NeurosctenceandBtobehamoralRevtews,Vol. 17, pp. 347-357, 1993 0149-7634/93$6.00 + .

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The Significance of the Metabolism of

the Neurohormone Melatonin:
Antioxidative Protection and
Formation of Bioactive Substances

R. H A R D E L A N D , ' R. J. R E I T E R , 2 B. P O E G G E L E R A N D D.-X. T A N

Department o f Cellular and Structural Biology, The University o f Texas Health Science Center,
San Antonio, T X 78284-7762

(Received 1 April 1992)

HARDELAND, R., R. J. REITER, B. POEGGELER AND D.-X. TAN. The significance of the metabolism of the
neurohormone melatonin: Antzoxldative protection and formatton of btoacttve substances. NEUROSCI BIOBEHAV REV
17(3) 347-357, 1993--Recent findings suggest that the ability of melatonin to enter all body tissues and to be metabolized,
enzymaticallyor nonenzymaucally, in any of them results in a spectrum of effects, which exceed substantially those transduced
by membrane receptors. These actions comprise the formation of various bioactive compounds such as N-acetylserotonin,
5-methoxytryptamlne, N,N-dimethyl-5-methoxytryptamine, 5-methoxytryptophol, cyclic 2-hydroxymelatonin, pinoline, and
5-methoxylated kynuramines. Apart from enzymaUcmetabohsm, nonenzymatic reactions with free radicals, in particular the
superoxide anion and the hydroxyl radical, represent a new and significant aspect of melatonin's biological role. Melatonin
represents the most potent physiological scavenger of hydroxyl radicals found to date, and recent findings suggest an essential
role of this indoleamine for protection from hydroxyl radical-induced carcinogenesisand neurodegeneration.

Aging B-Carbolines Carcinogenesis Free radicals Indoleammes Kynuramines

Pineal gland Psychotogens

INTRODUCTION Another reason for assuming further functions of melato-

THE pineal hormone melatonin has attracted attention
particularly with regard to its role as a mediator of the photo-
periodic information "darkness," a role that necessarily leads
to a kind of physiological pleiotropy. Melatonin controls cir-
cadian rhythmic organization (14,70,109,134) with its numer-
ous oscillatory functions at cellular, organ, and behavioral
levels, and it determines annual cyclicity, especially in seasonal
breeders (4,107-109,129). Most often, effects of melatonin
have been studied with regard to the hypothalamic-hypo-
physeal-gonadal axis, including aspects of inhibition of the
gonadotropin releasing hormone pulse generator, puberty,
aging, and various pathologies (4,83,84,86,91,99,107-111,
I 16,123,129,138). The spectrum of actions exerted by melato-
nin seems to exceed the purely periodic phenomena. One as-
pect of particular significance, the influence of melatonin on
the immune system, has now repeatedly been documented
(25,26,34,57,71).
nin results from its presence in nonpineal tissues, in particular,
the r e t i n a - f r o m which it appears not to be abundantly se-
c r e t e d - (3,13,32,93,142), the Harderian gland (46,74,93), the
gut (12,100,135), and ieukocytes (25). Temporal patterns and/
or kinetics of production and release are different in these
organs from those known for the pineal gland.
The pleiotropy of actions is obviously not limited to the
transduction of photoperiodic information by the molecule
melatonin itself; rather, a considerable sector within the spec-
trum of effects seems to be due to its metabolism to active
metabolites. With regard to the physiological consequences of
the biochemical reactions melatonin can undergo, and to the
substances formed by the respective pathways, two aspects of
particularly great potential significance can be distinguished.
On the one hand, melatonin possesses unique properties as a
radical scavenger; this capability strongly suggests a protec-
tive, especially, neuroprotective role. On the other hand, sub-
stances are generated from melatonin, both via enzymatic and

Present address: I. Zoologisches Institut der Univers~tatGottingen, Germany.

2 To whom requests for reprints should be addressed.

347
348 H A R D E L A N D ET AL.

nonenzymatic reactions, which act as physiological effectors
themselves (e.g., other methoxyindoles,/~-carbolines, and ky-
nuramines).
PHYSICOCHEMICALAND PHYSIOLOGICAL
PROPERTIES OF MELATONIN
Due to its hydrophobicity, melatonin can easily permeate
any biological membrane. Consequently, it appears in every
tissue and body fluid in concentrations of the same order of
magnitude as in blood plasma. The capability to enter all cells
may be of particular importance for the following reasons.
Although receptor proteins for melatonin have been identified
and characterized m and isolated from plasma membranes of
certain brain regions (23,65,113,125,126), these membrane-
bound receptors obviously transduce only part of the informa-
tion carried by the hormone. In this context, one should be
aware of the fact that the receptors mentioned have usually
been identified using iodinated melatonin, and, although they
efficiently bind melatonin in competition studies (23,65),
other binding sites may have a lower affinity for the iodinated
molecule or they may remain inaccessible to the ligand if they
are localized in the interior of the cell. Another type of binding
site with fairly high affinity has already been demonstrated to
stimulate Na+/K+ATPase (15), that is, an enzyme ubiqui-
tously distributed in neuronal and nonneuronal cells. More-
over, any effect exerted by melatonin metabolites would be
overlooked when considering exclusively the signal-transduc-
tion pathway associated with the previously described mem-
brane receptor.
Apart from its hydrophobicity, the melatonin molecule has
other properties which are of importance for metabolic con-
siderations. The 5-methoxy group, by which melatonin differs
from 5-hydroxylated and nonsubstituted indoles (Fig. 1), re-
sults in a considerable increase in radical-trapping capacity,
especially at physiological pH (42,132). Moreover, it shields
the molecule from other reactions (e.g., from dimerization)
presumably by preventing phenoxylic radical formation (47),
and it prevents the formation of quinone imine structures
(88,128). What the methyl group does not do, however, is
prevent interactions with serotonin receptors, because 5-
methoxytryptamine and some of its derivatives (e.g., N,N-
dimethyl-5-methoxytryptamine) efficiently bind to these re-
ceptors, though with different consequences (inhibitory or
stimulatory) at the various subtypes (31,67,13 I). The N-acetyl
group, therefore, obviously represents that portion of the mol-
ecule which prevents substantial binding to serotonin recep-
tors. Moreover, the N-acetyl group protects melatonin from
degradation by monoamine oxidase (MAO), a fact that distin-
guishes this indoleamine from its analogue, 5-methoxytrypta-
mine (30,31,106). Additionally, the acetyl group participates
in the cyclization of the aliphatic residue (136). Finally, as
will be discussed below, comparisons of reactivity between
melatonin and 5-methoxytryptamine reveal that the acetyl
group contributes much to the radical scavenging capacity,
though this may not be obvious at the first glance (Tan, D.-X.;
Chen, L.-D.; Poeggeler, B.; Manchester, L.C.; Reiter, R.J.,
unpubl.; Hardeland, R., unpubl.). The pyrrole ring represents
one of the most important reactive parts of the molecule,
because it can be opened by peroxidative cleavage, either enzy-
matically (43,45) or by nonenzymatic mechanisms involving
free radicals (41,42; Tan, D.-X., et al., unpubl.); opening of
the ring results in the formation of an interesting and poorly
understood class of biogenic amines, the kynuramines (41-
43,45).
When considering physiological functions of melatonin
and of its metabolites, the circadian profile of this neurohor-
mone must be taken into account. While in some tissues mela-
tonin concentrations are subject to only slight temporal varia-
tions, as in the Harderian gland (46,74,93), the circadian

HO~ CH2~CH2~N~ cH3 HO~ CHZ-c H'z--NH'z HO~ CH2"~CH2

'-'OH
NM'r MAO ADH
ID
9 4 v ~I $11t~b~r.n P
H
NAT

O
~ CHz--CH2--NH--C.--CH
3
v "N- N-i~d,
ylserolonm
HIOMT HIOMT H HIOMT

H3CO ~ II
I ~ ~ CH'~C~H3

H3 C O ~ CH~:T_CHL:~N~ cH3 H3CO

NUT ~ CH2-'C~z-N~ ~o ADH H3COi ~ ~ " ~ CHz--CH'z--~

~N/J NN-d~yl- CH3 ~ ~ ' ~ / " ~ N/ ~x'y-

H 5-me~oxyttyptamme H tryl:Caml
~ H uyp~

FIG. 1. Formation of bloaetive indole compounds from melatonin within the metabolic network of mdoleanunes. Thick arrows: catabo-
hsm of melatonm; thin arrows: concurrent metabohc pathways. The formation of reactive urinary metabolites has been omitted from the
scheme. AAA: aryl acylamidase; ADH: alcohol dehydrogenase; HIOMT: hydroxyindole O-methyltransferase; MAO: monoamine oxidase;
NAT. mdolamme ("serotonln") N-acetyltransferase; NMT: N-methyltransferase(s); ODM: O-demethylase.
MELATONIN METABOLISM AND FREE RADICALS
rhythm of pineal melatonin, with its exceptionally large ampli-
tude [a prominent nocturnal maximum and low values during
the day (107-I10)], determines the quantities of this substance
in body fluids and, presumably, its cyclicity in most tissues.
BIOACTIVE INDOLE METABOLITES
The classical view of melatonin metabolism assumes that
this neurohormone is predominantly converted in the liver to
6-hydroxymelatonin, which is excreted by the kidney as either
a sulphate or glucuronate conjugate. In fact, these conjugates
represent the major urinary metabolites derived from the hor-
mone (48,55,58). However, the amount of melatonin synthe-
sized per day in the body, including that in extrapineal
sources, especially the gut (which may contain as much as 100
x more melatonin than the pineal), are obviously consider-
ably higher than that represented by the 6-hydroxymelatonin
conjugates (100). Therefore, from a quantitative standpoint
the other metabolites must be considered as being at least
potentially important. We presume that tissue concentrations
of locally produced melatonin as well as its metabolites, in-
eluding those in the brain, will become an important theme
in the future, although, until recently, this aspect has been
essentially disregarded.
Among the various metabolites that are derived from mela-
tonin, several are indolic compounds (Fig. 1). The 5-methoxy
group is not necessarily retained. Surprisingly, melatonin can
be reconverted to its precursor, N-acetylserotonin, in mam-
mals including man and in birds (3,68,69,141). This pathway
seems to be of particular significance in the eye; the retina
contains much higher amounts of N-acetylserotonin than mel-
atonin. It has even been assumed that a rapid reconversion to
its precursor explains why significant amounts of melatonin
are not secreted from the retina, in contrast to the pineal (3).
The physiological role of retinal N-acetylserotonin remains,
however, to be elucidated. A temperature-dependent inhibi-
tion of pineal hydroxyindole O-methyltransferase (HIOMT)
has been described for the rainbow trout (77), but the sig-
nificance of this finding for other organisms has yet to be
determined. Occasionally, N-acetylserotonin can even exert
stronger effects than melatonin [e.g., in depressing body tem-
perature in the rat (76)]. After pinealectomy, a circadian
rhythm of plasma N-acetylserotonin has been shown to persist
in the rat (142), a fact clearly demonstrating the significance
of extrapineal sources of the amine, and it has been suggested
that N-acetylserotonin may be a hormone in its own right
(3,142).
Much better documented are the effects of 5-methoxylated
indoles; some of these may be regarded as physiological effect-
ors or even hormones. This holds, in particular, for 5-meth-
oxytryptamine. This substance can be produced by two meta-
bolic pathways: firstly, by direct O-methylation of serotonin,
either due to the action of HIOMT (30,39,78), which can, in
principle, occur in organs such as pineal, retina, and Har-
derian gland, or via unspecific O-methyltransferases found
in vegetative tissues (9). Secondly, 5-methoxytryptamine is
formed from melatonin by aryl acylamidase (10,13,32,39,114;
Fig. 1). In the pineal gland, 5-methoxytryptamine is produced
at a fairly high rate (11,30,102,104); however, its measurement
is complicated by the fact that this indoleamine is rapidly
degraded by MAO and can, therefore, be appropriately quan-
tiffed only in the presence of MAO inhibitors (30,105). Conse-
quently, all conclusions relating to concentrations and diurnal
patterns of this substance have to consider the blockade of its
catabolism. For the hamster pineal gland, a circadian rhythm
349
of 5-methoxytryptamine biosynthesis has been described,
which is, however, out of phase with the melatonin rhythm;
thus, pineal 5-methoxytryptamine levels reportedly peak dur-
ing the day (30,105).
Like melatonin, 5-methoxytryptamine represents a phylo-
genetically old and highly conserved molecule. It is found
already in a unicellular organism, the bioluminescent dino-
flagellate Gonyaulax polyedra, where it exhibits circadian
rhythmicity (8,100). It is formed by deacetylation of melato-
nin (39) and acts as an intracellular agonist stimulating light
emission and encystment of cells (6,7,39). Some effects of
5-methoxytryptamine are also reported in vertebrates, espe-
cially, in the Syrian hamster. Many actions described for mela-
tonin can also be seen after administration of the deacetylated
amine. This holds, in particular, for the antigonadotropic ac-
tivity (84,86,89-92,95-98,103,111,115), although whether 5-
methoxytryptamine is equipotent to melatonin in modulating
reproductive physiology is debated (111). Even a direct sup-
pression of luteinizing hormone- (LH)-stimulated steroidogen-
esis in isolated Leydig cells has been reported for 5-methoxy-
tryptamine (84), though the major antigonadal action seems
to be via central neuroendocrine mechanisms. Other effects
of 5-methoxytryptamine concern promotion of both rapid eye
movement (REM) and nonREM sleep (75), a suppression of
thyroid hormones (in a teleost fish, Clarias batrachus; Ref.
83), autoreceptor-mediated inhibition of serotonin release
(31), and the reduction of vasopressin immmunoreactivity in
the diagonal band of Broca, the lateral septum, medial amyg-
dala, and ventral hypothalamus (94).
With regard to these various effects, the relationship be-
tween melatonin and 5-methoxytryptamine represents an intri-
guing and important question. A thorough study considering
especially dosage dependence and circulating concentrations
of the two indoleamines led to the conclusion that the relative
efficacy of melatonin was superior to that of 5-methoxytrypta-
mine in terms of both antigonadotrophic and counter-antigon-
adotrophic effects in the Syrian hamster (111). Similar con-
clusions were drawn from other investigations (115,116).
Occasionally, actions of 5-methoxytryptamine are reported to
be more pronounced than those of the acetylated compound
[e.g., on sleep-promotion in rats (75)], and under certain con-
ditions and at a fixed dosage, even in terms of the induction
of regression of the gonads and accessory organs (86). In Go-
nyaulax, 5-methoxytryptamine is by far more potent (by sev-
eral orders of magnitude) as a stimulator of bioluminescence
and of encystment than is melatonin, especially in terms of its
actions on cyst formation that are independent of tempera-
ture, whereas melatonin is effective only below 160C (6,7,39).
5-Methoxytryptamine, when administered to Syrian hamsters,
arguably does not act by formation of melatonin via acetyla-
tion (97,103). Moreover, effects of 5-methoxytryptamine are
seen after pinealectomy (97). The potential independence of
at least some of the actions of melatonin and 5-methoxytryp-
tamine also seems to be supported by the fact that certain
functions are influenced by melatonin but not by 5-methoxy-
tryptamine [e.g., the reduction of body temperature in the rat
(76)]. Moreover, the fact that 5-methoxytryptamine some-
times proves to be highly potent, despite its sensitivity to deg-
radation by MAO, and despite a much lower affinity for mela-
tonin receptors (by 2-3 orders of magnitude: Refs. 23,65),
suggests independent actions of the two indoleamines, at least
with regard to several physiological functions related to mem-
brane receptors. Collectively, the findings indicate that 5-
methoxytryptamine may, like melatonin, be a pineal hormone
(cf. Refs. 89-92).
350
Another indole compound with biological activity is gener-
ated from 5-methoxytryptamine via its conversion by MAO
and alcohol dehydrogenase, namely, 5-methoxytryptophol
(Fig. 1). However, this substance can also be formed by O-
methylation of 5-hydroxytryptophol (93). In various mamma-
lian and other vertebrate species, 5-methoxytryptophol is pres-
ent in the pineal gland, the retina, as well as in the Harderian
glands (59,74,79,89,91,93,121-123); at least in the former two
organs, 5-methoxytryptophol may exhibit circadian (59,93,
121,123) and seasonal (59) rhythms; when circadian rhythms
of 5-methoxytryptophol are observed, peak levels usually oc-
cur at night (an exception was reported in Jaculus ortentahs
due to seasonal variations: Ref. 59). The possible significance
of 5-methoxytryptophol may also be seen in the context of its
ability to enter all types of cells due to its high hydrophobicity,
which almost equals that of melatonin. In contrast, the some-
what less hydrophobic 5-methoxytryptamine enters cells with
greater difficulty.
A variety of effects have been described for 5-methoxytryp-
tophol. Usually, investigators have preferentially examined its
actions with regard to the physiological role of melatonin.
This approach is certainly somewhat selective, and, therefore,
independent functions may have been overlooked. Neverthe-
less, the comparison with melatonin indicates again an over-
lapping role for the two pineal constituents. These studies
demonstrated especially antigonadal influences, including ef-
fects on isolated Leydig cells (86,89,91). An action more po-
tent than that of melatonin was reported for its ability to
decrease basal temperature in the rat, an effect which 5-
methoxytryptophol surprisingly shares with nonmethoxylated
compounds, in particular, N-acetylserotonin and 5-hydroxy-
tryptophol (76). 5-Methoxytryptophol is usually assumed to
be rapidly degraded to the presumably physiologically inert
5-methoxyindoleacetic acid, which is excreted as a urinary me-
tabolite (22).
A substance exerting particularly obvious effects when it
is overproduced and which in this regard is of interest for
psychopathology can derive from 5-methoxytryptamine by N-
methylation. The final product, N,N-dimethyl-5-methoxy-
tryptamine (Fig. 1), originally attracted the interest of investi-
gators because of its abundance in certain hallucinogenic
concoctions from tropical plants. However, there is sufficient
arylamine N-methyltransferase activity present in various
mammalian tissues to generate N,N-dimethylated indole-
amines from tryptamine, serotonin, and 5-methoxytrypt-
amine; the derivatives, N,N-dimethyltryptamine, bufotenin
( = N,N-dimethylserotonin), and N,N-dimethyl-5-methoxy-
tryptamine can be formed by unspecific N-methyltransferases
using either methyltetrahydrofolic acid or S-adenosylmethion-
ine as methyl donors. It is often assumed that the synthetic
pathway for N,N-dimethyl-5-methoxytryptamine begins with
the N-methylation of serotonin, giving rise to bufotenin,
which is subsequently O-methylated (9,72). However, the
existence of a more specific N-methyltransferase using S-
adenosylmethionine as a methyl donor has been demon-
strated; the enzyme acts rather specifically on 5-methoxytryp-
tamine and shows fairly high activity in rabbit and human
lung (72). With regard to the recently assumed higher con-
centrations of tissue melatonin, which can be deacetylated
and thereafter N-methylated, N,N-dimethyl-5-methoxytrypta-
mine may, in fact, now be regarded as a derivative of mela-
tonin, too.
N,N-Dimethyl-5-methoxytryptamine is an extremely po-
tent serotoninergic ligand (67,131). Studies on the serotoniner-
gic control of LH release revealed stimulatory actions via HT2
HARDELAND ET AL.
receptors and inhibitory effects via HT~ receptors (67). The
serotoninergic activity also is the reason for the strong psy-
chotogenic actions of this substance, which are seen in situa-
tions such as drug abuse (29,131), experimental administration
to nonhuman primates and other laboratory animals (119,
131), and endogenous overproduction (56,81,82). In schizo-
phrenics it is found, with the other N,N-dimethylated indole-
amines, in elevated concentrations in the urine (81). These
substances constitute a class of endogenous hallucinogens/
psychotogens, to which humans almost do not adapt. To elu-
cidate the causes for the overproduction of these compounds
in mental disorders remains an objective of high priority.
TRICYCLIC METABOLITES
The metabolism of 5-methoxylated indoleamines is not re-
stricted to modifications of the side residues. Especially the
aliphatic chain can undergo cyclization. In principle, two pos-
sibilities seem to exist (Fig. 2). One is the formation of a
second 5-atom ring occurring in conjunction with hydroxyla-
tion and saturation of the 2,3-double bond; this results in a
cyclic isomer of 2-hydroxymelatonin (136). This substance
has, in fact, been isolated from human and rat urine (136). Its
biological activity has not yet been conclusively demonstrated,
but according to structure-activity studies (28), a skin-
lightening effect has been suggested, as is the case with melato-
nin. Moreover, its structural similarity with fl-carbolines gave
rise to the assumption that it possibly has psychomimetic ef-
fects (136).
fl-Carbolines can also be formed from pineal indoles; this
represents the second possibility for cyclization, which re-
quires the incorporation of an additional C-atom. fl-Carbo-
lines are found in all organs which produce high amounts
of melatonin [i.e., pineal gland, retina, and Harderian gland
(1,2,49,50,61,63,66)]. Their metabolic generation under physi-
ological conditions is still unclear; it has been assumed that at
least a portion of these may represent artifacts occurring after
homogenization (62). However, formation of a fl-carboline
has been demonstrated as a consequence of peroxidation of
serotonin by the superoxide anion radical (133). Because anti-
oxidative protection seems to be a particularly important
property of 5-methoxylated indoleamines (see below), this
type of reaction may be of biological significance. The reac-
tion should not be possible with melatonin itself, due to the
presence of the N-acetyl group, but should occur with its me-
tabolite, 5-methoxytryptamine. Hence, O-methylation of a
hydroxylated, serotonin-derived fl-carboline is possible, but
not a prerequisite for the formation of an O-methoxylated
analog, fl-Carbolines are well-known for their psychomimetic
actions (2,61,85). Their effects relate to their interference with
two important neurobiological mechanisms. Firstly, they can
bind to the benzodiazepine receptor, where they interact with
the same histidine residue as the benzodiazepines (33,60). Sec-
ondly, they modulate the affinity of the imipramine binding
site related to the serotonin transporter, an effect which can
readily explain their psychomimetic activity, but which seems
to be of more general importance: The same mechanism of
action was demonstrated for ceils as different as neurons and
platelets (61-64). With regard to the serotoninergic innerva-
tion of the hypothalamus, fl-carbolines influence the secretion
of pituitary hormones, in particular, prolactin (I18,124).
Moreover, inhibition of MAO-A by fl-carbolines has been
detected in serotoninergic neurons (I). The methoxylated
fl-carboline, pinoline ( = 6-methoxy-l,2,3,4-tetrahydro-fl-car-
boline = 5-methoxytryptoline), has been shown to occur in bio-
MELATONIN METABOLISM AND FREE RADICALS 351

H3CO"-,~,,,~ H 3 C O ~

~L~_.J~N~,,,.../"NH
HOH I H
C=O
cyclic I pinoline
2-hydroxy- CH3
melatonin
FIG. 2. Tr]cyclic melatonin metabolites.

logical material and is of potential physiological significance eral indications for such a relationship exist: pinealectomy
because of its efficacy at nanomolar concentrations (1, disrupts the circadian rhythm of brain GABA and benzodiaze-
2,49,50,61-64,66,118,124). Its biological potency is usually pine binding; already low doses of exogenous melatonin coun-
considerably greater than that of the nonmethylated analog teract the pinealectomy-induced changes; chronic administra-
(61,85). Pinoline is reportedly formed from melatonin via tion of melatonin increases both GABA and benzodiazepine
5-methoxytryptamine (1). Binding sites for pinoline are found binding; melatonin increases GABA turnover as well as gluta-
at a particularly high density in the pineal gland, but exist also mate decarboxylase activity and chloride conductance.
in other brain regions as well as in both the adrenal cortex
and medulla (1). Interestingly, the existence of this substance MELATONINAS A RADICALSCAVENGER
has also been demonstrated in a unicell producing melatonin
The catalytic mechanism of indoleamine 2,3-dioxygenase
and 5-methoxytryptamine, Gonyaulax polyedra (100). More-
already demonstrates the capacity of melatonin to undergo an
over, this organism is sensitive to imipramine, to which it
iron-porphyrin-mediated reaction with the superoxide anion
responds with a dose-dependent increase or decrease of biolu-
minescence (Hardeland, R., unpubl.). Hence, /~-carbolines
and their respective binding sites may represent elements of
phylogenetically ancient cellular mechanisms. O
H3CO-,,,,,,,,,.,~",,~ II
5-METHOXYLATEDKYNURAMINES CH2~CH2,~N~H 3
Another important route of melatonin metabolism leads to
the formation of substituted kynuramines (= kynurenamines; melatomn
which are unrelated to kynurenic acid) due to oxidative cleav- H
age of the pyrrole ring. This reaction can be catalysed by
indoleamine 2,3-dioxygenase, a heme enzyme requiring super-
oxide anion radicals as a substrate (43). This type of reaction hemin, 0 2 =
also seems to occur in cells by two nonenzymatic, but biologi-
cally catalysed, mechanisms involving free radicals (see fol-
lowing section). The first metabolite generated by cleavage of
the pyrrole ring is NLacetyl-N2-formyl-5-methoxykynuramine O O
(AFMK), which is easily deformylated by arylamine formami- H3CO.. ~ II II
dase to give NLacetyl-5-methoxykynuramine (AMK). Both C~.CH2--C H2-..NH.--C--C H3
substances (Fig. 3) occur in the brain, and in particular, in the
pineal gland (45,51,53,54,73). In general, kynuramines repre- ~ N H - - - C H O
sent a class of biogenic amines which have rarely been investi-
gated but which possess high biological potencies (summarized
in Ref. 5). There is increasing evidence that some actions of
N1-acetyI-N2-formyl-
melatonin may be mediated by these compounds (51-54). In
terms of a chronobiological effect, entrainment was reported
following the administration of AFMK, but not of AMK (52).
It was shown that AMK is a biologically occurring ligand of
AFAl 5-methoxykynuramine

the benzodiazepine receptor, exhibiting equally strong binding

in preparations from hypothalamus, striatum, and mid-brain
(54,73). These findings are not only of interest from a more
H3CO~, O
II II
O

general point of view [e.g., with regard to sedative effects

[~NH2 ~H2--~H2-'NH-'-C'~CH3
exerted by melatonin (41)], but also for the understanding of
GABAergic actions as influenced by the pineal, including the
possibility of a dual influence of indole metabolites via /~-
carbolines and kynuramines. Because the chloride channel is N1-acetyt-5-methoxykynuramine
under the control of both a GABA and a benzodiazepine bind-
ing site, interactions between melatonin (or its metabolites) FIG. 3. Formation of two 5-methylated kynuramines from melato-
and GABAergic mechanisms are physiologically highly impor- nin. IDO: indoleamine 2,3-dioxygenase; AFA: arylamine formami-
tant. As pointed out by Rosenstein and Cardinali (117), sev- dase; 02- : superoxide anion.
352
(43). This type of reaction is, however, also possible nonenzy-
matically, as shown in a system generating this species of free
radicals by means of xanthine oxidase and using iron-EDTA
as catalyst (132). Nonenzymatic cleavage of the pyrrole ring
due this mechanism is generally possible with all indoles, but
melatonin exhibits an exceptionally high reactivity, especially
at physiological pH (42,132). The biological significance of
this finding was initially ignored but it has become more evi-
dent since hemin [i.e., a substance widely occurring in biologi-
cal material in both free and protein-bound forms, is an ex-
tremely efficient catalyst of this reaction (41; Fuhrberg, B.;
Hardeland, R., unpubl.)]. This could also be related to the
surprisingly high instability of melatonin in homogenates of
nonpineai sources, especially in the dinoflagellate Gonyaulax
polyedra (42,100). Even in cell extracts from this organism, in
which protein is precipitated by ethanol, melatonin is effi-
ciently degraded by superoxide anions induced by illumina-
tion, even at fairly low intensities of light (e.g., 240 Ix), which
contains negligible amounts of UV (42). However, no such
degradation was observed after many hours in the absence of
cell extract or without illumination. This type of reaction was
further corroborated by investigations on the heme-catalysed
interaction with superoxide anions derived from H202 at alka-
line pH (8.0 or 8.5; Ref. 41). Using this procedure it was
possible to turn over melatonin quantitatively in millimolar
concentrations (Fuhrberg, B.; Hardeland, R., unpubl.). The
most important consequence of these findings is that melato-
nin can be extremely unstable in certain types of biological
material when it contains substantial quantities of porphyrins
and is exposed to superoxide anions at sufficiently high tissue
concentrations. Superoxide anions are produced by flavine
enzymes (e.g., MAO), the presence of radical-generating leu-
kocytes, or irradiation by visible or ultraviolet light. Melato-
nin is thereby degraded to AFMK and other metabolites, such
as AMK, which in turn can elicit particular biological effects
(41,54,73). Because melatonin enters all cell types due to its
hydrophobicity, and because the heme-catalysed free-radical
reaction does not depend on the presence of a particular en-
zyme, this catabolic route may be relevant especially for the
previously underrated tissue melatonin (i.e., melatonin in the
brain and in other organs).
Melatonin is not only able to trap superoxide anions, but
also represents a very efficient scavenger of hydroxyl radicals
(Tan, D-X.; Chen, L.-D.; Poeggeler, B.; Manchester, L. C.;
Reiter, R.J., unpubl.). In experiments related to superoxide
anions this reaction was prevented by eliminating hydroxyl
radicals with ethanol, which was added to some incubation
mixtures in excess amounts. Scavenging of hydroxyl radicals
by melatonm was unequivocally demonstrated in experiments
by Tan et al. using the spin-trapping reagent 5,5-dimethylpyr-
roline N-oxide (DMPO) and the UV-photolysis of H202. This
mixture was devoid of ethanol. In these investigations, mela-
tonin turned out to be the most potent scavenger of hydroxyl
radicals ever detected, exceeding that of mannitol by more
than an order of magnitude. Moreover, a comparison of dif-
ferent indoleamines revealed the significance of the particular
structure of melatonin. 5-Methoxytryptamine had a similar
affinity for hydroxyl radicals, but showed a lower final rate of
turnover; 5-acetylserotonin was ineffective, whereas serotonin
surprisingly led to an enhanced production of hydroxyl radi-
cals. As for 5-methoxytryptamine, a similar structure/activity
relationship was found for the trapping of superoxide anions
(Hardeland, R., unpubl.).In the ethanol-free system, UV-
irradiation could be replaced by iron-EDTA, thereby generat-
ing hydroxyl radicals via the Fenton reaction (Tan, D.-X., et
HARDELAND ET AL.
al., unpubl.). Again, the possibility of greatly enhancing the
turnover of melatonin by complexed iron strongly suggests a
significant role of hemin or other porphyrins in the nonenzy-
matic degradation of melatonin. At least one of the products
formed upon the interaction of melatonin with hydroxyl radi-
cals seems to be AFMK, though by another chemical mecha-
nism (Tan, D.-X., et al., unpubl.); this results presumably
with the abstraction of an electron to give an indolyl cation
radical, which finally traps a superoxide anion (Fig. 4). The
particular capacity of melatonin as a radical scavenger is,
therefore, not only due to its extraordinarily high chemical
affinity for the hydroxyl radical, but also to the ability of
the indolyl cation radical to react directly, without requiring
another catalyst, with an additional superoxide anion.
The reaction mechanisms described identify an important
property of melatonin, namely, that it acts to definitively ter-
minate radical reaction chains, and that it does not participate
in redox cycling, such as other scavengers including ascorbate,
ct-D-tocopherol, and glutathione. Together with its extraordi-
narily high trapping capacity, this makes melatonin an ex-
tremely interesting substance, in terms of both physiological
and therapeutical protection from aggressive oxygen radicals.
THE SIGNIFICANCEOF ANTIOXIDATIVEPROTECTION
It has been hypothesized that the function of antioxidative
protection may be the primary role of melatonin in phylogeny
O
II
CH~CH 2 N ~ H 3
melatonin
H
~
, OH
OH
O
H3CO'-,..,,,,~ II
CH2~CH2---N~H 3
H indolyl cation radical
O O
H3CO.. ~ II II
"~ )~ ~H2~CH2---NI'I~C-~H3
~'NH--CHO
Nl-acetyI-N2-formyl-..-
5~rnethoxykynuramlne
FIG. 4. Presumed mechamsm of direct free-radical trapping by mel-
atonm and ~ts mdolyl cation radical. OH: hydroxyl radical; 02-: su-
peroxide anion.
MELATONIN METABOLISM AND FREE RADICALS
(40,42). This idea is particularly attractive with regard to cells
that are directly exposed to light/dark cycles and are, there-
fore, subjected to an extremely robust exogenous cyclicity of
oxygen radicals. This periodicity of exposure to radicals, de-
stroying melatonin during the day and preserving it at night,
may have made the molecule suitable as a carrier and mediator
of the information "darkness" (40,42).
The recent discovery that melatonin scavenges hydroxyl
radicals with extremely high efficacy (112,130; Tan, D.-X., et
al., unpubi.), along with the observation that melatonin is
present in substantial amounts in extrapineal tissues including
gut, liver, and brain (12,100,135; Menendez-Pelaez, A.; Re-
iter, R.J., unpubl.), indicates that this ancient role of melato-
nin has been conserved in higher organisms, and that the
transmittance of photoperiodic information is probably a sec-
ondary function of this neurohormone. This view is strongly
supported by several observations. Thus, in the rodent Hard-
erian gland, the circadian variation in melatonin is relatively
small (46,74), but there exists a marked sexual dimorphism
with much higher melatonin levels in the glands of females
than of males. This dimorphism in melatonin levels finds its
parallel in the larger amounts of porphyrins produced and
secreted by the female compared to that of the male Harderian
gland (46,74). The presence of melatonin in these glands as a
protective agent which is required because of the high concen-
trations of the hydroxyl-radical forming and superoxide-anion
transducing catalyst seems highly likely. There is also more
direct evidence for the involvement of melatonin in protection
from free oxygen radicals. In a study on DNA damage in-
duced by the hydroxyl radical-generating carcinogen safrole,
melatonin, given at a dosage 1,000 x lower than that of the
carcinogen, protects the DNA almost entirely from free radi-
cal damage (130). Other recent data strongly suggest binding
of melatonin to chromatin in both neural and nonneural tis-
sues (Menendez-Pelaez, A.; Reiter, R. J., unpubl.), a finding
that seems to indicate direct on-site protection of DNA.
Protection by melatonin from free radicals, especially from
the highly reactive, potentially mutagenic, carcinogenic and
cell-toxic hydroxyl radicals, is a very important feature which
leads to diverse implications for health, aging and, in particu-
lar, age-related neurogenerative disorders (112,130). The po-
tential role of the pineal gland in aging has repeatedly been
stressed (99,101,110,138). Especially the often observed age-
dependent decline of nocturnal melatonin may have consider-
able consequences for neurodegenerative processes. More-
over, sources of free-radical production can increase with age
[e.g., the activity of MAO-B (127,140)]. Many other indica-
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Our view on the physiological role of melatonin is about
to change considerably. Although its function as a mediator
of photoperiodic information is well established, and even
though this aspect has been confirmed in primitive organisms
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actions of hydroxyl radicals.
ACKNOWLEDGEMENTS
Work by the authors was supported in part by NSF grant IBN 91
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