Melatonin: circadian rhythm regulator, chronobiotic, antioxidant and ...

Clinics in Dermatology (2009) 27, 202–209
Melatonin: circadian rhythm regulator, chronobiotic,
antioxidant and beyond
Bruno Berra, PhD ⁎ , Angela Maria Rizzo, PhD
Institute of General Physiology and Biochemistry, “Giovanni Esposito”, University of Milan, Faculty of Pharmacy,
Via Trentacoste 2, Milan 20134, Italy
Abstract For many years, melatonin has been known to interact with circadian rhythms. New evidence
indicates that melatonin acts as a free radical scavenger and antioxidant. Moreover, melatonin prevents
apoptosis in different types of cells, because it induces mRNA levels of several antioxidant enzymes. It
is evident that melatonin is involved in the cellular bioenergetic system as a mechanism that counteracts
the progression of Alzheimer's disease.
© 2009 Elsevier Inc. All rights reserved.
Introduction
Melatonin, the major secretory product of the pineal gland,
has been known to interact with the neuroendocrine axis and
circadian rhythms. Recently, it has been reported that
melatonin acts as a free radical scavenger and antioxidant, 1,2
and thus as an antiapopotic agent. 3 Mayo and colleagues 4
reported that melatonin prevents apoptosis in undifferentiated
and differentiated PC12 cells and suggested that the
protective effect of melatonin may be associated with the
increase in the mRNA levels of several antioxidative
enzymes.
Pineal gland and melatonin
In humans, the pineal gland is 5 mm long, 1-4 mm thick,
and weighs about 100 mg, in men and women. 5 The pineal
gland contains two major cell types: neuroglial cells and the
predominant pinealocytes that produce melatonin. The
pineal gland is a central structure in the cardian system that
⁎ Corresponding author. Tel.: +39 02 50315777; fax: +39 02 50315775.
E-mail address: bruno.berra@unimi.it (B. Berra).
is innervated by a neural multi-synaptic pathway originating
in the suprachiasmatic nucleus (SCN) located in the anterior
hypothalamus. The SCN is the major circadian pacemaker of
the mammalian brain and plays a central role in the
generation and regulation of biological rhythms. 6 The pineal
gland produces melatonin in a marked circadian fashion, 7
reflecting signals originating in the SCN. The human SCN
innervates only a small number of hypothalamic nuclei
directly. 8 However, it may impose circadian fluctuations
indirectly on the organism by means of melatonin released
from the pineal gland. 9
Melatonin biosynthesis and its regulation
The biosynthetic pathway of melatonin has been studied
thoroughly. L-trytophan is taken from circulation and
converted to serotonin (5-HT) by tryptophan hydroxylase. 5-
HT is metabolized by the rate-limiting enzyme arylalkylamine
N-acetyltransferase (AA-NAT) to N-acetyl-5-hydroxytryptamine,
and in turn by hydroxyindole-O-methyltransferase to
melatonin. In all vertebrates, the activity of the rhythmgenerating
enzyme AA-NAT increases at night by a factor of
7-150, depending on the species. The molecular mechanisms
regulating AA-NAT also are remarkably different among
0738-081X/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.clindermatol.2008.04.003

Melatonin: circadian rhythm regulator, chronobiotic, antioxidant and beyond
203
species. For instance, in rats, pineal AA-NAT is regulated
at both the mRNA level and protein level; however, in sheep
and rhesus macaque, the mRNA pineal AA-RAT levels
show relatively little change over a 24-hr period, and changes
in AA-NAT activity are regulated primarily at the protein
level. 10 In the human pineal gland, significant daily fluctuations
in AA-NAT activity may be regulated mainly at the post
transcriptional level. 11
Light intensity is the main environmental control of the
pineal melatonin synthesis. Light perceived by the retina
reaches the SCN through the retinohypothalamic tract, which
has been revealed in the human hypothalamus. 12 The SCN
innervates the pineal gland, resulting in the rhythmic
secrection of melatonin. 13 The importance of ocular light
as a temporal clue has been demonstrated in circadian studies
of blind people, bilaterally enucleated, showing desychronized
melatonin and cortisol rhythm. 14 Abundant evidence
indicates that, in humans, the sympathetic stimulus is crucial
for melatonin secrection. The primary neurotransmitter
released from the postganglionic sympathetic terminals in
the pineal gland is norepinephrine (NE); during darkness at
night, NE is discharged onto the pinealocyte, where it
couples (especially with beta 1-adrenoceptors). This process
is potientated further by stimulations of alfa 1- adrenoceptors.
This leads to a marked rise in intracellular cAMP levels
to denovo protein synthesis and eventually to the stimulation
of AA-NAT.
Unlike other endocrine organs, the pineal does not store
melatonin for later release after its synthesis; melatonin
quickly diffuses out of the pinealocytes into the rich
capillary bed within the gland and directly into the
cerebrospinal fluid. Melatonin displays high lipid and
water solubility, which facilitates passage across cell
membranes. After release into the circulation, it gains access
to various fluids, tissues, and cellular compartments (saliva,
urine, cerebrospinal fluid, preovulatory follicles, semen,
amniotic fluid, and milk).
Three mammalian melatonin receptors have been
described: MT1, MT2, MT3. The first two are G-proteincoupled
receptors, and their activation modulates a wide
range of intracellular messangers (eg, cAMP, cGMP, or
calcium concentrations). The MT1 receptor, with high
affinity, is mainly expressed in the SCN and hypophyseal
pars-tuberalis. The MT1 receptor with low affinity is
expressed mainly in the retina. The MT3-binding site has
been identified as a quinone reductase protein, but its
physiological significance needs to be clarified.
A very large goiter may compress the superior cervical
ganglia (SCG), thus altering melatonin synthesis in
patients. 15 After bilateral T1-T2 ganglionectomy in a patient
with hyperhidrosis, melatonin levels in the cerebrospinal
fluid (CSF) and plasma were reduced, and the diurnal rhythm
was abolished. 16 A circadian rhythm of β1-adrenergic
receptors has been found in human pinealocytes. 17 Propanolol,
a β-adrenergic receptor antagonist, causes a dosedependent
decrease in melatonin levels or completely
abolishes nighttime surge. 18 In turn, melatonin elicits two
distinct, separable effects on the SCN: acute neuronal
inhibition and phase shifting. 19 The ability of melatonin to
phase-shift in the circadian system has been investigated
extensively in humans. 20
Jet lag
Jet lag is a considerable problem in the modern world
with widespread air travel. When the internal body clock
(or circadian rhythm) is not synchronized with the external
“local” time (light-dark cycle), jet lag is experienced. The
symptoms, which vary between individuals, include tiredness,
inability to sleep at the new bedtime, inability to
concentrate, disturbed sleep for several days after a long
flight, headache, and gastrointestinal problems. All of these
symptoms can interfere with normal activities. Jet lag also
may cause considerable problems for training and performance
in sports competitions and should be taken into
account when planning journey times for sportsmen and
women. Symptoms are more marked in older travellers,
when more time zones are crossed, and when travelling in
an easterly direction. 21
A recent Cochrane review 22 assessed 10 trials comparing
melatonin with a placebo and compared it with the hypnotic
zolpidem. In 9 out of 10 trials, melatonin decreased jet lag
resulting from crossing five or more time zones when taken
close to the desired bedtime at the destination (10 P.M. to
midnight). The time of dosing was very important, because if
it is taken at the wrong time, melatonin could cause a delay in
adaptation to the local time. The safety profile was very high
in these trials, and the authors concluded that melatonin can
be recommended safely to adults travelling across five or
more time zones, particularly those who have experienced jet
lag previously.
Delayed sleep phase disorder
According to the International Classifications of Sleep
Disorders, individuals with delayed sleep phase disorder
(DSPD) have difficulty falling asleep at their desired bedtime
and an inability to wake spontaneously at the planned
time in the morning. 23 Weitzman and colleagues 24 first
defined this disorder and described its characteristics: long
sleep onset latencies and late sleep onset times. This is
caused by a delay of the major sleep period.
There is considerable evidence that DSPD arises from a
delayed endogenous circadian rhythm. 25 Sleep parameters,
melatonin, and core body temperature rhythms have been
delayed in individuals with sleep onset insomnia and DSPD
when compared to control groups. 26 If the core body
temperature and melatonin circadian rhythms were phase
delayed, then the “wake maintenance zone” would be
delayed also. 27 Although circadian rhythm phase delay is
seen as the major contributor to DSPD, there are some

204 B. Berra, A.M. Rizzo
important behavioral and cognitive factors that should be
addressed to improve treatment effectiveness.
Treatments that change the circadian rhythm phase or
timing (such as morning bright light, exogenous melatonin,
and chronotherapy) have been effective in treating of delayed
circadian rhythm sleep disorders. Bright light is an effective
intervention for phase advancing circadian rhythm.
The timing and duration of the light stimulus and the
brightness and wavelength of light affects the magnitude of
phase shift. The human phase response curve to light
suggests that a phase advance of the circadian rhythm is
achieved when the light stimulus is presented immediately
after the normal circadian time (CT). 28
Exogenous melatonin administration also is capable of
shifting the circadian rhythm to a more desired time. 29 In
the evening phase, melatonin advances circadian rhythm
when combined with optimal time of administration and
greater doses: (3-5 mg) 4 to 8 hours prior to the onset
of endogenous melatonin and smaller doses (0.3-0.5 mg)
3 hours before the beginning of melatonin production. 30 A
recent study 31 showed that the addition of evening
melatonin administration to morning bright light therapy
produced a significantly greater phase advance than the
morning bright light alone, suggesting that the two
therapies are additive.
Although exogenous melatonin appears to be safe with
short-term use (less than 3 months), there is little
information available on its long-term administration. 32
Because typical doses (3-5 mg) in many studies can elevate
melatonin concentrations well above normal physiological
plasma levels, it would be prudent to use much lower
doses. Fortunately, the chronobiotic effects of low doses
(0.3-0.5 mg) appear to be sufficient without requiring
excessive supraphysiological levels. 31 Adverse side effects
reported following melatonin administration include headache,
dizziness, nausea, and drowsiness. 32 The effects of
exogenous melatonin on sleep was the object of a recent
meta-analysis report, 33 which states: “ this meta analysis
supports the hypothesis that melatonin decreases sleep
onset latensy, increases sleep efficiency and total sleep
duration. In spite of the heterogeneity of the data the
present meta analysis does land statistical support to the
notion that melatonin preparations can improve sleep
quality with regard to sleep onset latensy, sleep efficiency
and sleep duration.”
Melatonin as an antioxidant, radical scavenger,
and anti aging product
Melatonin is present in bacteria, plants, eukaryotes, fungi,
and all phyla of multicellular animals; its original evolutionary
role probably was to act as an antioxidant. 34 Its
antioxidant properties have been seen in tissue cultures and
intact animals. One problem still discussed is whether
melatonin acts in this manner directly or activates critical
pathways involved in the disposition of free radicals. 35 The
evidence for a direct effect is seen when melatonin acts as a
power-free radical scavenger in isolated cell-free-systems 36 ;
there are, however, reports that melatonin can act as a prooxidant
in such systems. 37
Melatonin is present within brain at a concentration of
only 5% of that found in serum. 38 Therefore, unless it is
highly concentrated in a localized area, it can contribute
little to scavenging when compared to predominant
antioxidant species like glutathione and alfa-tocopherol. 39
Melatonin receptors and enzyme induction
There are three major plasma membrane receptors for
melatonin in the brain. However, the presence of additional
melatonin binding sides in the nuclei of many cell types
suggests the existence of a mechanism different from the
mechanism mediated by the interaction with plasma
membrane receptors. 40 The specificity of melatonin may
reside in its properties as a neurohormone, which affects
transcriptional events in the CNS. 41
A wide range of antioxidant enzymes is induced by
melatonin (eg, glutathione peroxidase, catalase, and superoxide
dismutases). 42 These protein changes are paralleled by
altered levels of gene expression of oxidative emzymes. 43 In
addition, levels of some pro-oxidant enzymes (such as
lipoxygenase and nitric oxide synthetase) are depressed after
melatonin treatment. 44
Presently, melatonin functions seem to fall into three
categories: receptor-mediated, protein-mediated, and nonreceptor-mediated
effects. Receptor-mediated melatonin
events involve membrane and nuclear receptors. Although
membrane melatonin receptors are well-characterized in
humans, 45 some of the receptor-related antioxidant effects of
melatonin seem to be related to its nuclear receptors. 46 With
this information, the interaction between membrane and
nuclear melatonin signalling has been proposed. 47
Melatonin and aging
One of the most recent theoretical advances in basic
gerontology is the free radical theory of aging. This thoery
proposes that reactive ogygen species (ROS) (including
superoxide [O ● 2], hydroxyl (OH ● ) free radicals, hydrogen
peroxide [H 2 O 2 ] and possibly singlet oxygen) are generated
as by-products of cellular respiration and other
metabolic processes and damage cellular macromolecules.
This results in mutations and genome instability, and all of
these abnormalities can lead to the development of agerelated
pathological phenomena, including cancer, circulatory
diseases, immuno depression, brain disfunction, and
cataracts. 48 Much evidence indicates that aging is characterized
by a progressive deterioration of circadian time
keeping. 49

205
Melatonin: circadian rhythm regulator, chronobiotic, antioxidant and beyond
tissue and were compared with those of other known expression of apoptosis-related factors in vivo. 63
Besides the age-related decline of melatonin production, antioxidants. 54 After oxidative stress, virtually all GSH
age-related changes in the timing of the melatonin rhythm
have been reported. 50 The possible mechanism of age-related
melatonin changes were associated with changes in the
pineal gland morphology and its calcification; these data are
in agreement with the assertion of decreased melatonin
production with age. 51,52 These findings suggest that the
changes observed in the melatonin rhythm may be part of a
general effect of aging in particularly on the central clock
of SCN and its regulation.
in mitochondria is oxidized to GSSG, and the activity of
both GPx and GRd are reduced almost to zero. In this
situation, melatonin counteracted these effects, restored basal
levels of GHS and the normal activities of both GPx and
GRd. To characterize the effects of melatonin on mitochondrial
ETC activity, submitochondrial particles were used.
Melatonin increased the activity of the C-I and C-IV
complexes in a dose-dependent manner, as previously
shown on intact mitochondria.
These results suggest a direct effect of melatonin on
mitochondrial energy metabolism and provide a new
Melatonin, mitochondria, and
homeostatic mechanism for regulation of mitochondrial
function. The findings also identify a new mechanism of
cellular bioenergetics
action of melatonin at the mitochondrial level. Thus, melatonin
improves the bioenergetics of the cell, by providing
Aerobic cells use oxygen for the production of 90%-95% more efficient nuclear and mitochondrial genomic repair
of the total amount of ATP they use. The synthesis of ATP mechanisms, increasing GSH levels, elevating ATP production,
and improving ATP-dependent functions, including
is the result of electron transport along the mitochondrial
electron chain, resulting in the ultimate oxygen reduction neurotransmission.
and coupled to oxidative phosphorylation. Under normal
conditions, a small percentage of oxygen may be reduced
by one, two, or three electrons only, yielding superoxide
anion, hydrogen peroxide, and the hydroxyl radical, Melatonin and Alzheimer's disease
respectively. The main radical produced by mitochondria
is superoxide anion, and the intramitochondria antioxidant Alzheimer's disease (AD) is characterized by the presence
systems must scavenge this radical to avoid oxidative of β-amyloid deposits and neurofibrillary tangles (NFT) in
damage to the mitochondrial membrane, which leads to the brains of affected individuals. The development of early
impaired ATP production.
diagnostic tools and quantitative markers are crucial for
During aging and some neurodegenerative diseases, exploring promising therapeutic strategies. 55 Anti-inflammatory
agents, antioxidants, vaccinations, cholesterol-low-
oxidatively damaged mitochondria are unable to maintain
the energy demands of the cells, leading to a further ering agents, and hormone therapy are examples of new
increased production of free radicals. Both defective ATP approaches being developed for treating or delaying the
production and increased oxygen radical may induce progression of AD.
mitochondrial-dependent apoptic cell death.
Recent evidence indicates that melatonin reduces the
Melatonin has been reported to exert neuroprotective neuronal damage mediated by oxygen-based reactive
effects in several experimental and clinical situations involving
neurotoxicity and exocitotoxicity. Additionally, in a radical scavenger and antioxidant. 56,57 Several clinical
species in experimental models of AD by acting as a free
series of pathologies in which high production of free radicals studies also have indicated that melatonin levels are
is the primary cause of the diseases, melatonin is protective. decreased in AD patients. 58 Thus, melatonin's receptorindependent
scavenging effects and receptor-mediated
The common features of these diseases is the existence of
mitochondrial damage caused by oxidative stress.
influences on enzyme activities (which counteract the effect
of oxidative stress) may account for its possible beneficial
Melatonin and mitochondria
effects in AD.
Increased awareness of the role oxidative stress plays in
Two main considerations support melatonin's role in
mitochondrial homeostasis: 1) mitochondria produce high
the pathogenesis of AD has highlighted the issue of whether
oxidative damage is a fundamental step in pathogenesis or a
amounts of ROS and RNS, and 2) mitochondria depend on result of the disease-associated pathology. 59,60 Several
the GSH uptake from the cytoplasm, although they have GPx
and GRd to maintain GSH redox cycling. Thus, the
antioxidant effect of melatonin and its ability to increase
GSH levels may be of great importance for mitochondrial
physiology. 53
The protective effects of melatonin were analyzed in
isolated mitochondria prepared from rat brain and liver
studies have demonstrated the presence of lipid, protein,
and DNA oxidation products in postmortem examinations of
the brains of AD patients. 61,62
By observing the specific markers of in vivo oxidative
stress and the expression of apoptotic-related factors,
researchers demonstrated that melatonin suppresses brain
lipid peroxidation in transgenic mice, and reduces the

206 B. Berra, A.M. Rizzo
Inhibitory role of melatonin in tau protein
hyperphosphorylation
The cytoskeleton plays a crucial role in maintaining the
highly asymmetrical shape and structural polarity of neurons
essential for normal physiology. In AD, the cytoskeleton is
assembled abnormally into NFT, and impairment of
neurotransmission occurs. Microtubule-associated protein
tau is capable of binding to tubulin to form the micro-tubules,
essential structures for neuronal viability NFT.
The micro-tubule-stabilizing function of tau is diminished
greatly by its hyperphosphorylation, which results in
poor binging to tubulin. 64 Glycogen synthase kinase-3, a
downstream element of phosphoinositol-3 kinase, is one of
the most active enzymes in phosphorylating tau in vivo.
Protein kinase A (PKA) is another crucial kinase in ADlike
tau hyperphosphorylation. Isoproterenol, the specific
PKA activator, can induce tau hyperphosphorylation.
Furthermore, melatonin enhances SOD activity and
decreases the level of MDA. It has been suggested that
isoproterenol may induce abnormal hyperphosphorylation
of tau through not only the activation of PKA, but also by
increasing oxidative stress.
Circadian rhythm disruptions in Alzheimer's disease
The fragmented sleep-wake pattern that occurs in elderly
people is even more pronounced in AD patients. 65 Many
patients also suffer often from circadian system related
bahavioral disturbances, such as daytime agitation and
nightly restlessness. 66
Melatonin changes in preclinical and clinical
Alzheimer's disease
Many studies demonstrate that nocturnal melatonin levels
are selectively decreased in AD, 11 and daytime melatonin
levels are increased in AD patients. 67 A strong reduction was
observed in postmortem CSF melatonin levels of AD
patients; CSF melatonin levels in AD patients were only
one-fifth of those in control subjects. 68
The melatonin levels in CSF decrease with the progression
of AD neuropathology. 69 More strikingly, CSF
melatonin levels are already reduced in preclinical AD
patients, who are cognitively intact and are showing only the
earliest signs of AD neuropathology.
A significant high correlation exists between pineal melatonin
content and CSF melatonin levels 70 and between CSF
and plasma melatonin amount, 71 suggesting that reduced
melatonin levels may be an early marker for the first stages of
AD that could not be monitored another way. Melatonin
deficiency is not only a consequence of the AD process; it
may contribute to the pathogenesis of AD, because it acted as
an antioxidant and neuroprotector in in vitro and in vivo
experiments. 70,72
Mechanism underlying the melatonin changes in
the progression of Alzheimer's disease
The pineal gland shows molecular changes in preclinical
and clinical AD. However, cells or afferent fibers are clear of
the neuropathological hallmarks of AD (ie, neurofibrillary
tangles, accumulation of neurofilaments, and hyperphosphorylated
tau or β/A4 amyloid deposition). 73
The circadian melatonin rhythm disappears because of
decreased nocturnal melatonin levels in AD preclinical and
advanced patients. Moreover, the circadian rhythm of β1-
adrenergic receptor mRNA disappears in both patient
groups, which suggests a dysfunction of the SCN innervation
to the pineal. The biological SCN clock is affected
severely. It shows prominent degenerative changes 74 and
the typical cytoskeletal alterations caused by pretangles 75
and tangles. 76 These degenerative changes in the SCN
most likely result in a disrupted melatonin synthesis and
may underline the clinically common circadian rhythm
disorders in AD.
The input of environmental light to the circadian timing
system is also disrupted in AD. Besides the degenerative
changes that are present in the SCN, several factors attenuate
the input of environmental light to the circadian timing system;
AD patients are exposed to less environmental light than their
age-matched controls. 77 Furthermore, the retina and optic
nerve show degenerative changes, but without neurofibrillary
tangles, neuritic plaques, or amyloid angiopathy. 78,79
Melatonin supplementation
In AD patients, melatonin has been suggested to
improve circadian rhythmicity, decreasing agitated behavior,
confusion, and sundowning in uncontrolled studies.
80,81 Melatonin also may have beneficial effects on
memory, 82 possibly through protection against oxidative
stress and neuroprotective capabilities. However, these
suggestions need to be confirmed in well-controlled studies,
and a few randomized placebo-controlled trials of melatonin
administration to AD patients did not find improved in
the sleep-wake pattern. 83,84
Conclusions
This contribution summarizes the actions of melatonin in
reducing the effects of jet lag, delayed sleep phase disorder,
and molecular damage caused by free radicals. In
particular, melatonin has effects the reduction of oxidative
damage in the CNS as it cross the blood-brain barrier.
However, it is unlikely that all the actions by which melatonin
reduces free radical damage have been uncovered. The
simplest way to account for the multiple effects of melatonin
is to hypothesize that it modifies early alterations of
gene expression consisting in the depression of mRNAs for

Melatonin: circadian rhythm regulator, chronobiotic, antioxidant and beyond
207
immuno-related cytokines and in the elevation of mRNA
for antioxidant enzymes. Also, transcription factors are
activated after binding to cytoplasmic melatonin receptors
or by changes caused by melatonin acting directly on
molecular receptors.
The reported beneficial effects of melatonin on oxidative
stress-related damage were supported by the improvement
of mitochondrial function, which was achieved by
counteracting the oxidation of mitochondrial membrane
and resulting in the amelioration of cell bioenergetic. This
leads to a more efficient nuclear genomic repair mechanism,
to increased GSH levels, the elevation of ATP
production, and the improvement of ATP-dependent functions,
including neurotransmission. All these mechanisms
may represent the basis for the potential anti-aging
properties of melatonin and its effective use in neurodegenerative
diseases.
Melatonin could be considered an evolutionarily ancient
neurohormone that has a very low toxicity and no carcinogenic
properties which makes it a very safe compound that
is available at a low cost. However more research is needed
on the effects of therapeutic modulation of the melatoninergic
system on circadian haemodynamies and the possible
impact on morbility and mortality in humans.
Aknowledgment
The authors thank Francesca Tunesi for her collaboration
in the accurate preparation of this contribution.
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