Regulation of antioxidant enzymes: a significant role for melatonin
Aerobic organisms require ground state oxygen to live.
However, the use of oxygen during normal metabolism
produces reactive oxygen species (ROS), some of which are
highly toxic and deleterious to cells and tissues. The most
abundant ROS formed in the course of cellular metabolism
is the superoxide radical (O
). This radical is mainly
produced during electron transport in the mitochondria
and in the endoplasmic reticulum, although it is also a
byproduct in several enzymatic reactions (oxidases and
oxygenases); likewise, it is formed during the hepatic
metabolism of some molecules and also as a result of the
decomposition of oxyhemoglobin .
Dismutation of the O
gives rise to hydrogen peroxide
). This molecule is not a free radical per se but, in the
presence of transition metals via the Fenton reaction, it is
rapidly converted to the hydroxyl radical (
widely accepted as being the most damaging ROS produced
by cells . Free radicals in general and the
particular react with virtually every molecule in living cells
(i.e. lipids, sugars, amino acids, nucleotides)with very high
rate constants ; the resulting damage ultimately may lead
to diseases such as cancer, neurodegeneration and autoim-
mune conditions [4–6].
To protect cells from the damage caused by free radicals
and related reactants, organisms have evolved several
defense mechanisms to rapidly and eﬃciently remove
ROS from the intracellular environment. When the equi-
librium between free radicals (oxidants)and antioxidant
defense systems is imbalanced in favor of oxidants, the
condition causes what is known as oxidative stress. The
oxidants that are not directly scavenged or otherwise not
metabolized attack cellular components producing useless
molecular debris and sometimes cell death.
Antioxidant defense systems may be generally classiﬁed
into indirect enzymatic antioxidant enzymes and into small
molecular weight molecules which directly scavenge free
radicals and related reactants. The antioxidant enzymes
represent a ﬁrst line of defense against these toxic reactants
by metabolizing them to innocuous byproducts.
The ﬁrst enzymatic reaction in the reduction pathway of
oxygen occurs during the dismutation of two molecules of
when they are converted to hydrogen peroxide (H
and diatomic oxygen. The enzyme at this step is one of two
isoforms of superoxide dismutase (SOD); CuZnSOD is
present in the cytosol while (MnSOD)is located in the
mitochondrial matrix. These enzymes possess transition
, respectively)at their active sites;
this allows for the rapid exchange of electrons between the
Abstract: Antioxidant enzymes form the ﬁrst line of defense against free
radicals in organisms. Their regulation depends mainly on the oxidant status
of the cell, given that oxidants are their principal modulators. However,
other factors have been reported to increase antioxidant enzyme activity and/
or gene expression. During the last decade, the antioxidant melatonin has
been shown to possess genomic actions, regulating the expression of several
genes. Melatonin also inﬂuences both antioxidant enzyme activity and
cellular mRNA levels for these enzymes. In the present report, we review the
studies which document the inﬂuence of melatonin on the activity and
expression of the antioxidative enzymes glutathione peroxidase, superoxide
dismutases and catalase both under physiological and under conditions of
elevated oxidative stress. We also analyze the possible mechanisms by which
melatonin regulates these enzymes.
Juan C. Mayo
, Rosa M. Sainz
, Federico Herrera
and Russel J. Reiter
Departamento de Morfologı´a y Biologı´a
Instituto Universitario de
Oncologı´a del Principado de Asturias (IUOPA)
Facultad de Medicina, C/ Julian Claveria,
Department of Cellular and
Structural Biology, University of Texas Health
Science Center, San Antonio, TX, USA
Key words: antioxidant enzyme activity,
antioxidant enzyme gene expression,
antioxidant enzymes, melatonin, regulation
Address reprint requests to Carmen
Rodriguez, Departamento de Morfologı´a y
Biologı´a Celular, Facultad de Medicina,
c/Julian Claveria, 33006 Oviedo, Spain.
Received July 28, 2003;
accepted September 4, 2003.
J. Pineal Res. 2004; 36:1–9
Blackwell Munksgaard, 2004
Journal of Pineal Research
two superoxides. Although H
is not a radical itself, it is
reactive and it is rapidly converted into the highly reactive
OH in the presence of ferrous ion (Fe
)via the Fenton
reaction unless it is eﬃciently removed. Two enzymes
participate in the removal of H
from the cellular
environment, peroxidases and catalase. The most abundant
peroxidase is the glutathione peroxidase (GSH-Px), which
is present in both the cytosol and mitochondria. This
enzyme has the transition metal selenium at its active site
and uses reduced glutathione (GSH)as a substrate to
transfer electrons to H
(and other peroxides)thereby
converting it into two molecules of water. The second H
metabolizing enzyme is catalase (CAT); it is present mainly
in the peroxisomes, presents a molecule of ferric ion at its
active site and converts two molecules of H
molecule each of water and diatomic oxygen .
Antioxidant enzymes are regulated by multiple factors.
Oxidative status of the cell is the primary factor regulating
gene expression and activity of these enzymes [8–10]. Both
endogenous  and exogenous agents [12, 13] act as
oxidants and alter cellular oxidative equilibrium and
therefore antioxidant enzyme gene expression. There are,
however, several other factors which inﬂuence antioxidant
enzymes. In addition to developmental changes, diﬀerenti-
ation and aging inﬂuences [14–18], inﬂammation [19, 20]
and hormonal regulation of antioxidative enzymes have
been reported [21–23]. Additionally, several antioxidants
and cell protectors are believed to regulate gene expression
and antioxidant enzyme activity [24–29].
Although, melatonin is known to be an indole secreted
by the pineal gland, other organs may produce melatonin
where it has functions without being released. Besides its
properties as a circadian rhythm transducer , several
other actions for this interesting molecule have been in
uncovered in the last two decades [31, 32]. Its direct free
radical scavenging activity [33, 34] and its regulation of
gene transcription  for antioxidative enzymes are of
special interest in the present review. The antioxidant
properties of melatonin have been extensively studied and
the use of this molecule as a cell protector and as a potential
disease-preventing agent have been summarized [36–40].
Melatonin has been proven to be an eﬃcient oxidant
Hypothetical pathways involved in melatonin regulation of antioxidant enzyme gene expression and activity. (1)Melatonin acti-
vation of MT1/2 receptors, vı´a G inhibitory protein (Gi), inhibits adenylate cyclase and reduces cyclic AMP (cAMP). This results in
inhibition of protein kinase A (PKA)and cAMP response element binding protein/activation transcriptor factor (CREB-ATF). This
pathway could modulate immediate early gene (IEG)transcription and consequently gene transcription regulation and antioxidant enzyme
concentration. (2)MT1/2 binding by melatonin activates the phospholipase C pathway. The consequent increase in Ca
phosphorylate protein-kinase C (PKC)which activates CREB/ATF thereby increasing the transcription of IEG. Indeed, PKC activates
IEG. PKC activation may also activate NF kappa B (NFjB)and other transcription factors (TF). Melatonin may also, in other systems,
induce a Ca
decrease leading to inhibition of PKC. (3)MT1/2 activation may, through both inhibitory G (Gi)and other G proteins,
activate several mitogen activated protein kinases, i.e., extracellular regulated kinase (ERK)and Jun N-terminal kinase (JNK), which
regulate IEG activation and thereby gene transcription. (4)Melatonin may inhibit calcium-calmodulin (Ca-CaM)complex by direct binding
A lowered Ca
concentration mediated by MT1/2 receptors has been reported in some models. This would inhibit calmodulin-kinase
(CaMK), which in turn may regulate NFjB, the retinoid-related receptor (ROR)and other transcription factor activation, thereby
inﬂuencing gene transcription. Ca
-CaM inhibition may also regulate PKC. (5)Melatonin is a free radical scavenger. Although this eﬀect
is not receptor-mediated, its possible involvement in the regulation of antioxidant enzymes should not be ruled out. Changes in the cellular
redox state towards a more reduced environment produces protein reduction which may lead to enzyme activation (a). Also this envi-
ronment may induce translational changes which would increase enzyme concentrations (b). Finally, a decrease of free radicals would allow
repression of redox-sensitive transcription factors (i.e. NFjB, AP-1)which would regulate gene transcription (c). Continuous lines indicate
previously reported melatonin actions. Dashed lines indicate general cellular mechanisms previously known but not probed with melatonin.
*These eﬀects of melatonin have not been documented.
Rodriguez et al.
scavenger of a variety of radical and non-radical reactants
[37, 41]. Control of gene expression by melatonin was
initially suggested by Menendez-Pelaez et al. [42, 43].
Thereafter, the regulation of expression of several genes
related to antioxidative enzymes was reported [24, 44–58].
Herein, the literature related to the regulation of enzyme
activity and gene expression of antioxidant enzymes by
melatonin is reviewed.
Regulation of antioxidant enzymes
Regulation under basal oxidative stress
Reports documenting the inﬂuence of melatonin on anti-
oxidant enzyme activity were ﬁrst published in the mid-
1990s [59, 60]. These papers described the ampliﬁcation of
GSH-Px activity in the brain of rat and in several tissues
(500 lg/kg)[36, 59, 60]. Thereafter, several groups showed
that melatonin increases the activity of antioxidant enzymes
in other tissues and models. Thus, Ozturk et al.  found
increased SOD activity in rat liver after administration of
10 mg/kg of melatonin for 7 days, while Liu and Ng 
reported enhancement of SOD activity in rat kidney, liver
and brain after a single melatonin injection (5 mg/kg).
rhythms under normal light:dark conditions. This is true
both in terms of their activity and gene expression. These
changes with time suggested that these cycles might be
dependent on the circadian melatonin rhythm [63–65].
Abolition of endogenous melatonin cycle by exposure of
animals to constant light, in fact, also abolished the night-
time rise in antioxidative enzyme activity. This illustrates
that changes in physiological levels of melatonin are
adequate to alter the antioxidative defense system as
reﬂected in the level of activities of antioxidative enzymes.
Continuous exposure to light is known to abolish the
nocturnal melatonin rise; this was associated with a
reduction in the night-time increase in GSH-Px and SOD
activities in several tissues of chicks [64, 66]. These results
were subsequently conﬁrmed by others in rodents [67, 68].
Similarly, Baydas et al.  reported that melatonin
deﬁciency caused by pinealectomy reduced GSH-Px activity
levels in several tissues of rats.
Melatonin administration during pregnancy has also
been shown to stimulate antioxidant enzyme activity in the
fetuses. Okatani et al. [70, 71] have reported this ﬁnding in
both rats  and humans . They initially showed that
relatively high doses of melatonin (10 mg/kg), administered
to pregnant rats, caused incremental changes in the
concentration of the indole in both maternal serum and
fetal brain as early as 1 hr after its administration.
Concomitantly, GSH-Px and SOD activities were likewise
increased in fetal brain. This indicates that melatonin may
be potentially beneﬁcial in the treatment of stressful
conditions that involve free radical production such as
fetal hypoxia and preeclampsia. Subsequently, they admin-
istered much lower doses of melatonin (100 lg/kg bw)
to pregnant women before they underwent voluntary
interruption of pregnancy and they found an increase in
GSH-Px activity in chorionic homogenates with a peak 3 hr
after indole administration. This again supports the idea
that melatonin may have potential usefulness as a fetal
protector under conditions of elevated oxidative stress.
Melatonin has also been shown to inﬂuence antioxidant
enzyme gene expression. As ﬁrst reported by Antolin et al.
, melatonin causes incremental changes in mRNA levels
for both CuZnSOD and MnSOD in the Harderian gland of
female Syrian hamsters after its exogenous administration
(500 lg/kg). Increases in antioxidant enzyme gene expres-
sion following melatonin injections (50 and 500 lg/kg)were
later conﬁrmed by the same group  in rat brain cortex.
Finally, Mayo et al.  showed that mRNA levels for
antioxidant enzymes were elevated in non-diﬀerentiated
PC12 cells and the human neuroblastoma cells SK-N-SH
after melatonin was added to the medium in which the cells
were grown. These workers reported that the increases in
CuZnSOD and gene HnSOD expression were maximal at
24 and 6 hr, respectively, following melatonin administra-
tion. This eﬀect was induced with a melatonin concentra-
tion of 10
, the physiological levels of this indole in
night-time serum; conversely, no eﬀect was observed when
higher doses of the indole were used. Regulation of
antioxidant enzyme gene expression by melatonin is
dependent on new protein synthesis, as use of an inhibitor
of protein synthesis, i.e., cycloheximide, prevents mRNA
increases after melatonin administration. The indole also
reduced the half life of CuZnSOD and GSH-Px while it did
not aﬀect that of MnSOD indicating that a larger amount
of less stable mRNA may be generated for GSH-Px and
CuZnSOD. Finally, the presence of melatonin in the culture
medium for 1 hr only is suﬃcient to increase mRNA for
antioxidant enzymes 24 hr later, indicating a possible role
for melatonin receptors in the regulation of antioxidant
enzymes by this indole.
Regulation under elevated oxidative stress
When cells are exposed to oxidative stress they increase the
activity and expression of antioxidant enzymes as a
compensatory mechanism to better protect them from the
damage induced by free radicals. In many cases the number
of free radicals generated may be so great that even the
increased activity of the antioxidative enzymes are insuﬃ-
cient to counteract the potential damage. When antioxidant
enzyme activities and/or gene expression were examined
under highly elevated oxidative stress conditions, it was
found that they are sometimes diminished; thus, it has been
proposed that moderate levels of toxic reactants induce
rises in antioxidant enzymes while very high levels of
reactants reduce enzyme activities as a result of damage of
the molecular machinery that is required to induce these
enzymes [18, 73]. Melatonin has a lengthy history of
beneﬁcial actions. For example, almost two decades ago it
was reported as a protector against glucocorticoid damage
[74, 75], against some degenerative neurological conditions
, as an anticancer agent [31, 77–79], and also as an
enhancer of immune function [32, 79]. Subsequently, the
multiple antioxidant properties of melatonin were described
Melatonin regulation of antioxidant enzymes
[33, 34, 80, 81] and research on its protective eﬀects against
oxidative processes have now been identiﬁed under a very
wide range of conditions in both experimental animals [82–
84] and humans [85, 86]. Some of the earliest studies
documented the antioxidant properties of melatonin in the
central nervous system , in the prevention of cataract
formation , and in the reduction in the severity of colitis
. At roughly the same time, Pablos et al.  described
the regulation of antioxidant enzyme activities by melato-
nin; this was quickly followed by studies conﬁrming the
original ﬁndings and extending the observations of the
inﬂuence of melatonin on gene expression for antioxidative
Antioxidant enzyme regulation by melatonin has been
shown to occur concomitant with its protection against
elevated oxidative stress in numerous experimental situa-
tions. In the ﬁrst report to document this correlation it
was shown that melatonin increased GSH-Px activity and
simultaneously reduced free radical damage to the brain
and liver of rats treated with lipopolysaccharide (LPS)
. In this study, LPS increased total glutathione (tGSH)
levels as well as oxidized glutathione (GSSG)concentra-
tions while reducing the activity of GSH-Px. Melatonin
(4 mg/kg)given to LPS-treated rats enhanced tGSH above
basal levels and lowered GSSG concentrations while
stimulating the activity of GSH-Px. This indicated that
melatonin may act on several points in the antioxidant
defense system, not exclusively on GSH-Px. Subsequently,
Antolin et al.  reported rises in both CuZn and
MnSOD gene expression in the Harderian gland after
melatonin (500 lg/kg)was administered to female ham-
sters. The female hamster Harderian gland is in continual
jeopardy of experiencing oxidative stress which causes cell
damage because of the extremely high content of porphy-
rins in this organ. The administration of melatonin
lowered porphyrin synthesis and cell damage in this
extraorbital tissue and increased gene expression for both
isoforms of SOD. In a number of subsequent studies, the
activities of both GSH-Px and the SOD were repeatedly
shown to be regulated by melatonin with these changes
being concurrent with the ability of the indole to reduce
Multiple reports on neural protection by melatonin via
its antioxidant properties have appeared subsequent to the
initial reports of this action [81, 90, 91]. In several
experiments, antioxidant enzyme activity as well as expres-
sion was studied. Mayo et al.  found that in an
experimental model of Parkinson disease in which dop-
aminergic PC12 cells were treated with the neurotoxin
6-hydroxydopamine (6-OHDA), low doses of melatonin
)provided protection against apoptotic death
induced by the neurotoxin. In this study, melatonin also
prevented the reduction in gene expression for three
antioxidant enzymes, GSH-Px, CuZnSOD and MnSOD,
which followed 6-OHDA treatments. In vivo experiments
have provided results consistent with the in vitro ﬁndings.
When rodents (rats and mice)were treated with either beta-
amyloid peptide 25–35  or with d-galactose  both of
which cause oxidative damage to the brain, melatonin at
doses ranging from 0.1 to 10 mg/kg restored both SOD and
GSH-Px activities. Naidu et al.  reported reversal of
haloperidol-induced decreases in brain SOD and catalase
activities by 1–5 mg/kg melatonin. Melatonin (10 mg/kg or
2 lg/mL in drinking water, respectively)also has been
shown to be protective against oxidative stress in both fetal
 and aging brain of rodents , with these beneﬁcial
eﬀects being associated with increased GSH-Px activity.
In addition to the brain, antioxidant enzyme activity
regulation by melatonin has been shown to be involved in
the protection against oxidative damage in other tissues.
Restoration or even augmentation of antioxidant enzyme
activity by melatonin has been shown to be associated with
prevention of free radical damage induced by several toxins
[97–99]. For example, intestinal and gastric damage follow-
ing ischemia-reperfusion or drug administration [100–103],
multiple organ damage resulting from therapeutic and non-
therapeutic chemotherapeutic agents [104–110], ultraviolet
damage to tissues , free radical damage in experimental
diabetes [112, 113], as well as chemio- and radiotherapy
lesions [114, 115] are reduced by melatonin. Finally, it has
been recently shown that melatonin may retard aging of the
senescence-accelerated mouse with this being associated
with augmented antioxidant enzyme activity .
Intracellular pathways involved
in antioxidant enzyme regulation
Mayo et al.  provided an insight into the mechanisms by
which melatonin regulates antioxidant enzyme gene expres-
sion using cultured dopaminergic cells. They found that
melatonin induced synthesis of new protein as a condition
for regulation of gene expression of all the three antioxi-
dative enzymes, CuZnSOD, MnSOD and GSH-Px. Mela-
tonin also diminished the half-life of mRNAs coding for
both CuZnSOD and GSH-Px, without altering that of
MnSOD in this study. This indicates that, in the case of the
two former enzymes, melatonin in the medium probably
induced more abundant levels of mRNAs with shorter half-
lives. Finally, nanomolar concentrations of melatonin were
adequate to induce antioxidant gene expression with a 1-hr
exposure to melatonin being adequate to sustain elevated
mRNA levels 24 hr later. As noted above, this points to the
likelihood of receptors being involved in antioxidant
enzyme gene expression.
The mechanisms involved in the regulation of antioxid-
ant enzymes by melatonin in vivo have not been precisely
determined. It is known, however, that stimulation of
antioxidant enzyme gene expression occurs at nanomolar
concentrations of melatonin in cultured cells ; these
melatonin levels are equivalent to the serum concentration
of melatonin at its nocturnal peak in vivo. The quantities of
melatonin used in most of the in vivo experiments, however,
very likely caused circulating levels to exceed physiological
concentrations. Thus, melatonin in these studies may have
functioned as a direct radical scavenger thereby changing
the redox state of cells, which in turn may have altered the
speciﬁc activity of these enzymes or their level of translation
. Only twice, as far as could be determined, has gene
expression for antioxidative enzymes under the inﬂuence of
melatonin been analyzed in in vivo experiments [24, 52]
and, surprisingly, changes in enzyme activities after
Rodriguez et al.
melatonin treatment has not been examined in cell culture
Kotler et al.  found that after chronic administration
of melatonin (50 and 500 lg/kg)to rats, the lower dose
clearly had a greater stimulatory eﬀect on antioxidant
enzyme gene expression than did the 500 lg/kg dose.
Antolin et al.  reported melatonin protection against
in vivo neurotoxicity of MPTP using 500 lg/kg melatonin
(the presumed equivalent melatonin used to induce nano-
molar concentrations in serum may be roughly 25–
50 lg/kg). The work of Barlow-Walden et al.  using
500 lg/kg and Kotler et al.  using 50 and 500 lg/kg,
indicate that antioxidant enzyme activity and expression,
respectively, are elevated after the administration of mela-
What intracellular molecular pathways are involved in
the regulation of antioxidant enzyme gene expression and/
or activity by melatonin is presently unknown (Fig. 1). A
membrane G-protein-coupled melatonin receptor MT1 was
cloned and characterized by Ebisawa et al. . Subse-
quently, MT2 and Mel 1c receptors have also been
identiﬁed, the former mainly diﬀering from MT1 in terms
of the tissues in which it is expressed, while Mel 1c is not
found in mammals . Melatonin also has been tenta-
tively shown to activate a nuclear orphan receptor belong-
ing to the retinoid Z receptor b and a (RZR b and a)family.
Melatonin acts on RORa receptor repressing the expression
of the 5-lipoxygenase gene  and inhibiting growth of the
breast cancer MCF-7 cells . The results from Mayo
et al.  suggest that melatonin regulation of antioxidant
enzymes is receptor-mediated, thereby most likely implica-
ting the MT1/MT2 receptors via second messengers such as
cAMP, phospholipase C or intracellular calcium concen-
tration. In addition, binding of melatonin to membrane
receptors could stimulate MAP kinase cascades thereby
activating several transcription factors . The possibility
exists that RZR/ROR receptors could also mediate mela-
tonin eﬀects on antioxidative enzymes as suggested by the
results of Pablos et al. ; if so, the pathways involved in
their regulation obviously remain unknown. One possibility
may relate to MT1/MT2 melatonin binding that, through
second messengers and phosphorylation cascades, activates
RZR/ROR as reported by Ram et al. . Another
possibility by which melatonin may regulate RZR/ROR
receptors would be via modulation of the calcium/calmod-
ulin signaling pathway, either by changing intracellular
calcium concentrations by binding to MT1/MT2 receptors
, or by direct binding to calmodulin . The calcium/
calmodulin signaling pathway has been reported to regulate
transcriptional activity of RZR/ROR receptors via CaM
Antioxidant enzymes are known to be regulated by
several factors which induce oxidative stress [12, 13, 19,
126]; these factors presumably activate oxidative stress-
sensitive transcription factors. Also, transcriptional acti-
vation of antioxidant enzyme genes has been reported after
the treatment of cells with protective agents  where
non-oxidative stress-dependent transcription factors are
involved. Melatonin has been shown to regulate the
activation or repression of several transcription factors
[55, 127–130], all of them present in the promoter region of
the three-antioxidant enzymes reviewed herein. Thus,
subsequent experiments should be undertaken in order to
shed light on the intracellular pathways and transcription
factors involved in the regulation of antioxidant enzyme
gene expression and activity by melatonin.
This work was supported by the CICYT grant no. SAF00-
0010, the FICYT grants FC PB MED 01 12 and FC PC
REC 01 11, and ASTURPHARMA SA (CR). JCM and
VM were supported by FICYT. FH acknowledges a
fellowship of the Institute of Health Carlos III (FIS).
RMS was supported by a Fulbright Grant.
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