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

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Printedin the U.S.A. All rights reserved Copyright1993PergamonPress Ltd.

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-


nin results from its presence in nonpineal tissues, in particular,
THE pineal hormone melatonin has attracted attention the r e t i n a - f r o m which it appears not to be abundantly se-
particularly with regard to its role as a mediator of the photo- c r e t e d - (3,13,32,93,142), the Harderian gland (46,74,93), the
periodic information "darkness," a role that necessarily leads gut (12,100,135), and ieukocytes (25). Temporal patterns and/
to a kind of physiological pleiotropy. Melatonin controls cir- or kinetics of production and release are different in these
cadian rhythmic organization (14,70,109,134) with its numer- organs from those known for the pineal gland.
ous oscillatory functions at cellular, organ, and behavioral The pleiotropy of actions is obviously not limited to the
levels, and it determines annual cyclicity, especially in seasonal transduction of photoperiodic information by the molecule
breeders (4,107-109,129). Most often, effects of melatonin melatonin itself; rather, a considerable sector within the spec-
have been studied with regard to the hypothalamic-hypo- trum of effects seems to be due to its metabolism to active
physeal-gonadal axis, including aspects of inhibition of the metabolites. With regard to the physiological consequences of
gonadotropin releasing hormone pulse generator, puberty, the biochemical reactions melatonin can undergo, and to the
aging, and various pathologies (4,83,84,86,91,99,107-111, substances formed by the respective pathways, two aspects of
I 16,123,129,138). The spectrum of actions exerted by melato- particularly great potential significance can be distinguished.
nin seems to exceed the purely periodic phenomena. One as- On the one hand, melatonin possesses unique properties as a
pect of particular significance, the influence of melatonin on radical scavenger; this capability strongly suggests a protec-
the immune system, has now repeatedly been documented tive, especially, neuroprotective role. On the other hand, sub-
(25,26,34,57,71). 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 especially at physiological pH (42,132). Moreover, it shields
themselves (e.g., other methoxyindoles,/~-carbolines, and ky- the molecule from other reactions (e.g., from dimerization)
nuramines). presumably by preventing phenoxylic radical formation (47),
and it prevents the formation of quinone imine structures
PHYSICOCHEMICALAND PHYSIOLOGICAL (88,128). What the methyl group does not do, however, is
PROPERTIES OF MELATONIN prevent interactions with serotonin receptors, because 5-
methoxytryptamine and some of its derivatives (e.g., N,N-
Due to its hydrophobicity, melatonin can easily permeate dimethyl-5-methoxytryptamine) efficiently bind to these re-
any biological membrane. Consequently, it appears in every ceptors, though with different consequences (inhibitory or
tissue and body fluid in concentrations of the same order of stimulatory) at the various subtypes (31,67,13 I). The N-acetyl
magnitude as in blood plasma. The capability to enter all cells group, therefore, obviously represents that portion of the mol-
may be of particular importance for the following reasons. ecule which prevents substantial binding to serotonin recep-
Although receptor proteins for melatonin have been identified tors. Moreover, the N-acetyl group protects melatonin from
and characterized m and isolated from plasma membranes of degradation by monoamine oxidase (MAO), a fact that distin-
certain brain regions (23,65,113,125,126), these membrane- guishes this indoleamine from its analogue, 5-methoxytrypta-
bound receptors obviously transduce only part of the informa- mine (30,31,106). Additionally, the acetyl group participates
tion carried by the hormone. In this context, one should be in the cyclization of the aliphatic residue (136). Finally, as
aware of the fact that the receptors mentioned have usually will be discussed below, comparisons of reactivity between
been identified using iodinated melatonin, and, although they melatonin and 5-methoxytryptamine reveal that the acetyl
efficiently bind melatonin in competition studies (23,65), group contributes much to the radical scavenging capacity,
other binding sites may have a lower affinity for the iodinated though this may not be obvious at the first glance (Tan, D.-X.;
molecule or they may remain inaccessible to the ligand if they Chen, L.-D.; Poeggeler, B.; Manchester, L.C.; Reiter, R.J.,
are localized in the interior of the cell. Another type of binding unpubl.; Hardeland, R., unpubl.). The pyrrole ring represents
site with fairly high affinity has already been demonstrated to one of the most important reactive parts of the molecule,
stimulate Na+/K+ATPase (15), that is, an enzyme ubiqui- because it can be opened by peroxidative cleavage, either enzy-
tously distributed in neuronal and nonneuronal cells. More- matically (43,45) or by nonenzymatic mechanisms involving
over, any effect exerted by melatonin metabolites would be free radicals (41,42; Tan, D.-X., et al., unpubl.); opening of
overlooked when considering exclusively the signal-transduc- the ring results in the formation of an interesting and poorly
tion pathway associated with the previously described mem- understood class of biogenic amines, the kynuramines (41-
brane receptor. 43,45).
Apart from its hydrophobicity, the melatonin molecule has When considering physiological functions of melatonin
other properties which are of importance for metabolic con- and of its metabolites, the circadian profile of this neurohor-
siderations. The 5-methoxy group, by which melatonin differs mone must be taken into account. While in some tissues mela-
from 5-hydroxylated and nonsubstituted indoles (Fig. 1), re- tonin concentrations are subject to only slight temporal varia-
sults in a considerable increase in radical-trapping capacity, 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 349

rhythm of pineal melatonin, with its exceptionally large ampli- of 5-methoxytryptamine biosynthesis has been described,
tude [a prominent nocturnal maximum and low values during which is, however, out of phase with the melatonin rhythm;
the day (107-I10)], determines the quantities of this substance thus, pineal 5-methoxytryptamine levels reportedly peak dur-
in body fluids and, presumably, its cyclicity in most tissues. ing the day (30,105).
Like melatonin, 5-methoxytryptamine represents a phylo-
BIOACTIVE INDOLE METABOLITES genetically old and highly conserved molecule. It is found
already in a unicellular organism, the bioluminescent dino-
The classical view of melatonin metabolism assumes that flagellate Gonyaulax polyedra, where it exhibits circadian
this neurohormone is predominantly converted in the liver to rhythmicity (8,100). It is formed by deacetylation of melato-
6-hydroxymelatonin, which is excreted by the kidney as either nin (39) and acts as an intracellular agonist stimulating light
a sulphate or glucuronate conjugate. In fact, these conjugates emission and encystment of cells (6,7,39). Some effects of
represent the major urinary metabolites derived from the hor- 5-methoxytryptamine are also reported in vertebrates, espe-
mone (48,55,58). However, the amount of melatonin synthe- cially, in the Syrian hamster. Many actions described for mela-
sized per day in the body, including that in extrapineal tonin can also be seen after administration of the deacetylated
sources, especially the gut (which may contain as much as 100 amine. This holds, in particular, for the antigonadotropic ac-
x more melatonin than the pineal), are obviously consider- tivity (84,86,89-92,95-98,103,111,115), although whether 5-
ably higher than that represented by the 6-hydroxymelatonin methoxytryptamine is equipotent to melatonin in modulating
conjugates (100). Therefore, from a quantitative standpoint reproductive physiology is debated (111). Even a direct sup-
the other metabolites must be considered as being at least pression of luteinizing hormone- (LH)-stimulated steroidogen-
potentially important. We presume that tissue concentrations esis in isolated Leydig cells has been reported for 5-methoxy-
of locally produced melatonin as well as its metabolites, in- tryptamine (84), though the major antigonadal action seems
eluding those in the brain, will become an important theme to be via central neuroendocrine mechanisms. Other effects
in the future, although, until recently, this aspect has been of 5-methoxytryptamine concern promotion of both rapid eye
essentially disregarded. movement (REM) and nonREM sleep (75), a suppression of
Among the various metabolites that are derived from mela- thyroid hormones (in a teleost fish, Clarias batrachus; Ref.
tonin, several are indolic compounds (Fig. 1). The 5-methoxy 83), autoreceptor-mediated inhibition of serotonin release
group is not necessarily retained. Surprisingly, melatonin can (31), and the reduction of vasopressin immmunoreactivity in
be reconverted to its precursor, N-acetylserotonin, in mam- the diagonal band of Broca, the lateral septum, medial amyg-
mals including man and in birds (3,68,69,141). This pathway dala, and ventral hypothalamus (94).
seems to be of particular significance in the eye; the retina With regard to these various effects, the relationship be-
contains much higher amounts of N-acetylserotonin than mel- tween melatonin and 5-methoxytryptamine represents an intri-
atonin. It has even been assumed that a rapid reconversion to guing and important question. A thorough study considering
its precursor explains why significant amounts of melatonin especially dosage dependence and circulating concentrations
are not secreted from the retina, in contrast to the pineal (3). of the two indoleamines led to the conclusion that the relative
The physiological role of retinal N-acetylserotonin remains, efficacy of melatonin was superior to that of 5-methoxytrypta-
however, to be elucidated. A temperature-dependent inhibi- mine in terms of both antigonadotrophic and counter-antigon-
tion of pineal hydroxyindole O-methyltransferase (HIOMT) adotrophic effects in the Syrian hamster (111). Similar con-
has been described for the rainbow trout (77), but the sig- clusions were drawn from other investigations (115,116).
nificance of this finding for other organisms has yet to be Occasionally, actions of 5-methoxytryptamine are reported to
determined. Occasionally, N-acetylserotonin can even exert be more pronounced than those of the acetylated compound
stronger effects than melatonin [e.g., in depressing body tem- [e.g., on sleep-promotion in rats (75)], and under certain con-
perature in the rat (76)]. After pinealectomy, a circadian ditions and at a fixed dosage, even in terms of the induction
rhythm of plasma N-acetylserotonin has been shown to persist of regression of the gonads and accessory organs (86). In Go-
in the rat (142), a fact clearly demonstrating the significance nyaulax, 5-methoxytryptamine is by far more potent (by sev-
of extrapineal sources of the amine, and it has been suggested eral orders of magnitude) as a stimulator of bioluminescence
that N-acetylserotonin may be a hormone in its own right and of encystment than is melatonin, especially in terms of its
(3,142). actions on cyst formation that are independent of tempera-
Much better documented are the effects of 5-methoxylated ture, whereas melatonin is effective only below 160C (6,7,39).
indoles; some of these may be regarded as physiological effect- 5-Methoxytryptamine, when administered to Syrian hamsters,
ors or even hormones. This holds, in particular, for 5-meth- arguably does not act by formation of melatonin via acetyla-
oxytryptamine. This substance can be produced by two meta- tion (97,103). Moreover, effects of 5-methoxytryptamine are
bolic pathways: firstly, by direct O-methylation of serotonin, seen after pinealectomy (97). The potential independence of
either due to the action of HIOMT (30,39,78), which can, in at least some of the actions of melatonin and 5-methoxytryp-
principle, occur in organs such as pineal, retina, and Har- tamine also seems to be supported by the fact that certain
derian gland, or via unspecific O-methyltransferases found functions are influenced by melatonin but not by 5-methoxy-
in vegetative tissues (9). Secondly, 5-methoxytryptamine is tryptamine [e.g., the reduction of body temperature in the rat
formed from melatonin by aryl acylamidase (10,13,32,39,114; (76)]. Moreover, the fact that 5-methoxytryptamine some-
Fig. 1). In the pineal gland, 5-methoxytryptamine is produced times proves to be highly potent, despite its sensitivity to deg-
at a fairly high rate (11,30,102,104); however, its measurement radation by MAO, and despite a much lower affinity for mela-
is complicated by the fact that this indoleamine is rapidly tonin receptors (by 2-3 orders of magnitude: Refs. 23,65),
degraded by MAO and can, therefore, be appropriately quan- suggests independent actions of the two indoleamines, at least
tiffed only in the presence of MAO inhibitors (30,105). Conse- with regard to several physiological functions related to mem-
quently, all conclusions relating to concentrations and diurnal brane receptors. Collectively, the findings indicate that 5-
patterns of this substance have to consider the blockade of its methoxytryptamine may, like melatonin, be a pineal hormone
catabolism. For the hamster pineal gland, a circadian rhythm (cf. Refs. 89-92).
350 HARDELAND ET AL.

Another indole compound with biological activity is gener- receptors and inhibitory effects via HT~ receptors (67). The
ated from 5-methoxytryptamine via its conversion by MAO serotoninergic activity also is the reason for the strong psy-
and alcohol dehydrogenase, namely, 5-methoxytryptophol chotogenic actions of this substance, which are seen in situa-
(Fig. 1). However, this substance can also be formed by O- tions such as drug abuse (29,131), experimental administration
methylation of 5-hydroxytryptophol (93). In various mamma- to nonhuman primates and other laboratory animals (119,
lian and other vertebrate species, 5-methoxytryptophol is pres- 131), and endogenous overproduction (56,81,82). In schizo-
ent in the pineal gland, the retina, as well as in the Harderian phrenics it is found, with the other N,N-dimethylated indole-
glands (59,74,79,89,91,93,121-123); at least in the former two amines, in elevated concentrations in the urine (81). These
organs, 5-methoxytryptophol may exhibit circadian (59,93, substances constitute a class of endogenous hallucinogens/
121,123) and seasonal (59) rhythms; when circadian rhythms psychotogens, to which humans almost do not adapt. To elu-
of 5-methoxytryptophol are observed, peak levels usually oc- cidate the causes for the overproduction of these compounds
cur at night (an exception was reported in Jaculus ortentahs in mental disorders remains an objective of high priority.
due to seasonal variations: Ref. 59). The possible significance
of 5-methoxytryptophol may also be seen in the context of its TRICYCLIC METABOLITES
ability to enter all types of cells due to its high hydrophobicity,
which almost equals that of melatonin. In contrast, the some- The metabolism of 5-methoxylated indoleamines is not re-
what less hydrophobic 5-methoxytryptamine enters cells with stricted to modifications of the side residues. Especially the
greater difficulty. aliphatic chain can undergo cyclization. In principle, two pos-
A variety of effects have been described for 5-methoxytryp- sibilities seem to exist (Fig. 2). One is the formation of a
tophol. Usually, investigators have preferentially examined its second 5-atom ring occurring in conjunction with hydroxyla-
actions with regard to the physiological role of melatonin. tion and saturation of the 2,3-double bond; this results in a
This approach is certainly somewhat selective, and, therefore, cyclic isomer of 2-hydroxymelatonin (136). This substance
independent functions may have been overlooked. Neverthe- has, in fact, been isolated from human and rat urine (136). Its
less, the comparison with melatonin indicates again an over- biological activity has not yet been conclusively demonstrated,
lapping role for the two pineal constituents. These studies but according to structure-activity studies (28), a skin-
demonstrated especially antigonadal influences, including ef- lightening effect has been suggested, as is the case with melato-
fects on isolated Leydig cells (86,89,91). An action more po- nin. Moreover, its structural similarity with fl-carbolines gave
tent than that of melatonin was reported for its ability to rise to the assumption that it possibly has psychomimetic ef-
decrease basal temperature in the rat, an effect which 5- fects (136).
methoxytryptophol surprisingly shares with nonmethoxylated fl-Carbolines can also be formed from pineal indoles; this
compounds, in particular, N-acetylserotonin and 5-hydroxy- represents the second possibility for cyclization, which re-
tryptophol (76). 5-Methoxytryptophol is usually assumed to quires the incorporation of an additional C-atom. fl-Carbo-
be rapidly degraded to the presumably physiologically inert lines are found in all organs which produce high amounts
5-methoxyindoleacetic acid, which is excreted as a urinary me- of melatonin [i.e., pineal gland, retina, and Harderian gland
tabolite (22). (1,2,49,50,61,63,66)]. Their metabolic generation under physi-
A substance exerting particularly obvious effects when it ological conditions is still unclear; it has been assumed that at
is overproduced and which in this regard is of interest for least a portion of these may represent artifacts occurring after
psychopathology can derive from 5-methoxytryptamine by N- homogenization (62). However, formation of a fl-carboline
methylation. The final product, N,N-dimethyl-5-methoxy- has been demonstrated as a consequence of peroxidation of
tryptamine (Fig. 1), originally attracted the interest of investi- serotonin by the superoxide anion radical (133). Because anti-
gators because of its abundance in certain hallucinogenic oxidative protection seems to be a particularly important
concoctions from tropical plants. However, there is sufficient property of 5-methoxylated indoleamines (see below), this
arylamine N-methyltransferase activity present in various type of reaction may be of biological significance. The reac-
mammalian tissues to generate N,N-dimethylated indole- tion should not be possible with melatonin itself, due to the
amines from tryptamine, serotonin, and 5-methoxytrypt- presence of the N-acetyl group, but should occur with its me-
amine; the derivatives, N,N-dimethyltryptamine, bufotenin tabolite, 5-methoxytryptamine. Hence, O-methylation of a
( = N,N-dimethylserotonin), and N,N-dimethyl-5-methoxy- hydroxylated, serotonin-derived fl-carboline is possible, but
tryptamine can be formed by unspecific N-methyltransferases not a prerequisite for the formation of an O-methoxylated
using either methyltetrahydrofolic acid or S-adenosylmethion- analog, fl-Carbolines are well-known for their psychomimetic
ine as methyl donors. It is often assumed that the synthetic actions (2,61,85). Their effects relate to their interference with
pathway for N,N-dimethyl-5-methoxytryptamine begins with two important neurobiological mechanisms. Firstly, they can
the N-methylation of serotonin, giving rise to bufotenin, bind to the benzodiazepine receptor, where they interact with
which is subsequently O-methylated (9,72). However, the the same histidine residue as the benzodiazepines (33,60). Sec-
existence of a more specific N-methyltransferase using S- ondly, they modulate the affinity of the imipramine binding
adenosylmethionine as a methyl donor has been demon- site related to the serotonin transporter, an effect which can
strated; the enzyme acts rather specifically on 5-methoxytryp- readily explain their psychomimetic activity, but which seems
tamine and shows fairly high activity in rabbit and human to be of more general importance: The same mechanism of
lung (72). With regard to the recently assumed higher con- action was demonstrated for ceils as different as neurons and
centrations of tissue melatonin, which can be deacetylated platelets (61-64). With regard to the serotoninergic innerva-
and thereafter N-methylated, N,N-dimethyl-5-methoxytrypta- tion of the hypothalamus, fl-carbolines influence the secretion
mine may, in fact, now be regarded as a derivative of mela- of pituitary hormones, in particular, prolactin (I18,124).
tonin, too. Moreover, inhibition of MAO-A by fl-carbolines has been
N,N-Dimethyl-5-methoxytryptamine is an extremely po- detected in serotoninergic neurons (I). The methoxylated
tent serotoninergic ligand (67,131). Studies on the serotoniner- fl-carboline, pinoline ( = 6-methoxy-l,2,3,4-tetrahydro-fl-car-
gic control of LH release revealed stimulatory actions via HT2 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 HARDELAND ET AL.

(43). This type of reaction is, however, also possible nonenzy- al., unpubl.). Again, the possibility of greatly enhancing the
matically, as shown in a system generating this species of free turnover of melatonin by complexed iron strongly suggests a
radicals by means of xanthine oxidase and using iron-EDTA significant role of hemin or other porphyrins in the nonenzy-
as catalyst (132). Nonenzymatic cleavage of the pyrrole ring matic degradation of melatonin. At least one of the products
due this mechanism is generally possible with all indoles, but formed upon the interaction of melatonin with hydroxyl radi-
melatonin exhibits an exceptionally high reactivity, especially cals seems to be AFMK, though by another chemical mecha-
at physiological pH (42,132). The biological significance of nism (Tan, D.-X., et al., unpubl.); this results presumably
this finding was initially ignored but it has become more evi- with the abstraction of an electron to give an indolyl cation
dent since hemin [i.e., a substance widely occurring in biologi- radical, which finally traps a superoxide anion (Fig. 4). The
cal material in both free and protein-bound forms, is an ex- particular capacity of melatonin as a radical scavenger is,
tremely efficient catalyst of this reaction (41; Fuhrberg, B.; therefore, not only due to its extraordinarily high chemical
Hardeland, R., unpubl.)]. This could also be related to the affinity for the hydroxyl radical, but also to the ability of
surprisingly high instability of melatonin in homogenates of the indolyl cation radical to react directly, without requiring
nonpineai sources, especially in the dinoflagellate Gonyaulax another catalyst, with an additional superoxide anion.
polyedra (42,100). Even in cell extracts from this organism, in The reaction mechanisms described identify an important
which protein is precipitated by ethanol, melatonin is effi- property of melatonin, namely, that it acts to definitively ter-
ciently degraded by superoxide anions induced by illumina- minate radical reaction chains, and that it does not participate
tion, even at fairly low intensities of light (e.g., 240 Ix), which in redox cycling, such as other scavengers including ascorbate,
contains negligible amounts of UV (42). However, no such ct-D-tocopherol, and glutathione. Together with its extraordi-
degradation was observed after many hours in the absence of narily high trapping capacity, this makes melatonin an ex-
cell extract or without illumination. This type of reaction was tremely interesting substance, in terms of both physiological
further corroborated by investigations on the heme-catalysed and therapeutical protection from aggressive oxygen radicals.
interaction with superoxide anions derived from H202 at alka-
line pH (8.0 or 8.5; Ref. 41). Using this procedure it was THE SIGNIFICANCEOF ANTIOXIDATIVEPROTECTION
possible to turn over melatonin quantitatively in millimolar
concentrations (Fuhrberg, B.; Hardeland, R., unpubl.). The It has been hypothesized that the function of antioxidative
most important consequence of these findings is that melato- protection may be the primary role of melatonin in phylogeny
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 O
concentrations. Superoxide anions are produced by flavine II
enzymes (e.g., MAO), the presence of radical-generating leu-
CH~CH 2 N ~ H 3
kocytes, or irradiation by visible or ultraviolet light. Melato-
nin is thereby degraded to AFMK and other metabolites, such melatonin
H

~
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 , OH
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 OH
brain and in other organs).
Melatonin is not only able to trap superoxide anions, but O
also represents a very efficient scavenger of hydroxyl radicals
H3CO'-,..,,,,~ II
(Tan, D-X.; Chen, L.-D.; Poeggeler, B.; Manchester, L. C.;
CH2~CH2---N~H 3
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 H indolyl cation radical
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 O O
than an order of magnitude. Moreover, a comparison of dif- H3CO.. ~ II II
ferent indoleamines revealed the significance of the particular "~ )~ ~H2~CH2---NI'I~C-~H3
structure of melatonin. 5-Methoxytryptamine had a similar
affinity for hydroxyl radicals, but showed a lower final rate of ~'NH--CHO
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
Nl-acetyI-N2-formyl-..-
relationship was found for the trapping of superoxide anions
5~rnethoxykynuramlne
(Hardeland, R., unpubl.).In the ethanol-free system, UV- FIG. 4. Presumed mechamsm of direct free-radical trapping by mel-
irradiation could be replaced by iron-EDTA, thereby generat- atonm and ~ts mdolyl cation radical. OH: hydroxyl radical; 02-: su-
ing hydroxyl radicals via the Fenton reaction (Tan, D.-X., et peroxide anion.
MELATONIN METABOLISM AND FREE RADICALS 353

(40,42). This idea is particularly attractive with regard to cells tions exist for the involvement of free radicals in neurodegen-
that are directly exposed to light/dark cycles and are, there- eration, including aspects of stress, calcium overload in con-
fore, subjected to an extremely robust exogenous cyclicity of nection with excitatory neurotransmitters such as glutamate
oxygen radicals. This periodicity of exposure to radicals, de- (16,19,20,35-38,80,87,101,127,137), and presence of tissue
stroying melatonin during the day and preserving it at night, iron as a catalyst for free-radical formation (37,38,139,140).
may have made the molecule suitable as a carrier and mediator Progressive damage of neurons by hydroxyl radicals may even
of the information "darkness" (40,42). be regarded as one of the major irreversible processes of aging
The recent discovery that melatonin scavenges hydroxyl (21,24,44,101,120,143). Additionally, aging is associated with
radicals with extremely high efficacy (112,130; Tan, D.-X., et an increase in radical-induced changes at the DNA level,
al., unpubi.), along with the observation that melatonin is which, if not already lethal to the individual cell, may cause
present in substantial amounts in extrapineal tissues including dysfunctions or cancer (18,24,27,44,101). Melatonin, how-
gut, liver, and brain (12,100,135; Menendez-Pelaez, A.; Re- ever, seems to counteract the impairment produced by free
iter, R.J., unpubl.), indicates that this ancient role of melato- radicals, both with regard to enzyme function and in terms of
nin has been conserved in higher organisms, and that the the protection of DNA (17,101,112,130; Tan. D.-X. et al.,
transmittance of photoperiodic information is probably a sec- unpubl.) The presence of melatonin in sufficiently high con-
ondary function of this neurohormone. This view is strongly centrations, especially in neuronal tissues and, in particular
supported by several observations. Thus, in the rodent Hard- in cell nuclei, may represent a substantial precondition for
erian gland, the circadian variation in melatonin is relatively longevity and health of the elderly.
small (46,74), but there exists a marked sexual dimorphism
with much higher melatonin levels in the glands of females PERSPECTIVES
than of males. This dimorphism in melatonin levels finds its
parallel in the larger amounts of porphyrins produced and Our view on the physiological role of melatonin is about
secreted by the female compared to that of the male Harderian to change considerably. Although its function as a mediator
gland (46,74). The presence of melatonin in these glands as a of photoperiodic information is well established, and even
protective agent which is required because of the high concen- though this aspect has been confirmed in primitive organisms
trations of the hydroxyl-radical forming and superoxide-anion such as the dinoflagellate Gonyaulax, our understanding of
transducing catalyst seems highly likely. There is also more this indoleamine's biological significance is expanding sub-
direct evidence for the involvement of melatonin in protection stantially. On the one hand, evidence is increasing for melato-
from free oxygen radicals. In a study on DNA damage in- nin as a source of various biologically active metabolites; some
duced by the hydroxyl radical-generating carcinogen safrole, of these are candidates for regulators of GABA-dependent
melatonin, given at a dosage 1,000 x lower than that of the chloride channels while others act as powerful modulators of
carcinogen, protects the DNA almost entirely from free radi- serotoninergic receptors. In future work, this aspect of metab-
cal damage (130). Other recent data strongly suggest binding olism deserves more attention, especially with regard to the
of melatonin to chromatin in both neural and nonneural tis- ubiquitous distribution of melatonin and to the fact that its
sues (Menendez-Pelaez, A.; Reiter, R. J., unpubl.), a finding catabolism is not exclusively restricted to the pineal itself and
that seems to indicate direct on-site protection of DNA. to liver, as previously believed. Metabolites derived from mel-
Protection by melatonin from free radicals, especially from atonin should also be more often considered as pharmacologi-
the highly reactive, potentially mutagenic, carcinogenic and cal tools produced by nature. Moreover, melatonin itself
cell-toxic hydroxyl radicals, is a very important feature which promises to become a powerful pharmacological agent with
leads to diverse implications for health, aging and, in particu- its unique properties as a nontoxic, highly effective radical
lar, age-related neurogenerative disorders (112,130). The po- scavenger which provides protection eventually from neurode-
tential role of the pineal gland in aging has repeatedly been generation as well as from the mutagenic and carcinogenic
stressed (99,101,110,138). Especially the often observed age- actions of hydroxyl radicals.
dependent decline of nocturnal melatonin may have consider-
able consequences for neurodegenerative processes. More- ACKNOWLEDGEMENTS
over, sources of free-radical production can increase with age Work by the authors was supported in part by NSF grant IBN 91
[e.g., the activity of MAO-B (127,140)]. Many other indica- 21262.

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