; this causes
the high molecular mass compounds in the matrix to exert
an osmotic effect and water reaches the matrix causing its
swelling. As a result, mitochondrial cristae straighten and
the outer membrane is broken since it is much smaller that
the inner membrane. The loss of outer membrane integrity
causes all the intermembrane proteins to be released into the
cytosol including some involved in apoptosis including cyt
c, apoptosis-inducing  factor (AIF) and some procaspases [3,
27, 28]. Cyt c and AIF form a complex with the cytosolic
Apaf-1 and ATP. The complex hydrolyzes inactive
procaspase 9 to active caspase 9, which in turn hydrolyzes
procaspase 3 to caspase 3. Caspase 3 attacks some other key
proteins resulting in controlled cell death [29]. To avoid
apoptosis, the bcl-2 family of proteins block death by
preventing the mitochondrial release of the intermembrane
proteins [29, 30]. Once the process gets past the
mitochondria, the anti-apoptotic proteins are not effective.
[22].

Neurodegenerative Pathologies

Neurodegenerative diseases of different etiologies may

share mitochondrial dysfunction as a final common  pathway.
Recent studies using cybrid cell lines certainly support this
possibility [41]. Parkinson’s disease (PD) is characterized by
bradykinesia, rigidity and tremor. Mitochondrial
involvement in PD was suggested by deficiencies in
mitochondrial C-I in the substantia nigra [42], with a
parallel reduction in GSH levels, suggesting the existence of
oxidative stress. In platelets of PD patients, C-I activity is
also reduced, and in some cases are accompanied by C-II, C-
III and C-IV deficiencies. Studies with cybrids have shown
that alterations in C-I is due to a defect in the mtDNA [42].
This defect is accompanied by an alteration in the expression
of C-IV activity and a decreased 

∆ψ

m

, which lowers the

apoptotic threshold.

Mitochondrial Pathologies

The functional activity of mitochondria depends on a

precise cross-talk between two different genetic systems, i.e.,
nuclear and mitochondrial genomes [31]. A series of

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Castroviejo et al.

Environmental factors influence PD, as shown by  the C-I

inhibitory effects of MPTP and paraquat. The C-I inhibition
is prevented by free radical scavengers indicating oxidative
damage to C-I. MPTP also stimulates NMDA-dependent
nNOS activity thereby increasing nitric oxide (NO)
production [43], and decreasing the content of mtDNA [44].

lipids, proteins and nucleic acids. This suggests that the use
of antioxidants such as vitamin E, melatonin and estrogens
may be beneficial in AD [45, 47].

Epilepsy may involve mitochondrial dysfunction which

may contribute to neuronal damage during seizures, as in the
case of myoclonic epilepsy and generalized tonic-clonic
seizures. The OXPHOS defects, reduced ATP production,
free radical generation and altered Ca

2+

  handling may all

contribute to neuronal damage and epileptogenesis [45].

Huntington’s disease (HD) is a neurodegenerative

disorder characterized by ataxia, chorea and dementia. It is
known to be caused by alterations in a gene for nDNA
encoding huntinin, a widely expressed protein of unknown
function. The pathology of HD involves mainly the GABA-
containing neurons of the caudate nucleus [42].
Excitotoxicity has been suggested to play an important role
in this disease. This includes activation of NMDA-
dependent neuronal nitric oxide synthase (nNOS) and NO
production. NO and particularly peroxynitrite (ONOO¯)
mediate the oxidative damage. There are also deficiencies in
the activities of mitochondrial C-II, C-III and C-IV in
caudate and in a lesser extend in putamen in HD. Aconitase,
an iron-sulphur-containing enzyme is particularly susceptible
to inhibition by superoxide and NO/ONOO¯, as are C-II and
C-III, which are FeS-containing enzymes [42, 45]. The
subsequent oxidative damage to proteins, lipids and mtDNA
reduces 

∆ψ

m

 and induces apoptosis.

Neuromuscular Disorders

Whereas nuclear mutations can affect genes encoding

enzymatic or structural mitochondrial proteins, translocases,
mitochondrial protein importation, and intergenomic
signalling, mtDNA mutations fall in three main categories:
sporadic rearrangements (deletions/duplications), maternally
inherited rearrangements (duplications), and maternally
inherited point mutations.

Disorders Due to Defects of mtDNA

Several mtDNA-related diseases are frequent. The

mitochondrial genetic code differs from Mendelian genetic
code in several ways: polyplasmy, which means there are
several genomes in each mitochondrin (5-10); heteroplasmy,
means that a mutation may affect all or only one mtDNA
genome; threshold effect, which requires a minimal critical
number of mutant mtDNAs to express an alteration; mitotic
segregation, since at cell division, the proportion of mutant
mtDNA in daughter cells may vary, as does maternal
inheritance. These alterations include: a) sporadic
rearrangements of mtDNA, which are single deletions of
duplications. There are three main clinical syndromes:
Kearns-Sayre syndrome, a subtype of progressive external
ophthalmoplegia (PEO) with early onset (before 20 years),
limb weakness and fatigue; Pearson syndrome, which is
manifested in infancy as a severe hematopoietic disorder
with sideroblastic anemia and exocrine pancreas dysfunction,
and sporadic PEO with ragged-red fibers (RR) [48, 49]; b)
maternally inherited rearrangements of mtDNA. Although
there is no evidence that single mtDNA deletions are
inherited, there are a few disorders in which
duplications/deletions are maternally transmitted. These
conditions are usually associated with diabetes and
myopathy [48]; c) point mutations of mtDNA.

Hereditary spastic paraparesis (HSP) is another hereditary

disease involving a nDNA mutation. It may be present in
children or adults. A new gene defect has been recently
described encoding paraplegin which contains an N-terminus
sequence and which is imported into mitochondria. Muscle
biopsies show mitochondrial alterations including
cytochrome oxidase negative fibers.

Wilson’s disease, usually present in children and

adolescences, shows liver failure with movement disorders
(dystonia, parkinsonism), and is caused by a mutation in the
gene encoding a mitochondrial P-type ATPase, leading to
copper accumulation and ROS generation [42, 45].

Friedreich’s ataxia (FA) is an adolescent autosomal

disease with progressive ataxia, dysarthria, skeletal
deformations, hyporeflexia, pyramidal features and
cardiomyopathy. The pathology also includes distal
axonopathy affecting the large sensory axons of the dorsal
root ganglia and the spinocerebellar and pyramidal tracts in
the cord with loss of neuronal parikarya. The genetic defect
results in a deficiency of frataxin protein whose function is
not known. Since it has a N-terminus sequence and is
associated with mitochondrial membranes, a role of
mitochondria was proposed. There are several deficiencies of
complexes I-III and in the Krebs cycle enzyme aconitase.
There is a parallel increase in the mitochondrial iron levels
and, through the Fenton reaction, oxidative damage to
mtDNA may also result [42, 45].

Over 50 pathological point mutations have been

documented, including myoclonus epilepsy with RR fibers,
characterized by myoclonus, generalized seizures, cerebellar
ataxia, and myopathy. In this condition, muscle biopsy
shows RRF, which are typically COX negative; also
included in this group is mitochondrial encephalomyopathy,
lactic acidosis, and stroke-like episodes (MELAS),
characterized by stroke-like episodes with hemiparesia or
hemianopia, before 40 years and often in childhood.
Common features include generalized seizures, migraine-like
headache and vomiting, and dementia. There is also a
mutation in the tRNA

Leu(UUR)

 associated with myoclonic

epilepsy with ragged red fibers (MERRF) showing
myopathy and myoclonus and generalized seizures. An

Alzheimer’s disease (AD), is associated with a decrease

in mRNA expression of mtDNA encoding cytochrome
oxidase (COX) subunit II, although it has been proposed
that other nDNA-encoded COX subunits may be also altered
[46]. Also, 

β

-amyloid peptide generates ROS in a metal-

catalyzed reaction inducing neuronal cell death in a ROS-
mediated process resulting in damage to neuronal membrane

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   137

alteration in tRNA

Lys

 produces neuropathy, ataxia, retinitis

pigmentosa (NARP) consisting in a multisystem disorder of
young adult life comprising neuropathy, ataxia, seizures,
dementia and retinitis pigmentosa. An alteration in ATPase
6 leads to the maternally inherited Leigh syndrome (MILS)
or a more severe condition than NARP  syndrome manifested
in infancy by developmental delay, hypotonia, seizures,
pyramidal signs, ataxia, retinitis pigmentosa, and with the
neurological factors of LS. There are several biochemical
conditioning including a pyruvate dehydrogenase deficiency
and OXPHOS defects, and Leber hereditary optic neuropathy
(LHON), which causes loss of vision in young adults, and is
associated with a C-I deficiency [48, 49].

from of this disease is the MNGIE (mitochondrial
neurogastrointestinal encephalomyopathy) syndrome
showing both strong gastrointestoinal dysfunction and
peripheral neuropathy [48].

In all mitochondrial encephalomyopathies, cells die

because the lack of an adequate energy supply and the
decrease in 

∆ψ

m

 which triggers PTP and apoptosis,

although small amounts of the non-mutant genome seem to
be sufficient to protect tissue from defects of ETC. In
neurons, inability to maintain adequate ATP levels leads to
a partial neuronal depolarization and excitotoxicity, and
muscle cells seem to die mainly by apoptosis [48, 50].

Mitochondria and Aging

Disorders due to defects of nDNA

This group of pathologies includes: a) defects of genes

encoding enzymatic or structural proteins. Many
neuromuscular or generalized syndromes are due to
mutations of mtDNA-encoded subunits of the ETC.
Examples of this situation are three pediatric syndromes
associated with C-IV deficiency. One of them is Leigh
syndrome, a devastating encephalopathy with
characteristically symmetric lesions of the basal ganglia and
the brain stem. COX deficiency appears to be the most
common [48]. Two other syndromes associated with COX
deficiency are tissue-specific and cause severe generalized
myopathy in infancy:  one of them is invariably fatal within
a year (fatal infant myopathy), whereas the other is
spontaneously reversible (benign infantile myopathy),
implying a COX subunit. Another Mendelian disease which
is due to an ETC chain defect, a primary ubiquinone
(CoQ10) deficiency, responds well to CoQ10 administration
[48]; b) disorders due to defects in translocases, proteins that
are essential for the trafficking of metabolites across the
inner membrane. Most frequent is the defect of carnitine-
acylcarnitine translocase and deficiencies in ANT, mainly of
the subunit ANT1. Children with dysmorphic features,
hypotonia, developmental delay, seizures and hydrocephalus
show deficits in the outer membrane protein porin, also
called the voltage-dependent anion channel (VDAC). In this
case, pyruvate oxidation and ATP synthesis are decreased in
muscle mitochondria. [48]; c) disorders due to defects in
mitochondrial protein importation. Transporting proteins to
mitochondria is accomplished through targeting signals
localized at the N-terminus of polypeptides. Some
alterations involving this disorder have been described,
although considering the complexity of the multistep
process involving protein import, it seems likely more
defects will be uncovered [48]; d) disorders due to defects of
intergenic signalling. In this group, the primary genetic
lesion is in the nDNA but the consequence of the nuclear
mutation is an abnormality of mtDNA. Included are defects
in mtDNA replication, including deletions that cause
congenital myopathy or hepatopathy. The severity depends
on the quantity of mtDNA remaining. Depletion of 60-80%
mtDNA is usually associated with myopathy starting at 1
year of age and causing respiratory failure and death in
childhood. Serum creatine kinase is usually elevated, which
is an unusual finding in other mitochondrial myopathies.
Another group includes multiple mtDNA deletions, with
paralysis of eye muscles and associated in some cases to
exercise intolerance, hearing loss and psychosis. A special

Two views have been inked to genetic models of aging.

One is that aging is a genetically programmed event.
Specific aging genes, functioning as hierarchical clocks,
might exist to cause aging and death of the individual. The
alternative view is that environmental insults and/or
endogenous ROS and reactive nitrogen species (RNS) may
cause genetic damage and mutations [51]. The proposal that
free radicals, produced by normal aerobic metabolism, cause,
at subcellular locations, random tissue damage that impairs
cellular function and proliferative capacity was proposed as a
cause of aging by Harmon in 1956 [52]. Aging is then the
result of the failure of various protective mechanisms to
counteract the radical-induced damage [51].

The mitochondrial theory of aging states that the

accumulation of impaired mitochondria is the driving force
of the aging process [53-56]. This theory has gained new
support in recent years with the discovery of age-related
mtDNA delections. The mtDNA inherited variability could
play a role in successful aging and longevity in humans
[57], whereas continuous damage to mtDNA leads to a
bioenergetic crisis. It has been demonstrated that the levels
of mitochondrial transcripts in Drosophila during aging are
significantly reduced, which means that the ability of
mtDNA to perform transcriptional activity decreases [58].
However, an increase in mtDNA damage in response to
oxidative stress in human cells has been recently reported
[59]. Experimental accumulation of mtDNA deletion
mutations have been observed in several species including
mice and humans [60]. Additionally, some genes encoded
by nDNA involved in aging have been identified [51].

There is increasing consensus that ROS and RNS are a

major cause of aging [55, 61]. Aging is accompanied by
structural changes in mitochondria including their reduction
in number and increase in size (97), and a decrease in C-IV
and C-V activities. These changes may impair energy-
dependent neurotransmission, contributing to senescent
decline in memory and other brain functions [62, 63]. The
mutation rate of mtDNA is much higher than that of nDNA
because expression of the whole genome is essential for the
maintenance of mitochondrial bioenergetic function, while
only about 7% of the nuclear genome is expressed at any cell
differentiation [63]. Oxidative injury is not limited to
mtDNA but also occurs in mitochondrial membranes. This
may lead to a progressive lipid peroxidation and

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Castroviejo et al.

crosslinking damage, with concomitant changes in
respiration rate, ATP synthesis, membrane fluidity and
permeability, Ca

2+

 homeostasis and apoptosis. The free

radical theory of aging provides a rationale for intervention
by means of antioxidant administration [64, 65]. In fact,
mitochondrial aging may be due to chronic oxidative stress,
with and the O

2

¯

 generated by mitochondria leading to the

formation of other ROS/RNS [66, 67]. All these oxidants
reduce GSH availability thereby producing oxidative damage
to mtDNA, lipids and proteins, which is manifested as
mitochondrial aging and in turn, cell aging.

mouse and human mitochondria. The formamidopyridine
DNA glycosylase, an enzyme that deletes 8-oxo-dG, has
been reported in rat hepatic mitochondria. Removal of 4-
nitroquinoline lesions from mtDNA, have also been
reported; these are generally removed via NER pathways.
However, NER as it exists in the nucleus, does not exist in
mitochondria, and thus, the role of NER protein in
mitochondrial repair remains unclear [69]. It was found that
the endonucleolytic activity of the enzyme that specifically
cleaves 8-oxo-dG oligonucleotides is higher in 12 and 23-
month old rats than in 6-month old rats. Thus, the
mitochondrial capacity to repair 8-oxo-dG seems to increase
with age [69].

Mitochondria repair mechanisms

Melatonin and mitochondrial pathology

The increase in mitochondrial mass and mtDNA content

are the early molecular events of human cells in response to
oxidative stress [59]. Most of the O

2

 taken up by cells is

reduced to water via the action of mitochondrial C-IV by the
addition of 4 electrons per O

molecule. The intermediate

steps of O

2

 reduction are formation of O

2

¯

 , H

2

O

2

 and

hydroxyl radical (HO

), corresponding to reduction by one,

two and three electrons, respectively. NO and its metabolite
ONOO¯ are RNS produced in the mitochondria.
Mitochondrial DNA is not protected by histones and lies in
close proximity to the free radical-producing ETC. Primary
(mtDNA) or secondary (nDNA) mitochondrial mutations
and/or changes in ROS production may induce
mitochondrial damage which is the basis of aging and
several conditions including neurodegenerative diseases and
mitochondriopathies.

In the last decade, an increasing amount of evidence

supports new roles and mechanisms for the actions of
melatonin. The actions of melatonin depend on receptor- and
non-receptor-mediated processes, the latter accounting for the
antioxidant properties of melatonin. Receptor-mediated
events for melatonin involve both membrane and nuclear
receptors [70-73], and the existence of a membrane-nuclear
signaling pathway has been proposed [74]. Some of the
protective effects of melatonin on the cell seem to be
mediated by genomic regulation, and some genes, including
the 5-lipoxygenase gene in the human B lymphocyte, seem
to be regulated by melatonin [75]. In addition, the
expression of some genes, mainly related to the cell redox
and inflammatory responses including GPx, GRd, SOD,
inducible nitric oxide synthase (iNOS) and cytokines are
also under genomic regulation by melatonin [76-78]. In
addition, the specific binding of melatonin to Ca

2+

-

calmodulin (CaCaM) appears to regulate some CaCaM-
dependent enzymes such as nNOS [79, 80]. The recent
discovery of the mitochondrion being a target for melatonin
action opens new perspectives to understand the mechanism
of action of melatonin, and may help to explain the
antiapoptotic and thermogenic effects of the indoleamine
[81, 82].

A large number of DNA base modifications caused by

oxidative stress have been detected. On of the most widely
studied is 8-hydroxydeoxyguanosine (8-oxo-dG). This
mutagenic lesion also accumulates with age. Mitochondria
defend against oxidative stress by two main mechanisms:
eliminating ROS/RNS (antioxidants and scavengers) and
repairing the damaged molecules. The former include SOD
which actively dismutates O

2

¯

 to H

2

O

2

, which in turn is

transformed to water by glutathione peroxidase (GPx). In
this process, GSH is oxidized to GSSG and the enzyme
GRd restores GSH levels. The glutathione recycling system
is very active in mitochondria because these organellos do
not synthetize GSH and they do not possess catalase. Thus,
they mainly depend on their own GSH pool, although they
can also import GSH from cytosol. ROS/RNS are involved
in tissue injury associated with aging and a number of
inflammatory and neurodegenerative diseases. In addition,
mitochondrial respiratory chain deficiencies lead to
overexpression  of antioxidant enzymes [68]. Thus, under
physiological conditions, an equilibrium should exist
between the mechanisms generating and scavenging O

2

¯

 and

other ROS.

In a number of experimental and clinical situations, a

beneficial effect of melatonin has been reported in those
pathologies which involve mitochondria dysfunction as a
primary or secondary cause of the disease, including ROS-
induced DNA damage, excitotoxicity and neurodegenerative
diseases such as PD, AD and epilepsy, and aging [83-88].
Melatonin’s ability to counteract excitotoxicity and ROS-
induced DNA damage has been described under a variety of
different experimental paradigms. Melatonin prevents DNA
damage in human blood cells exposed to ionizing radiation,
and reduces genetic damage in lymphocytes which were
exposed to ionizing radiation after their removal from
individuals who had consumed melatonin [89]. Oxidation of
guanine bases in DNA from rat liver induced by whole body
ionizing radiation also was prevented by melatonin
administration [90]. Furthermore, the DNA damage caused
by the chemical carcinogen safrole or by chromium is
reduced by melatonin [91, 92]. Using the comet assay, it
was shown that treatment with melatonin reduced neural
DNA fragmentation induced by exposure in rats to extremely
low frequency magnetic fields [93]. Melatonin counteracts

Endogenous metabolic processes generate ROS yielding

oxidized bases that are removed from the DNA mainly by
the base excision repair (BER) pathway. Adducts due to UV
exposure are removed by the nucleotide excision repair
(NER) pathway [69]. Mitochondria are able to carry out
BER. The first repair enzyme detected was uracil DNA
glycosylase. Homologes of the yeast repair enzymes, OGG1,
which excise 8-oxo-dG from DNA, have been found in

Melatonin-Mitochondrial Interactions

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   139

paraquat-induced genotoxicity in mice [94], as well as ferric
nitrilotriacetate-induced DNA damage and H

2

O

2

-induced

DNA damage in U-937 cells [95, 96]. The protective effects
of melatonin against DNA damage was shown by measuring
the 8-oxo-dG levels in the brain of kainic acid-treated rats
[97].

Electrophysiological experiments document the

antagonism of melatonin on the N-methyl-D-aspartate
(NMDA) receptor involved in excitotoxicity [113-116]. The
effect of melatonin was specific, dose-dependent and was
independent of melatonin receptors. Thus, an intracellular
action of melatonin in inhibiting NMDA-dependent
excitotoxic events was further demonstrated with synthetic
kynurenamines suggesting an inhibition of the NOS/NO
system, the main mediator of glutamate-dependent
excitotoxicity [117-119]. The effects of melatonin against
brain excitotoxicity were the basis for the clinical use of
melatonin in infantile seizures [120, 121]. Melatonin also
protects against excitotoxicity by reducing the autoxidation
of dopamine (DA) which occurs in some degenerative
diseases such as PD [122]. These effects were documented in
1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP)-
induced PD in mice [123, 124]. The effect of melatonin in
reducing DA autoxidation was tested elsewhere and it
showed a greater potency than other antioxidants including
vitamin E and C, and that of L-deprenyl, a monoamine
oxidase (MAO) B inhibitor which also has antioxidant
properties [122].

When DNA repair mechanisms are induced, the

activation of the nuclear enzyme PARS triggers an energy-
consuming repair cycle reducing cellular NAD

+

 levels. This

also occurs in rats treated with zymosan, a non-bacterial
agent causing cellular injury by inducing the production of
ONOO¯ and consequent PARS activation. In this case there
is also an inhibition of mitochondrial respiration due to
ONOO¯. The administration of melatonin protects against
cellular energy depletion and prevents the appearance of
DNA damage [98]. Renal and hepatic DNA damage induced
by the carcinogen 

δ

-aminolevulinic acid was assessed by

measuring the levels of 8-oxo-dG; these levels were reduced
by melatonin [99, 100]. Rat lung and spleen concentrations
of 8-oxo-dG induced by 

δ

-aminolevulinic acid are also

lowered by melatonin [101].

Interesting findings were described when the effect of

melatonin on mitochondrial membrane fluidity was tested.
Mitochondrial membrane fluidity decreased after the animals
were treated with 

δ

-aminolevulinic acid with these changes

being prevented by melatonin co-treatment [99]. However,
no changes in mitochondrial membrane lipid peroxidation
(LPO) levels were reported and thus, the effects of melatonin
on mitochondrial membrane fluidity may be independent of
its ability to counteract lipid damage [99, 100]. The effects
of melatonin in maintaining optimal membrane fluidity of
mitochondrial membranes may depend on its ability to
localize into the membrane itself, in a superficial position in
lipid bilayers near the polar heads of membrane
phospholipids. In this position melatonin would be near the
mitochondrial proteins which then would be protected from
ROS. It should be note that 

δ

-aminolevulinic acid damage

to mitochondria results in the disruption of the 

∆ψ

m

 and

enhanced permeability [101, 102] leading to decrease of
ATP, PTP opening and apoptosis. Thus, melatonin may
protect protein complexes in the inner mitochondrial
membrane and thereby improve ETC.

The neuroprotective effects of melatonin were also tested

against neurodegenerative manifestations in AD [125]. When
neuroblastoma cells were incubated with 

β

-amyloid, more

than 80% of the neurons died due to apoptosis, but the
presence of melatonin reduced cellular death and DNA
damage in a dose-related manner [126]. In human platelets
melatonin also protected against AB-induced damage [127,
128]. The protective properties of melatonin were
extensively tested in models of aging, which involves
extensive cell damage. In different models of aging and age-
related diseases including cancer and cataracts, melatonin
administration has been shown to be protective. The fact that
melatonin decreases with age was then suggested as one of
the potential causes of aging in mammals [87, 129-137].

The importance of melatonin as an antioxidant depends

from several characteristics; it is both lipophilic and
hydrophilic and it passes all bio-barriers with ease. It is
available to all tissues and cells, where it scavenges free
radicals [138-141]. Melatonin distributes in all subcellar
compartments, being especially high in the nucleus and
mitochondria. This means that melatonin is available at the
sites in which free radicals are being generated, thus
decreasing their toxicity [142, 143]. Melatonin, identified by
Lerner as a product of the mammalian pineal gland [144], is
also found in several tissues including the retina, cells of the
immune system, gut, bone marrow, ovary and testes [145-
152]. It seems that these tissues may produce melatonin
required for antioxidant regulation [153], since this
melatonin does not enter the circulation. Also, most of these
tissues have much higher levels of melatonin than those in
the blood. Levels of melatonin 2-3 orders of magnitude
higher than maximal blood melatonin concentrations are
present in bile [154]. Another fluid that contains very high
levels of melatonin is the cerebrospinal fluid (CSF) [155].

A recent series of experiments have provided strong

evidence for the antiexcitotoxic properties of melatonin both
in vivo and in vitro. Anticonvulsant activity of melatonin
was initially shown to be related to its effects on both brain
GABA-benzodiazepine receptor complex and Na

+

, K

+

-

ATPase activity [103-107]. However, due to the inhibitory
effect of melatonin on the NOS/NO system, an effect of the
indoleamine on glutamate-induced excitotoxicity was soon
proposed. A melatonin deficiency was related to increased
brain damage after stroke or excitotoxic seizures in rats
[108], and an anticonvulsant activity of melatonin against
seizures induced by a variety of drugs in mice was reported
[109]. Melatonin protects cultured cerebellar neurons from
kainate excitotoxicity [110]. Quinolinic acid, a neuroactive
metabolite of tryptophan implicated in some
neurodegenerative diseases [111], induces neuronal
degeneration when injected into animals, an effect
counteracted by melatonin administration [112].

It was recently reported that expression of the genes of

the key enzymes for melatonin synthesis, i.e., N-
acetyltransferase (NAT) and hydroxyindole-O-methyl-
transferase (HIOMT), are found in many organs [156]. Thus,

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Castroviejo et al.

these organs may synthesized their own melatonin and do
not depend on the circulation to provide this indoleamine.
This suggests that each organ may in part produce the
melatonin that it needs independently of its the circulating
levels. Thus, the concept of what constitutes a physiological
level of melatonin is changing, and physiological levels
must be defined based on specific fluids and subcellular
organelles.

from impending cell death, which may have important
consequences in neurodegenerative diseases involving the
nigrostriatal pathway such as in PD [166].

The relation of melatonin to cell death was tested in

several cell cancer models as well. In an ovarian carcinoma
cell line it was found that melatonin exerts an oncostatic
action linked to a nuclear effect of the indoleamine, since the
melatonin nuclear receptor agonist CGP 52608 caused a
similar effect [167]. Interestingly, melatonin seems to
enhance apoptosis in carcinoma cells, as has been
demonstrated with Ehrlich ascites carcinoma cells. In this
case, changes in GSH were not detected during the
proapoptotic effects of melatonin [168]. Similarly, in colon
mucosa and colon tumors induced by 1,2-dimethylhydrazine
in rats, melatonin behaves as a potent stimulator of
apoptosis [169]. It was recently shown that melatonin also
inhibited LOOH-triggered cell death, in a similar manner to
that of CsA, an inhibitor of the permeability transition pore
[170]. While melatonin counteracted H

2

O

2

-induced DNA

damage in U-937 cells [96], other authors were unable to
confirm the antiapoptotic role of melatonin against 7-
ketocholesterol-induced apoptosis in the same cell type,
although melatonin prevented O

2

¯

 generation by

mitochondria [171]. In general, it seems that the antioxidant
and to some extend the GSH-enhancing effects of melatonin
may account for melatonin’s antiapoptotic activity in non-
cancerous cells.

Melatonin and Apoptosis

The observation that melatonin influences apoptotic cell

death is a documented regulatory effect of melatonin on cell
survival. The possibility that the antioxidant properties of
melatonin account for inhibition of apoptosis was
investigated in vivo and in vitro by measuring DNA
fragmentation. These experiments showed that melatonin
administration counteracts apoptosis in rat thymus. In
cultured thymocytes, 1 nM of melatonin decreased cell death
by 20% [157]. It was suggest that melatonin down-regulates
the glucocorticoid receptor in thymocytes, which may
explain its antiapoptotic effect in the thymus [158]. In
primary cultures of cerebellar granule neurons, melatonin
protects them from singlet oxygen-induced apoptosis [159].
Melatonin also inhibited pre-B-cell apoptosis during
lymphopoiesis in mouse bone marrow; this has implications
for neoplasia since boosting the formed B cells would have
effects on humoral immunity [160]. Melatonin was also
shown to protect bovine cerebral endothelial cells from
hyperoxia-induced DNA damage and apoptotic death [161].

Melatonin Actions on Mitochondria

Since apoptosis is a possible mechanism involved in the

neuronal death described in several neurodegenerative
diseases such as PD, AD and epilepsy, it would be expected
that melatonin may exert antiapoptotic effects in these
diseases. In fact, in neuroblastoma cells exposed to the
Alzheimer 

β

-amyloid peptide, melatonin prevented cell death

[126]. Melatonin also prevents apoptosis induced by MPTP
in the mouse [124] and by 6-hydroxydopamine in PC12
cells [162]; these finding could be of potential clinical
importance in the treatment of PD. Melatonin also abrogated
cell death induced by cystamine pretreatment of PC12 cells;
cystamine treatment involves mitochondrial iron
sequestration [163]. The age-associated accumulation of
redox-active iron in subcortical astrocytes may facilitate the
bioactivation of DA to neurotoxic free radical intermediates
and thereby predispose the nervous system to PD and other
neurodegenerative diseases. Melatonin counteracts very
efficiently DA autoxidation, by reducing iron-dependent
ROS production by mitochondria [122]. In rats injected with
kainic acid to produced excitotoxicity-induced apoptotic cell
death, melatonin significantly attenuated apoptosis, an effect
linked to the reduction in oxidative damage and an increased
GSH content [164]. In a spontaneous, age-induced model of
apoptosis using cerebellar granule cells, it was shown that
melatonin and Ca

2+

-channel blockers such as amlodipine,

inhibited spontaneous apoptosis [165]. The antagonism
between melatonin and Ca

2+

-channels was also demonstrated

in electrophysiological and binding experiments [116].
Striatal neurons growing in low density culture on serum-
free medium and in the absence of glia die within 3 days by
apoptosis. The presence of melatonin rescues striatal neurons

Three main considerations suggest a role for melatonin in

mitochondrial homeostasis. First, the mitochondrion is the
organelle with the highest ROS/RNS production in the cell,
and melatonin is a powerful scavenger of ROS and RNS.
Secondly, mitochondria depend on GSH uptake from the
cytosol, although they have GPx and GRd to maintain the
GSH redox cycling; melatonin improves GSH redox cycling
and increases GSH content by stimulating its synthesis in
the cytosol. Third, melatonin exerts important antiapoptotic
effects (Fig. 1) and most of the apoptotic signals originate
from the mitochondria [81].

The relationships between melatonin and mitochondria

have been known for several years, but to date the existence
of a specific role of the indoleamine in mitochondrial
homeostasis remains enigmatic. Following the lines of
evidence that an aerobic organism entered an anaerobic one,
the subsequent symbiosis had beneficial consequences for
the two organisms [172]. However, the anaerobic one had an
unexpected problem, i.e., oxygen is highly toxic and
oxidizes many molecules of the host. The hybrid organisms
had to acquire new antioxidant mechanisms not only to
protect itself from O

2

 toxicity but also to preserve the

enzymatic machinery required to produce ATP highly
efficiently. Also, when ROS produced by the guest were
excessive, the host organism evolved a trigger to initiate
mitoptotic signals to eliminate the damaging symbiotic
organelle [3]. Since melatonin was present in the invading
unicellular organisms, what was the function of melatonin in
the mitochondria of multicellular organisms?

Melatonin-Mitochondrial Interactions

Current Topics in Medicinal Chemistry,  2002, Vol. 2, No. 2 

 

   141

Fig. (1). Schematic representation of the effects of melatonin on the mitochondrion. Nuclear and/or mitochondrial DNA mutations
lead to a defective electron transport chain (ETC) and subsequent energy depletion. Energy depletion can also result from free radical-
induced mtDNA and/or ETC damage. Oxidative stress and ATP depletion favor opening of the mitochondrial permeability transition
pore (PTP), which in turn induces mitochondrial swelling and release of proapoptotic factors such as cytochrome c and AIF
(apoptosis-inducing factor). These proapoptotic factors activate a caspase cascade in the cytosol leading to cell death. Melatonin
scavenges free radicals and other reactive species including H

2

O

2

 and the peroxynitrite anion (ONOO

-

) and reduces lipid peroxidation

(LPO), thus lowering free radical damage to mtDNA and the ETC. Moreover, melatonin improves the activity of the ETC complexes I
and IV and ATP synthesis. Finally, melatonin increases transcriptional activity of mtDNA, improving mitochondrial physiology. As a
consequence of the effects of melatonin, the mitochondrion is protected and recovers almost normal function thereby avoiding PTP
opening and apoptosis.

Chronic melatonin administration increases the number

and size of mitochondria in the pineal and ependymal
epithelium of choroids plexuses [173, 174]. Binding
experiments with 

125

Iodomelatonin also revealed a high

percentage of specific binding sites in the mitochondrial
fraction of the pigeon brain and in spleens of guinea pigs
[175, 176]. In the hamster hypothalamus, higher binding of

125

Iodomelatonin was recorded in the mitochondrial pellet

than in the nuclear pellet [177]. Soon thereafter, it was
shown that melatonin influenced mitochondrial activity
throughout the circannual cycle [178]. Milczarek [179]
showed that melatonin inhibits NADPH-dependent lipid
peroxidation in human placental mitochondria. Melatonin
protects fetal rat brain against oxidative mitochondrial
damage [180]. Finally, a protective effect for melatonin
against the MPP

+

-induced inhibition of C-I of the ETC was

also shown [181].

mitochondria when melatonin was given simultaneously
with ruthenium red [182].

To further test the antioxidant ability of melatonin

against mitochondrial oxidative stress, in vitro experiments
with isolated mitochondria prepared from rat brain and liver
were performed. Oxidative stress was induced by incubation
of these mitochondria with t-butyl hydroperoxide (t-BHP),
which oxidizes pyridine nucleotides and depletes the
mitochondrial GSH pool and inhibits both GPx and GRd
activities [183]. In this situation, 100 nM melatonin
counteracted these effects, by restoring basal levels of GSH
and the normal activities of both GPx and GRd. N-
acetylcysteine (NAC) and vitamins E and C were unable to
exert any significant effect on t-BHP-induced oxidative
stress in mitochondria despite the high doses of these
compounds used [184]. Interestingly, melatonin increased
the activity of C-I and C-IV in a dose-dependent manner, the
effect being significant at 1 nM melatonin [184]. Melatonin
was also able to counteract cyanide-induced inhibition of the
C-IV, and restored the levels of cyt a+a

3

. Melatonin also

increased the activity of isolated C-I by blue native
polyacrylamide gel electrophoresis (PAGE). These effects of
melatonin are of physiological significance since the
indoleamine increased the ETC activity coupled to
OXPHOS, which was reflected in an increase of ATP

The ability of melatonin to influence mitochondrial

homeostasis was initially tested in vivo. In this study it was
shown that the injection of melatonin into normal rats
significantly increased the activity of the complex C-I and
C-IV of the mitochondrial ETC measured in mitochondria
obtained from brain and liver, whereas the C-II and C-II were
unaffected [182]. Melatonin also counteracted ruthenium red-
induced inhibition of the C-I and C-IV in brain and liver

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Castroviejo et al.

synthesis, either in normal mitochondria or in mitochondria
depleted of ATP by cyanide incubation (D. Acuña-
Castroviejo et al., unpub-lished observations).

peaking at 90 min after melatonin injection. This time-
dependence of melatonin action agrees well with the time-
dependent changes in complex IV activity after melatonin
injection into rats as reported elsewhere [182]. Thus, it
seems that mtDNA transcriptional activity and complex IV
enzyme activity are two melatonin-related events. To further
analyze the effects of melatonin, another set of rats was
intraperitoneally injected with melatonin (10 mg/kg) or
vehicle for 10 days; at this point their livers were used for
mitochondria preparations. Fresh mitochondria were
incubated as described [190], and incorporation of labelled
UTP into mRNA was analyzed. The results showed that the
animals treated with melatonin have lower mRNA levels
than the controls, an effect partially counteracted in
pinealectomized animals. Given the effect of melatonin on
ATP production [81] and the effect of ATP on  mitochondrial
mRNA synthesis [190], a 10-day regimen of melatonin
treatment in rats may increase significantly ATP production
which in turn inhibits mRNA synthesis.

These results suggest a direct effect of melatonin on

mitochondrial energy metabolism (Fig. 1), providing a new
homeostatic mechanism regulating mitochondrial function
[81, 137, 182, 184]. First, melatonin scavenges H

2

O

2

 [140]

the most important ROS produced into the mitochondria
from O

2

¯

, This reduces the loss of the intramitochondrial

GSH pool and lowers mitochondrial damage  [99, 100]. This
effect is also supported by the observation that melatonin
increases mitochondrial membrane fluidity thereby at least
partially protecting against protein oxidative damage. Due to
the high content of proteins in the inner mitochondrial
membrane, this effect of melatonin may also account for the
improvement in ETC activity. Second, improving
mitochondrial respiration and ATP synthesis increases the
rate of electron transport across the ETC and reduces ROS
production. Due to the high redox potential of melatonin
[141], this molecule may donate an electron to C-I of the
ETC. Thus, melatonin improves ETC and reduces
mitochondrial oxidative damage. These effects reflect an
ability  of melatonin to reduce the harmful decrease in 

∆ψ

m

that may trigger PTP opening and the apoptotic cascade.
Another important consequence of the effects of melatonin
on mitochondria is its role in thermogenesis [82]. Since the
data indicated that melatonin exerts an opposite effect to
UCPs, melatonin reduces heat production by mitochondria
and induces a more efficient use of substrates in terms of
ATP production.

CONCLUDING REMARKS

The observations described herein suggest that melatonin

acts as a coupling agent in mitochondria to reduce heat
production, to increase ATP synthesis, and to increase
mtDNA expression. These effects may be the basis for the
alleged anti-aging properties of melatonin. The reduction of
melatonin levels during aging [191] presumably promotes an
increase in oxidative stress [135] that impairs mitochondria
metabolism, favoring apoptosis. Additionally, the age-
dependent reduction in mtDNA transcriptional activity may
also partially depend on the age-dependent loss of
melatonin. Additionally, the neuroprotective properties of
melatonin in many degenerative disorders which exhibit
mitochondrial alterations may also relate to melatonin’s
mitochondrial homeostatic role. Considering this, melatonin
may be useful for the treatment of some mitochondrial
dysfunctions which involve mtDNA damage and/or other
mitochondriopathies. The mitochondria are now considered a
potentially important target for drug delivery, and strategies
to prevent mitochondrial damage or to manipulate
mitochondrial function may provide new therapies for these
disorders [192]. Moreover, the application of antioxidant
therapy in oxidative stress-related diseases is now of
increasing clinical interest [193, 194]. On the basis of data
summarized in this report, melatonin becomes an interesting
pharmacological tool in mitochondrial-related diseases since
it easily reaches the mitochondria, it regulates the
mitochondrial redox status and mtDNA transcriptional
ability, and it is metabolized to other compounds with
strong antioxidant ability [195]. Finally, the lack of
significant toxic effects of melatonin treatment at
pharmacological doses [120, 196], allows for a wide margin
of safety in clinical trials.

An important question becomes apparent from these data.

If melatonin improves OXPHOS and ATP synthesis, does
melatonin exert some effect on mtDNA transcriptional
and/or translational activity? It was shown in tumor cell
studies that melatonin exerts an oncostatic effect unrelated to
nascent DNA synthesis [185]. However, when melatonin
was added to cultured J774 macrophages, the indoleamine
reduced the suppression of mitochondrial respiration and
inhibited the development of DNA single strand breaks in
response to ONOO¯ [186]. In another set of experiments, it
was shown that melatonin administration prevented
oxidative degradation of mtDNA and reduction of mtDNA
transcripts in several tissues including liver, heart, skeletal
muscle and brain [187, 188]. In addition, a direct effect of
melatonin on mitochondrial genome expression in brown
adipocytes of Siberian hamster was documented [189].

Because of these findings, we performed a series of

experiments to analyze the possible effects of melatonin of
the expression on the mtDNA encoded polypeptide subunits
of the C-IV in both in vivo and in vitro. Starting from the
mtDNA-encoded subunits I, II and III of the C-IV, a
quantitative analysis of the mRNAs of these subunits by
means of quantitative reverse transcription polymerase chain
reaction (RT-PCR) was performed (D. Acuña-Castroviejo et
al.
, unpublished). Rats were intraperitoneally injected with
melatonin (10 mg/kg body weight) or vehicle and sacrificed
at different times after treatment to obtain the livers used for
the determinations. The results show a significant increase in
the expression of the mRNAs for the three subunits tested.
The increases in the mRNA content were time-dependent,

ACKNOWLEDGEMENTS

This work was partially supported by grant SAF98-0156

from the Ministerio de Educación y Cultura, Spain. JL is a
postdoctoral fellow of the Ministerio de Educación y
Cultura, Spain.