PHYSIOLOGY 29: 325–333, 2014; doi:10.1152/physiol.00011.

2014
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Melatonin: Exceeding Expectations Russel J. Reiter,1 Dun Xian Tan,1 and
Annia Galano2
1
Department of Cellular and Structural Biology, UT Health
Melatonin is a small, highly conserved indole with numerous receptor-medi- Science Center, San Antonio, Texas; and 2Departamento de
Quimica, Universidad Autonoma Metropolitana-Iztapalapa,
ated and receptor-independent actions. Receptor-dependent functions include Mexico D.F., Mexico
reiter@uthscsa.edu
circadian rhythm regulation, sleep, and cancer inhibition. The receptor-inde-
pendent actions relate to melatonin’s ability to function in the detoxification of
free radicals, thereby protecting critical molecules from the destructive effects
of oxidative stress under conditions of ischemia/reperfusion injury (stroke,
heart attack), ionizing radiation, and drug toxicity, among others. Melatonin
has numerous applications in physiology and medicine.

When Aaron B. Lerner and his associates (29) iso-
lated and characterized the methoxy derivative of
serotonin from bovine pineal tissue near the mid-
dle of the last century, in their most optimistic
outlook they would never have envisioned the
multiple functions this molecule would eventually
display. As dermatologists, they had engaged in
this effort because, based on a publication that
appeared 50 years earlier (35), they knew there was
something in the pineal gland that blanched cuta-
neous chromatophores and thereby lightened the
skin of amphibians. Indeed, the name they se-
lected for their newly discovered molecule, i.e.,
melatonin, is in part based on its effect on skin
pigmentation (“mela” from melanin and “tonin”
from serotonin).
After melatonin’s discovery in pineal tissue, the
image of the gland quickly changed from that of a
functionless vestige to one of an active organ of
internal secretion. Biochemists identified the steps
in the synthetic pathway of melatonin (4, 41, 67)
(FIGURE 1), and the physiologists described a now
well known function of the pineal, i.e., the regula-
tion of seasonal reproduction in photoperiod-sen-
sitive mammals (22, 24). By the end of what has
been referred to as the Decade of Transformation
(mid-1950s to mid-1960s) (7), the pineal gland and
melatonin were both recognized as legitimate
components of the endocrine system: the pineal
was known to synthesize melatonin (67), the gland
influenced seasonal reproduction in photosensi-
tive species (23, 24), and it required its sympathetic
innervation to remain functional (43, 66).
Research within the last 60 years, however, has
revealed that melatonin is functionally much more
diverse than being only a regulator of the neuroen-
docrine-reproductive axis in photoperiod-depen-
dent, seasonally breeding mammals. Also, the
conflating of melatonin exclusively with the pineal
gland has been shown to be untenable (47).
Herein, we briefly summarize the data document-
ing the heterogeneity of melatonin’s multiple
actions and the evidence that melatonin is pro-
duced in all plants and animals that have been
studied.
Unconventional Aspects of the
Pineal and Melatonin
In vertebrates, the pineal gland synthesizes mela-
tonin in a circadian fashion, with nighttime dark-
ness being a requirement for maximal production
(57); because of this, melatonin is referred to as the
chemical expression of darkness (42). The influ-
ence of the prevailing photoperiod on circadian
melatonin production is accomplished by a re-
stricted bandwidth of visible light, i.e., wavelengths
in the range of 460 – 480 nm (blue light) (FIGURE 1).
These wavelengths account for the inhibition of
melatonin synthesis in the pineal gland during the
day or, if they occur, at night. For the retinal pro-
cessing of blue wavelengths, the mammalian reti-
nas have up to five highly specialized subtypes of
intrinsically photosensitive retinal ganglion cells
(ipRGCs), which use a specialized photopigment,
melanopsin, to respond to light (31, 68). The
message related to the light environment is then
transferred to the central biological clock, the
suprachiasmatic nuclei (SCN), in the hypothala-
mus via the retinohypothalamic tract, which is em-
bedded in the optic nerve. The SCN is a critical
relay center that conveys a neural signal to the
pineal gland (39) (FIGURE 1). The neural message
arrives at the pinealocytes from the SCN via the
central and peripheral sympathetic nervous sys-
tem. This is one of several features that distin-
guishes the pineal from classic endocrine organs,
i.e., its output is governed by a neural input, which,
if destroyed, renders the gland totally inept (43);
likewise, feedback effects from peripherally de-
rived hormonal signals are usually not considered
to be major factors in changing the quantity of
melatonin produced or altering the phasing of its
rhythm (11). Hence, the melatonin rhythm is more

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REVIEWS
or less independent of endogenous influences, and
melatonin synthesis is determined almost exclu-
sively by the prevailing photoperiod, thereby mak-
ing the pineal an unconventional endocrine gland.
There is also nothing phylogenetically that ties
melatonin production to the pineal gland. Mela-
tonin is not unique to vertebrates but is also pro-
duced in invertebrates, unicells, and plants, none
of which possess a pineal gland and some of which,
i.e., unicells, have no organs whatsoever (37, 50,
51). These nonvertebrates need melatonin for
some of the same reasons vertebrates do, e.g., as an
antioxidant (37). Some plants already have been
genetically engineered to produce elevated quan-
tities of melatonin, which protects them from free
radical-producing environmental stressors (tem-
perature extremes, drought, disease) (8, 9).
Melatonin probably evolved more than 2.5 billon
years ago, likely in purple nonsulfur bacteria, pre-
sumably in Rhodospirillum rubrum (61), to protect
them from an oxidizing environment that was a
consequence of increasing atmospheric oxygen
concentrations associated with the Great Oxygen
Event (the Oxygen Catastrophe). These melatonin-
producing bacteria were subsequently phagocy-
tized by eukaryotes, a process referred to as
endosymbiosis, where they eventually evolved into
mitochondria. Because of this, we have speculated
that mitochondria in all eukaryotic cells may have
retained the ability to produce melatonin (61); if

FIGURE 1. A diagrammatic representation of the means by which light wavelengths regulate

the central circadian pacemaker
A diagrammatic representation of the means by which 460- to 480-nm light wavelengths (blue light), de-
tected by the intrinsically photoreceptive retinal ganglion cells (ipRGC), regulate the central circadian pace-
maker, the suprachiasmatic nucleus (SCN), and the transfer of photoperiodic information to the pineal gland
for the regulation of melatonin synthesis from tryptophan. This vertebrate pathway involves synapses in the
paraventricular nucleus (PVN) of the hypothalamus, the intermediolateral cell column (ILCC) at thoracic cord
levels 1 and 2, and the superior cervical ganglia (SCG). Melatonin released into the cerebrospinal fluid (CSF)
and blood act on the SCN to modulate its circadian activity and on circadian oscillations in peripheral tissues.
Besides influencing circadian rhythms, melatonin has many other functions, as summarized in the text. In the
pinealocyte depicted at the top right, melatonin synthesis as it occurs in the mammalian pineal gland is
shown. Based on currently available data, the synthetic pathway for melatonin production in plant cells dif-
fers slightly from that in animals. In plants, tryptophan is initially decarboxylated to tryptamine, which is then
hydroxylated to form serotonin. The final two steps are similar to those in the mammalian pineal gland.
AANAT, arylalkylamine N-acetyltransferase; AC, adenylate cyclase; ASMT, acetylserotonin methyl transferase;
CaMK, calcium/calmodulin protein kinase; cAMP, cyclic adenosine monophosphate; CREB, cAMP response
element-binding protein; CSF, cerebrospinal fluid; NE, norepinephrine; NMDA, N-methyl-D-aspartate recep-
tor; NO, nitric oxide; PACAP, pituitary adenylate cyclase-activating peptide; PKA, protein kinase A; RHT, reti-
nohypothalamic tract.

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so, all tissues in multicellular organisms generate
melatonin for their own use (64).
Of interest in this regard is that melatonin con-
centrations in the mitochondria of rat hepatocytes
greatly exceed blood levels, and they do not drop
after pinealectomy, which depletes melatonin from
the blood (64). Many extrapineal organs, as sites of
melatonin production, have been identified in ver-
tebrates. Unlike the pineal gland, most of these
organs (exception, the retinas) may not synthesize
melatonin in a circadian manner nor do they re-
lease it into the blood in any significant amount.
Rather, in these organs, melatonin functions as an
antioxidant, as an autocoid, or as a paracoid to
regulate intracellular events.
Another misconception that should be dispelled
is that blood melatonin levels are representative of
its concentrations throughout the body. This is
clearly not the case given that the bile (58) and the
cerebrospinal fluid (54) have melatonin concentra-
tions that exceed those in the blood by several
orders of magnitude, and, as already noted, intra-
cellular levels are also higher than those in the
blood. Thus, because of its unequal distribution,
blood melatonin concentrations should not be
used to judge melatonin levels in other body fluids
or within subcellular compartments (47).
Melatonin, Its Potpourri of Actions
Receptor-Mediated Actions
Melatonin has an uncommonly large skill set,
and, considering that melatonin is billions of
years old, it has had ample time to hone its
functions. For several decades after its discovery,
melatonin was assumed to mediate all of its ac-
tions via receptors that were bound to membranes
of a limited number of cells, e.g., neurons of the
SCN (56). Two membrane receptors were pharma-
cologically characterized, subsequently cloned,
and are now identified as the MT1 (Mel1a) and
MT2 (Mel1b) receptors. These are both members
of the superfamily of transmembrane, G-protein-
coupled receptors. The MT1 receptor is 350 amino
acids in length, whereas MT2 consists of 363 amino
acids; the receptors share 60% homology (14).
There is also an orphan molecule referred to as a
melatonin-related receptor (MRR; also called
GPR50) that shares 45% homology to MT1 and
MT2; little is known of its function. In contrast to
the original assumption that the membrane mela-
tonin receptors were located on only a few cells,
subsequent investigations have in fact localized
them on many cells (55), and they may exist on the
membranes of all cells.
There is also what is referred to as an MT3 re-
ceptor located in the cytosol of a few cells. It is not
coupled to a G protein and exhibits low affinity for
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iodo-melatonin; it may be equivalent to quinone
reductase 2, a detoxification enzyme (21). Finally,
melatonin may carry out some of its activities by
an interaction with a group of nuclear receptors
referred to as retinoid orphan receptors (ROR) or
retinoid Z receptors (RZR). The superfamily mem-
bers that reportedly bind melatonin include the
ROR␣, RZR␣, ROR␣2, and RZR␤. These nuclear
receptors contain an NH2-terminal domain, a DNA
binding domain, a ligand binding domain (in the
COOH terminal), a zinc double finger, and a hinge
region. The nuclear receptors may be differentially
distributed among tissues (1) and perhaps are best
functionally described in the immune system (12,
27). In most cases, their specific functions are un-
der debate.
Receptor-Independent Actions
In addition to its abundant actions mediated by its
multiple receptors described above, melatonin
also directly detoxifies reactive oxygen species
(ROS) and reactive nitrogen species (RNS) by non-
receptor-mediated means (45). Like some other
classic radical scavengers, major chemical mech-
anisms by which melatonin ensnares radicals include
single electron transfer and hydrogen transfer, but
other yet to be defined processes may be involved
(15). Consistent with its presumed high intramito-
chondrial concentrations, melatonin improves the
activities of several respiratory chain complexes,
thereby reducing electron leakage and free-radical
generation (34), a process referred to as radical
avoidance (20). Moreover, metabolites of melatonin
[cyclic-3-hydroxymelatonin (c3OHM), N1-acetyl-N2-
formyl-5-methoxykynuramine (AFMK), and N1-acetyl-
5-methoxykynuramine (AMK)] (FIGURE 2) provide
additional protection against oxidative damage by
the same mechanisms described above for mela-
tonin and perhaps also by radical adduct forma-
tion (16, 17, 48, 59, 62).
The relative efficacies of melatonin, c3OHM,
AFMK, and AMK as scavengers of the hydroxyl
radical (·OH) and the hydroperoxyl radical (·OOH)
have been examined in several settings. These re-
sults were generated using a variety of techniques,
including functional density theory. Although
there are slight differences in their relative efficien-
cies, each of the four molecules scavenge the
highly reactive ·OH at diffusion-controlled rates,
regardless of the polarity of the environment. Thus
they are all highly effective scavengers of the dev-
astatingly reactive ·OH (17, 59, 62).
With regard to the ·OOH, neither melatonin,
AFMK, nor AMK are particularly effective in neu-
tralizing this agent. This is consistent with mela-
tonin per se being an inefficient chain-breaking
antioxidant (33) but inconsistent with the data
showing that both in vitro and in vivo melatonin
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REVIEWS
markedly reduces lipid peroxidation (45). This
action was then assumed to be a consequence
of melatonin’s ability to prevent the onset of
lipid peroxidation by neutralizing the initiating
agents, ·OH and the peroxynitrite anion. Subse-
quently, it was found, however, that c3OHM, in
contrast to the other three molecules, is highly
effective in scavenging the ·OOH (17, 59). More-
over, c3OHM was found to detoxify the ·OOH al-
most 100 times faster than trolox (water soluble
vitamin E). This is remarkable since vitamin E is
considered the primer peroxyl radical scavenging
agent and inhibitor of lipid peroxidation. The
FIGURE 2. Melatonin and its metabolites that
are formed when the parent molecule scav-
enges toxic radicals
This series of reactions is referred to as melatonin’s anti-
oxidant cascade, and it permits melatonin to directly
and indirectly detoxify multiple radicals. These mole-
cules differ somewhat in terms of their efficacies toward
the radicals they scavenge.
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conclusion of these studies is that melatonin both
limits the initiation of lipid breakdown and re-
duces its propagation when the metabolite c3OHM
scavenges the ·OOH.
Melatonin also reportedly detoxifies the strong
oxidizing agent, the peroxynitrite anion (ONOO⫺)
and hypochlorous acid (20). Whether melatonin’s
metabolites likewise function in these capacities
remains untested.
To illustrate the importance of melatonin as an
antioxidant, throughout evolution, no organism
that has been investigated has lost the ability to
synthesize this indoleamine, as is the case with
some other radical scavengers, e.g., vitamin C. Ad-
ditionally, besides being endogenously produced,
melatonin is ingested in the diet of all animal spe-
cies given that it is produced in plants (28). These
dual routes ensure that melatonin is always avail-
able, again unlike some other radical scavengers.
Finally, at least in some animal and plant spe-
cies, melatonin synthesis is upregulated under
circumstances where free-radical generation is
exaggerated (3). This greatly increases its protec-
tive activities.
Related to melatonin’s capability of reducing
molecular damage due to free radicals are its ef-
fects on anti- and pro-oxidative enzymes (45).
These actions are prominent and highly repro-
ducible but, mechanistically, not well investi-
gated, although preliminary evidence suggests
the involvement of the membrane receptors MT1
and MT2 (49). The major antioxidative enzymes
that are stimulated by melatonin under basal condi-
tions include the intracellular superoxide dismutases
(CuZnSOD and MnSOD), the selenium-containing
glutathione peroxidases (GPX1, GPX2 and GPX3),
and catalase (CAT) (FIGURE 3). Conversely, under
toxic conditions of high oxidative stress, these pro-
teins are protected from free-radical damage, and
their enzymatic activity is preserved. Melatonin
also maintains the activities of enzymes that en-
hance intracellular levels of reduced glutathione
(GSH), an important intracellular antioxidant.
Thus melatonin promotes glutathione reductase
(GRd) activity, which reduces glutathione disulfide
(GSSG) to the sulfhydryl form of GSH and ␥-glu-
tamylcysteine ligase (GCL), the rate-limiting en-
zyme in GSH synthesis (63). The pro-oxidative
enzymes inhibited by melatonin include nitric ox-
ide synthase, myeloperoxidase, and eosinophil
peroxidase (20).
Melatonin Doses and Timing of
Administration
When melatonin is to be used, the dose and
timing of when it is given may be critical in
determining its efficacy as a treatment. The
doses typically used in animal/human studies for

membrane receptor-mediated circadian rhythm
regulation are usually lower than those used for
defeating free radicals, an action that is membrane
receptor independent. Moreover, when melatonin
is used in situations where an influence on circa-
dian processes is a factor, e.g., sleep in humans,
melatonin should be taken in the evening before
sleep. Conversely, if the purpose of melatonin use
is to harness free radicals, melatonin would be
most effective if given when the free radical event,
e.g., stroke, occurs, regardless of the time of day.
The statement has been made that melatonin
functions as a free-radical scavenger only when
given in amounts that are considered pharmaco-
logical. This is incorrect. For any scavenger, even a
single molecule is capable of neutralizing a radical.
To combat the massive number of free radicals
generated under conditions of severe oxidative
stress, however, pharmacological amounts of any
antioxidant must be given to neutralize the over-
whelming numbers of these toxic brigands that are
produced. Vitamins C and E, for example, are often
taken in gram quantities, even under conditions
where severe oxidative damage is not a consider-
ation. Thus, when melatonin is used to reduce
free-radical destruction, it is given in pharmaco-
logical doses since the free radicals are also being
produced at “pharmacological” levels.
Melatonin Isomers
Isomers of melatonin have been identified in plant
products (19, 65), and we have predicted they will
eventually be found throughout the plant and an-
imal kingdoms (60). Isomers are molecules with
the identical molecular size as melatonin; they also
possess the same methoxy residue and the ali-
phatic side chain as melatonin itself, but they are
in different locations on the indole nucleus. Pres-
ently published reports show that the isomers bind
to classic membrane melatonin receptors and
function in free-radical detoxification (60).
Melatonin: Functionally, a Swiss
Army Knife
Like the multi-tooled device bearing the Swiss coat
of arms, melatonin has a bewildering array of func-
tions and employs a variety of means to carry them
out (FIGURE 4). These actions likely impact every
cell in every organism throughout the plant and
animal kingdoms. As a consequence, the physio-
logical and pathophysiological actions of mela-
tonin are numerous.
Melatonin is best known for its mediation of
circannual variations in metabolism and reproduc-
tive competence in photosensitive species (5), its
ability to influence circadian processes that are
ubiquitous in organisms and in cells, and its sleep-
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promoting activity (10). Each of these functions
relies on the circadian message provided by the
pineal-derived blood and cerebrospinal fluid mela-
tonin rhythms that are transferred to cells that
“have a need to know.” Melatonin is not impeded
by any of the known morphophysiological barriers,
e.g., blood-brain barrier, blood-testis barrier, etc.,
and it readily crosses the placenta in an unaltered
form to impact the fetus (46). Melatonin’s concen-
tration in some bodily fluids, e.g., CSF, bile, etc.,
exceeds that in the serum by several orders of
magnitude. Likewise, at least in those cells where
measurements have been made, subcellular organ-
elle melatonin levels are greater than in the blood
and may not rely on the blood as a source of their
melatonin (36, 64). We have speculated that all
cells have the capability of synthesizing melatonin,
particularly in their mitochondria. This locally pro-
duced melatonin is for protection from free radi-
cals and from cells in the neighborhood via
autocoid and paracoid actions and is not normally
released into the blood.
Receptor-independent actions of melatonin and
its metabolites relate to their ability to directly
quench free radicals and nonradical, but toxic,
FIGURE 3. The relationship of molecular
oxygen to the formation of reactive oxy-
gen species, reactive nitrogen species,
and the production of hypochlorous acid
The relationship of molecular oxygen (O2) to the
formation of reactive oxygen species (O2·⫺; 1O2;
H2O2; ·OH), reactive nitrogen species (NO·;
ONOO⫺), and the production of hypochlorous
acid (HOCl), a toxic chloride species. Also
shown are the enzymes that are either stimu-
lated (1) or inhibited (2) by melatonin. CAT,
catalase; GCL, glutamylcysteine ligase; GPx, glu-
tathione peroxidase; GRd, glutathione reduc-
tase; MPO, myeloperoxidase; NOS, nitric oxide
synthase; SOD, superoxide dismutase.
PHYSIOLOGY • Volume 29 • September 2014 • www.physiologyonline.org 329
REVIEWS
species. Excessive free-radical generation is noto-
riously destructive and kills cells secondary to mas-
sive oxidative damage, which induces cellular
apoptosis or necrosis. The loss of cells due to pro-
grammed cell death is a consequence and/or con-
tributes to many diseases as well as to age-related
deterioration. Melatonin’s ability to prevent mo-
lecular damage meted out by free radicals and the
cellular mutilation becomes manifested when the
indole protects against that destruction. Such
damage can be produced by ultraviolet and ioniz-
ing radiation–ingestion of toxins, heavy metals, al-
cohol, and prescription drugs–smoking, ischemia/
reperfusion injury, which occurs during a heart
attack or stroke, severe inflammation, neurodegen-
erative diseases, and many other pathophysiologi-
cal situations (45).
Similarly, aging is associated with the progres-
sive accumulation of oxidative debris, which
contributes to functional inefficiency of cellular
processes, thereby inducing additional free radi-
cals to be produced. Thus aging becomes a vi-
cious cycle. As molecular processes fail, oxidative
damage accumulates, which leads to additional
physiological collapse, further exaggerating the
production of free radicals. Melatonin, because of
its ability to neutralize radicals, defers age-related
dysfunction of several organs (22).
Increased age leads to a gradual diminished
melatonin production such that, in the elderly, the
nocturnal melatonin rise in the circulation is either
greatly attenuated or it no longer exists. The con-
sequences of this reduction may be highly signifi-
cant in terms of health (44). Since the cycle of
melatonin strengthens circadian rhythms by ac-
ting at the SCN and also on peripheral slave
oscillators, normal circadian rhythms deterio-
rate, and, considering their importance for opti-
mal health, the dysregulation of these rhythms,
e.g., the sleep/wake cycle, negatively impacts or-
ganisms. Additionally, the loss of melatonin during
aging contributes to the accelerated accumulation
of oxidative stress due to the reduced availability of
this important antioxidant. This presumably con-
tributes to the progression of diseases that have a
free-radical component, e.g., neurodegenerative
diseases (52), cardiovascular disease (13), skin de-
terioration (25), and metabolic syndrome (38),

FIGURE 4. Some of the numerous actions of melatonin in mammals

Melatonin has both receptor-dependent and receptor-independent actions. The indole binds to well
known membrane receptors (MT1 and MT2) and, via several signal transduction pathways, influences a
host of physiological effects. MT1 and MT2 may homo- and/or heterodimerize in some cases, and
they may interact with nuclear receptors (binding sites). There is considerable debate regarding the
existence/function of the orphan nuclear melatonin receptors ROR and RZR. The cytosolic receptor,
MT3, is a detoxifying enzyme, quinone reductase 2. Receptor-independent actions are mediated by
the ability of melatonin and its metabolites to scavenge reactive oxygen (ROS) and reactive nitrogen
species (RNS). These actions allow melatonin to protect against a wide variety of toxins and processes
that generate highly toxic reactants. Any cell can simultaneously respond to melatonin by both its re-
ceptor-mediated and receptor-independent actions. Many of the documented physiological and mo-
lecular actions of melatonin are not listed in this figure. Additionally, the figure does not include
melatonin functions in nonvertebrates or in plants.

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among others. In experimental studies, supple-
menting aging animals with melatonin defers
some of these debilitating changes (22).
Some functions of melatonin may depend on
both receptor-mediated events and on receptor-
independent processes. As an example, melatonin
inhibits the proliferation of many cancer cells (40)
as well as limiting tumor metastases (32). The abil-
ity of melatonin to forestall cancer cell division and
the molecular processes associated with the relo-
cation of cancer cells to a secondary site may in-
volve the classic membrane receptors that these
cells possess as well as its receptor-independent
antioxidative functions; the latter also may account
for melatonin’s ability to promote apoptosis of
cancer cells (6). This pro-apoptotic action of mela-
tonin in cancer cells is diametrically opposite to its
anti-apoptotic function in normal cells, a differen-
tial action that has been difficult to explain (6).
Melatonin, Its Special Association
With Mitochondria
Mitochondria are a major site of free-radical gen-
eration since electrons that are fumbled during
their transfer through the electron transport chain
reduce molecular oxygen to the superoxide anion
radical (O2·⫺). This oxidizing agent is quickly dis-
mutated to form hydrogen peroxide (H2O2), which
is transformed, in the presence of transition met-
als, to the ·OH (FIGURE 3). Alternatively, O2·⫺ may
couple with nitric oxide to produce the highly toxic
ONOO⫺. If the prediction that melatonin is pro-
duced in mitochondria is valid, its generation in
these organelles would be a major advantage for
reducing mitochondrial damage and preserving
energy production.
Melatonin is highly effective as an antioxidant
at the mitochondrial level (2). When compared
with synthetically produced, mitochondrial-tar-
geted antioxidants Mito Q and Mito E, which are
concentrated up to 500-fold in mitochondria (18),
melatonin still performed better in reducing every
aspect of damage to this organelle (30). The impli-
cation is that melatonin, when administered via
any route, is absorbed and makes its way to mito-
chondria in sufficiently high amounts where it is
more effective than the synthetic antioxidants,
which are known to accumulate in the matrix of
mitochondria, in preserving the function of these
critically important organelles. Thus, besides being
produced in mitochondria, when it is taken as a
supplement or consumed in the diet it is readily
available to mitochondria. Thus melatonin may be
classified as a naturally occurring, mitochondrial-
targeted antioxidant. Melatonin may have regula-
tory actions on mitochondrial uncoupling protein,
but these functions remain poorly defined.
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Conclusions and Perspectives
Melatonin’s flummoxing array of physiological ac-
tions as well as the numerous means by which
these actions are mediated supports the idea that
melatonin is a multitasking molecule. Regarding
both these features, melatonin has certainly ex-
ceeded the expectations of the most ardent mela-
tonin devotees. Yet, there is likely more to come.
Stable circadian rhythms, which melatonin
aids in regulating, are crucial for the optimal
generalized physiology of organisms and for the
microphysiology of molecular functions. Likewise,
melatonin’s ability to modulate oxidative pro-
cesses and protect against free-radical mutilation
of essential molecules is indispensible for flawless
cellular function.
Research on melatonin seems to be at the “end
of the beginning.” Obviously, extensive research
has been performed regarding the actions of mela-
tonin in experimental cells, plants, and animals. Its
uncovered actions are uniformly beneficial, al-
though not all the specific mechanisms have been
described. In addition to its multiple positive phys-
iological actions, melatonin has an uncommonly
high safety profile. Thus it is time to advance this
research to the next level, i.e., to more extensively
test its use in clinical trials (26). Although small
steps are being taken in this direction (53), it is
essential that multi-centered, blinded, well controlled
studies be performed using adequate amounts of
melatonin and for appropriate durations to deter-
mine melatonin’s ability to defer or prevent certain
diseases, as suggested by the numerous experi-
mental studies that have been performed. This is
especially important since prevention always
trumps treatment. 䡲
No conflicts of interest, financial or otherwise, are de-
clared by the author(s).
Author contributions: R.J.R. and D.-X.T. analyzed data;
R.J.R., D.-X.T., and A.G. interpreted results of experi-
ments; R.J.R. drafted manuscript; R.J.R. and D.-X.T. edited
and revised manuscript; R.J.R., D.-X.T., and A.G. approved
final version of manuscript; A.G. prepared figures.
References
1. Acuna-Castroviejo D, Reiter RJ, Menendez-Pelaez A, Pablos
MI, Burgos A. Characterization of high-affinity melatonin
binding sites in purified cell nuclei of rat liver. J Pineal Res 16:
100 –112, 1994.
2. Acuna-Castroviejo D, Lopez LC, Escames G, Lopez A, Garcia
JA, Reiter RJ. Melatonin-mitochondria interplay in health and
disease. Curr Top Med Chem 11: 221–240, 2011.
3. Antolin I, Obst B, Burkhardt S, Hardeland R. Antioxidative
protection in a high-melatonin organism: the dinoflagellate
Gonyaulax polyedra is rescued from lethal oxidative stress by
strongly elevated, buy physiologically possible concentra-
tions of melatonin. J Pineal Res 23: 182–190, 1997.
4. Axelrod J, Weissbach H. Enzymatic O-methylation of N-ace-
tylserotonin to melatonin. Science 131: 1312, 1960.
PHYSIOLOGY • Volume 29 • September 2014 • www.physiologyonline.org 331
REVIEWS
5. Barrett P, Bolborea M. Molecular pathways in-
volved in seasonal body weight and reproductive
responses governed by melatonin. J Pineal Res
52: 376 –388, 2012.
6. Bizzarri M, Proietti S, Cucina A, Reiter RJ. Molec-
ular mechanisms of the pro-apoptotic actions of
melatonin in cancer: a review. Expert Opin Ther
Targets 17: 1483–1496, 2013.
7. Brainard GC. Pineal research, the decade of
transformation. J Neural Transm Suppl 13: 3–10,
1978.
8. Byeon Y, Park S, Kim YS, Back K. Microarray
analysis of genes differentially expressed in mela-
tonin-rich transgenic rice expressing a sheep se-
rotonin. N-acetyltransferase. J Pineal Res 55:
357–363, 2013.
9. Byeon Y, Lee K, Park YI, Park S, Back K. Molecular
cloning and functional analysis of serotonin N-
acetyltransferase from the cyanobacterium Syn-
echocystis sp. PCC 6803. J Pineal Res 55: 371–
376, 2013.
10. Campos Costa I, Nogueira Carvalho H, Fer-
nandes L. Aging, circadian rhythms and depres-
sive disorders: a review. Am J Neurodegener Dis
29: 228 –246, 2013.
11. Cano P, Jimenez-Ortega V, Larrad A, Reyes Toso
CF, Cardinali DP, Esquifino AI. Effect of high-fat
diet on 24-h pattern of circulating levels of pro-
lactin, luteinizing hormone, testosterone, corti-
costerone, thyroid-stimulating hormone and
glucose and pineal melatonin content, in rats.
Endocrine 33: 118 –125, 2008.
12. Carrillo-Vico A, Guerrero JM, Lardone PL, Reiter
RJ. A review of the multiple actions of melatonin
on the immune system. Endocrine 27: 189 –200,
2005.
13. Dominguez-Rodriguez A, Abreu-Gonzalez P,
Avanzas P. The role of melatonin in acute myo-
cardial infarction. Front Biosci 17: 2433–2441,
2012.
14. Dubocovich ML, Markowska M. Functional MT1
and MT2 melatonin receptors in mammals. Endo-
crine 27: 101–110, 2005.
15. Galano A. On the direct scavenging activity of
melatonin towards hydroxyl and series of peroxyl
radicals. Phys Chem Chem Phys 13: 7178 –7188,
2011.
16. Galano A, Tan DX, Reiter RJ. On the free radical
scavenging activities of melatonin’s metabolites,
AFMK and AMK. J Pineal Res 54: 245–257, 2013.
17. Galano A, Tan DX, Reiter RJ. Cyclic 3-hy-
droxymelatonin, a key metabolite enhancing the
peroxyl radical scavenging activity of melatonin.
Roy Soc Chem Adv 4: 5220 –5227, 2014.
18. Galley HF. Bench-to-beside review: targeting an-
tioxidants to mitochondria in sepsis. Crit Care 14:
230 –238, 2010.
19. Gomez FJV, Raba J, Cerutti S, Silva MF. Monitor-
ing melatonin and its isomers in Vitis vinifera cv.
Malbec by UHPLC-MS/MS from grape to bottle.
J Pineal Res 52: 349 –355, 2012.
20. Hardeland R. Antioxidative protection by mela-
tonin: multiplicity of mechanisms from radical de-
toxification to radical avoidance. Endocrine 27:
119 –130, 2005.
21. Hardeland R. Melatonin: signaling mechanisms of
a pleiotropic agent. Biofactors 35: 183–192,
2009.
22. Hardeland R. Melatonin and the theories of ag-
ing: a critical appraisal of melatonin’s role in an-
tiaging mechanisms. J Pineal Res 55: 325–356,
2013.
23. Hoffman RA, Reiter RJ. Pineal gland: influence on
gonads of male hamsters. Science 148: 1609 –
1611, 1965.
332 PHYSIOLOGY • Volume 29 • September 2014 • www.physiologyonline.org
Downloaded from journals.physiology.org/journal/physiologyonline (110.137.101.246) on March 4, 2022.
24. Hoffman RA, Reiter RJ. Responses of some en-
docrine organs of female hamsters to pinealec-
tomy and light. Life Sci 5: 1147–1151, 1966.
25. Kim TK, Kleszczynski K, Janjetoric Z, Sweatman
T, Lin Z, Li W, Reiter RJ, Fischer TW, Slominski
AT. Metabolism of melatonin and biological ac-
tivity of intermediates of melatoninergic pathway
in human skin cells. FASEB J 27: 2742–2755,
2013.
26. Korkmaz A, Reiter RJ, Topal T, Manchester LC,
Oter S, Tan DX. Melatonin: an established anti-
oxidant worthy of use in clinical trials. Mol Med
15: 43–50, 2009.
27. Lardone PJ, Guerrero JM, Fernandez-Santos JM,
Rubio A, Martin-Lacave I, Carrillo-Vico A. Mela-
tonin synthesized by T-lymphocytes as a ligand
of the retinoic acid-related orphan receptor. J
Pineal Res 51: 454 – 462, 2011.
28. Lei Q, Wang L, Tan DX, Zhao Y, Zheng XD, Chen
H, Li QT, Zuo BX, Kong J. Identification of genes
for melatonin synthetic enzymes in ‘Red Fuji’ ap-
ple (Malus domestica Borkh. cv Red) and their
expression and melatonin production during fruit
development. J Pineal Res 55: 443– 451, 2013.
29. Lerner AB, Case JD, Takahashi Y, Lee TH, Mori
W. Isolation of melatonin, the pineal factor that
lightens melanocytes. J Am Chem Soc 80: 2587–
2592, 1958.
30. Lowes DA, Webster NR, Murphy MP, Galley HF.
Antioxidants that protect mitochondria reduce
interleukin-6 and oxidative stress, improve mito-
chondrial function, and reduce biochemical mark-
ers or organ dysfunction in a rat model of acute
sepsis. Br J Anaesth 110: 472– 480, 2013.
31. Lucas RJ, Peirson SN, Berson DM, Brown TM,
Cooper HM, Czeisler CA, Figuero MG, Gamlin
PD, Lockley SW, O’Hagan JB, Price LL, Provencio
I, Skene DJ, Brainard GC. Measuring and using
light in the melanopsin age. Trends Neurosci 37:
1–9, 2014.
32. Mao L, Dauchy RT, Blask DE, Slakey LM, Xiang S,
Yuan L, Dauchy EM, Shan B, Brainard GC, Hanifin
JP, Frasch T, Duplessis TT, Hill SM. Circadian
gating of epithelial-to-mesenchymal transition in
breast cancer cells via melatonin-regulation of
GSK3␤. Mol Endocrinol 26: 1808 –1820, 2012.
33. Marshall KA, Reiter RJ, Poeggeler B, Aruoma OI,
Halliwell B. Evaluation of the antioxidant activity
of melatonin in vitro. Free Radic Biol Med 21:
307–315, 1996.
34. Martin M, Macias M, Escames G, Reiter RJ,
Agapito MT, Ortiz GG, Acuna-Castroviejo D.
Melatonin-induced increased activity of respira-
tory chain complexes I and IV can prevent mito-
chondrial damage induced by ruthenium red in
vivo. J Pineal Res 28: 242–248, 2000.
35. McCord CP, Allen FP. Evidences associating pi-
neal gland function with alterations in pigmenta-
tion. J Exp Zool 23: 207–224, 1917.
36. Menendez-Pelaez A, Reiter RJ. Distribution of
melatonin in mammalian tissues: the relative im-
portance of nuclear versus cytosolic localization.
J Pineal Res 15: 59 – 69, 1993.
37. Mercolini L, Mandrioli R, Raggi MA. Content of
melatonin and other antioxidants in grape-re-
lated foodstuffs: measurement using a MEPS-
HPLC-F method. J Pineal Res 53: 21–28, 2012.
38. Nduhirabandi F, du Toit EF, Lochner A. Mela-
tonin and the metabolic syndrome: a tool for
effective therapy in obesity-associated abnor-
malities? Acta Physiol (Oxf) 205: 209 –223, 2012.
39. Owens L, Buhr E, Tu DC, Lamprecht TL, Lee J,
Van Gelder RN. Effect of circadian clock gene
mutations on nonvisual photoreception in the
mouse. Investig Ophthalmol 53: 454 – 460, 2012.
40. Proietti S, Cucina A, Reiter RJ, Bizzarri M. Molec-
ular mechanisms of melatonin’s inhibitory actions
on breast cancer. Cell Mol Life Sci 70: 2139 –
2157, 2013.
41. Quay WB. Circadian rhythms in rat pineal sero-
tonin and its modifications by the estrous cycle
and photoperiod. Gen Comp Endocrinol 3: 473–
479, 1963.
42. Reiter RJ. Melatonin: the chemical expression of
darkness. Mol Cell Endocrinol 79: C153–C158,
1991.
43. Reiter RJ, Hester RJ. Interrelationships of the
pineal gland, the superior cervical ganglia and
the photoperiod in the regulation of the endo-
crine systems of hamsters. Endocrinology 79:
1168 –1170, 1966.
44. Reiter RJ, Guerrero JM, Garcia JJ, Acuna-
Castroviejo D. Reactive oxygen intermediates,
molecular damage, and aging: relation to mela-
tonin. Ann NY Acad Sci 854: 410 – 424, 1998.
45. Reiter RJ, Paredes SD, Manchester LC, Tan DX.
Reducing oxidative/nitrosative stress: a newly-
discovered genre for melatonin. Crit Rev
Biochem Mol Biol 44: 175–200, 2009.
46. Reiter RJ, Tan DX, Korkmaz A, Rosales-Corral SA.
Melatonin and stable circadian rhythms optimize
maternal, placental and fetal physiology. Human
Reprod Update 20: 293–307, 2014.
47. Reiter RJ, Tan DX, Rosales-Corral SA, Man-
chester LC. The universal nature, unequal distri-
bution and antioxidant functions of melatonin
and its derivatives. Mini Rev Med Chem 13: 373–
384, 2013.
48. Ressmeyer AR, Majo JC, Zelosko V, Sainz RM,
Tan DX, Poeggeler B, Antolin I, Zsizsik BK, Reiter
RJ, Hardeland R. Antioxidant properties of the
melatonin metabolite N1-acetyl-5-methoxykynu-
ramine (AMK): scavenging of free radicals and
prevention of protein destruction. Redox Rep 8:
205–213, 2003.
49. Rodriguez C, Mayo JC, Sainz RM, Antolin I, Her-
rera F, Martin V, Reiter RJ. Regulation of antiox-
idant enzymes: a significant role for melatonin. J
Pineal Res 36: 1–9, 2004.
50. Rodriguez-Naranjo MI, Torija MJ, Mas A, Cantos-
Villar E, Garcia-Parilla MC. Production of mela-
tonin by Saccharomyces strains under growth
and fermentation conditions. J Pineal Res 53:
219 –224, 2012.
51. Roopin M, Levy O. Temporal and histological
evaluation of melatonin patterns in a ‘basal’
metazoan. J Pineal Res 53: 259 –269, 2012.
52. Rosales-Corral SA, Acuna-Castroviejo D, Coto-
Montes A, Boga JA, Manchester LC, Fuentes-
Broto L, Korkmaz A, Ma S, Tan DX, Reiter RJ.
Alzheimer’s disease: pathological mechanisms
and the beneficial role of melatonin. J Pineal Res
52: 167–202, 2012.
53. Sanchez-Barcelo E, Mediavilla MD, Tan DX, Re-
iter RJ. Clinical uses of melatonin: evaluation of
human trials. Curr Med Chem 17: 2070 –2095,
2010.
54. Skinner DC, Malpaux B. High melatonin concen-
trations in third ventricular cerebrospinal fluid are
not due to Galen vein blood circulating through
the choroid plexus. Endocrinology 140: 4399 –
4405, 1999.
55. Slominski RM, Reiter RJ, Schlobritz-Loutsevitch
N, Ostrom RS, Slominski AT. Melatonin mem-
brane receptors in peripheral tissues: distribution
and functions. Mol Cell Endocrinol 35: 152–166,
2012.
56. Stankov B, Reiter RJ. Melatonin receptors: cur-
rent status, facts and hypothesis. Life Sci 46:
971–982, 1990.
57. Stehle JH, Saade A, Rowashdeh O, Ackermann K,
Jilg A, Sebesteny T, Maronde E. A survey of
molecular details in the human pineal gland in the
light of phylogeny, structure, function and chro-
nobiological diseases. J Pineal Res 51: 17– 43,
2011.

58. Tan DX, Manchester LC, Reiter RJ, Qi W, Hanes
MA, Farley NJ. High physiological levels of mela-
tonin in the bile of mammals. Life Sci 65: 2523–
2529, 1999.
59. Tan DX, Manchester LC, Reiter RJ, Plummer BF.
Cyclic 3-hydroxymelatonin: a melatonin metabo-
lite generated as a result of hydroxyl radical scav-
enging. Biol Signals Recpt 8: 70 –74, 1999.
60. Tan DX, Hardeland R, Manchester LC, Rosales-
Corral SA, Coto-Montes A, Boga JA, Reiter RJ.
Emergence of naturally occurring melatonin iso-
mers and their proposed nomenclature. J Pineal
Res 53: 113–121, 2012.
61. Tan DX, Manchester LC, Liu X, Rosales-Corral SA,
Acuna-Castroviejo D, Reiter RJ. Mitochondria
and chloroplasts as the original sites of melatonin
synthesis: a hypothesis related to melatonin’s pri-
mary function and evolution in eukaryotes. J Pi-
neal Res 54: 127–138, 2013.
Downloaded from journals.physiology.org/journal/physiologyonline (110.137.101.246) on March 4, 2022.
62. Tan DX, Hardeland R, Manchester LC, Galano A,
Reiter RJ. Cyclic-3-hydroxymelatonin (c3HOM), a
potent antioxidant, scavenges free radicals and
suppresses oxidative reactions. Curr Med Chem
21: 1557–1565, 2014.
63. Urata Y, Honma S, Goto S, Todoroki S, Iida T,
Cho S, Honma K, Kondo T. Melatonin induces
gamma-glutamylcysteine synthetase mediated
by activator protein-1 in human vascular endo-
thelial cells. Free Radic Biol Med 27: 838 – 847,
1999.
64. Venegas C, Garcia JA, Escames G, Ortiz F, Lopez
A, Doerrier C, Garcia-Corzo L, Lopez LC, Reiter
RJ, Acuna-Castroviejo D. Extrapineal melatonin:
analysis of its subcellular distribution and daily
fluctuations. J Pineal Res 52: 217–227, 2012.
PHYSIOLOGY • Volume 29 • September 2014 • www.physiologyonline.org
REVIEWS
65. Vitalini S, Gardana C, Simonetti P, Fico G, Iriti M.
Melatonin, melatonin isomers and stilbenes in
Italian traditional grape products and their anti-
radical activity. J Pineal Res 54: 322–333, 2013.
66. Wurtman RJ, Axelrod J, Fischer JE. Melatonin
synthesis in the pineal gland: effect of light me-
diated by the sympathetic nervous system. Sci-
ence 143: 1328 –1330, 1964.
67. Wurtman RJ, Axelrod J, Phillips LS. Melatonin
synthesis in the pineal gland control by light.
Science 142: 1071–1073, 1963.
68. Zhoa X, Stafford BK, Godin AL, King WM, Wong
KY. Photoresponse diversity among the five
types of intrinsically photosensitive retinal gan-
glion cells. J Physiol 592: 1619 –1636, 2014.
333