Further insights into the reaction of melatonin with hydroxyl radical

Article abstract of DOI:10.1016/S0045-2068(02)00511-4

Recent interest has focused on the use of exogenous melatonin as an antioxidant, particularly to scavenge the highly cytotoxic hydroxyl radical (HO^?) which may be generated in many pathological conditions. However, in vitro and in vivo studies aimed at assessing the antioxidant properties of melatonin have produced conflicting results. While it is known that HO^? reacts with melatonin at a diffusion limited rate, very little is known about the products of this reaction. In this investigation it is shown that incubation of melatonin with a Fenton-type HO^?-generating system at pH 7.4 forms a complex mixture of primary products. These include 2-hydroxymelatonin, which was isolated as its more stable oxindole tautomer, 4- and 6-hydroxymelatonin, N-acetyl-N²-formyl-5-methoxykynurenine and 7,7′-bi-(5-methoxy-N-acetyltryptamine-4-one). Reaction pathways that might lead to these products are described. The differing biological effects of these products, while currently incompletely understood, might account for the controversy concerning the antioxidant properties of melatonin.

Full text of DOI:10.1016/S0045-2068(02)00511-4

BIOORGANIC
CHEMISTRY
Bioorganic Chemistry 30 (2002) 371–382
www.elsevier.com/locate/bioorg
Further insights into the reaction of
melatonin with hydroxyl radical
Joseph A. Horstman, Monika Z. Wrona, and Glenn Dryhurst*
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019, USA
Received 27 June 2002
Abstract
Recent interest has focused on the use of exogenous melatonin as an antioxidant,
Å
particularly to scavenge the highly cytotoxic hydroxyl radical (HO ) which may be
generated in many pathological conditions. However, in vitro and in vivo studies aimed
at assessing the antioxidant properties of melatonin have produced conflicting results.
Å
While it is known that HO reacts with melatonin at a diffusion limited rate, very little is
known about the products of this reaction. In this investigation it is shown that in-
Å
cubation of melatonin with a Fenton-type HO -generating system at pH 7.4 forms a
complex mixture of primary products. These include 2-hydroxymelatonin, which was
isolated as its more stable oxindole tautomer, 4- and 6-hydroxymelatonin, N-acetyl-
2
0
N -formyl-5-methoxykynurenine and 7,7 -bi-(5-methoxy-N-acetyltryptamine-4-one).
Reaction pathways that might lead to these products are described. The differing
biological effects of these products, while currently incompletely understood, might
account for the controversy concerning the antioxidant properties of melatonin.
Ó 2002 Published by Elsevier Science (USA).
Keywords: Melatonin; Hydroxyl radical; Antioxidant; Methamphetamine; MPTP
1
. Introduction
Brain melatonin (N-acetyl-5-methoxytryptamine) is biosynthesized from 5-
hydroxytryptamine (5-HT; serotonin) in the pineal gland [1,2]. Melatonin levels follow
*
Corresponding author: Fax: (405)-325-6111.
E-mail address: gdryhurst@ou.edu (G. Dryhurst).
0045-2068/02/$ - see front matter Ó 2002 Published by Elsevier Science (USA).
PII: S0045-2068(02)00511-4

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J.A. Horstman et al. / Bioorganic Chemistry 30 (2002) 371–382
a cyclic pattern with peak concentrations being achieved at night [3]. This variation
of melatonin concentration with time appears to be a major factor associated with
the maintenance of circadian rhythmicity [4,5]. While endogenously formed mela-
tonin may play a number of physiological roles, much recent interest has focused on
its potential role as an antioxidant that scavenges and detoxifies reactive oxygen
Å
species (ROS), particularly the highly cytotoxic hydroxyl radical (HO ) [6,7], and
reactive nitrogen species (RNS) such as peroxynitrite and nitric oxide [8,9]. However,
Å
while melatonin reacts very rapidly with HO [10] its physiological concentrations in
blood plasma, cerebrospinal fluid and various tissues (<1 nM) [11–13] are much
lower than those of other endogenous antioxidants such as glutathione (GSH), a-
tocopherol, and ascorbate. Thus, it is unlikely that a major biological function of
melatonin in vivo is as an antioxidant [9], particularly under pathological conditions
in which excessive production of ROS and RNS contribute to tissue damage.
A number of in vitro and in vivo studies, however, have reported that exogenously
administered melatonin provides protection against oxidative stress [14–17]. This, in
Å
part, may be due to the ability of melatonin to directly scavenge HO and other ROS
but also by upregulation of endogenous antioxidant defense mechanisms. For ex-
ample, melatonin increases the activity of glutathione peroxidase [18] and superoxide
dismutase [19]. However, other studies suggest that melatonin is a relatively inef-
fective antioxidant [20–22] or may even have prooxidant properties [23].
There is substantial evidence that the selective nigrostriatal dopaminergic
þ
neurotoxicity of 1-methyl-4-phenylpyridinium (MPP ), the active metabolite of
Å
1
generation [24,25]. Furthermore, elevation of brain levels of HO scavengers such as
-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the brain, involves HO
Å
þ
GSH [26] or salicylate [27] protect against MPTP/MPP -induced neurotoxicity.
Some studies also indicate that administration of large doses of melatonin to mice
and rats fully [28,29] or partially protect dopaminergic neurons against MPTP/
þ
Å
MPP -induced toxicity by scavenging HO [30]. However, other studies fail to
þ
demonstrate that melatonin protects against the neurotoxicity of MPTP/MPP [31–
33]. Methamphetamine (MA) also evokes neurotoxic effects on nigrostriatal dopa-
mine neurons and serotonergic terminals throughout the brain [34] by a mechanism
Å
that appears to involve HO generation [35,36]. Again, however, while some studies
report that large doses of melatonin provide full [33] or partial [37] protection
against the neurotoxic effects of MA, others suggest that melatonin exacerbates its
dopaminergic and serotonergic neurotoxicity [38]. Reports that melatonin fails to
þ
block the neurotoxicity of MPTP/MPP [31–33], exacerbates the neurotoxic effects
of MA [38] and may be a prooxidant [23] raise the possibility that under certain
Å
circumstances it may react with HO forming products that potentiate the neurotoxic
effects of these drugs. Indeed, administration of high doses of melatonin to human
cancer patients induces a form of eosinophilia-myalgia (ESM) [39,40], a syndrome
associated with neurotoxic consequences in the brain [41]. It may also be of relevance
that partially characterized oxidized forms of melatonin present as contaminants in
commercial preparation of this drug may be associated with ESM [42].
Å
Although HO attack on melatonin in vitro and in vivo forms a number of products,
evidence for the structure of only one product, a cyclized form of 3-hydroxymelato-
nin (1,2,3,3a,8,8a-hexahydro-1-acetyl-5-methoxy-3a-hydroxypyrrolo[2,3-b]indole), has

J.A. Horstman et al. / Bioorganic Chemistry 30 (2002) 371–382
373
been presented [43]. The present communication describes additional products formed
Å
by the HO -mediated oxidation of melatonin.
2
. Materials and methods
Melatonin, ethylenediaminetetraacetic acid (disodium salt, Na EDTA), ascorbic
2
acid, 5-hydroxy-2-oxindole, and deuterated acetonitrile (CD CN) were obtained
3
from Sigma (St. Louis, MO), ferrous ammonium sulfate from Mallinckrodt (St.
2 2
Louis, MO), HPLC grade methanol (MeOH) and H O (30% by weight) from EM
Science (Gibbstown, NJ), HPLC grade MeCN from Fisher Scientific (Fairlawn, NJ)
and trifluoroacetic acid (TFA) from Arcos (NJ).
2
.1. Preparative HPLC
A Gilson (Middleton, WI) binary gradient HPLC system was used equipped with
two model 712 pumps, 10 mL injection loop, preparative reversed phase column
(Xpertek Bakerbond C-18, 250 Â 22:5-mm, P.J. Cobert Associates, St. Louis, MO)
and a Holochrome UV detector set at 254 nm.
HPLC method I was used for the initial separation of the products formed by the
Å
HO -mediated oxidation of melatonin. Solvent A was prepared by adding 100 mL of
MeCN to 900 mL of deionized water; the pH of the resulting solution was then
adjusted to 2.4 with concentrated TFA. Solvent B was prepared by adding 100 mL of
MeOH and 400 mL of MeCN to 500 mL of deionized water; the pH of this solution
was then adjusted to 2.4 with TFA. The following gradient was used: 0–3 min, linear
gradient from 100% solvent A to 50% solvent B; 3–10 min, linear gradient to 60%
solvent B; 10–20 min, linear gradient to 100% solvent B; 21–25 min, 100% solvent B.
À1
The flow rate was 10 mL min .
Individual products of the HO -mediated oxidation of melatonin were further
Å
purified using HPLC method II. This method differed from HPLC method I only
with respect to the mobile phase gradient employed which was: 0–3 min, 100% sol-
vent A; 3–53 min, linear gradient to 100% solvent B; 53–60 min, 100% solvent B. The
À1
flow rate was 10 mL min .
2
.2. Hydroxyl radical-mediated oxidation of melatonin
A stock solution of melatonin (7.6 mM) was prepared by dissolving 52.86 mg of
the solid in 30 mL of deionized water with gentle heating and stirring. This stock
solution was stable for at least one week when stored in the dark at 4 °C.
A recycling Fenton reaction system developed by Udenfriend et al. [44] was em-
Å
ployed to generate HO which employed a freshly prepared solution of ascorbic acid
(
2 2 2
9.5 mM), Na EDTA (26.6 mM), ferrous ammonium sulfate (2.0 mM), and H O
8.70 mM). The sequence of additions and final concentrations for these reagents was
(
as follows: 7.8 mL of pH 7.4 phosphate buffer (50 mM), 1.5 mL of melatonin stock
solution (0.8 mM), 1.5 mL of ascorbic acid solution (1.0 mM), 0.15 mL Na EDTA
solution (1.1 mM) to give a
2
2þ
(
0.28 mM), 1.5 mL Fe (0.21 mM) and 1.8 mL of H O
2 2

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J.A. Horstman et al. / Bioorganic Chemistry 30 (2002) 371–382
total volume of14.25 mL. The resulting solution was stirred at ambienttemperaturefor
1
5 min. Preliminary studies revealed that the latter reaction time was optimal for
Å
generation of primary reaction products of the HO -mediated oxidation of melatonin
with minimal secondary reactions which occurred at longer reaction times.
The reaction mixture was filtered through a Millipore (Bedford, MA) type HA
(0.45 lm) filter and then injected into the preparative HPLC system and products
initially separated using method I. Fractions eluted under each chromatographic
peak were collected separately and immediately frozen in a dry-ice/acetone bath. In
order to obtain sufficient amounts of each product to permit spectroscopic structure
elucidation and other studies, it was necessary to carry out the preceding procedure
>
50 times. Combined fractions collected under each peak were then freeze-dried and
then redissolved in the minimum volume of solvent A and purified using preparative
HPLC method II. This latter process was usually repeated several times in order to
obtain pure fractions. The solution containing each purified compound was then
freeze-dried and subsequently stored in a dessicator at )80 °C.
2
.3. Spectroscopy
Electrospray ionization mass spectrometry (ESMS) was carried out on either a
Micromass (Beverly, MA) Q-TOF or PE Biosystems (Foster City, CA) Sciex API III
1
spectrometer. H NMR spectra were obtained on a Varian (Palo Alto, CA) XL-400
spectrometer. UV–visible spectra were recorded on a Hewlett-Packard (Palo Alto,
CA) model 8452A diode array spectrophotometer.
Spectroscopic evidence in support of the proposed structures of major products
Å
formed following the HO -mediated oxidation of melatonin is presented below. The
1
assignments of proton resonances observed in H NMR spectra of melatonin and its
reaction products were confirmed by two-dimensional correlated spectroscopy
(COSY) experiments.
2
.4. Melatonin
The UV spectrum of melatonin dissolved in 5% aqueous MeOH at pH 7.4 gave
þ
bands at k
¼ 295 (sh), 278 and 228 nm. ESMS gave m=z ¼ 233 (MH , 100%).
max
1
H NMR ðCD CNÞ gave d: 8.96 (bs, 1H, N(1)–H), 7.29 (d, 1H, J ¼ 8:8 Hz, C(7)–
3
H), 7.09 (d, 1H, J ¼ 2:4 Hz, C(4)–H), 7.07 (d, 1H, J ¼ 2:0 Hz, C(2)–H), 6.79 (dd, 1H,
J ¼ 8:8 Hz, J ¼ 2:4 Hz, C(6)–H), 6.42 (bs, 1H, side-chain N–H), 3.82 (s, 3H, OCH
3
3
),
).
3
.42 (m, 2H, CðbÞ–H
2
), 2.86 (t, 2H, CðaÞ–H
2
), and 1.84 (s, 3H, side-chain –COCH
2
.5. 6-Hydroxymelatonin
This compound was isolated as an off-white solid that, when dissolved in pH 7.4
phosphate buffer, exhibited a UV spectrum with bands at kmax ¼ 300, 280 (sh) and
þ
1
2
1
20 nm. ESMS gave m=z ¼ 249 (MH , 100%). H NMR ðCD
H, N(1)–H), 7.10 (s, 1H, C(7)–H), 6.91 (d, 1H, J ¼ 2:4 Hz, C(2)–H), 6.85 (s, 1H,
), 3.41 (m, 2H, CðbÞ–H ),
3
CNÞ gave d: 8.76 (bs,
C(4)–H), 6.40 (bs, 1H, side-chain N–H), 3.90 (s, 3H, OCH
.83 (m, 2H, CðaÞ–H ), 1.84 (s, 3H, side-chain –COCH ).
3
2
2
2
3

J.A. Horstman et al. / Bioorganic Chemistry 30 (2002) 371–382
.6. 5-Methoxy-3-(N-acetyl)-ethylamino-2-oxindole (5-MeO)
375
2
This compound was isolated as a white solid. At pH 7.4 it exhibited a UV spectrum
þ
with bands at kmax ¼ 298 and 258 nm. ESMS gave m=z ¼ 249 (MH , 100%).
1
H NMRðCD CN) gave d: 8.34 (s, 1H, N(1)–H), 6.97 (d, 1H, J ¼ 2:4 Hz, C(4)–H),
3
6
.81 (d, 1H, J ¼ 8:4 Hz, C(7)–H), 6.77 (dd, 1H, J ¼ 8:4, 2.4 Hz, C(6)–H), 6.58 (bs, 1H,
side-chain N–H), 3.76 (s, 3H, –OCH ), 3.42 (t, 1H, J ¼ 6:0 Hz, C(3)–H), 3.30 (m, 2H,
3
CðbÞ–H ), 2.01 (m, 2H, CðaÞ–H ), 1.83 (s, 3H, side-chain –COCH ).
2
2
3
2
.7. 4-Hydroxymelatonin
This compound was isolated as an off-white solid. In pH 7.0 phosphate buffer it
had a UV spectrum with bands at kmax ¼ 308 and 238 nm. ESMS gave m=z ¼ 249
þ
(
MH , 100%). It was not possible to isolate sufficient quantities of this compound to
1
obtain a reliable H NMR spectrum. However, its molecular mass (248 g) clearly
indicates that this compound is a monohydroxylated derivative of melatonin. Since
the keto tautomer of 2-hydroxymelatonin (i.e., 5-MeO) and 6-hydroxymelatonin
were unequivocally identified, it seems probable that this compound is 4-hydrox-
ymelatonin. The UV spectrum of this compound is also very similar to that of
4
4
,5-dihydroxytryptamine (kmax at pH 7.0 ¼ 304 and 220 nm) and 7-S-glutathionyl-
,5-dihydroxytryptamine [45,46] providing additional indirect support for the con-
clusion that it is 4-hydroxymelatonin.
0
0
2
.8. 7,7 -Bi-(5-methoxy-N-acetyltryptamine-4-one) (7,7 -D)
This product was isolated as a dark brown/purple solid that gave bright pink–
purple solutions between pH 2–8. The UV–visible spectrum of this compound dis-
solved in MeOH gave bands at kmax ¼ 566, 378, 302 and 226 nm. ESMS of a freshly
þ
purified solution of this compound (HPLC method I) gave m=z ¼ 493 (MH , 100%).
1
Attempts to obtain a H NMR spectrum of this compound, however, were unsuc-
cessful owing to its very low solubility and instability in all solvents employed.
However, in recent studies, to be described in detail elsewhere, we have isolated and
fully characterized a structurally related compound formed from tryptamine-4,5-
0
dione in acidic MeOH solution, 7,7 -bi-(5-methoxytryptamine-4-one). The UV–
0
visible spectrum of 7,7 -bi-(5-methoxytryptamine-4-one) in MeOH, kmax ¼ 554, 372,
0
2
96 and 226 nm is virtually identical to that of 7,7 -D. Furthermore, the molar mass
0
0
of 7,7 -D(492 g) differs from that of 7,7 -bi-(5-methoxytryptamine-4-one) (408 g) by
that expected for two acetyl residues substituted on the ethylamino side-chains of the
former compound. We have also synthesized a compound with identical properties
to 7,7 -Dutilizing the same method employed to obtain 7,7 -bi-(5-methoxytrypta-
mine-4-one). Briefly, N-acetyl-5-hydroxytryptamine (30 mg) was oxidized with po-
tassium nitrosodisulfonate (60 mg) in 20 mL of deionized water for 3 h. The product
of this oxidation, N-acetyltryptamine-4,5-dione (ꢀ11 mg), was purified by pre-
parative HPLC method II and then freeze-dried. The latter dry product was dis-
solved in MeOH (5 mL) containing 50 lL of concentrated TFA and stirred for
0
0
4
5 min. The resultant product solution was injected into the preparative HPLC

376
J.A. Horstman et al. / Bioorganic Chemistry 30 (2002) 371–382
system and separated using HPLC method I. The major product of this reaction had
0
an identical retention time (t
R
), UV–visible and ESMS spectra to 7,7 -D.
2
2
.9. N-Acetyl-N -formyl-5-methoxykynurenamine (AFMK)
This compound was isolated as a pale yellow solid which in solution at pH 7.4 ex-
hibited a UV–visible spectrum with bands at kmax ¼ 342, 262 (sh) and 234 nm. ESMS
þ
gave m=z ¼ 265 (MH , 100%). These properties are in agreement with those expected
for AMFK [47].
3
. Results
An HPLC chromatogram (preparative method I) of the product mixture formed
Å
following incubation of melatonin (0.8 mM) for 15 min with the HO -generating sys-
Å
tem employed is presented in Fig. 1. This chromatogram reveals that the HO -mediated
oxidation of melatonin at pH 7.4 forms a complex mixture of products. Preliminary
studies revealed that all of the major product peaks in the chromatogram shown in Fig.
1
the final reactant (H O ) that initiates HO generation. During the succeeding 15 min
were present when the reaction solution was sampled within 1 min after addition of
Å
2
2
each of these major product peaks reached their maximal height. After longer periods,
however, secondary reactions began to occur as indicated by the decline of some major
product peaks and the appearance of new peaks in chromatograms. Accordingly, in
Å
order to gain some insights into the initial steps in the HO -mediated oxidation of
Å
melatonin, several major products formed after a 15 min incubation with the HO -
generating system were isolated and identified.
R
The major compound eluted under the peak having a t of 15 min (Fig. 1), using
HPLC method I, was 4-hydroxymelatonin. However, subsequent chromatography
of the species eluted under this peak using HPLC method II revealed that 4-
hydroxymelatonin was contaminated with two additional compounds although these
were not formed in sufficient quantities to permit their isolation and spectroscopic
identification. Similarly, the peak at eluting t
into one major component, AMFK, and two unidentified minor components using
R
¼ 19 min (Fig. 1) could be resolved
HPLC method II.
The molar mass of the compound eluted at t
it contains one more oxygen substituent than melatonin (molar mass 232 g). The UV
R
¼ 17 min was 248 g indicating that
spectrum of this compound was virtually identical to that of 5-hydroxy-3-ethyl-
Å
amino-2-oxindole (5-HEO), the major product of the HO -mediated oxidation of
5
-HT at pH 7.4 [48], and an authentic sample of 5-hydroxy-2-oxindole. Furthermore,

J.A. Horstman et al. / Bioorganic Chemistry 30 (2002) 371–382
377
Fig. 1. HPLC chromatogram (preparative method I) of the product mixture formed following
Å
incubation of melatonin (0.8 mM) with a recycling Fenton-type HO generating system.
1
the characteristic triplet (1H, 3.42 ppm) observed in the H NMR spectrum of the
¼ 17 min and in that of 5-HEO (1H, 3.52 ppm), which are
compound eluted at t
R
both coupled to the side-chain CðbÞ–H protons, clearly indicates that both com-
2
pounds possess a hydrogen atom substituted at C(3). Furthermore, there are no
changes in the N(1)–H, side-chain N–H, CðaÞ–H and C(b)–H resonances of 5-MeO
2
2
compared to melatonin indicating that free rotation of the N-acetylethylamino side-
chain is preserved thus excluding the possibility of a cyclized structure. Taken to-
gether, the mass, NMR and UV spectra of the compound that eluted at t
and their similarity to those of 5-HEO indicate that the former is the keto tautomer
of 2-hydroxymelatonin, i.e., 5-MeO.
R
¼ 17 min
The ESMS of the compound eluted under the peak at t
cates it has a molar mass of 492 g suggesting it consists of two residues formed by an
R
¼ 21 min (Fig. 1) indi-

378
J.A. Horstman et al. / Bioorganic Chemistry 30 (2002) 371–382
þ
overall 2e=2H oxidation of a hydroxymelatonin (molar mass 248 g). Unfortunately,
1
attempts to obtain the H NMR spectrum of this compound were not successful.
Nevertheless, comparison of the spectral properties of this compound with those of
0
7
and methods) supports the proposed structure of 7,7 -D.
,7 -bi-(5-hydroxytryptamine-4-one) and an independent synthesis (see Materials
0
Attempts to isolate and characterize the products eluted under the peaks prior to
-hydroxymelatonin (Fig. 1) have not yet been successful owing to their instability.
4
4
. Discussion
Despite a growing, albeit controversial, literature concerning the antioxidant
properties of melatonin, surprisingly little is known about the chemistry of its
Å
reaction with HO , the most cytotoxic ROS. Previous studies have established that
Å
10
À1 À1
HO reacts very rapidly with melatonin in aqueous solution (k ¼ 2:7 Â 10 M s )
[
3
10] although evidence for only a single product of this reaction, a cyclized form of
-hydroxymelatonin, is available [43]. The present investigation demonstrates that
Å
the HO -mediated oxidation of melatonin in buffered aqueous solution at pH 7.4
results in formation of a complex mixture of primary products. The products that
have been identified are clearly formed as a result of hydroxylation of the indolic
ring system of melatonin. Monohydroxylated products are 2-hydroxymelatonin
(isolated as its more stable 2-oxindole tautomer, 5-MeO), 4- and 6-hydroxymela-
tonin. Hardeland et al. [49] have proposed a mechanism for the initial reaction of
Å
melatonin with HO in which an electron is abstracted from the aromatic indolic
ring system to form a radical cation intermediate. However, a subsequent study
concluded that such a reaction is not thermodynamically feasible [50]. More
Å
probable pathways involve either abstraction of an indolic hydrogen by HO , to
Å
form a neutral radical intermediate, or direct addition of HO to the indolic moiety
Å
[
and 5-HT [48]. Thus, it is proposed that HO initially attacks melatonin to form
50]. The latter reaction has been proposed for the attack of HO on catechols [51]
Å
the neutral radicals 1a–1d (Scheme 1). Subsequent hydrogen atom abstraction from
Å
1
a, 1b, and 1c by a second HO , with elimination of H O, results in formation of 6-
2
hydroxy-, 4-hydroxy-, and 2-hydroxymelatonin, respectively, the latter tautomer-
izing to 5-MeO. In order to account for formation of AMFK, it seems plausible to
Å
suggest that the initial attack by HO on melatonin forms radical 1d in which a
hydroxyl group is added to the C(3)-position. Subsequent hydrogen atom ab-
Å
straction by HO would then form 3-hydroxymelatonin (Scheme 1). The latter
compound is a possible precursor of cyclized 3-hydroxymelatonin [44] although in
the present study neither 3-hydroxymelatonin nor its cyclized forms were identified
Å
Å
as a primary product of the HO -mediated oxidation of melatonin. Attack by HO
on 3-hydroxymelatonin would be expected to form radical 5, containing a second
Å
hydroxyl substituent at C(2), which is further oxidized by HO to 2-keto-3-
hydroxymelatonin (6, Scheme 1). Facile ring opening of 6 would then account for
formation of AMFK.
A plausible route leading to formation of 7,7 -Dinvolves hydrogen atom ab-
0
Å
straction by HO from 4-hydroxymelatonin forming radical intermediate 3 which

J.A. Horstman et al. / Bioorganic Chemistry 30 (2002) 371–382
379
Scheme 1.
dimerizes to 4a/4b. Subsequent oxidation of 4 by molecular oxygen would then form
0
7
,7 -D(Scheme 1).
Previous studies have reported that 6-hydroxymelatonin is the major metabolite
of melatonin in the liver and kidney [52] whereas in brain melatonin is metabolized
to AMFK and thence to N-acetyl-5-methoxykynurenamine [53]. The present in-
vestigation demonstrates for the first time that 6-hydroxymelatonin and AMFK are
Å
also formed by non-enzymatic oxidation of melatonin by HO .
As noted earlier, there is considerable controversy in the literature concerning the
antioxidant properties of melatonin. Such controversies are particularly evident in
studies aimed at assessing the protective effects of melatonin against the neurotox-
þ
Å
icity of MPTP/MPP and MA in which HO generation appears to be an important
participant in the neurotoxic mechanism evoked by these drugs [24,35,36]. One
Å
product of the HO -mediated oxidation of melatonin, AMFK, based on the present
investigation, might be expected to be formed in the brain in response to MPTP/
MPP and MA administration. Furthermore, AMFK has been shown to be a potent
antioxidant [54]. Thus, formation of AMFK would be expected to potentiate
the neuroprotective properties of melatonin. However, another product of the
þ

380
J.A. Horstman et al. / Bioorganic Chemistry 30 (2002) 371–382
Å
HO -mediated oxidation of melatonin, 6-hydroxymelatonin, can function not only as
Å
Å
a scavenger of HO but also as a promoter of HO generation [10]. Thus, in neutral
2þ
aqueous solution in the presence of Fe and molecular oxygen, 6-hydroxymelatonin
Å
can generate large quantities of HO [10]. By analogy, it is conceivable that another
Å
product of the HO -mediated oxidation of melatonin, 4-hydroxymelatonin, may also
Å
potentiate HO generation by similar reaction pathways. Because of its quinoidal
0
structure, 7,7 -Dwould also be expected to redox cycle in the presence of endogenous
reductants such as ascorbate and/or glutathione with resultant generation of ROS
ÀÅ
Å
such as O , H O and thence HO by Fenton/Haber–Weiss-type chemistry [44].
2
2
2
Thus, melatonin might potentiate oxidative stress as a consequence of formation of
products that mediate ROS generation. Such chemistry may contribute to the con-
troversial results concerning the antioxidant properties of melatonin. It may also be
of relevance that oxindoles structurally similar to 5-MeO can evoke a number of
pathophysiological effects in vivo including impairment of locomotor activity, hy-
potension, loss of righting reflex and coma [55]. Furthermore, incompletely identified
hydroxylated forms of melatonin that contaminate commercial preparations of this
compound have been case-implicated with ESM [42] and possible neurotoxic con-
sequences [41]. Taken together, the controversies concerning the antioxidant prop-
erties of melatonin in vitro and in vivo may be dependent on a variety of factors
including the doses employed and the nature and relative yields of products formed
Å
consequent to its reactions with HO or other ROS and RNS.
Acknowledgments
This work was supported by National Institutes of Health Grant No. GM32367.
Additional support was provided by the Vice President for Research and the Honors
College at the University of Oklahoma. The research reported in this communication
resulted from an undergraduate Senior Thesis (JAH).
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