ТОМ 4, СТ. 77 (стр. 113-119) // Март, 2003 г.
EXPERIMENTAL OBSERVATIONS RELATED TO THE UTILITY OF MELATONIN IN ATTENUATING AGE-RELATED DISEASES
"Успехи геронтологии", 1999г., выпуск 3
Russel J. Reiter
Department of Cellular and Structural Biology
The University of Texas Health Science Center
7703 Floyd Curl Drive
San Antonio, TX 78284-7762
Running title: Melatonin and age-related diseases
For: Advances in Gerontology
Melatonin was recently discovered to be free radical scavenger; in this capacity it
very effectively detoxifies the most highly toxic oxygen-based
radical, that is, the hydroxyl radical, while also directly
neutralizing nitric oxide, the peroxynitrite anion, singlet oxygen,
and to some degree the peroxyl radical. Besides its direct free
radical scavenging activities, melatonin also may stimulate the
activity of several antioxidative enzymes including the superoxide
dismutases, glutathione peroxidase, glutathione reductase, and
glucose-6-phosphate dehydrogenase. Conversely it inhibits neural
nitric oxide synthase, a pro-oxidative enzyme. In both in vitro
studies melatonin has been shown to reduce oxidative damage
to membrane lipids, proteins and nuclear DNA. It readily crosses all
morphophysiological barriers and gets into every cell with ease. The
bulk of the studies performed to date have used what seem to be
pharmacological levels of melatonin to achieve antioxidative
protection, but there is also evidence that physiological melatonin
concentrations contribute to the total antioxidant status of the
organism. In model systems of age-related diseases (cancer,
cataracts, Alzheimer's disease, Huntington's disease,
Parkinsonism) melatonin has been shown to be protective. Melatonin
levels diminish with age in mammals including man. The association
between the age-related reduction in melatonin and the onset of free
radical-related diseases in the aged is currently being investigated.
Aging is the accumulation of adverse changes which lead to functional
deterioration and increase the chances of death (47). These changes
are generally considered responsible for a wide variety of diseases,
which typically occur after middle age and often result in the death
of an individual. The physiological alterations that accompany aging
are attributable to a variety of factors including genetic,
developmental, environmental, and the inborn processes referred to as
Many theories have been advanced to explain the process of aging and their
contribution to age-associated diseases. While no one theory has
proven to be the definitive explanation for aging, one that has
received a great deal of attention is the free radical theory (38,
39, 47). The free radical theory states that aging and the
associated diseases are a consequence of the life long destruction
and accumulation of essential macromolecules damaged by toxic free
radicals. A corollary of this theory is that administration of
molecules that either prevent the formation of radicals or neutralize
them once they are formed would defer the rate of aging and reduce
the incidence of age-associated diseases (44, 46, 94, 96, 97). To
date, studies carried out to achieve this result have met with
limited success. Molecules which reduce the damage inflicted by
radicals are referred to as free radical scavengers and antioxidants
of which there are many (68, 111).
One newly discovered agent that is a highly effective antioxidant and
which has already been widely tested for its ability to protect
against free radical-related processes is
N-acetyl-5-methoxytryptamine, commonly known as melatonin (36,
37, 86, 95, 105). The purpose of this review is to summarize what is
known about melatonin as a free radical scavenger and antioxidant and
to discuss these findings relative to the free radical theory of
Melatonin as a Free Radical Scavenger
Many free radicals are derived from the use of molecular oxygen (O2)
by aerobic organisms. While roughly 97% of the O2 inhaled
is utilized by mitochondria for the generation of energy, i.e., ATP,
the remainder is reduced to free radicals and reactive oxygen
intermediates (ROI) which have varying degrees of toxicity (Fig. 1)
By definition a free radical is a species that has an unpaired electron
(e-) in its outer orbital. Since e- are
normally paired in molecules, this feature makes the molecules that
possess them highly reactive (19). ROI on the other hand, do not
possess an unpaired e- but they may, however, be highly
reactive and, furthermore, when metabolized they generate free
radicals. One of the most widely investigated ROI is H2O2;
its importance stems from its metabolism to the most reactive and
toxic free radical, the hydroxyl radical (·OH)
(57). The ·OH is generally
considered the most damaging of the oxygen-based free radicals that
are produced and it is believed to account for an estimated 50% of
the total damage induced by free radical mechanisms (42). Thus, any
molecule that would efficiently scavenge the ·OH
and importantly be in the vincinity of where ·OH
is produced intracellularly would be considered an important
antioxidant. The importance of the free radical scavenger being at
the site where the ·OH is
generated is important since, due to its very high reactivity, the
·OH travels no more than
several Angstroms before it interacts with another molecule.
This distance has been referred to as the reaction age of the ·OH
and a free radical scavenger must be within this "cage"
to neutralize the radical (15). To put this in the context of a
cell, if an antioxidant is confined to the lipid-rich membrane of a
cell because of its specific solubility, it will be ineffective in
reducing ·OH damage to DNA
in the nucleus.
In 1993, it was shown that melatonin is highly efficient ·OH
scavenger in vitro (117). The definitive test to verify the ability
of a free radical scavenger to neutralize a radical is considered to
be the identification of the end product by electron spin resonance
spectroscopy (ESR). Tan and colleagues (117) used ESR to verify that
melatonin reduced adduct formation, in this case DMPO-·OH,
in the following reaction:
Normally, the exposure of hydrogen peroxide (H2O2) to 254
nm ultraviolet light causes the formation of the ·OH.
However, ·OH rapidly
interact with each other because they each possess an unpaired e- in
what is referred to as termination reactions. Thus, a spin trapping
agent, in this case 5-5-dimethyl-pyrroline N-oxide or DMPO,
was added. The spin trap forms an adduct with the generated ·OH
(DMPO-·OH) which is
quantified by ESR (90). The number of DMPO-·OH
adducts formed is used as an index of ·OH
generation. In this scheme, the addition of melatonin to the
reaction mixture reduced the formation of the DMPO-·OH
adducts illustrating the indole scavenged the ·OH
(117). Furthermore, this group showed that melatonin was more
effective than either reduced glutathione, an endogenously
synthesized ·OH radical
scavenger, or mannitol, an exogenously produced ·OH
scavenger, in reducing DMPO-·OH
These seminal observations led to a series of studies in which the ·OH
scavenging activity of melatonin was confirmed. Thus, also using ESR
technology, Matuszek et al. (58) and Susa and colleagues (116)
re-affirmed the high efficiency of melatonin as a ·OH
radical scavenger. Likewise, Stasica and co-workers (114) also
reported that melatonin reduces ·OH
generation in vitro. That indoles with a structure similar to
melatonin may also have free radical scavenging activity, although
less than that of melatonin, was demonstrated in the studies by Tan
and co-workers (117), Poeggeler et al. (85) and Matuszek et al. (58)
The ability of melatonin to scavenge the ·OH
was also shown by Pahkla et al. (73) who used yet another
endpoint to estimate the ·OH
scavenging activity of melatonin. Thus, they used terephthalic acid
(THA) as a chemical index of ·OH
formation since it forms an adduct, i.e., THA-·OH,
which can be quantified. Melatonin, in a concentration-dependent
manner, inhibited the formation of THA-·OH
indicating reduced ·OH
availability. In this system the concentration of melatonin required
to inhibit ·OH formation by
50%, i.e., the IC50, was 11.4 +/-
1.0 µM (73). This is similar to
the IC50 value (21 µM)
reported by Tan et al. (117) where melatonin was found to reduce
DMPO-·OH adduct formation.
Melatonin has also been shown to be a powerful inhibitor of ·OH-mediated
macromolecular damage (94, 103, 104). Hence, H2O2
toxicity, which is widely accepted to be a consequence of the
conversion of H2O2 to ·OH
(61), is readily suppressed by the addition of melatonin to tissue
homogenates (25, 109, 112). This is consistent with the observation
of Li and co-workers (50) who showed that, in vivo, melatonin
scavenges the ·OH as
indicated by melatonin's suppression of dihydrobenzoic acid
(DHBA) in the microdialysate collected from brains undergoing
Even more definitive proof of melatonin's in vivo ·OH
scavenging activity was provided by the recent observation that the
administration of melatonin to animals subjected to ionizing
radiation increased the amount of cyclic 3-hydroxymelatonin in their
urine (118). Using a combination of mass spectrometry (MS), proton
nuclear magnetic resonance (lH-NMR), thermodynamic
stability studies and high performance liquid chromatography with
electrochemical detection (HPLC-EL), we showed cyclic
3-hydroxymelatonin to be an end product of the interaction of
melatonin with two hydroxyl radicals (Fig. 2). This end product was
found to be excreted in the urine of man and rats and when the latter
species was exposed to ionizing radiation, a procedure known to
generate the ·OH in vivo
(70), the quantity of cyclic 3-hydroxymelatonin increased
incrementally in the urine. This provides direct evidence that when
·OH are generated in vivo,
melatonin neutralizes them resulting in the formation of a metabolite
that is excreted in the urine and is a footprint of ·OH
generation (118). This will likely become a useful method for
estimating in vivo ·OH
production, something that has been difficult to access to date. The
findings are also consistent with the repeated demonstration that
melatonin reduces chromosomal damage induced by ionizing radiation
Besides scavenging the most toxic of the radicals produced, i.e., the ·OH,
melatonin may also directly quench some other reactive oxygen
metabolites. Singlet oxygen (1O2), which is
formed by the addition of energy to ground state O2 (Fig.
1), although not a free radical, exhibits considerable toxicity and
is capable of oxidizing polyunsaturated fatty acids and DNA (31). In vitro
melatonin has been shown to reduce oxidative damage to neural
lipids induced as a result of treatment of the animals with a
photosensitizer, rose bengal, followed by the exposure of the neurons
to bright light (20). Although this evidence is indirect, certainly
the most likely explanation for melatonin's ability to reduce
the products of lipid peroxidation and apoptosis in this model is its
ability to quench 1O2 (86).
The peroxynitrite anion (ONOO-) is a non-radical but highly
toxic molecule that is formed from the combination of the superoxide
anion radical (O2•-) (Fig. 1) and nitric oxide (NO·)
(Fig. 3) (107). Besides its intrinsic toxicity, ONOO-
degrades into other agents that are equally or more highly toxic than
the parent molecule (91); one of these products may be the ·OH.
That melatonin effectively neutralizes the ONOO- was
recently shown by Gilad and co-workers (34). This group found that
the ability of ONOO- to oxidize dihydrorhodamine 123 was
reduced in the presence of melatonin in a dose-dependent manner.
They further found, using J774 macrophages, that melatonin reduced
DNA strand breaks and reversed the inhibition of mitochondrial
respiration resulting from ONOO- treatment. When compared
with other ONOO- scavengers, i.e., reduced glutathione and
cysteine, melatonin was found to be equally as effective. These
studies were further extended by Cuzzocrea et al. (26, 27) who
demonstrated that melatonin greatly reduced the inflammatory response
when animals are treated with carrageenan, where both ONOO-
and NO· are believed to be
the mediators of the inflammation (10). That melatonin scavenged the
ONOO- was demonstrated by a highly significant reduction
in the formation of nitrotyrosine, a footprint molecule in ONOO-
formation (26, 27).
has both positive and negative actions in vivo. It is known to
function as an important endothelium relaxing factor in small blood
vessels and as a gaseous neurotransmitter in the brain (29).
However, in high concentrations it may be responsible for the
damaging effects of ischemia-reperfusion injury and, as noted above,
it can combine with O2·-
to produce the ONOO-, a molecule with well known toxicity.
Besides scavenging ONOO-, as discussed above, Noda and
colleagues (67) recently reported that melatonin directly scavenges
NO· in vitro. While the
significance of this finding has not yet been demonstrated in vivo,
considering the widespread actions of NO·,
especially in the central nervous system, the ability of melatonin to
scavenge it may be significant particularly in terms of reducing its
A major damaging agent of cellular physiology is the peroxyl radical
(LOO·) which is generated
during the peroxidation of lipids which are in high concentrations in
cellular membranes (23). The LOO·
has devastating consequences that are in part due to the fact that
once formed during the process of lipid peroxidation it is
sufficiently reactive so as to re-initiate (propagate) the processes;
thus, theoretically at least once underway the process of lipid
peroxidation would continue until all the lipid molecules in the
membrane are oxidized (Fig. 4). This chain reaction can be
interrupted by antioxidants such as vitamin E which readily scavenges
the LOO· and protects
against lipid peroxidation. Damaged lipids have marked consequences
on membrane functions since they alter electrolyte transport, the
activity of membrane-bound enzymes, channel functions, and membrane
receptor processes. As the lipid moities of membranes become
increasingly oxidized the membranes themselves become less fluid (32,
33). This rigidity of the membrane is a common feature of aging
Melatonin's role in reducing lipid peroxidation is assured by the fact that it
scavenges 1O2, ONOO- and ·OH,
all of which are sufficiently reactive to initiate the peroxidative
process. Evidence has also been presented, however, that melatonin
functions as a chain breaking antioxidant by directly scavenging the
LOO· (81, 82). Indeed, it
was this group's conclusion that melatonin is twice as
effective in scavenging the LOO·
as is vitamin E, the primer chain-breaking antioxidant. This
finding, if valid, would make melatonin the most effective chain
breaking antioxidant discovered to date.
Confirmation of this finding, however, has not been forthcoming although several
reports have shown that melatonin may have some LOO·
scavenging activity. According to Scaiano (108), melatonin is
probably equivalent to vitamin E in neutralizing the peroxyl radical.
In studies by Marshall and colleagues (56) the high efficacy of
melatonin in scavenging the LOO·
as indicated by Pieri et al (81, 82) was not totally confirmed. The
results of these reports are, however, difficult to directly compare
since very different model systems were used to evaluate the ability
of melatonin to scavenge the LOO·.
A recent study also concluded that melatonin's ability to
scavenge the LOO· is limited
and probably not on a par with vitamin E (5).
While the efficacy of melatonin as a direct LOO·
scavenge is debated, there is no doubt, especially in vivo, that it
markedly inhibits lipid peroxidation due to a wide variety of toxins
(21, 28, 63, 64, 89, 97). Whether this ability to limit the
breakdown of lipids is solely the result of melatonin's ability
to scavenge the initiating radicals or whether it also functions as a
chain breaking antioxidant remains to be resolved. Besides reducing
damaged lipid products in cellular membranes melatonin also prevents
the changes in membrane fluidity which accompany the breakdown of
polyunsaturated fatty acids (32, 33), thereby helping to preserve
normal membrane and cellular physiology.
Besides the ability of melatonin to detoxify a variety of reactive,
oxygen-based free radicals, it also indirectly reduces molecular
damage by stimulating the metabolism of potentially toxic molecules
to non-toxic products. At least two antioxidative enzymes metabolize
H2O2, the precursor of the ·OH,
to H2O. These enzymes are not equally distributed in
cells but both catalytically remove H2O2 from
the intracellular environment. One of these, glutathione peroxidase
(GSH-Px) (Fig. 5) is selenium dependent and has been repeatedly shown
to be stimulated by melatonin (8, 71, 72). When activated GSH-Px
oxides reduced glutathione (GSH) to its disulfide form (GSSG) and in
the process it uses H2O2 and other
hydroperoxides as substrates, thereby reducing their concentrations.
GSSG is converted back to GSH by the enzyme GSSG reductase (GSSG-Rd),
a reaction that depends on the availability of NADPH. GSSG-Rd is
also reportedly stimulated by melatonin ensuring that GSSG is
recycled back to GSH (72). Finally, NADPH is replenished by the
action of glucose-6-phosphate dehydrogenase (G-6PD), another
antioxidative enzyme reported to increase its activity in response to
melatonin (84). Besides removing H2O2 from
cells, GSH-Px also functions as a peroxynitrite reductase thereby
metabolizing ONOO- to a non-toxic product.
A major antioxidative family of enzymes which metabolizes O2·-
are the superoxide dismutases (SOD). These rapidly convert O2·-
to H2O2 thereby restricting the interaction of
O2·- with NO·
and limiting the inherent toxicity of O2·-.
Melatonin has now been shown to stimulate both mRNA for SOD as well
as increasing the activity of the enzyme (4, 48). In so doing,
melatonin would be expected to increase the intracellular
concentrations of H2O2 which would normally
enhance the formation of the highly toxic ·OH
[this is what happens normally in individuals with trisomy 21 (Down
syndrome) where SOD activity is increased 1.5-fold]. However,
besides stimulating SOD activity, as already mentioned, melatonin
also augments GSH-Px activity and reduces H2O2
thereby decreasing the likelihood of ·OH
formation by concurrently increasing the formation of H2O2
from O2·- and
augmenting its metabolism to non-toxic products. In this way
melatonin can avert the macromolecular damage which results when only
SOD activity is increased.
There is also one potentially pro-oxidative enzyme, nitric oxide synthase
(NOS), which is inhibited by melatonin. As noted above NO, which is
synthesized from arginine under the influence of NOS, has some
intrinsic toxicity and furthermore it degrades into the ONOO-
(when it combines with O2·-).
Thus, the formation of NO is sometimes a pro-oxidative process. At
both physiological and pharmacological concentrations in vivo
melatonin is known to inhibit neuronal NOS activity and
consequentially the accumulation of excessive NO (13, 87, 88). This
action of melatonin contributes to the indirect means by which the
indole is capable of reducing oxidative stress, at least in the
central nervous system.
Age-related Changes in Melatonin
While there may be a variety of organs that synthesize melatonin, the
concentrations of this indole in the blood are usually considered to
be primarily or wholly derived from the pineal gland under usual
conditions (92). This is quite remarkable since the retinas also
produce melatonin in a rhythm reminiscent of that seen in the pineal
gland, but unlike the pineal, they are incapable of releasing the
synthetic product or it is locally metabolized before it is released.
In the pineal gland, melatonin is produced almost exclusively during the
night and it seems to be immediately released after its synthesis
resulting in nocturnal blood levels that are substantially higher at
night than during the day (92). This rhythmic pattern of melatonin
production and release is common to all vertebrates. While the
rhythm is typically not present in newborns, it appears soon
thereafter. From infancy to middle-aged the melatonin rhythm is
detectable in the blood of mammals although in humans their may be
large variations in the nocturnal melatonin increase even among
individuals of the same age (6).
Absolute levels of melatonin in the blood are usually measured in pg•ml-l
with these values being below 20 pg•ml-I during the
day up to 150-200 pg•ml-l at night. Relatively
little is known about intracellular concentrations of melatonin
although under some circumstances these values can exceed those
measured in the blood by a wide margin (62). The low values of
melatonin in the blood would seem to detract from the possibility
that physiological concentrations of the indole are relevant in terms
of the total antioxidative capacity of the organism. When the total
antioxidant status of the serum of rats and humans are compared with
the concentration of melatonin in the same samples, however, there is
a very high correlation (11, 12). This is consistent with the
observations that endogenously produced melatonin is sufficient to
provide partial protection against agents or processes which greatly
increase oxidative damage (55, 120). Thus, the antioxidative
capacity of melatonin seems not to be a phenomenon exclusively
related to the administration of pharmacological concentrations of
the indole, but rather the amount of melatonin produced endogenously
is important in providing significant protection against the
devastating actions of free radicals.
In light of the seemingly relative importance of endogenously produced melatonin
in terms of antioxidative defense, depression in melatonin production
due to any cause could be significant relative to protection from
free radicals. One factor that leads to a reduction in melatonin is
aging (93). In every mammalian species where the melatonin synthetic
and secretory capability of the pineal gland have been measured they
diminish in advanced age and this is
associated with low levels of melatonin in the blood even at night. Of the
mammals studied to date the rat (75, 98), Syrian hamster (74, 101)
(Fig. 6) and Mongolian gerbil (99) have all exhibited a reduction in
the pineal synthesis of melatonin after middle age. Similar studies
in humans using either blood levels of melatonin or the excretion of
the chief enzymatic metabolite of melatonin, 6-hydroxymelatonin
sulfate, as endpoints have yielded similar findings (41, 66, 106,
123). The conclusion from these studies is that elderly individuals
produce less pineal melatonin and, due to the loss of this
antioxidant, they are increasingly vulnerable to free radical damage.
The drop in melatonin in advanced age may come at a highly
non-propitious time since it has been shown that free radical
generation increases in aged individuals (129). Thus in a since
melatonin may be lost when it is needed most. Whether in fact the
reduced melatonin production is consequential in terms of the obvious
age-associated, free radical-based diseases that commonly occur
simultaneously is unknown but is being actively investigated.
Furthermore, although the suggestion has been made that secretory
products of the pineal gland may be able to defer aging per se (69,
83) the experimental data are in complete and, at this point, not
compelling (100, 102).
Melatonin and Age-related Diseases
There are a host of age-related diseases that are in part related to free
radical damage and, therefore, could possibly be deferred by the use
of a proper antioxidant regimen. Only a small number of these
diseases will be considered here.
One of the most common surgical procedures performed in the elderly is
the replacement of the opaque lenses of the eyes. The opaqueness,
i.e., cataracts, are classical free radical damage to macromolecules
in the lens which leads to molecular deterioration and cloudiness
causing these individuals to loose their sight (43). Since cataracts
are free radical-based, one may anticipate that their development
would be deferred by antioxidants. We have in fact tested an animal
model of cataracts and found that indeed melatonin can prevent
experimental cataract induction (1, 51). In this model newborn rats
are treated with the drug buthionine sulfoximine (BSO) which inhibits
gamma-glutamyl cysteine synthase, the rate limiting enzyme in the
production of an important antioxidant, reduced glutathione (GSH).
As a result of this treatment the animals develop grossly observable
cataracts by the time they are 2 weeks of age, i.e., several days
after their eye lids open. Newborn rats, however, produce very
little melatonin (at least in their pineal gland) during the first 2
weeks of life. Thus, in the experiment described, the newborn rats
were actually deficient in two important antioxidants, GSH and
melatonin. This being the case, we surmised that if newborn rats who
developed cataracts because they were GSH deficient (and melatonin
deficient) were given melatonin daily during the experimental period,
the cataracts would not form. This predicted outcome was in fact
realized. Melatonin given as a single daily injection prevented the
development of cataracts in newborn rats that had been treated with
BSO to deplete their GSH. Thus, the antioxidant melatonin had
adequately substituted for the antioxidant, GSH thereby preventing
free radical damage to macromolecules in the lens and inhibiting the
development of cataracts (1, 51).
On the basis of these studies, it may be theorized that cataractogenesis
in humans, which typically occurs in advanced age when endogenous
melatonin levels are depleted, may be related to the loss of this
important antioxidant. Since no tests have yet been performed to
examine the effects on melatonin on human cataractogenesis, however,
the assumption that opaquification of the human lens is related to
the reduction in melatonin remains precisely that, an assumption, and
would seem to be worthy of examination.
Many types of cancer increase in frequency as individuals age. In many
cases, the initiating events, i.e., the damage to nuclear DNA, is
mediated by free radicals (3, 52). If the damaged DNA undergoes
mutation, cancer can be a consequence. Not only are free radicals
often involved in the initiation of cancer but their promotion may
also involve free radicals (22). Finally, recent evidence suggests
that the transformation of benign cancers to their more malignant
forms may relate to the continuing oxidative damage sustained by
nuclear DNA (54).
Since melatonin has been found in the nucleus (30, 62), it is in a position
to scavenge free radicals that could potentially damage DNA. The
initial studies which examined melatonin's ability to reduce
oxidative damage to DNA were published roughly 5 years ago (119,
120). In this case, rats were treated with the chemical carcinogen
safrole with and without concurrent melatonin administration. When
damaged DNA adducts were measured in the liver of these rats,
melatonin had proven highly effective in reducing the quantity of DNA
adducts that had formed. The presumed mechanism of the reduced DNA
damage related to the ability of melatonin to detoxify the free
radicals generated during the metabolism of the carcinogen before
they mutilated the nearby DNA.
If in fact melatonin is an effective ·OH
scavenger as has been claimed, one would expect it to protect against
genomic damage that accompanies the exposure of animals to ionizing
radiation. Ionizing radiation is known to cause the homolytic
scission of H2O molecules resulting in the formation of
·OH which then go on to
damage DNA as well as other cellular macromolecules (70). In all
experiments performed to date, melatonin has proven highly effective
in reducing cytogenetic damage in cells exposed to either ionizing
radiation (125-128) or radiomimetics (7, 65). Furthermore, the death
rate in mice given a lethal dose of radiation has been shown to be
reduced when animals are pretreated with melatonin (124). These
findings are consistent with melatonin being an effective
radioprotective agent in part related to its ability to curtail
oxidative damage to nuclear DNA. In so doing, melatonin would also
be expected to suppress cancer initiation. Using the same endpoints
to assess DNA damage, Sewerynek and colleagues (110) and Melchiorri
et al. (60) have shown that melatonin also protects against the
genotoxic actions of bacterial lipopolysaccharide and paraquat,
Still other scientists have used other techniques to estimate the efficacy
of melatonin in protecting against oxidative damage to DNA.
According to Susa and colleagues (116), DNA strand breaks normally
associated with the exposure of hepatocytes to chromium (VI) were
prevented when melatonin was also present in the incubation medium.
In this case the authors concluded that melatonin was a potent
protector of DNA against such damage. Finally, using measures of
8-hydroxydeoxyguanosine (8OHdG), a documented damaged DNA product,
Tang and co-workers (121) found melatonin to reduce in vivo 8OHdG
levels in rat liver and brain after their treatment with the free
radical generating agent kainic acid.
Again, these studies like those summarized in the preceding paragraphs,
suggest that melatonin would decrease the incidence of cancer which
is initiated as a result of free radical damage. Heretofore it has
been assumed that melatonin's protective effect against cancer
related to its ability to reduce the growth of growing cancers, a
phenomenon that has been thoroughly investigated (14, 76). In light
of the data summarized above, however, it seems that reducing both
the initiation and promotion of tumors may be within the capability
of melatonin. These findings should then be considered in the
context of the aged individual where endogenous melatonin production
may be minimal.
Neurodegenerative diseases occur more frequently now than earlier in the century. The
major reason for this presumably is that humans are living
progressively longer allowing these diseases of the aged to become
manifested. This being the case, it is anticipated that the
prevalence of these conditions will continue to rise inasmuch as
people are surviving longer and longer. While neurodegenerative
conditions often have complex etiologies there is widespread
agreement that many of the disease processes involve free radicals
(Table 2). In the current review only the association of melatonin
with models of Alzheimer's disease, Huntington's disease
and Parkinsonism will be considered.
Alzheimer's disease, formally referred to as presenile dementia, afflicts
approximately one of 20 people over 65 years of age while by 85 this
increases to one in three individuals. This disorder is extremely
debilitating and is characterized by a gradual decline in virtually
all brain functions including memory, judgement, behavior,
personality, abstract thinking, language and motor skills. The
deterioration can be slow (over a 7-10 year period) and less
frequently rapid. In the latter stages of the disease the subjects
often have a feeling of anxiety, restlessness and agitation in the
later afternoon, a collection of signs referred to as sundowning. A
large amount of research suggests that the deposition of a product
called -amyloid peptide is a
causative factor in Alzheimer's disease. The toxicity of
-amyloid has been linked to the
generation of oxidative stress. While -amyloid
peptide is a 39-43 amino acid chain, the 25-35 amino acid residue is
the active portion of the molecule in terms of free radical
In a series of two reports we (79, 80) we found that melatonin readily
inhibited apoptosis of neurons incubated in the presence of either
the 25-35 amino acid residue of -amyloid
or the complete peptide. All measures performed on the incubated
neurons revealed that melatonin had prevented the oxidative damage
associated with the exposure of the cells to amyloid-.
These findings are perhaps of particular significance because of the
recent finding that melatonin levels are very low in the
cerebrospinal fluid of Alzheimer's patients (53).
Besides reducing the toxicity of -amyloid,
melatonin may interfere with the formation of -sheets
and amyloid fibrils which make the peptide especially toxic (49, 78).
Of these two reports, that of Pappolla and colleagues is more
complete and relevant because of the concentrations of melatonin
used. Nevertheless, both these studies suggest an additional
mechanism (besides the direct scavenging of free radicals) by which
melatonin may reduce neuronal death in the Alzheimer's brain.
A couple of reports have appeared where melatonin has actually been
given as a supplement to individuals suffering with Alzheimer's
disease. The studies included a rather small number of subjects and
they were not conducted in a double-blind manner. Significantly,
however, both of these studies indicated that giving as little as 6
mg melatonin daily over a 36 month period delayed the progression of
the disease, based on their behavioral assessment on standardized
tests, and improved the general well being of the Alzheimer's
patients (16, 17).
Huntington's disease (or Huntington's chorea) is an autosomal dominant
condition in which neurodegeneration is a major feature (122). This
disease usually first manifests itself in middle-aged individuals and
is characterized by motor disturbances, behavioral changes and
dementia. Commonly, brain weight may be reduced by 20% when these
individuals die (122).
A causative factor for this debilitating disease is believed to be the
excitotoxic tryptophan metabolite, quinolinic acid (9). In the study
in question, quinolinic acid was injected directly into the striatum
of the rat, a treatment which reproduced the neuropathyology observed
in the brains of individuals' who died with Huntington's
disease. These observations have led to the wide spread use of
quinolinic acid administration as an experimental model for this
condition. As an excitatory neurotoxin, quinolinic acid is believed
to damage and kill neurons via free radical mechanisms after it
interacts with the N-methyl-D-aspartate (NMDA)
receptor. It is especially damaging since it is not metabolized and
therefore it acts on the receptors for a prolonged period (115).
Given that free radicals are the presumed destructive neuronal agents in
the Huntington's disease model, Southgate and colleagues (113)
assumed that melatonin may provide protection against the toxicity of
quinolinic acid much like it did in the models of Alzheimer's
disease. After the injection of quinolinic acid directly into the
hippocampus of adult rats, the excitotoxin caused distinctive
morphological changes in the pyramidal neurons with 5 days.
Additionally, they measured a reduction in glutamate receptor number
in the hippocampus following quinolinic acid administration. When
melatonin was given just prior to excitotoxin administration, neither
the morphological changes nor the alterations in glutamate receptor
numbers were apparent; the authors theorized that melatonin's
antioxidant activity accounted for its ability to protect against
Although the cause of Parkinson's disease remains to be clarified, it is
clearly age-related with an age of onset being between 40-65 years.
This disorder is characterized by tremor, rigidity, slowness of
movement and difficulty in walking. That oxygen free radicals are
involved in the destruction of neurons in individuals with
Parkinsonism is now widely accepted. The neurons that exhibit the
most marked reduction are the dopaminergic neurons of the pars
compacta of the substantial nigra.
For the experimental induction of Parkinson's disease,
1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine (MPTP) is the agent most
frequently used. After its uptake by glial cells, MPTP is
metabolized to the methyl-4-phenyl pyridinium cation (MPP+) which is
then released and taken up by dopaminergic neurons where it generates
destructive free radicals. This model has been used by
Acuna-Castroviejo et al (2) to test the efficacy of melatonin
in curtailing the toxicity of MPTP. Melatonin was found to limit the
loss of dopamine and the reduction of striatal tyrosine hydroxylase
activity observed in the MPTP-treated mice not given melatonin.
Likewise, the neural lipid peroxidation induced by MPTP was prevented
by melatonin. These findings are consistent with the antioxidative
actions of the indole. When the same biochemical endpoints were
measured, Kim et al. (45) also showed that melatonin reduced the
damage as well as the biobehavioral changes in rats treated with
6-hydroxydopamine (6OHDA); this agent also destroys dopaminergic
neurons and induces Parkinson-like signs in experimental animals.
Besides the in vivo studies described above, two reports that used in vitro
methods have confirmed the neuroprotective effects of melatonin
against MPP+ and 6OHDA toxicity (40, 59). Both these studies
monitored similar endpoints and concluded that melatonin's
protective actions most likely related to its ability to detoxify
free radicals induced by the respective toxins.
This review is by no means exhaustive in terms of agents and processes,
all of which are believed to involve free radicals, against which
melatonin has been found to be protective. Rather, the examples
summarized herein were chosen to illustrate how melatonin may relate
to the progression of age-related diseases and to aging itself. It
is obvious from the reports discussed herein that the bulk of the
studies have involved experimental animals and that the number of
clinical studies is small. Considering the overwhelmingly positive
nature of melatonin's protective actions in these experimental
studies, it would seem clinical trials are now warranted. This would
seem justified considering the virtual absence of toxicity of
melatonin and its apparent numerous beneficial effects. Furthermore,
several of the diseases against which it could be tested have no
known effective treatments. Finally, the importance of these studies
is further emphasized by the fact that humans are surviving
progressively longer and, as a result, some of the most devastating
age-related diseases will continue to increase in frequency. Unless
successful treatments can be found for these conditions the burden to
society, both from the standpoint of effective care as well as
financially, may become insurmountable. Studies to date indicate
melatonin may be able to relieve part of this burden.
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Fig. 1. The generation
of oxygen-based free radicals and reactive oxygen species from ground
state molecular oxygen (O2) involves a variety of
permutations as illustrated in this figure. A single electron (e-)
reduction of O2 produces the superoxide anion radical
(O2-·) which can
either combine with nitric oxide (NO·)
to produce the peroxynitrite anion (ONOO-) or it is
dismutated (by a family of enzymes, the superoxide dismutases) to
generate hydrogen peroxide (H2O2). In the
presence of a transition metal, such as Fe2+ or Cu1+,
H2O2 is converted, via the Fenton reaction, to
the hydroxyl radical (·OH).
Another toxic O2 by-product is singlet oxygen (1O2)
which is formed by the addition of energy to O2.
Fig. 2. Melatonin is a
highly efficient scavenger of the hydroxyl radical (·OH),
generally considered the most toxic by-product of oxygen metabolism.
We have recently shown, in fact, that each melatonin molecule has the
capability of scavenging two ·OH
with the "footprint" molecule being cyclic
3-hydroxymelatonin. This product is then excreted in the urine and
its amount can be used as an index of ·OH
generation in vivo.
Fig. 3. Nitric oxide
(NO·) readily combines with
the superoxide anion radical to produce the peroxynitrite anion
(ONOO-). Although not a free radical, ONOO- is
highly toxic to a variety of macromolecules and furthermore it
degrades into dangerous by-products including the hydroxyl radical
Fig. 4. The
peroxidation of polyunsaturated fatty acids (PUFA), many of which are
located in the membranes of cells, is extremely devastating to
cellular physiology. During the initiation phase a hydrogen atom
(H+) is extracted from a PUFA to produce a lipid radical (L·)
which eventually leads to the generation of the peroxyl radical
(LOO·). The peroxyl radical
is sufficiently reactive that it can oxidize an adjacent PUFA, i.e.,
it propagates the process of lipid peroxidation. Thus, because of
this recurring cycle the oxidation of a single PUFA could
theoretically lead to the breakdown of all the fatty acids in a cell.
The best known scavenger of the LOO·
is vitamin E. How effective melatonin is as a chain breaking
antioxidant is being debated.
Fig. 5. Hydrogen
peroxide (H2O2), while not being a free
radical, is highly dangerous because it degrades into the hydroxyl
radical (·OH) (see Fig. 1).
Fortunately, much of the H2O2 generated is
metabolized to H2O by the actions of two detoxifying
enzymes, catalase and glutathione peroxidase. Melatonin has been
shown to stimulate the activity of glutathione peroxidase which
converts reduced glutathione (GSH) to its oxidized form (GSSG). GSSG
is catalytically metabolized back to GSH by the enzyme glutathione
reductase, which is also reportedly stimulated by melatonin. A major
source of H2O2 in cells is produced by the
dismutation of the superoxide anion radical (O2-·),
a conversion that requires one of a family of superoxide dismutases
Fig. 6. In those
mammals where it has been studied, pineal melatonin production
diminishes with increased age. Shown here are the pineal melatonin
rhythms in 2 month old and 18 month old female Syrian hamsters.
Clearly, in the old animals the nocturnal peak of melatonin is
attenuated. A similar reduction in pineal melatonin production has
been observed in the human.
Table 1. Calculated rate
constants (Kr) for the scavenging of the ·OH
by melatonin and related methoxylated and hydroxylated indoles. Data
from Tan et al. (1993), Poeggeler and colleagues (1996) and Matuszek
et al. (1997).
2.7 +/- 0.3
2.3 +/- 0.1
1.95 +/- 0.1
1.7 +/- 0.3
1.1 +/- 0.3
2. Neurodegenerative conditions, most of which are associated with
the aging population, are believed in many cases to involve free
radicals as part of the disease process. It should be noted that
whereas the following list of conditions can be at least in part
ameliorated by appropriate free radical scavengers and antioxidants,
it is not likely that a single antioxidant will totally prevent any
of these conditions.
Amyotrophic lateral sclerosis
Spinal cord injury
Vitamin E deficiency
Xenobiotic-induced nerve injury