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Arch Toxicol
DOI 10.1007/s00204-015-1475-z

REVIEW ARTICLE

Protective role of melatonin in mitochondrial dysfunction 

and related disorders

Giuseppe Paradies · Valeria Paradies · 
Francesca M. Ruggiero · Giuseppe Petrosillo 

Received: 14 January 2015 / Accepted: 9 February 2015 
© Springer-Verlag Berlin Heidelberg 2015

through which melatonin exerts its protective role in mito-
chondrial dysfunction and related disorders are reviewed.

Keywords  Melatonin · Mitochondria bioenergetics · 
Cardiolipin · Oxidative stress · Physiopathology

Introduction

Melatonin (N-acetyl-5-methoxytryptamine) is a highly 
conserved molecule derived from tryptophan that is found 
in all organisms from unicells to vertebrates (Hardeland 
and Fuhrber

1996

; Hardeland et al. 

2006

Tan et al. 

2007

). 

Melatonin and its metabolites can function as endogenous 
free radical scavengers and broad-spectrum antioxidants 
(Tan et al. 

2002

; Reiter et al. 

2008

). Due to its small size 

and amphiphilic nature, melatonin can reach numerous 
cellular and subcellular compartments, particularly mito-
chondria (Menendez-Pelaez and Reiter 

1993

), raising the 

possibility of functional significance for this targeting with 
involvement in situ in mitochondrial bioenergetic processes 
(Leon et al. 

2004

; Paradies et al. 

2010a

). Most of the ben-

eficial consequences resulting from melatonin administra-
tion may depend on its effect on mitochondrial physiology 
(Acuña-Castroviejo et al. 

2007

2011

).

Mitochondrial dysfunction is considered an important 

contributing factor in a variety of physiopathological situ-
ations such as neurodegenerative and cardiovascular dis-
eases, diabetes, various forms of hepatic disorders, skeletal 
muscle disorders, as well as in aging (Beal 

1998

Wallace 

1999

; Schon et al. 

2010

). Alterations in mitochondrial 

function such as defects in the electron transport chain 
(ETC) activity and oxidative phosphorylation (OXPHOS) 
have all been suggested as the primary causative factors in 
the pathogenesis of these disorders.

Abstract  Mitochondria are the powerhouse of the eukar-
yotic cell through their use of oxidative phosphorylation to 
generate ATP. Mitochondrial dysfunction is considered an 
important contributing factor in a variety of physiopatho-
logical situations such as aging, heart ischemia/reperfusion 
injury, diabetes and several neurodegenerative and cardio-
vascular diseases, as well as in cell death. Increased forma-
tion of reactive oxygen species, altered respiratory chain 
complexes activity and opening of the mitochondrial per-
meability transition pore have been suggested as possible 
factors responsible for impaired mitochondrial function. 
Therefore, preventing mitochondrial dysfunction could be 
an effective therapeutic strategy against cellular degenera-
tive processes. Cardiolipin is a unique phospholipid located 
at the level of inner mitochondrial membrane where it 
plays an important role in mitochondrial bioenergetics, as 
well as in cell death. Cardiolipin abnormalities have been 
associated with mitochondrial dysfunction in a variety of 
pathological conditions and aging. Melatonin, the major 
secretory product of the pineal gland, is a well-known 
antioxidant agent and thus an effective protector of mito-
chondrial bioenergetic function. Melatonin was reported to 
prevent mitochondrial dysfunction from oxidative damage 
by preserving cardiolipin integrity, and this may explain, 
at least in part, the beneficial effect of this compound in 
mitochondrial physiopathology. In this article, mechanisms 

G. Paradies (*) · V. Paradies · F. M. Ruggiero 
Department of Biosciences, Biotechnologies 
and Biopharmaceutics, University of Bari, Bari, Italy
e-mail: g.paradies@biologia.uniba.it

G. Petrosillo (*) 
Institute of Biomembranes and Bioenergetics, National Research 
Council, Bari, Italy
e-mail: g.petrosillo@ibbe.cnr.it

 

Arch Toxicol

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Mitochondria are considered the main intracellular 

source of reactive oxygen species (ROS), as well as the 
major target of free radical attach. ROS are generated at 
very low levels during mitochondrial respiration under 
normal physiological conditions, while the level of these 
oxidants increases in several pathological conditions and 
aging. ROS produced by the mitochondrial ETC attach 
mitochondrial constituents including proteins, lipids and 
mitochondrial DNA (mtDNA). ROS-induced alterations to 
mitochondrial membrane constituents may lead to a decline 
of the mitochondrial bioenergetic function, and this may 
contribute to the etiology of a variety of pathological con-
ditions, including heart ischemia/reperfusion (Chen and 
Zweier 

2014

), aging and age-related cardiovascular and 

neurodegenerative diseases (Boveris and Navarro 

2008

DiMauro and Schon 

2003

; Raha and Robinson 

2000

).

Recent studies have demonstrated that melatonin plays 

an effective role in preserving mitochondrial homeosta-
sis (Martín et al. 

2002

; López et al. 

2009

; Paradies et al. 

2010a

; Acuña-Castroviejo et al. 

2011

; Navarro-Alarcón 

et al. 

2014

), which may explain the protective effect of this 

molecule in several physiopathological conditions includ-
ing neurological (Patki and Lau 

2011

; Pandi-Perumal et al. 

2013

; Cardinali et al. 

2013

) and cardiovascular disorders 

(Dominguez-Rodriguez and Abreu-Gonzalez 

2010

; Yang 

et al. 

2014

) as well as aging (Bondy et al. 

2004

; Dong 

et al. 

2010

), all of which are associated with mitochon-

drial dysfunction. This protective effect of melatonin may 
be explained, at least in part, on its antioxidant and free 
radical scavenging properties, thus preserving the stability, 
integrity and function of mitochondrial membranes (García 
et al. 

2014

; Reiter et al. 

2014

).

Cardiolipin (CL), a unique phospholipid located at the 

level of the inner mitochondrial membrane (IMM), has 
been shown to play a central role in the mitochondrial func-
tion (Hoch 

1992

; Houtkooper and Vaz 

2008

Ren et al. 

2014

; Paradies et al. 

2014a

). Abnormalities in CL not only 

alter fluidity and folding of the IMM, but can profoundly 
alter the organization and function of the respiratory chain 
complexes and/or their organization in supramolecular 
structure, such as supercomplexes (Musatov and Robinson 

2012

; Mileykovskaya and Dowhan 

2014

). In particular, 

oxidation and depletion of CL have been associated with 
mitochondrial dysfunction in several metabolic and degen-
erative diseases (Chicco and Sparagna 

2007

; Paradies et al. 

2009

b

). Recently, melatonin was reported to preserve 

mitochondrial CL from oxidative damage, and this may 
explain, at least in part, the beneficial effect of this mol-
ecule in mitochondrial dysfunction and associated disor-
ders (Petrosillo et al. 

2009c

; Paradies et al. 

2010a

). In this 

review, we discuss the several mechanisms through which 
melatonin can exert its protective role in mitochondrial 
dysfunction and related disorders.

Mitochondrial function and ROS generation

Mitochondria contain multiple copies of circular genome 
known as mtDNA as it has been characterized in humans 
(Anderson et al. 

1981

). The majority of mitochondrial pro-

teins needed for normal bioenergetic processes are encoded 
by nuclear DNA, while some proteins essential for ETC 
and OXPHOS are encoded by mtDNA. Human mtDNA 
encodes for 13 polypeptides of subunits of complexes I, 
III and IV and ATP synthase, 22 tRNA and two ribosomal 
nucleic acids.

Mitochondria play a central role in energy-generating 

processes within the cell through the ETC, the primary 
function of which is ATP synthesis through the OXPHOS 
process. The ETC, which is located in the IMM, comprises 
a series of electron carriers grouped into four enzyme com-
plexes, namely complex I (CI) (NADH-ubiquinone reduc-
tase), complex II (CII) (succinate-ubiquinone reductase), 
complex III (CIII) (ubiquinol-cytochrome c reductase) 
and complex IV(CIV) (cytochrome c oxidase) (Lenaz and 
Genova 

2010

). The electrons are transferred to molecular 

oxygen via the electron transport complexes, resulting in 
the reduction of oxygen to water at complex IV. During this 
process, protons (H

+

) are pumped by CI, CIII and CIV into 

the intermembrane space to form an electrochemical gra-
dient, which is the major contributor to the mitochondrial 
inner membrane potential. Complex V (CV) (ATP syn-
thase) utilizes the stored energy of this proton gradient to 
drive the formation of ATP from ADP and inorganic phos-
phate. ATP formed is then transferred by the ADP/ATP car-
rier (ANT) to the intermembrane space in exchange with 
ADP.

It is estimated that around 0.2–2 % of the oxygen taken 

up by the cell is converted by mitochondria to ROS, mainly 
through the production of O

2

·−

 (Boveris and Chance 

1973

). 

OXPHOS, however, comes with an additional cost, the pro-
duction of potentially harmful ROS. Mitochondrial ETC is 
considered the main source of ROS production. The pri-
mary ROS generated into the mitochondria is superoxide 
anion (O

2

·−

), which is then converted to hydrogen peroxide 

(H

2

O

2

) by spontaneous dismutation or by superoxide dis-

mutase (SOD). Hydrogen peroxide in turn is broken down 
into water by glutathione peroxidase or catalase; other-
wise, H

2

O

2

 can undergo Fenton’s reaction in the presence 

of divalent cations such as iron to produce hydroxyl radical 
(·OH), which can be even more harmful to the mitochon-

drial biomolecules. The sites of superoxide anion produc-
tion along the ETC have been the subject of many studies 
(for a recent review, see Murph

2009

). The two major sites 

of O

2

·−

 production are complex I and complex III. Mito-

chondria can produce O

2

·−

, predominantly from complex 

I, when the matrix NADH/NAD+ ratio is high, leading to 

a reduced FMN site on complex I, and when they have a 

Arch Toxicol 

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high proton-motive force and a reduced coenzyme Q pool, 
leading to reverse electron transport. The site of superoxide 
production at complex III is probably the unstable ubisem-
iquinone molecules (Turrens et al. 

1985

) or cytochrome b 

(Nohl and Stolze

1992

). The production of O

2

·−

 at complex I 

is believed to occur at the matrix site of the IMM. At com-
plex III, O

2

·−

 is released to both the matrix and the cytosolic 

sides of the IMM. The relative contributions of complex I 
and III to ROS production appear also to be dependent on 
types of tissues, species and experimental conditions. The 
rate of ROS production is also affected by mitochondrial 
metabolic conditions. O

2

·−

 production is highest under state 

4 respiration; when oxygen consumption is low, the proton-
motive force is high and ETC complexes are in reduced 
state (Skulache

1996

; Korshunov et al. 

1997

). ROS are 

also produced to a lesser extent outside of the mitochon-
drion. Examples of extra-mitochondrial ROS producing 
reactions include xanthine oxidase, D-amino oxidase, the 
P-450 cytochromes and proline and lysine hydroxylase.

Mitochondria can also produce nitric oxide (NO) from 

mitochondrial nitric oxide synthase (Ghafourifar and Rich-
ter 

1997

; Giulivi et al. 

1998

), located in the IMM, from 

l

-arginine. NO can be then converted to various reactive 

nitrogen species (RNS) such as nitroxyl anion (NO

) or the 

toxic peroxynitrite (ONOO

). The oxidizing reactivity of 

ONOO

 is generally considered equivalent to that of 

·

OH. 

NO strongly interferes with components of the ETC, in 
particular with cytochrome c oxidase (Mander and Brown 

2004

). NO in combination with ONOO

 not only interferes 

with respiratory complexes, but can also trigger free radi-
cal-mediated chain reactions that in turn damage proteins, 
lipids and DNA molecules (Rubbo et al. 

1994

; Levine and 

Stadtman 

2001

). Damage to the mitochondrial respiratory 

chain can cause a collapse of membrane potential with fur-
ther generation of free radicals, triggering a vicious cycle 
that ultimately leads to cell death.

Mitochondrial antioxidant defense systems

Mitochondria are equipped with an intricate array of enzy-
matic and nonenzymatic antioxidant defense systems 
poised to detoxify the ROS/RNS production. Nonenzy-
matic components of the system include hydrophilic and 
lipophilic radical scavengers, such as cytochrome c, alpha-
tocopherol, ascorbate, ubiquinone, glutathione and mela-
tonin. Another specific mitochondrial defense mechanism 
is the mild uncoupling that prevents marked increase in 
membrane potential and hence O

2

·−

 formation. Enzymatic 

components of the antioxidant systems include manganese-
superoxide dismutase (Mn-SOD), catalase, glutathione 
peroxidase and phospholipid hydroperoxide glutathione 
peroxidase. Within the mitochondrial matrix, Mn-SOD 

converts O

2

·−

 to H

2

O

2

, which can be further metabolized 

by glutathione peroxidase (Gpx I) and peroxiredoxine 
(Prx III) or diffuse from the mitochondria into the cytosol. 
Part of the O

2

·−

 produced by the mitochondrial ETC can be 

released into the inner membrane space where it can be 
converted to H

2

O

2

 by Cu–Zn SOD. The O

2

·−

 present in the 

intermembrane space could be scavenged by the oxidized 
form of the cytochrome c or diffuse into the cytosol through 
the voltage-dependent anion channel (VDAC; Madesh and 
Hajnoczky 

2001

). O

2

·−

 may also react with nitric oxide 

(NO) to form highly reactive ONOO

. Glutathione (GSH) 

and multiple GSH-linked antioxidant enzymes exert also 
an important mitochondrial antioxidant protection. Among 
GSH-linked enzymes involved in mitochondrial antioxi-
dant defense are Gpx 1 located predominantly in the cyto-
sol and Gpx 4, also known as phospholipid hydroperoxide 
glutathione peroxidase, which is associated with contact 
sites of the two mitochondrial membranes. These enzymes 
catalyze the reduction of H

2

O

2

 and of lipid hydroperoxides. 

Gpx 4 reduces hydroperoxide groups on phospholipids, 
lipoproteins and cholesteryl esters. Gpx 4 is considered to 
be the primary enzymatic defense mechanism against oxi-
dative damage to cellular membranes.

The redox cycling in the mitochondria is very active and 

serves to prevent significant loss of GSH. Melatonin pro-
motes de novo synthesis of GSH by stimulating the activ-
ity of the enzyme γ-glutamyl-cisteine synthase (Urata et al. 

1999

) and also through its effect on gene expression of 

Gpx, SOD and catalase (Antolín et al. 

1996

; Reiter et al. 

2000

), thus favoring the recycling of GSH and maintain-

ing high GSH/GSSG ratio. These effects of melatonin may 
have important implications in mitochondrial physiology 
(Escames et al. 

2010

).

Melatonin and mitochondrial oxidative stress

Mitochondria are the most powerful intracellular source 
of ROS and also the primary target for their damaging 
effects. The interaction of ROS with mitochondrial compo-
nents impairs the function of these organelles and directly 
affects cell viability and triggers cell death. ROS-induced 
structural and functional modification of proteins is one of 
the hallmarks of aging and several pathological disorders 
in biological systems (Stadtman 

2002

). One important tar-

get of ROS is mtDNA, which encodes polypeptides that 
are essential for ETC and ATP generation by OXPHOS. 
MtDNA is particularly susceptible to attack by ROS 
because of its proximity to the ETC and lack of protective 
histones. MtDNA therefore represents a critical cellular tar-
get for oxidative damage that could lead to lethal cell injury 
through the disruption of electron transport, mitochondrial 
membrane potential and ATP generation. ROS-induced 

 

Arch Toxicol

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mtDNA damage is probably a major source of mitochon-
drial genomic instability responsible for the mitochondrial 
dysfunction. The instability of mtDNA is thought to be one 
of the most important factors in aging (Wei and Lee 

2002

).

In addition to mtDNA and proteins, mitochondrial 

membrane lipids are highly susceptible to oxidative dam-
age. Phospholipids are the most abundant lipid compo-
nents of the cellular and subcellular membranes, includ-
ing mitochondria. Phospholipids are essential structural 
components of the mitochondrial membranes where they 
play multiple roles. They define the essential membrane 
permeability barrier and modulate the proper membrane 
fluidity, which is required for the optimal functional activi-
ties of proteins and enzymes. Polyunsaturated fatty acids 
(PUFAs) are essential components of mitochondrial phos-
pholipids. The presence of a methylene bridge between 
two double bonds renders the PUFAs particularly sensitive 
to ROS attack, enabling them to participate in long free 
radical chain reactions generating hydroperoxides as well 
as endoperoxides. These lipid peroxidation products can 
undergo fragmentation to produce a broad range of reactive 
intermediates, among them are malondialdehyde (MDA) 
and the most reactive, 4-hydroxy-trans-2-nonenal (HNE). 
Oxidation of membrane phospholipids is considered one of 
the major causes of mitochondrial dysfunction in a variety 
of physiopathological situations and aging. In fact, lipid 
peroxidation alters the structural and functional organiza-
tion of the lipid bilayer, changing membrane fluidity and 
permeability, thereby affecting respiration and OXPHOS 
process, maintenance of mitochondrial membrane potential 
and mitochondrial Ca

2+

 buffer capacity (Pamplona 

2008

).

Data accumulated in the last decade indicate that mela-

tonin plays an important role in antioxidant defense pre-
serving mitochondrial homeostasis, reducing free radical 
generation and stimulating ETC complex activity (Martín 
et al. 

2002

; López et al. 

2009

; Paradies et al. 

2010a

; Acuña-

Castroviejo et al. 

2011

; Navarro-Alarcón et al. 

2014

). The 

earliest evidence of the antioxidant capacities of mela-
tonin was reported in 1993 (Tan et al. 

1993

). Melatonin 

scavenges two molecules of 

·

OH, and in the process, it is 

converted to cyclic 3-hydroxymelatonin (Tan et al. 

1998

). 

This later compound was detected by mass spectra analy-
sis and carbon and proton–nuclear magnetic resonance in 
the urine of human and rats under oxidative stress condi-
tions and treated with melatonin (Tan et al. 

1998

). It was 

reported that melatonin does not directly scavenge H

2

O

2

 

in vitro, while a direct interaction of melatonin with H

2

O

2

 

occurs only in the presence of traces of the transition metal 
ions (Fowler et al. 

2003

), and this may have important 

implications in vivo under stress condition. Besides its 
effects on ROS, melatonin is also a powerful scavenger of 
RNS. Nitric oxide is produced by several forms of NOS. In 
the mitochondria, two NOS isoforms, namely constitutive 

(c-mtNOS) and inducible (i-mtNOS), have been reported 
(Ghafourifar and Richter 

1997

). NO strongly interferes 

with components of the respiratory chain in particular 
cytochrome c oxidase (Mander and Brown 

2004

). NO in 

combination with ONOO

 not only interferes with respira-

tory chain complexes, but can trigger free radical-mediated 
chain reactions that in turn damage proteins, lipids and 
DNA molecules (Rubbo et al. 

1994

; Stadtman and Levine 

2003

).

Polyunsaturated fatty acids, the main constituents of 

phospholipids (PLs), are particularly sensitive to peroxida-
tion. This process is considered to proceed via a sequence 
of steps, including the abstraction of a hydrogen atom from 
unsaturated fatty acids, forming an alkyl radical (PL

·

), fol-

lowed by a rapid addition of oxygen to form the peroxyl 
radical (PLOO

·

), and then formation of a hydroperoxide 

(PLOOH) via abstraction of a hydrogen from another acyl 
chain; as a consequence, the reaction is repeated and the 
whole process continues in a free radical chain reaction. 
Due to this auto-oxidative chain reaction, a single initiation 
event could theoretically lead to the oxidation of all lipids 
in a cellular organelle or in a cell. Other reactive species 
which initiate lipid peroxidation include ONOO

 and sin-

glet oxygen 

1

O

2

. Because of the highly destructive struc-

tural and functional nature of lipid peroxidation, there is 
great interest in identifying molecules which reduce the ini-
tiation and/or progression of the denaturation of PUFAs. It 
has been well documented that melatonin and its derivatives 
exert a protective effect against lipid peroxidation induced 
by oxidative stress in mitochondrial membranes (Reiter 
et al. 

2014

). The ability of melatonin to protect against lipid 

peroxidation has been repeatedly documented either in ani-
mal or in plant tissues under various oxidizing conditions 
such as ionizing radiation, heavy metal toxicity, and drug 
metabolism. (García et al. 

2014

). The precise mechanism 

by which melatonin and its derivatives affect lipid peroxi-
dation is not yet established. Melatonin has been shown 
to scavenge the peroxyl radical PLOO

·

 (Pieri et al. 

1994

Livrea et al. 

1997

), which is produced during lipid peroxi-

dation and being sufficiently reactive to propagate the chain 
reactions. The efficacy of melatonin to function as a PLOO

·

 

scavenger was evaluated by measuring inhibition of metal 
ion-, radiation- or human macrophage-mediated oxidation 
of human low-density lipoprotein (Abuja et al. 

1997

). Mel-

atonin was shown to be more effective than vitamin E in 
neutralizing PLOO

·

 and inhibiting lipid peroxidation (Pieri 

et al. 

1994

1996

). Another major contributor to lipid per-

oxidation is ONOO

, which is a powerful initiator of lipid 

breakdown. Due to the ability of melatonin to neutralize 
ONOO

, this is another means whereby melatonin may 

protect membrane lipids (Cuzzocrea et al. 

1997

). It is pre-

sumed that melatonin inhibits lipid peroxidation by inter-
fering with the radicals that initiate this process, especially 

Arch Toxicol 

1 3

the 

·

OH and ONOO

, and by positioning itself in a superfi-

cial location in membrane lipids layers near the polar heads 
of these molecules (Ceraulo et al. 

1999

). Its small molecu-

lar size and its amphiphilic properties facilitate melatonin’s 
penetration into subcellular compartments. In vitro assays 
showed that melatonin inhibits lipid peroxidation in rat 
brain homogenates, brain and liver microsomes and mito-
chondria treated with an ascorbate-Fe

2+

 system (Teixeira 

et al. 

2003

). Melatonin has been found to protect against 

lipid peroxidation in many experimental models (Maharaj 
et al. 

2006

; Parlakpinar et al. 

2002

).

Mitochondrial membranes, which are rich in phospho-

lipids containing PUFAs, are fluid structures, and optimal 
membrane fluidity is required for their proper function. 
When PUFAs are oxidized, mitochondrial membranes 
become more rigid; thus, their protection from oxidation 
is essential for optimal function of these organelles. Since 
the degree of lipid breakdown in mitochondrial membranes 
generally correlates with the fluidity of these organelles, it 
could be predicted that melatonin, by preventing lipid per-
oxidation, could preserve the proper fluidity and function of 
the membranes. Aging is characteristically associated with 
elevated cell membrane rigidity. Depressed level of mela-
tonin naturally occurring with aging leads to elevated levels 
of lipid peroxidation and to more rigid cellular membranes 
(Reiter et al. 

1999

Hardeland 

2013

). Likewise, treatment 

of senescence-accelerated prone mice with melatonin pre-
serves mitochondrial membranes in a more fluidity state 
(García et al. 

2014

).

Melatonin and mitochondrial function

Melatonin is highly lipophilic molecule that crosses cell 
membranes to easily reach cellular compartments including 
mitochondria, where it seems to accumulate in high con-
centration (Acuña-Castroviejo et al. 

2003

). Several stud-

ies have shown that melatonin can influence mitochondrial 
homeostasis by stabilizing mitochondrial inner membrane, 
thereby improving ETC activity and mitochondrial func-
tion (López et al. 

2009

; Martín et al. 

2002

). Melatonin 

was reported to increase the activity of C I and C IV in a 
time-dependent manner in mitochondria isolated from rat 
brain and liver tissues, while having no effect on C II and 
C III (Martín et al. 

2002

). Melatonin administration also 

prevented the inhibitory effect of ruthenium red on C I and 
C IV activities as well as on GPx enzyme in rats (Martín 
et al. 

2000

). Melatonin (1 nM) significantly increases the C 

I and C IV activities in rat liver mitochondria, while higher 
concentrations of this compound are required to stimulate 
the activity of these complexes in rat brain mitochondria. 
The effects on C I were also studied using BN-PAGE his-
tochemical procedure to measure change in its activity 

induced by melatonin. This study documented an increase 
in C I activity following melatonin treatment. Melatonin 
also counteracted cyanide-induced inhibition of C IV, 
as shown by the increase in the ETC activity coupled to 
OXPHOS and ATP synthesis, both either in normal or in 
rat brain and liver mitochondria depleted of ATP by cya-
nide treatment (Martín et al. 

2002

). The stimulatory effect 

of melatonin on the C I and C IV activities does not only 
rely on the antioxidant role of this indoleamine. Because 
of its high redox potential (0.94 V; Tan et al. 

2000

), mel-

atonin may interact with the ETC complexes by donating 
and accepting electrons, thereby increasing electron flux, 
an effect not shared by other antioxidants.

Recently, another effect of melatonin on mitochondrial 

bioenergetic parameters was described (López et al. 

2009

). 

Experiments carried out in vitro with normal mitochondria 
demonstrate that this indoleamine protected the mitochon-
drial function from oxidative damage, decreasing oxygen 
consumption in the presence of ADP in a concentration-
dependent manner and reducing the membrane potential, 
thereby inhibiting the production of O

2

·−

 and H

2

O

2

 (López 

et al. 

2009

). In addition, melatonin maintained the respira-

tory control ratio, the efficiency of the OXPHOS and ATP 
synthesis, while enhancing the activity of ETC complexes 
(mainly C I, C III and C IV). These effects of melatonin 
probably depend on a direct interaction of this indoleamine 
with mitochondria, as shown by the fact that mitochondria 
take up melatonin in a time- and concentration-dependent 
manner, and thus, the effects of melatonin were due to its 
presence within the mitochondria. The ability of mitochon-
dria to accumulate melatonin is of great pharmacological 
interest because it means that, following exogenous admin-
istration in vivo, melatonin enters the mitochondria and 
exerts its beneficial action on mitochondrial function.

Cardiolipin and mitochondrial function

Cardiolipin is commonly referred to as the signature phos-
pholipid of mitochondria. This phospholipid is associated 
with membranes designed to generate an electrochemi-
cal gradient that is used to produce ATP, such as bacterial 
plasma membrane and the IMM (Ren et al. 

2014

). This 

association between CL and energy transducing mem-
branes suggests an important role for CL in bioenergetic 
processes (Schlame et al. 

2000

; Ren et al. 

2014

). In fact, 

CL has been shown to interact with a number of IMM pro-
teins including, among others, the ETC complexes involved 
in OXPHOS (Musatov and Robinson 

2012

; Schlame et al. 

2000

; Houtkooper and Vaz 

2008

; Schlame and Ren 

2009

and the anionic substrates carriers (Klingenberg 

2009

). 

Indeed, CL is required for optimal activity of C I (Sharpley 
et al. 

2006

), C III (Fry and Green 

1981

), C IV (Robinson 

 

Arch Toxicol

1 3

1993

and C V (Eble et al. 

1990

). Crystallographic stud-

ies have shown the presence of a few tightly bound CL 
molecules in each of the crystal structures of the C III, C 
IV and ADP/ATP carrier (Lange et al. 

2001

; Ozawa et al. 

1982

; Eble et al. 

1990

). These results suggest that CL is an 

integral component of these proteins, the presence of which 
is critical for their proper folding. CL seems to facilitate the 
association and stabilization of respiratory chain complexes 
into supercomplexes (Zhang et al. 

2002

; Schagger 

2002

). 

Such supercomplexes formation is thought to improve the 
efficiency of OXPHOS by eliminating the need for diffu-
sion of substrates and products between individual ETC 
component (Genova and Lenaz 

2014

). CL is also required 

for the interaction between ADP/ATP carrier proteins and 
respiratory supercomplexes (Claypool 

2009

).

Another important role of CL is its participation in the 

process of apoptosis in animal cells through the interac-
tion with a variety of death-inducing proteins, including 
cytochrome c (Gonzalvez and Gottlieb 

2007

; Ott et al. 

2007a

b

). This hemoprotein is believed to acts as per-

oxidase, which reacts quite specifically with CL, causing 
oxidation and then hydrolysis of the product CL hydrop-
eroxide (Kagan et al. 

2005

). The consequence is that the 

cytochrome c is released into the intermembrane space, 
while the oxidized CL is translocated to the outer mito-
chondrial membrane and participates in the opening of the 
mitochondrial permeability transition pore (Petrosillo et al. 

2006a

). The opening of this pore facilitates the release of 

several proapoptotic factors, including cytochrome c, from 
mitochondria into the cytosol where they trigger apopto-
sis. CL appears to play an important role in mitochondrial 
morphology and dynamics including fusion and fission 
processes (DeVay et al. 

2009

; Ban et al. 

2010

), as well as 

in the protein insertion and assembly into the mitochondria 
(Marom et al. 

2009

). Given the role played by CL in mito-

chondrial bioenergetic processes as well as in apoptosis 
and in mitochondrial membrane stability and dynamics, it 
is conceivable that abnormalities in CL structure, content 
and acyl chains composition may have important implica-
tions in mitochondrial dysfunction associated with several 
physiopathological states and diseases (Chicco and Spar-
agna 

2007

; Paradies et al. 

2014b

c

). Alterations in mito-

chondrial CL profile may occur mainly as a consequence 
of: (1) loss of the CL content due to the changes in the CL 
synthase activity; (2) changes in acyl chain composition 
due to altered CL remodeling and (3) CL oxidation due to 
ROS attack.

Melatonin and cardiolipin oxidation

Oxygen free radicals lead to primary reaction and damage 
in the immediate surroundings of where they are generated, 

due to their high chemical reactivity. Therefore, the effect 
of these reactive species should be greatest at the level of 
mitochondrial membrane components such as phospho-
lipid molecules particularly rich in PUFAs. Among phos-
pholipids, CL molecules are particularly sensitive to oxi-
dation, either because of their location in IMM near to the 
site of ROS production, or because of their high content of 
PUFAs. In fact, CL molecules are rich in unsaturated fatty 
acyl chains, particularly linoleic acid in heart and liver, 
or docosahexanoic and arachidonic acids in brain tissue 
mitochondria. In addition, CL molecules are located in the 
mitochondrial membrane near to the site of ROS produc-
tion, mainly represented by the respiratory chain complexes 
I and III to which CL molecules are associated.

As described above, melatonin and its derivatives exert 

a protective effect on lipid peroxidation in mitochondrial 
membranes (Catala´ 

2007

; García et al. 

2014

; Reiter et al. 

2014

). Recently, we have studied the ability of melatonin 

to inhibit CL oxidation in isolated mitochondria (Petrosillo 
et al. 

2009c

). CL oxidation in mitochondria was induced 

by treating these organelles with t-butylhydroperoxide 
(t-BuOOH), a lipid-soluble hydroperoxide that closely 
resembles endogenous lipid hydroperoxides generated dur-
ing oxidative stress. Treatment of rat heart and brain mito-
chondria with t-BuOOH in the presence of micromolar 
concentrations of copper ions resulted in a loss of CL con-
tent and in an increase in the level of oxidized CL, the lat-
ter being detected by a normal phase HPLC technique with 
UV detection at 235 nm, indicative of conjugated dienes. 
Melatonin, at micromolar concentration, was able to pre-
vent the oxidation/depletion of CL. This inhibitory effect of 
melatonin on CL oxidation in mitochondria can be reason-
ably explained on the ability of this indoleamine to inhibit 
the peroxidation of linoleic acyl chains, which are the main 
constituents of CL molecules. In fact, previous results 
have demonstrated the antioxidant effect of melatonin on 
linoleate oxidation initiated by HO

·

 free radical generated 

by water gamma radiolysis (Mekhloufi et al. 

2005

). Using 

linoleate micelles as lipid model, two index of peroxidation 
have been measured, i.e., conjugated dienes and hydroper-
oxides. The results obtained in this study demonstrate that 
melatonin displays strong in vitro lipid peroxyl radicals 
(LOO

·

) scavenging properties, as shown by its inhibitory 

effect on the radiation-induced peroxidation of linoleate.

Emerging insights have linked CL oxidation/depletion 

to mitochondrial dysfunction associated with a variety of 
diseases and physiopathological settings including heart 
ischemia/reperfusion, diabetes, aging and age-associated 
disorders (Chicco and Sparagna 

2007

; Paradies et al. 

2009

2014b

c

). CL oxidation is also emerging as a key player in 

the regulation of several of the mitochondrial steps of cell 
death and in mitochondrial dynamics (Ott et al. 

2007a

b

Paradies et al. 

2014b

). Therefore, the ability of melatonin 

Arch Toxicol 

1 3

to prevent CL oxidation in mitochondria may have impor-
tant implications in mitochondrial dysfunction and related 
disorders.

Cardiolipin and MPTP

Mitochondrial permeability transition pore (MPTP) is 
defined as the sudden increase in IMM permeability to 
low molecular weight metabolites (

˂1.5 kDa) in response 

to many stimuli, including high levels of Ca

2+

 and oxidant 

stress (Crompton 

1999

; Leung and Halestrap. 

2008

). Open-

ing of the MPTP promoted by elevated matrix Ca

2+

 levels, 

associated with high phosphate, low adenine nucleotide 
concentrations and oxidative stress, induces the collapse 
of transmembrane ion gradients, resulting in membrane 
depolarization and uncoupling of OXPHOS. This causes 
irreversible damage to mitochondria, resulting in cell death 
predominantly through necrosis. A number of molecules 
were accepted as key structural components of the MPTP, 
including, Cyp-D in the matrix, ANT and phosphate car-
riers in the IMM and VDAC (also known as porin) in the 
outer membrane (Halestrap 

2009

). More recently, dimers 

of the F

0

F

1

 ATP synthase were suggested to be new puta-

tive components of the MPTP. In fact, reconstituted dimers 
of F

0

F

1

 ATP synthase, incorporated into lipid bilayers, form 

Ca

2+

-activated channels with properties similar to those of 

the mitochondrial mega-channel, the electrophysiological 
equivalent of the MPTP (Giorgio et al. 

2013

).

Release of cytochrome c from mitochondria into cyto-

sol is considered a central event in the induction of apop-
totic cascade that ultimately leads to cell death (Ott et al. 

2007b

). Cytochrome c is normally bound to the outer 

surface of the IMM primarily to CL molecules (Rytomaa 
et al. 

1992

). Oxidation of CL promotes the detachment of 

cytochrome c from mitochondrial membrane and its release 
into the extramitochondrial space (Petrosillo et al. 

2003b

Ott et al. 

2007a

). It has been proposed that the release of 

cytochrome c from the mitochondria takes place by a two-
step process involving, first, the dissociation of this hemo-
protein from the IMM, followed by permeabilization of 
the outer membrane probably through its association with 
Bcl2 family proteins, such as Bax and Bid, and/or through 
the MPTP opening (Ott et al. 

2007b

). CL oxidation may be 

involved in the permeabilization of the outer mitochondrial 
membrane, probably through its association with Bcl2 fam-
ily proteins such as Bax and Bid (Kagan et al. 

2004

; Ott 

et al. 

2007a

; Jiang et al. 

2008

).

We have shown that exogenously added oxidized CL to 

mitochondria sensitizes these organelles to Ca

2+

-induced 

MPTP opening (Petrosillo et al. 

2006a

). This synergistic 

effect of Ca

2+

 and oxidized CL on the induction of MPTP 

opening suggests that both these compounds could play a 

coordinated role in this process by interacting with compo-
nents of the MPTP, probably with adenine nucleotide car-
rier and/or with ATP synthase dimers. The involvement of 
oxidized CL in MPTP opening is further demonstrated by 
our recent study, showing that oxidation of intramitochon-
drial CL molecules results in MPTP induction (Petrosillo 
et al. 

2009c

). Interestingly, the induction of MPTP opening 

by oxidized CL and Ca

2+

 is associated with the release of 

cytochrome c from mitochondria.

Melatonin and MPTP

Studies carried out in our and other laboratories have dem-
onstrated a role of melatonin in the modulation of MPTP 
opening (Andrabi et al. 

2004

; Hibaoui et al. 

2009

; Pet-

rosillo et al. 

2009c

; Jou 

2011

). A direct MPTP inhibition by 

melatonin has been reported (Andrabi et al. 

2004

). Mela-

tonin diminished MPTP current with an IC

50

 of 0.8 μM, 

a concentration which would require accumulation of 
melatonin within mitochondria. Indeed, it has been dem-
onstrated that melatonin due to its amphiphilic nature can 
be actively accumulated by mitochondria (Messner et al. 

1998

; López et al. 

2009

). The direct MPTP inhibition by 

melatonin should be interpreted on the basis of a low affin-
ity binding site. This effect may contribute to the anti-apop-
totic properties of melatonin.

Our studies have shown that melatonin, at micromolar 

concentrations, inhibited both the Ca

2+

/t-BuOOH-induced 

CL peroxidation and MPTP opening, as indicated by the 
protective effect this indoleamine on matrix swelling, ΔΨ 
collapse and release of preaccumulated Ca

2+

 Petrosillo 

et al. 

2009c

). These results suggest that melatonin, by pre-

venting endogenous CL peroxidation, inhibits MPTP open-
ing. In addition, our results demonstrate that the release of 
cytochrome c from mitochondria associated with the MPTP 
opening induced by oxidative stress is almost completely 
prevented by melatonin. This effect of melatonin can be 
explained on its ability to inhibit CL peroxidation, thereby 
preventing both cytochrome c detachment from the IMM 
and MPTP opening. The ability of melatonin to prevent 
MPTP opening may have important implications in those 
physiopathological situations characterized by alterations 
of Ca

2+

 homeostasis and accumulation of peroxidized CL 

in mitochondria, such as heart ischemia/reperfusion, aging 
and other degenerative diseases.

Melatonin’s action in preventing MPTP opening induced 

by oxidative stress caused by t-BuOOH was shown in 
another study carried out in primary skeletal muscle cul-
tures (Hibaoui et al. 

2009

). Melatonin (1–100 µM) fully 

prevented myotube death induced by t-BuOOH as assessed 
by acid phosphatase, caspase 3 activities and cellular 
morphological changes. Using fluorescence imaging, it 

 

Arch Toxicol

1 3

was shown that the mitochondrial protection provided by 
melatonin was associated with an inhibition of t-BuOOH-
induced ROS generation. In isolated mitochondria, mel-
atonin desensitized the MPTP to Ca

2+

 and prevented 

t-BuOOH-induced mitochondrial swelling, pyridine nucle-
otide and GSH oxidation. The inhibition of MPTP opening 
by melatonin was suggested as an explanation for the pro-
tective action of this indoleamine against oxidative stress in 
myotubes (Hibaoui et al. 

2009

).

MPTP may occur in the cell through two modes, the 

transient MPTP and the prolonged MPTP, having different 
outcomes of survival or death. The transient MPTP protects 
mitochondria, whereas the prolonged MPTP triggers the 
“point of no return” for apoptosis or necrosis. It has been 
shown that melatonin targets mitochondrial Ca

2+

-mediated 

MPTP for protection during mitochondrial Ca

2+

 medi-

ated apoptosis in astrocytes (Jou 

2011

). With the applica-

tion of fluorescence laser scanning imaging microscopy, it 
was demonstrated that melatonin does not inhibit the MPT 
pore, rather it preserves the pore in its protective mode of 
transient MPT during mitochondrial Ca

2+

 stress. In addi-

tion, the melatonin-preserved transient MPT allowed mito-
chondria to release the toxic overloaded Ca

2+

 to sublethal 

levels, thus preventing Ca

2+

-mediated fission of mitochon-

dria, Ca

2+

-dependent prolonged MPT and possibly improv-

ing Ca

2+

-dependent ATP synthesis through activation of 

mitochondrial dehydrogenases. This unique melatonin-
dependent modulation of MPTP has been suggested to have 
important therapeutic potential in the treatment of mito-
chondrial Ca

2+

-mediated astrocyte-associated neurodegen-

erative disorders (Jou 

2011

).

Melatonin and mitochondrial dysfunction in heart 
ischemia/reperfusion

Ischemia/reperfusion (I/R) leads to myocardial dysfunction 
and irreversible damage characterized by cardiomyocyte 
hypercontracture, reduction of left ventricular pressure and 
elevated incidence of ventricular fibrillation. Mitochon-
dria are known to be involved in the processes that lead to 
cell death following I/R and are therefore potential target 
for protective intervention (Camara et al. 

2011

). ROS are 

recognized as an important factor in producing lethal cell 
injury associated with cardiac I/R (Chen and Zweier 

2014

). 

Mitochondria isolated from I/R rat heart exhibit altered 
bioenergetic function, associated with CL abnormalities. 
In fact, results obtained in our laboratory have shown that 
mitochondria isolated from I/R rat heart exhibit decreased 
rate of mitochondrial oxygen consumption, reduced activ-
ity of C I and CIII and increased basal rate of H

2

O

2

 pro-

duction (Petrosillo et al. 

2003a

; Paradies et al. 

2004

). These 

changes in the mitochondrial bioenergetic parameters were 
associated with an oxidation/depletion of CL. The defect in 
the respiratory chain complexes activity observed in mito-
chondria isolated from I/R rat heart has been ascribed, at 
least in part, to ROS-induced oxidation of CL, a phospho-
lipid required for the optimal activity of these enzymatic 
complexes. Melatonin treatment had strong protective 
effect against I/R-associated mitochondrial bioenergetic 
alterations (Petrosillo et al. 

2006b

). In fact, melatonin 

administration to I/R rat heart counteracted the reduction in 
C I and C III activity and the associated decrease in state 
3 respiration in isolated rat heart mitochondria as well as 
CL alterations. Similarly, melatonin prevented alterations 
to C I and C III as well as to CL in in vitro experiments 
on isolated rat heart mitochondria subjected to oxidative 
stress conditions. The melatonin’s protective effect against 
I/R-induced mitochondrial dysfunction could be ascribed 
to its ability to preserve CL integrity and/or to directly 
improve the activity of the respiratory chain complexes. 
This effect was associated with an improvement of post-
ischemic hemodynamic function of the heart. These results 
emphasize that melatonin-induced mitochondrial adaptive 
changes are likely of great value for the cardioprotective 
actions of the indoleamine (Dominguez-Rodriguez et al. 

2012

; Yang et al. 

2014

).

A large body of experimental evidence supports a cru-

cial role of MPTP in cardiomyocyte cell death occurring 
with I/R (Halestrap 

2009

; Ong et al. 

2015

). It has been 

suggested that MPTP remains closed during the ischemic 
period, because of the low pH due to lactate accumula-
tion. At reperfusion, there is an uptake of Ca

2+

 by mito-

chondria, a burst of ROS production and rapid correction 
of acidosis, all events that contribute to increase the like-
lihood of MPTP opening. Pharmacological intervention 
aimed to protect the heart from the damaging effect of 
MPTP opening are of considerably importance in attenu-
ating mitochondrial dysfunction associated with I/R injury. 
Recent studies have shown that melatonin and several of its 
metabolites have significant protective actions against car-
diac damage and altered physiology during I/R contributing 
to the rehabilitation of the heart contractile function dur-
ing reperfusion (Sahna et al. 

2005

; Petrosillo et al. 

2006b

Paradies et al. 

2010a

b

; Dominguez-Rodriguez et al. 

2012

Lochner et al. 

2013

). Other studies demonstrated that mel-

atonin plays a role in the mitochondrial adaptive changes 
and that cytochrome c is a significant mediator of this 
process (Giacomo and Antonio 

2007

). Very recently, we 

have demonstrated that melatonin protects against mito-
chondrial dysfunction associated with heart I/R injury by 
inhibiting MPTP opening (Petrosillo et al. 

2009a

). Mela-

tonin treatment significantly improves the functional 
recovery of Langendorff hearts on reperfusion, reduces 

Arch Toxicol 

1 3

the infarct size and decreases necrotic damage, as shown 
by the reduced release of lactate dehydrogenase. All these 
effects were accompanied by the inhibition of the MPTP 
opening as detected in situ by the mitochondrial release of 
NAD

+

. Furthermore, melatonin desensitizes mitochondria 

isolated from melatonin-reperfused heart to Ca

2+

-induced 

MPTP opening, as assessed by the CRC (calcium reten-
tion capacity), a sensitive and quantitative measure of the 
ability of mitochondria to open the MPTP in response to of 
Ca

2+

 uptake. Together, these results demonstrate that mela-

tonin protects against heart I/R injury by inhibiting MPTP 
opening, thus improving the post-ischemic hemodynamic 
function of the heart. The possible mechanism underlying 
the inhibition of MPTP opening during I/R by melatonin 
treatment was also investigated (Petrosillo et al. 

2009a

). It 

is now accepted that, in addition to Ca

2+

 overload, other 

factors may contribute to the MPTP opening during heart 
I/R. As described above, oxidized CL, together with Ca

2+

promotes the induction of MPTP in isolated rat heart mito-
chondria, suggesting that an increased level of oxidized 
CL may increase the probability of MPTP opening during 
I/R. We found an increased level of oxidized CL in mito-
chondria isolated from rat heart subjected to I/R, which 
was prevented by melatonin treatment. Thus, it is plausi-
ble that CL oxidation, together with Ca

2+

 overload, syner-

gistically contribute to MPTP opening during I/R and that 
melatonin treatment inhibits MPTP opening by preserv-
ing CL integrity by ROS attack. Melatonin was reported 
to inhibit linoleate peroxidation in a model system in vitro 
(Mekhloufi et al. 

2005

). The inhibitory effect of melatonin 

on mitochondrial CL peroxidation during reperfusion can 
be reasonably explained on the ability of this indoleamine 
to inhibit the oxidation of linoleic fatty acid which is the 
main constituent of heart CL.

It has been shown that CL binds cytochrome c to the 

outer surface of the IMM (Rytomaa et al. 

1992

). Oxida-

tion of CL results in the detachment of this hemoprotein 
from mitochondrial membrane and this event is considered 
an important initial step in the cytochrome c release from 
mitochondria (Petrosillo et al. 

2001

; Ott et al. 

2007a

b

). 

Our results have shown that, in addition to inhibit MPTP 
opening, melatonin prevents also the release of cytochrome 
c from mitochondria upon I/R. This effect of melatonin can 
be explained on the ability of this indoleamine to inhibit CL 
oxidation, thus preventing cytochrome c detachment from 
IMM. The inhibitory effect of melatonin on cytochrome c 
release and MPTP opening may contribute to the protective 
effect exerted by this indoleamine against mitochondrial 
dysfunction associated with I/R. These beneficial effects 
of melatonin reinforce the therapeutic potential of this 
compound to combat a variety of oxidative stress-induced 
oxidative dysfunctions as well as mitochondrial-mediated 
apoptosis in various cardiovascular disorders.

Melatonin and mitochondrial dysfunction in aging

Aging is a multifactorial process, which is genetically 
determined and influenced epigenetically by environment 
(Kirkwood 

2005

). This biological process is characterized 

by impairment of bioenergetic functions, increased oxi-
dative stress and increased risk of contracting age-associ-
ated diseases. Mitochondria are intimately involved in the 
aging process because these organelles are recognized as 
the main intracellular source of ROS as well as the major 
target of their oxidative attack. According to the mitochon-
drial theory of aging, ROS produced by the mitochondrial 
respiratory chain, attack mitochondrial constituents includ-
ing proteins, lipids and mitochondrial DNA (mtDNA; Har-
man 

1972

; Miquel et al. 

1980

; Pak et al. 

2003

). As mtDNA 

encodes essential components of OXPHOS and protein 
synthesis machinery, accumulation of mtDNA mutations 
may lead to impairment of either the assembly or the func-
tion of the respiratory chain, leading to further ROS gen-
eration and subsequent accumulation of more mtDNA 
mutations. This triggers a vicious cycle which leads to the 
progressive decline of mitochondrial and cellular bioener-
getics functions as results of insufficient supply of energy 
and/or increased susceptibility to cell death (Judge and 
Leeuwenburgh 

2007

; Paradies et al. 

2010b

). Although there 

is a large consensus on the mitochondrial free radical the-
ory of aging (Harman 

1972

), recent findings, obtained in 

particular in Caenorhabditis elegans and in rodents, suggest 
that ROS generation may not be the main factor involved in 
the aging process (Hekimi et al. 

2011

).

A number of studies have shown a decreased electron 

transport activity in mitochondria isolated from rat and 
mouse tissues upon aging (Navarro and Boveris 

2007

Judge and Leeuwenburgh 

2007

Paradies et al. 

2010b

2011

). Of the five respiratory chain complexes, C I and C 

IV show a selectively reduced enzymatic activity in mito-
chondria isolated from various tissues of rats and mice 
upon aging (Lenaz et al. 

1997

; Navarro and Boveris 

2007

Petrosillo et al. 

2008b

2009b

). One possible factor respon-

sible for the age-related impairment of C I and C IV activ-
ity might be the oxidation/depletion of mitochondrial CL as 
supported by the following experimental observations. The 
content of normal CL declines, while the level of oxidized 
CL increases with aging (Petrosillo et al. 

2008b

2009b

). 

Cardiolipin molecules are specifically bound to C I and 
C IV of the respiratory chain and required for their func-
tional activity (Lange et al. 

2001

; Musatov and Robinson 

2012

). In addition, mitochondrial-mediated ROS genera-

tion affects C I and C IV activity through CL peroxidation 
in beef heart submitochondrial particles (Paradies et al. 

2000

2002

). These results suggest that the age-associated 

defects in mitochondrial C I and C IV activities could be 
ascribed, at least in part, to ROS-induced oxidative damage 

 

Arch Toxicol

1 3

to mitochondrial CL. Complex I is considered a rate-limit-
ing factor in the mitochondrial respiratory chain and also 
the main source of ROS during the aging process. Thus, the 
impairment of mitochondrial C I activity, in addition to that 
of C IV, may increase the electron leak from the ETC, gen-
erating more superoxide radical, triggering a cycle of oxi-
dative damage that ultimately leads to mitochondrial bio-
energetic decay in aging (Judge and Leeuwenburgh 

2007

Paradies et al. 

2010b

2011

). The age-associated impair-

ment of mitochondrial C I e C IV activity observed in heart 
and brain tissues may have important implications in the 
etiopathology of age-associated cardiovascular and neuro-
degenerative disorders and may represent an important tar-
get for the development of potential therapeutic strategies 
(Beal 

2005

; Boveris and Navarro 

2008

).

Growing evidence indicate that the individual compo-

nents of the mitochondrial ETC may exist as large macro-
molecular assemblies, or so-called supercomplexes (Zhang 
et al. 

2002

; Schagger 

2002

; Genova and Lenaz 

2014

). A 

general role for CL in respiratory supercomplexes forma-
tion and stability in mammalian mitochondria has been 
suggested (Zhang et al. 

2002

; Bazán et al. 

2013

). Recently

age-associated destabilization of rat heart mitochondrial 
supercomplexes has been reported (Gómez and Hagen 

2012

). Due to the oxidation/depletion of CL with aging, it 

is possible that abnormalities in CL might be involved in 
the destabilization and dysfunction of mitochondrial respir-
atory supercomplexes, thus contributing to mitochondrial 
bioenergetics decay with aging.

Several properties of melatonin indicate that this com-

pound may have beneficial effects in aging. Serum levels of 
melatonin significantly decrease in aged animals compared 
with young animals (Reiter et al. 

1980

1981

). In humans, 

the total antioxidative capacity of serum correlates well 
with its melatonin levels. Thus, the decreased level of mel-
atonin in aging has been associated with the increased oxi-
dative damage observed in the elderly. The mechanism of 
aging process can be studied in experimental animal mod-
els like the senescent accelerated mouse (SAM; Takeda 

1999

). Recently, using SAM mice, the effects of chronic 

administration of physiological doses of melatonin on 
mitochondrial oxidative stress and mitochondrial function 
in heart tissue were investigated (Rodríguez et al. 

2007

). 

Mitochondrial oxidative stress was determined by measur-
ing the levels of lipid peroxidation, GSH and GSSG and the 
activities of GPx and GRd. The activities of several mito-
chondrial bioenergetic parameters including ETC com-
plexes and ATP levels were also evaluated. The results of 
these studies showed an age-dependent mitochondrial oxi-
dative damage in the heart tissue of SAM mice, which was 
associated with a reduction in the ETC complexes activity 
and in ATP levels. Chronic melatonin administration nor-
malized these age-associated alterations in mitochondrial 

bioenergetic parameters and increased ATP level. Moreo-
ver, melatonin treatment had beneficial effect on longev-
ity of SAM mice (Rodríguez et al. 

2008

). Together, these 

studies indicate that melatonin treatment counteracts age-
related oxidative damage and mitochondrial dysfunction in 
heart tissue of SAM mice by improving mitochondrial bio-
energetic function as reflected by increase in ATP produc-
tion and prolonged longevity.

Experimental evidence indicates that mitochondrial 

decay is a major contributor to brain tissue alterations asso-
ciated with aging (Navarro and Boveris 

2007

). Aged mam-

malian brain has a decreased capacity to produce ATP by 
OXPHOS and it is considered that this decreased capacity 
for energy production becomes limiting under physiologi-
cal conditions in aged individuals. The current knowledge 
indicates that the impairment of brain mitochondrial func-
tion in aging is mainly due to decreased electron transfer 
rates by C I and C IV, among other decreased mitochon-
drial activities (Navarro and Boveris 

2007

; Paradies et al. 

2011

). Impaired mitochondrial respiration with NAD-

dependent substrates has been consistently observed in 
brain mitochondria isolated from aged rats and mice (Nav-
arro and Boveris 

2007

; Petrosillo et al. 

2008a

). Given the 

brain’s high energy requirements, the impairment in brain 
ETC complexes activity could have a significant impact on 
brain function in aging and on the etiology of age-associ-
ated neurodegenerative disorders (Beal 

2005

).

A potential role of melatonin in mitigation of mitochon-

drial decay in brain aging has been described (Bondy et al. 

2004

; Bondy and Sharman 

2007

). Moreover, melatonin 

has been identified as a potential mitochondria-targeted 
protector against several oxidative stress-associated brain 
disorders (Caballero et al. 

2008

). Results obtained in our 

laboratory have shown that brain aging is associated with 
an impairment in C I and C IV activity, decrease in oxygen 
consumption and membrane potential and with an increase 
in mitochondrial ROS production (Petrosillo et al. 

2008a

b

Paradies et al. 

2011

). These age-related mitochondrial bio-

energetic alterations were largely attenuated by melatonin 
administration. Melatonin administration did not affect 
these bioenergetic parameters when administered to young 
rats, suggesting that the observed protective effects of this 
indoleamine are related to changes produced by aging. The 
melatonin’s ability to prevent the age-related alterations of 
mitochondrial bioenergetic parameters in rat brain could be 
ascribed, at least in part, to its protective effect against CL 
peroxidation (Paradies et al. 

2011

) as also supported by in 

vitro experiments on isolated rat brain mitochondria (Pet-
rosillo et al. 

2008a

).

Other studies carried out in brain mitochondria iso-

lated from male and female SAM mice indicated that there 
was significant age-dependent mitochondrial dysfunction 
with a reduced efficiency of the ETC and diminished ATP