Nitrite- and peroxide-dependent oxidation pathways of dopamine: 6- nitrodopamine and 6-hydroxydopamine formation as potential contributory mechanisms of oxidative stress- and nitric oxide-induced neurotoxicity in neuronal degeneration

Article abstract of DOI:10.1021/tx990121g

In the presence of nitrite ions (NO₂^-) in phosphate buffer (pH 7.4) and at 37 °C, dopamine was oxidized by a variety of hydrogen peroxide (H₂O₂)-dependent enzymatic and chemical systems to give, in addition to black melanin-like pigments via 5,6-dihydroxyindoles, small amounts of the potent neurotoxin 6-hydroxydopamine (1) and of 6-nitrodopamine (2), a putative reaction product of dopamine with NO-derived species. Treatment of 0.5 or 1 mM dopamine with horseradish peroxidase (HRP) or lactoperoxidase (LPO) in the presence of 1 or 2 mM H₂O₂ with NO₂^- at a concentration of 0.5-10 mM resulted in the formation of 1 and 2 in up to 8 and 2 μM yields, respectively, depending on the substrate concentration and the NO₂^-: H₂O₂ ratio. Nitration and hydroxylation of 0.1 mM dopamine was observed with 1 mM NO₂^- using HRP and the D-glucose/glucose oxidase system to generate H₂O₂ in situ. In the presence of NO₂^, Fe²⁺-, or Fe²⁺/EDTA-promoted oxidations of dopamine with H₂O₂ also led to the formation of 1 and 2, the apparent product ratios varying with peroxide concentration and the partitioning of the metal between EDTA and catecholamine chelates. In the presence of NO₂^-, Fe²⁺-promoted autoxidation of dopamine gave 2 but no detectable 1. When injected into the brains of laboratory rats, 2 caused sporadic behavioral changes, indicating that it could elicit a neurotoxic response, albeit to a lower extent than 1. Model experiments using tyrosinase as an oxidizing system and mechanistic considerations suggested that formation of 2 does not involve reactive nitrogen radicals but results mainly from nucleophilic attack of NO₂^- to dopamine quinone. Generation of 1, on the other hand, may be derives from different H₂O₂-dependent pathways. Collectively, these results outline a complex interplay of NO₂^-- and peroxide-dependent oxidation pathways of dopamine, which may contribute to impair dopaminergic neurotransmission and induce cytotoxic processes in neurodegenerative disorders.

Full text of DOI:10.1021/tx990121g

Chem. Res. Toxicol. 1999, 12, 1213-1222
1213
Nitr ite- a n d P er oxid e-Dep en d en t Oxid a tion P a th w a ys of
Dop a m in e: 6-Nitr od op a m in e a n d 6-Hyd r oxyd op a m in e
F or m a tion a s P oten tia l Con tr ibu tor y Mech a n ism s of
Oxid a tive Str ess- a n d Nitr ic Oxid e-In d u ced
Neu r otoxicity in Neu r on a l Degen er a tion
Anna Palumbo,† Alessandra Napolitano,‡ Paolo Barone,§ and Marco d’Ischia*,‡
Laboratory of Biochemistry, Zoological Station, I-80121 Naples, Italy, Department of Organic and
Biological Chemistry, University of Naples “Federico II”, via Mezzocannone 16,
1-80134 Naples, Italy, and Department of Neurological Sciences, University of Naples “Federico II”,
via Pansini 5, 1-80131 Naples, Italy
Received J uly 7, 1999
In the presence of nitrite ions (NO2-) in phosphate buffer (pH 7.4) and at 37 °C, dopamine
was oxidized by a variety of hydrogen peroxide (H2O2)-dependent enzymatic and chemical
systems to give, in addition to black melanin-like pigments via 5,6-dihydroxyindoles, small
amounts of the potent neurotoxin 6-hydroxydopamine (1) and of 6-nitrodopamine (2), a putative
reaction product of dopamine with NO-derived species. Treatment of 0.5 or 1 mM dopamine
with horseradish peroxidase (HRP) or lactoperoxidase (LPO) in the presence of 1 or 2 mM
-
H2O2 with NO2 at a concentration of 0.5-10 mM resulted in the formation of 1 and 2 in up
-
to 8 and 2 µM yields, respectively, depending on the substrate concentration and the NO2
:
-
H2O2 ratio. Nitration and hydroxylation of 0.1 mM dopamine was observed with 1 mM NO2
using HRP and the D-glucose/glucose oxidase system to generate H2O2 in situ. In the presence
of NO2--, Fe2+-, or Fe2+/EDTA-promoted oxidations of dopamine with H2O2 also led to the
formation of 1 and 2, the apparent product ratios varying with peroxide concentration and the
-
partitioning of the metal between EDTA and catecholamine chelates. In the presence of NO2
,
Fe2+-promoted autoxidation of dopamine gave 2 but no detectable 1. When injected into the
brains of laboratory rats, 2 caused sporadic behavioral changes, indicating that it could elicit
a neurotoxic response, albeit to a lower extent than 1. Model experiments using tyrosinase as
an oxidizing system and mechanistic considerations suggested that formation of 2 does not
-
involve reactive nitrogen radicals but results mainly from nucleophilic attack of NO2 to
dopamine quinone. Generation of 1, on the other hand, may be derives from different H2O2-
dependent pathways. Collectively, these results outline a complex interplay of NO2-- and
peroxide-dependent oxidation pathways of dopamine, which may contribute to impair dopa-
minergic neurotransmission and induce cytotoxic processes in neurodegenerative disorders.
Whether and by what modalities elevated fluxes of
reactive oxygen species can affect the structural and
functional integrity of dopamine and impair dopaminer-
gic nigrostriatal connectivities has not yet been assessed
in sufficient detail. Frequently cited theories advocate
aberrant oxidation pathways of the labile catechol func-
tionality as the leading cause of dopamine-mediated
neurotoxicity (8). These pathways may proceed through
either electron transfer steps, to give dopamine semi-
quinone and o-quinone via subsequent disproportion-
ation, or direct electrophilic attack to the aromatic ring
by a reactive oxygen species, e.g., the OH• radical (9).
In tr od u ction
Functional abnormalities of dopaminergic neuronal
pathways, due to selective degenerative changes and the
loss of pigmented nigrostriatal neurons, and a progres-
sively severe depletion of dopamine constitute the pri-
mary neurochemical correlates of Parkinson’s disease and
related neurodegenerative disorders, and are currently
interpreted as arising from a chronic oxidative stress
condition (1-4) associated with a progressive siderosis
with abnormal iron load (5) and decreased ferritin levels
(6), promoting hydroxyl radical (OH•) formation from
superoxide (O2-•) and H2O2 through Fenton-type reac-
tions (7).
Whatever the actual mechanism(s), these reactions
may generate a spectrum of potentially toxic oxidative
metabolites, which may interact with critical cellular
ingredients, including thiol compounds such as cysteine,
glutathione, and sulfhydryl-containing enzymes (10-13).
Among the putative mediators of dopamine-dependent
neurotoxicity in vivo, 6-hydroxydopamine (1) has tradi-
tionally occupied a prominent position because of its
established ability to destroy dopaminergic neurons and
* To whom correspondence should be addressed: Department of
Organic and Biological Chemistry, University of Naples “Federico II”,
Via Mezzocannone, 16 I-80134 Naples, Italy. Phone: +39-081-7041207.
Fax: +39-081-5521217. E-mail: dischia@cds.unina.it.
† Zoological Station.
‡ Department of Organic and Biological Chemistry, University of
Naples “Federico II”.
§ Department of Neurological Sciences, University of Naples
“Federico II”.
10.1021/tx990121g CCC: $18.00 © 1999 American Chemical Society
Published on Web 11/18/1999

1214 Chem. Res. Toxicol., Vol. 12, No. 12, 1999
Palumbo et al.
cause Parkinsonism, possibly through oxygen radical
production and alteration of zinc metallothionein (1, 2,
14). Extensive investigations aimed at identifying pos-
sible routes from dopamine to 1 under physiologically
relevant conditions have highlighted several candidate
mechanisms, including reaction of dopamine with OH•
radicals derived from the Fenton reagent and related
ascorbate-dependent hydroxylating systems (9, 15, 16);
the nucleophilic addition of H2O2 to dopamine quinone
produced by peroxidase/hydrogen peroxide (17); iron- and
manganese-promoted autoxidation (18, 19); and a novel
hydroxylation of the dopamine-Fe3+ chelate mediated by
lipid hydroperoxides (20).
Novel hints of potential mechanisms of dopamine-
related neurotoxicity have been derived from the recent
outbreak of nitric oxide (nitrogen monoxide, NO) as a
central transducer and effector in a bewildering array of
physiological and pathophysiological processes (21, 22).
Despite a generally protective role of NO in inflammatory
processes (23), an excessive production by upregulated
activity of the various NO synthase isoforms may amplify
and exacerbate host tissue injury and cell damage.
Putative mediators of the adverse effects of NO include
nitrogen dioxide (NO2), formed by aerobic oxidation,
which has been shown to effectively react with dopamine
and related catecholamines to give 6-nitrodopamine (2)
and its analogues (24, 25), and peroxynitrite (ONOO-),
a most powerful nitrating and hydroxylating agent (26-
29) produced by coupling of NO with O2-. An additional
pathway of NO-dependent toxicity involves interaction
with H2O2 promoting hydroxylation of aromatic sub-
strates (30).
assessing on a merely chemical ground the relative
importance of hydroxylation versus nitration routes
leading to 1 and 2, respectively. The factors favoring one
pathway over the other are also addressed, and the
results are integrated into an expanded mechanistic
scheme of dopamine oxidation and oxidative stress-
dependent neurotoxicity.
Exp er im en ta l P r oced u r es
Ma ter ia ls. Dopamine hydrochloride, 2,4,5-trihydroxyphen-
ylethylamine hydrobromide (6-hydroxydopamine), ascorbic acid,
D-glucose, reduced glutathione, ferrous sulfate heptahydrate,
potassium thiocyanate, EDTA disodium salt, and nonstabilized
hydrogen peroxide (35% solution in water) were used as they
were obtained. Horseradish peroxidase (HRP) (donor:H2O2
oxidoreductase, EC 1.11.1.7) type II (167 pyrogallol units/mg,
RZ E430/E275 ) 2.0), lactoperoxidase (LPO) from bovine milk (80
pyrogallol units/mg, RZ E412/E280 ) 0.76), superoxide dismutase
(SOD) from bovine liver (H2O2:H2O2 oxidoreductase, EC 1.11.1.6;
9740 units/mg), glucose oxidase from Aspergillus niger (â-D-
glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) type II (20 000
units/mg), and mushroom tyrosinase (o-diphenol:O2 oxidoreduc-
tase, EC 1.14.18.1; 6300 units/mg) were used. All enzymes were
used without further purification. 6-Nitrodopamine (2) was
prepared from dopamine by reaction with sodium nitrite/sulfuric
acid as reported previously (24). 5-S-Glutathionyldopamine (11)
and 5,6-dihydroxyindole (17) were synthesized according to
reported procedures.
An a lytica l Meth od s. Analytical HPLC was performed with
an instrument equipped with a UV detector set at 254 nm.
Octadecylsilane-coated columns (4.6 mm × 250 mm or 10 mm
× 250 mm, 5 µm particle size) were used for analytical or
preparative runs, respectively. Flow rates of 1 or 6 mL/min were
used. The elution conditions included 0.05 M phosphate buffer
(pH 3.0) containing 10 mM sodium 1-octanesulfonate/acetoni-
trile [93:7 v/v (eluant A) or 88:12 v/v (eluant B)] or 5% formic
acid/acetonitrile [85:15 v/v (eluant C)]. The within-run reproduc-
ibilities were determined on four different samples, each
analyzed in triplicate (CV values in the range of 3-5%). The
detection limit was 50 nM for
1 and 2 at the detection
wavelength. Data are reported as mean values from three
separate experiments (SD < 5%). The pKa of the nitrocatechol
ring of 2 was determined in 0.1 M phosphate buffer by plotting
the absorbance at 422 nm versus pH in the pH range of 3-11.
ES/MS spectra were recorded with a VG BIO-Q triple-quadru-
pole mass spectrometer.
Bioch em ica l Assa ys. Production of H2O2 by glucose oxidase
was quantitated by oxidation of Fe(II) and formation of an Fe-
(III)-thiocyanate complex (29). The amount of nitrite was
determined spectrophotometrically at 543 nm using the Griess
reagent (1% sulfanylamide and 0.1% naphthylethylenediamine
in 2.5% phosphoric acid) (33).
En zym a tic Oxid a tion of Dop a m in e. To solutions of dopa-
mine (0.5-1.0 mM) in 0.1 M phosphate buffer (pH 7.4) in a
water bath thermostated at 37 °C were added sodium nitrite
(5-10 mM), HRP (0.4 unit/mL, final concentration), and 0.6%
hydrogen peroxide (1-3 mM final concentration) in that order
under vigorous stirring. Aliquots of the reaction mixture were
periodically withdrawn, treated with freshly prepared sodium
borohydride solution in water up to a final concentration of 10
mM to terminate oxidation, and filtered through Millipore
filters, and the filtrates were analyzed by HPLC using eluant
A or C. Similar experiments were performed in which hydrogen
peroxide was generated in situ using D-glucose (0.28-2.8 mM)
and glucose oxidase (0.01-0.1 unit/mL, final concentration) with
dopamine at a concentration of 0.1-1 mM in the presence of
nitrite (1-10 mM). In other experiments where mammalian
LPO was used, the enzyme (0.53 unit/mL, final concentration)
was added to the incubation mixture containing dopamine (1
mM) and when necessary nitrite (0.5-10 mM) followed by
hydrogen peroxide (2 mM). Additives, i.e., potassium thiocyanate
Increasing interest, in this context, is currently focused
to the possible role of nitrite (NO2-), the end product of
the hydrolysis of NO-derived nitrogen oxides (31, 32), as
an intrinsically less aggressive yet potentially toxic (33)
metabolite, capable of mediating phenolic nitration and
other reactions upon oxidative activation by inflamma-
tory oxidants (e.g., HOCl) or enzymes such as peroxidase
and superoxide dismutase (SOD)1 (34-37). Significant
concentrations of NO2- are found in physiological fluids
(29), in stimulated substantia nigra neurons (38), and,
notably, in the cerebrospinal fluid of patients with
various neurologic diseases (39), including Parkinson’s
disease (40), where abnormally elevated levels possibly
reveal an upregulated production of NO in degenerating
dopaminergic neurons (41).
The potential variety of NO-dependent cell-damaging
mechanisms emerging from these studies warranted a
detailed elucidation of the many possible routes of
dopamine-mediated neurotoxicity associated with exces-
sive production of NO. In this paper, we report a
-
systematic survey of the effects of NO2 on the reaction
behavior of dopamine with a variety of oxidizing agents
reported to induce tyrosine nitration and supposedly
implicated in neuronal degeneration, with a view to
1
Abbreviations: SOD, superoxide dismutase; HRP, horseradish
peroxidase; LPO, lactoperoxidase; ES/MS, electrospray mass spec-
trometry.

Dopamine Nitration and Hydroxylation
Chem. Res. Toxicol., Vol. 12, No. 12, 1999 1215
(1-10 mM), reduced glutathione (1 mM), ascorbic acid (1 mM),
sodium hydrogencarbonate (25 mM), and SOD (300 units/mL),
were added to the incubation mixtures containing dopamine at
a
concentration of 1 mM. In those experiments in which
tyrosinase was used as the oxidant, the enzyme (75 units/mL,
final concentration) was added to the solution of dopamine (1
mM) and sodium nitrite (10 mM) in 0.1 M phosphate buffer (pH
7.4). When necessary, hydrogen peroxide at a concentration of
1 mM was added to the oxidation mixture prior to the enzyme.
Identification and quantitation of dopamine and reaction prod-
ucts were carried out by comparing retention times and inte-
grated peak areas with external calibration curves for authentic
samples.
Ir on -P r om oted Oxid a tion of Dop a m in e. A stirred solution
of dopamine (1.0 mM) and sodium nitrite (1-10 mM) in 0.1 M
phosphate buffer (pH 7.4) in a water bath thermostated at 37
°C was treated sequentially with H2O2 and the Fe(II)-EDTA
complex obtained by premixing (NH4)2Fe(SO4) and EDTA at
final concentrations of 0.7 and 0.8 mM, respectively, to start
the reaction. Aliquots of the reaction mixture were periodically
withdrawn, treated with sodium borohydride, and analyzed as
described above. In other experiments, EDTA was omitted. In
the iron-catalyzed aerial oxidation, dopamine solutions (1 mM)
containing nitrite (1-10 mM) and (NH4)2Fe(SO4) (10 µM) were
allowed to stand at 37 °C for 18 h under vigorous stirring.
Oxidation of dopamine (1 mM) by ferricyanide (2 mM) in 0.1 M
phosphate buffer (pH 7.4) was carried out as described above
and analyzed at the 20 min reaction time.
F igu r e 1. HPLC elution profile of the products formed by HRP/
H2O2 oxidation of dopamine in the presence and in the absence
of NO2-. Dopamine (1 mM) in 0.1 M phosphate buffer (pH 7.4)
was oxidized with 0.4 unit/mL HRP and 2 mM H2O2 in the
absence (A) and in the presence (B) of 10 mM NO2-. Elutogram
C was obtained from the same reaction mixture as elutogram
B but spiked with authentic 2. All elutograms were registered
after 10 min by stopping the oxidation with sodium borohydride.
mM H2O2 proceeded rapidly to give a transient red
chromophoric phase, denoting dopaminochrome, and soon
after a dark melanin-like material. After treatment of
the mixture with sodium borohydride to destroy unre-
acted H2O2 and reduce quinone intermediates, HPLC
analysis at the melanin stage (i.e., after about 10 min)
revealed small amounts of 1 along with 5,6-dihydroxy-
indole, the rearrangement product of the aminochrome,
P r od u ct Id en tifica tion or Isola tion . Compound 1 was
identified after sodium borohydride reduction by comparison of
the UV spectrum and retention time with eluants A and C with
those of a commercial sample. For isolation of 2, a large-scale
reaction mixture obtained by HRP/H2O2 oxidation of 1 mM
as main detectable products (Figure 1). When the reac-
-
-
dopamine (20 mg) in the presence of NO2 (10 mM) was
tion was carried out in the presence of an excess of NO2
,
fractionated by semipreparative HPLC using eluant C. The
appropriate fraction eluting with a tR of 20 min was concentrated
at room temperature with a rotary evaporator, care being taken
to avoid evaporation to dryness, and analyzed within 24 h by
electrospray ionization mass spectrometry (ES/MS), giving a
pseudomolecular ion peak at m/z 199. The identity of the
compound was secured by comparison of the chromophore at
acidic and alkaline pHs and the reactivity toward dithionite of
the HPLC fraction and those of a synthetic sample. 5-S-
Glutathionyldopamine was identified by co-injection with a
synthetic sample with eluants B and C.
P r elim in a r y An im a l Exp er im en ts. In all experiments, the
animal welfare guidelines set forth by the National Institutes
of Health (42) were adhered to. Male Sprague-Dawley rats
(weighing 250-300 g) were anesthetized with choral hydrate
(400 mg/kg ip) positioned in a David Kopf stereotaxic apparatus
and injected into the left medial forebrain bundle [coordinates
A, -2.2; L, -1.5; V, -7.4; according to the atlas of Pellegrino et
al. (43)] with 8 µg of 1 or 2 dissolved in 4 µL of saline containing
0.05% ascorbic acid as described previously (44). The animals
were housed in groups of four per cage on a 12 h light/dark cycle
(from 7:00 a.m. to 7:00 p.m.) with free access to food and water.
Fourteen days after the surgery, apomorphine (0.05 mg/kg) was
injected intraperitoneally and the rats were observed in indi-
vidual cages for rotational behavior, and repeatedly tested for
the presence of hypokinesia, postural abnormality, stiffness of
tail, and other behavioral patterns. The circling responses were
assessed 30 min after the apomorphine challenge for at least
60 min.
a novel product that eluted at relatively high retention
times was clearly detectable. This product was identified
as 2 by comparison of the chromatographic properties
with those of authentic 2 (24) under different elution
conditions. The identity of the product was secured by
isolation from a large-scale reaction mixture by HPLC
fractionation and electrospray ionization mass spectrom-
etry (ES/MS) coupled with UV analysis. Compound 2
(pKa ) 6.2) is easily monitored by its characteristic pH-
dependent chromophore, with maximum absorbance at
351 nm in acidic solution and at 422 nm in alkaline
solution, as well as by its facile reduction with sodium
dithionite at neutral or alkaline pH, but not with sodium
borohydride. All of these properties were shared by the
synthetic 2 used as the standard in this work and by the
isolated product of dopamine nitration.
The remainder of the oxidation mixture apparently
consisted of polymeric, ill-defined dark material resem-
bling melanin. In control experiments, no detectable
dopamine nitration was observed by incubating the
substrate for at least 30 min with various NO2- concen-
trations in the absence of HRP or H2O2, which ruled out
possible artifactual formation of 2 in the acidic eluant
used for HPLC. In all the experiments, attention was
paid to carefully avoid dropping of the pH below 7.4, to
prevent nitration of dopamine by the HNO2 formed in
the acidic medium. This precluded use of acids to halt
dopamine oxidation, as in previous studies, and sug-
gested reduction with sodium borohydride as the most
advisable procedure.
Resu lts
-
In a survey of the effects of NO2 on dopamine
oxidation chemistry, the horseradish peroxidase (HRP)/
H2O2 system was initially chosen because of its estab-
lished ability to promote effective substrate conversion
and formation of 1 (17). In 0.1 M phosphate buffer (pH
7.4) and at 37 °C, oxidation of 1 mM dopamine with 2
Periodic monitoring of the HRP-promoted oxidation
reaction in the presence of NO2- revealed a higher yield
of formation of 1 with respect to 2, although the former
product was relatively more labile in the oxidizing
reaction medium and was largely oxidized after 20 min,

1216 Chem. Res. Toxicol., Vol. 12, No. 12, 1999
Palumbo et al.
F igu r e 2. Time course of dopamine oxidation and product
formation by reaction with HRP/H2O2 and NO2-. Dopamine (1
mM) was incubated in 0.1 M phosphate buffer (pH 7.4) in the
-
presence of 0.4 unit/mL HRP, 2 mM H2O2, and 10 mM NO2
.
At various times, aliquots of the reaction mixture were with-
drawn, treated with sodium borohydride, as described in
Experimental Procedures, and analyzed by HPLC (eluant A).
Data are means of experiments carried out in triplicate. SD <
8%. Left axis (dashed lines): dopamine (2) and 5,6-dihydroxy-
indole ([). Right axis (solid lines): 1 (b) and 2 (9).
F igu r e 4. Dopamine hydroxylation and nitration by HRP/
-
-
H2O2/NO2 as a function of NO2 concentration. Dopamine [1
mM (A) or 500 µM (B)] was incubated with HRP (0.4 unit/mL)
and the indicated concentrations of NO2-. Data are means of
three experiments, and SD < 7%. Left axis: 1 (b). Right axis:
2 (9).
and are not corrected on the basis of reacted substrate.
Since in most of the experiments significant amounts of
unreacted starting material could be detected, concentra-
tions of recovered dopamine are indicated whenever
possible to permit an estimate of the actual yields of
formation of the products.
The results indicated a consistent dependence of the
yield of 1 on the substrate and oxidant concentrations,
but no significant effect of NO2-, as evidenced by com-
-
parative experiments carried out in the absence of NO2
(Figure 3A).
A similar, though less linear, dependence on H2O2
concentration was observed in the case of 2, substantial
decomposition of the product occurring apparently at the
highest oxidant concentration (Figure 3B). Portionwise
addition of H2O2 to a 1 mM solution of dopamine in the
presence of HRP under the conditions described above
did not result in any appreciable variation in product
distribution compared with the bolus addition experi-
ments.
The yields of formation of 2 versus 1 were found to vary
also with NO2- levels with fixed amounts of H2O2. At two
different dopamine concentrations, formation of 2 in-
creased with increasing NO2- concentrations (Figure 4),
whereas the level of generation of 1 was slightly de-
creased.
Reduced glutathione, at a concentration of 1 mM,
virtually suppressed formation of both 1 and 2 by reaction
of 1 mM dopamine with HRP/H2O2/NO2-, whereas ascor-
bate under the same conditions caused significant inhibi-
tion. Notably, in the presence of 1.0 mM reduced glu-
tathione, dopamine was converted to a major reaction
product, which was identified as 5-S-glutathionyldopa-
F igu r e 3. Dopamine hydroxylation and nitration by HRP/
-
H2O2/NO2 as a function of H2O2 concentration. Dopamine [1
mM (solid line) or 500 µM (dashed line)] was incubated with
HRP (0.4 unit/mL), with (black symbols) or without (white
symbols) NO2- (10-fold molar excess with respect to dopamine)
and the indicated concentrations of H2O2. Data are means of
three experiments, and SD < 5%. A: 1 (b or O). B: 2 (9).
when 2 still persisted (Figure 2). This was confirmed in
separate experiments in which authentic 1 (as the
quinone) and 2 were exposed to the HRP/H2O2 system
and were found to be rapidly oxidized, the former at a
rate approximately 2.5 times faster than the latter.
In subsequent experiments, the relative yields of
formation of 1 and 2 were determined with varying H2O2
concentrations using either 1 mM dopamine and 10 mM
NO2- or 500 µM dopamine and 5 mM NO2- (Figure 3A,B).
It should be noted that, throughout this study, yields
indicate the absolute concentrations of products formed

Dopamine Nitration and Hydroxylation
Chem. Res. Toxicol., Vol. 12, No. 12, 1999 1217
Ta ble 1. Effect of Ad d itives on Dop a m in e Hyd r oxyla tion
Ta ble 2. Dop a m in e Hyd r oxyla tion a n d Nitr a tion by
- a
- a
a n d Nitr a tion by HRP /H2O2/NO2
LP O/H2O2/NO2
yieldb (µM)
unreacted
yieldb (µM)
-
[NO2
]
dopamineb
(mM)
additive
1
2
(mM)
additive
1
2
-
7.2
1.1
4.3
-
6.9
1.7
0.5
1.5
-
1.5
-
-
5.0
4.8
5.2
4.9
3.8
3.2
3.9
2.9
-
0.60
0.62
0.53
0.59
0.55
0.57
0.66
0.70
ascorbate (1 mM)
0.5
-
0.60
0.82
0.92
1.2
HCO3- (25 mM)
glutathione (1 mM)
SOD (300 units/mL)
1.0
2.0
5.0
10.0
10.0
10.0
-
-
-
a
-
1.6
1.6
1.1
Dopamine (1 mM) in 0.1 M phosphate buffer (pH 7.4) was
SCN- (1 mM)
SCN- (10 mM)
incubated with HRP (0.40 unit/mL) and H2O2 (2 mM) as described
in detail in Experimental Procedures. b Mean of three experiments,
and SD < 5%.
a
Dopamine (1 mM) in 0.1 M phosphate buffer (pH 7.4) was
incubated with LPO (0.53 unit/mL) and H2O2 (2 mM) as described
in detail in Experimental Procedures. b Mean of three experiments,
and SD < 5%.
F igu r e 5. Time course of dopamine hydroxylation and nitration
by the HRP/glucose/glucose oxidase/NO2- system. Dopamine (1
mM) was incubated with HRP (0.4 unit/mL), NO2- (1 mM), 280
µM D-glucose, and glucose oxidase (50 milliunits/mL, generating
4.0 µM H2O2/min). Data are means of four experiments, and
SD < 6%. Left axis (dashed line): residual dopamine (2). Right
axis (solid lines): 1 (b) and 2 (9).
F igu r e 6. Dopamine nitration by Fe2+-promoted autoxidation
-
-
in the presence of NO2 as a function of NO2 concentration.
Dopamine (1 mM) in 0.1 M phosphate buffer (pH 7.4) was
incubated with Fe2+ (10 µM); the indicated concentrations of
NO2- were taken under vigorous stirring, and the mixture was
analyzed by HPLC with a 18 h reaction time. Data are means
of three experiments, and SD < 6%. Left axis (dashed line):
unreacted dopamine (2). Right axis (solid line): 2 (9).
mine by co-injection with an authentic sample produced
-
by tyrosinase-catalyzed oxidation (11). The HCO3 ion,
a pH-dependent source of carbon dioxide reported to
enhance peroxynitrite-mediated nitrations (45, 46), in-
duced only a modest inhibition of the hydroxylation
reaction, but had little or no effect on the nitration
reaction. SOD also had no detectable effect. Significant
hydroxylation and nitration of 1 mM dopamine promoted
product formation, significant yields of 1 and 2 being
obtained with a SCN- concentration as high as 10 mM.
In another series of experiments, the abilities of
various nonenzymatic Fe2+-dependent oxidizing systems
to promote dopamine conversion to 2 in the presence of
-
NO2 were compared. In all cases, the yields of 1 were
-
by HRP in the presence of 10 mM NO2 was achieved
also determined. When 1 mM dopamine was left to
autoxidize in air-equilibrated 0.1 M phosphate buffer (pH
7.4) at 37 °C in the presence of 10 µM Fe2+, a relatively
high yield of 2 compared to the yield of 1 was obtained
using D-glucose/glucose oxidase as a source of low,
constant fluxes of H2O2 (Figure 5). Notably, yields of 2
at concentrations of up to 100 nM with a 2 h reaction
time were obtained when dopamine was used at a
-
after 18 h, increasing linearly with NO2 concentration
-
concentration of 0.1 mM in the presence of 1 mM NO2
.
and exceeding that observed with the various peroxidase/
H2O2 systems (Figure 6). Addition of higher concentra-
tions of Fe2+, however, considerably decreased the level
of formation of 2 despite comparable or enhanced rates
of dopamine consumption (not shown). In control experi-
ments carried out under similar reaction conditions but
in the absence of O2, Fe2+ as well as Fe3+ proved to be
unable to induce oxidation and/or nitration of 1 mM
For comparative purposes, L-tyrosine (1 mM) was
-
reacted with 10 mM NO2 in the presence of HRP/H2O2
and was found to give 40 µM 3-nitrotyrosine with a 60
min reaction time. A mammalian peroxidase, lactoper-
oxidase (LPO), proved to be likewise effective in convert-
ing dopamine to 1 and 2 in the presence of H2O2 and
-
NO2 (Table 2).
-
Especially worthy of note is the ability of LPO to induce
dopamine in the presence of NO2 (data not shown).
formation of detectable amounts of 2 from 1 mM dopa-
mine with a NO2 concentration of 0.5 mM, conditions
Interesting variations in the hydroxylation:nitration
ratios were observed when the Fe2+/H2O2 system was
used for dopamine oxidation in the presence and in the
absence of EDTA as a chelating agent (Figure 7). In the
former case, a significant nitration of dopamine leading
to 2 was observed, which was partially inhibited by the
OH radical scavenger mannitol (45 ( 4% inhibition with
respect to control, n ) 3). Careful product analysis failed
-
under which HRP proved to be virtually uneffective.
Since thiocyanate (SCN) is a presumed major physiologi-
cal substrate for LPO (29), its effect on the LPO-promoted
oxidation of 1 mM dopamine in the presence of 10 mM
NO2- was also investigated. The data in Table 2 indicated
only a modest concentration-dependent inhibition of

1218 Chem. Res. Toxicol., Vol. 12, No. 12, 1999
Palumbo et al.
F igu r e 7. Dopamine hydroxylation and nitration by Fe2+
-
EDTA/H2O2/NO2 and Fe2+/H2O2/NO2-. Dopamine (1 mM) in
0.1 M phosphate buffer (pH 7.4) was treated with Fe2+ (0.7 mM)
in the presence (black symbols) or in the absence (white symbols)
of EDTA (0.8 mM) as described in Experimental Procedures.
The reaction mixture was analyzed by HPLC with a 10 min
reaction time. Data are means of three experiments, and SD <
5%. Left axis: 1 (b or O). Right axis: 2 (9 or 0).
-
to reveal the presence of hydroxydopamines other than
1. In particular, 5-hydroxydopamine, a product known
to be formed by dopamine hydroxylation with Uden-
friend- and Fenton-type systems in the presence of
ascorbate (9), was not detected.
F igu r e 8. Dopamine hydroxylation and nitration by tyrosinase/
O2/NO2- as a function of NO2- concentration. Dopamine (1 mM)
In the absence of EDTA, on the other hand, under
conditions where most of the iron in the ferric form was
chelated by the catechol functionality of dopamine (purple
complex λmax ) 575 nm), the level of formation of 2 was
somewhat decreased and 1 was by far prevailing. These
latter reactions were only slightly susceptible to inhibi-
tion by added mannitol.
in 0.1 M phosphate buffer (pH 7.4) was incubated with tyro-
-
sinase (75 units/mL) and the indicated concentrations of NO2
,
and the mixture was analyzed by HPLC with a 30 min reaction
time. Data are means of three experiments, and SD < 5%. A:
left axis (dashed line), unreacted dopamine (2); and right axis
(solid line), 2 (9). B: like panel A, with H2O2 (1 mM), 1 (white
bars) and 2 (black bars).
temporary restlessness lasting for about 1-2 min. These
episodes usually preceded periods in which the animals
remained motionless. None of the treated rats exhibited
significant postural abnormalities. By contrast, animals
treated with the same doses of 1 exhibited turning
behavior lasting 1 h with an intensity of approximately
25 turns/5 min.
Control animals (n ) 6) treated with the same volume
of saline survived without unusual behavioral responses.
Apomorphine-induced circling is a behavior strongly
correlated to dopamine denervation induced by 1 (48),
whereas the absence of circling is an indirect indicator
of the absence of denervation.
With a view to gaining an insight into the mechanism
of catecholamine nitration, in further experiments dopa-
mine was subjected to aerial oxidation in the presence
of tyrosinase, a copper enzyme causing direct two-
electron oxidation of catecholic substrates to the corre-
sponding o-quinones (47), and various concentrations of
-
NO2 and H2O2. In the presence of NO2-, 2 was formed
in yields comparable to those obtained with the HRP/
H2O2 system, but no detectable 1 was observed (Figure
8A). Addition of H2O2 inhibited formation of 2 to some
extent, leading to significant amounts of 1, which de-
-
creased however with increasing NO2 concentrations
(Figure 8B).
Oxidation of 1 mM dopamine with 2 mM ferricyanide
and 10 mM NO2- also afforded 2 in yields comparable to
those obtained with tyrosinase (3 µM), but no detectable
1.
Discu ssion
Increasing lines of evidence support the possible gen-
eration of 6-nitrocatecholamines by NO-dependent mech-
anisms both in vitro (24, 49) and in vivo (50). In this
frame, the results reported in this paper provide a
chemical background for postulating that the generation
of 2, in addition to 1, is a potential contributory pathway
accounting for aberrant dopamine conversion under
conditions of oxidative stress. Interestingly, while most
of the NO2--containing oxidizing systems used in this
study have been reported to bring about NO2-mediated
free radical nitrations when acting on tyrosine and other
monophenolic substrates, they seem to nitrate dopamine
to 2 through different mechanisms, not mutually exclu-
sive, which may have been overlooked in the general
panorama of biologically relevant nitrations.
Finally, in preliminary biological experiments, the
relative neurotoxicity of 2 versus that of 1 was assessed
by stereotaxic injection of the same amounts of the pure
compounds into the substantia nigra of laboratory rats,
using isotonic saline as a control. At the low dose used
(8 µg), 2 was not lethal (n ) 6). Behavioral responses
evoked by stimulation with the dopaminergic agonist
apomorphine after injection of 2 included sporadic epi-
sodes of circling contralateral to the site of injection
(partial or complete turnings at irregular intervals in the
range of 3-6 min over a period of at least 60 min) and
hypokinesia with moderate inability to move and walk
over a period of g1 h, but no evidence of tremor, rolling,
or jumping. Occasionally, animals stood on their limbs
trying apparently to escape from cages and exhibited
A typical case in point is provided by the peroxidase/
H2O2/NO2--mediated nitration. Ample experimental evi-

Dopamine Nitration and Hydroxylation
Chem. Res. Toxicol., Vol. 12, No. 12, 1999 1219
Sch em e 1
In this scheme, the quinone 4 partitions among three
main competitive routes, the major involving intramo-
lecular cyclization leading to melanin-like pigments via
dopaminochrome (5) and 5,6-dihydroxyindole (6) (path
A) and the minor ones accounting for hydroxylation and
nitration (paths B and C, respectively). The ability of
NO2- and H2O2 to add nucleophilically to quinones is well
documented in the literature (57), and the generation of
dopamine quinone in the peroxidase-promoted reaction
is indirectly supported by the reaction with reduced
glutathione, leading to the S-conjugate in accord with the
commonly held quinone-based mechanism (10). In this
context, the finding that SCN-, which can efficiently
-
inhibit phenolic nitration by LPO/H2O2/NO2 (29), does
not suppress dopamine nitration even at concentrations
as high as 10 mM, despite its affinity for the enzyme
-
being greater than that of NO2 (29), would also not
support NO2- oxidation by peroxidase compound I as the
main mechanism of dopamine nitration. Most likely, the
observed variations in the abilities of HRP and LPO to
bring about dopamine nitration may be ascribed to
slightly different substrate specificities or oxidation
mechanisms. Similar differences are reported in the case
of tyrosine oxidation by HRP and LPO (58).
-
The reaction of NO2 with dopamine quinone can
conceivably be invoked also to account for the effective
nitration of dopamine observed during autoxidation, as
well as tyrosinase- and ferricyanide-promoted oxidations.
This, indeed, can be taken as a most convincing piece of
dence indicates that HRP and LPO active forms are
thermodynamically competent in bringing about oxida-
tion of NO2 to NO2 (E° ) -0.99 V) (51), which, in the
-
evidence for the proposed ultimate involvement of NO2
-
in dopamine nitration, since the reaction conditions used
would not result in a significant oxidation to NO2. It
should be mentioned, in this connection, that the genera-
tion of 2 by autoxidation of dopamine in the presence of
NO2- and iron ions has recently been described by other
authors (49), though the emphasis in that study was
mainly on the HNO2-dependent reaction. In the case
presence of enzymatically produced tyrosyl radicals, gives
rise to 3-nitrotyrosine (29, 34). In the case of dopamine,
however, mechanistic arguments would apparently rule
out the analogous coupling of NO2 with the semiquinone
radical as being the sole or the main reaction pathway.
The higher susceptibility to oxidation of dopamine com-
pared to tyrosine would account for a faster and more
efficient oxidation by peroxidase. As a consequence,
-
presented here, i.e., with up to 10 mM NO2 at pH 7.4,
estimated equilibrium concentrations of HNO2 (pKa )
3.25) (59) would be on the order of e10-6 M and would
not induce significant dopamine nitration, as is apparent
also from reported control experiments.
-
dopamine would be expected to compete with NO2 for
oxidation by peroxidase compounds I and II more ef-
ficiently than tyrosine, making generation of NO2 less
significant. In accord with this view, for example, rate
constants of 1.8-2.4 × 104 M-1 s-1 have been determined
for dopamine oxidation by HRP compound II versus
values of 4 × 102 to 1.8 × 103 M-l s-1 for L-tyrosine (52)
and much lower (ca. 102 M-1 s-1) for NO2- (53). Further-
more, a free radical nitration mechanism of dopamine
by NO2 would not be in line with the results of recent
studies underscoring the greater susceptibility to free
radical nitration of monophenolic substrates than of
catechols (54-56), the latter being rather prone to
oxidation in the presence of reactive nitrogen radicals.
Alternate mechanisms involving peroxynitrite or a per-
An interesting finding which emerges from this study
is the role of iron chelation in affecting the relative extent
of hydroxylation and nitration routes in the Fe2+/H2O2-
induced oxidations. In the presence of EDTA, i.e., under
conditions in which most of the Fe3+ produced by oxida-
tion of Fe2+ is sequestered by the chelating agent (20),
formation of 2 is relatively more significant and may
occur at least in part by Fenton-type chemistry involving
OH radicals or related mannitol-scavengeable species (60)
(Scheme 2).
Given the comparable rate constants for true OH
-
radical attack on NO2 (k ) 1.3 × 109 M-1 s-1) (61) and
+
dopamine (k ) 5.9 × 109 M-1 s-1) (62), it seems plausible
that OH• radicals and related species produced by Fen-
ton-type reagents can bring about competitive attack on
oxidase-bound NO2 species as the actual nitrating
agents have been ruled out in previous studies on the
LPO-promoted nitration of tyrosine (29), and the same
arguments should hold in the case of dopamine nitration
as well. In view of the foregoing considerations and the
comparable extents of nitration observed in the peroxi-
dase- and tyrosinase-catalyzed reactions in the presence
of NO2-, a mechanistic scheme is proposed in which 1
-
dopamine and NO2 and, hence, that formation of 2 via
radical coupling of NO2 with dopamine semiquinone may
become a significant route at the high concentrations
used in the study presented here. However, the rapid
conversion of dopamine semiquinone to the o-quinone by
disproportionation or oxidation by the Fenton-type re-
agent (oxidation by O2 seems of little importance; see ref
52), and the subsequent nucleophilic attack by NO2- may
also contribute to the overall formation of 2. Unfortu-
-
and 2 arise mainly via nucleophilic attack of NO2 and
H2O2 on dopamine o-quinone (4) generated by dispropor-
tionation of enzymatically produced semiquinone 3
(Scheme 1).

1220 Chem. Res. Toxicol., Vol. 12, No. 12, 1999
Palumbo et al.
Sch em e 2
6-position and favoring attack on the labile O-O bond.
Whether a similar mechanism, represented by route (iii),
applies also to the H2O2-induced hydroxylation of the
Fe3+-dopamine chelate (in the absence of EDTA) re-
ported in this study is an attractive yet open issue. Direct
OH radical attack on dopamine or a dopamine-derived
species to give 1, as in route (iii), though possible, seems
to be a dominant route in neither of the reported systems,
mainly because of the failure to detect other positional
isomers of 1 reported to arise from OH radical attack (9).
A comment about the pathophysiological relevance of
the chemistry described in this paper seems appropriate.
In most of the experiments reported in this study, high
concentrations of dopamine, H2O2, and NO2 were neces-
sary for obtaining detectable nitration of dopamine,
compared to that of tyrosine (29). This reflects (a) the
higher susceptibility of catechols to oxidation and polym-
erization with respect to monophenols, implying that
fastly generated, reactive free radical intermediates can
partition among different oxidative pathways and are less
susceptible to interaction with nitrating agents; and (b)
the relatively high instability to oxidation of nitrocat-
echols compared to that of nitrophenols (data not shown),
which accounts for significant product decomposition
during the reaction. Indeed, although the electron-
withdrawing nitro group stabilizes the catechol ring to
some extent, compound 2 proved to be unexpectedly facile
to oxidation by the HRP/H2O2 system, due probably in
part to the lowered pKa of the phenoxyl groups relative
to that of dopamine (59) which results in an increased
proportion of the more oxidizable ionized form at pH 7.4.
The limitations described above would in principle
preclude a fast and efficient oxidative nitration of dopa-
mine by the peroxidase/H2O2/NO2- system which, on the
other hand, is rather effective in promoting nitration of
tyrosine residues. Nonetheless, formation of 2 may
become significant at sites where NO synthase is over-
activated under conditions of oxidative stress and severe
antioxidant depletion, whereby dopamine is slowly oxi-
dized in the presence of excess NO2-. Thus, in the case
of dopamine and other catechols, NO2--dependent nitra-
tion routes seem chemically to be more plausible than
free radical routes promoted by transient NO-derived
nitrogen species, like NO2 and ONOO-, because of the
nately, the relative importance of the radical and ionic
pathways, not mutually exclusive, could not be assessed
on the basis of available evidence.
In the absence of EDTA and other strong chelating
agents, most of the iron is converted under oxidizing
conditions to the Fe3+ form which is efficiently chelated
by the o-diphenolic group of dopamine, as evidenced by
development of the purple complex (63). Under these
-
conditions, oxidation of NO2 to NO2 by H2O2 might not
be so significant as in the presence of EDTA, and a
decreased rate of generation of the o-quinone caused by
metal chelation, e.g., through stabilization of the precur-
sor semiquinone (52), could be invoked to account for the
reduced susceptibility to nucleophilic attack by NO2- and
a decreased level of formation of 2.
In the Fe2+-promoted autoxidation, on the other hand,
the Fe3+ produced in the mixture in the presence of O2
is chelated by dopamine and the resulting chelate is
smoothly converted to the o-quinone by O2 and species
derived from it, thus becoming susceptible to attack by
NO2-. In the absence of O2, Fe3+ proved to be unable to
oxidize the catecholamine to the quinone and, hence, to
induce nitration in the presence of NO2
-
.
More complex mechanistic options must be considered
for the hydroxylation of dopamine to 1 observed in the
study presented here. Using the schemes presented above
as a simplified format for discussion, it appears that 1,
in the form of the relatively stable quinone, may arise
by at least three different routes which are not mutually
exclusive and that strictly require H2O2 as the oxygenat-
ing agent, namely, (i) the nucleophilic addition of H2O2
to dopamine quinone followed by carbonyl-forming cleav-
age of the resulting hydroperoxide adduct, (ii) the direct
interaction of the peroxide with a dopamine-iron chelate,
and (iii) hydroxylation by OH radicals or related Fenton-
type species. Whenever sufficient concentrations of dopa-
mine quinone are generated, e.g., by peroxidase/H2O2,
tyrosinase, or ferricyanide or during autoxidation, forma-
tion of 1 should expectedly involve mainly nucleophilic
attack of H2O2 as in route (i) (17).
-
expectedly higher levels of NO2 that can accumulate
under (patho)physiological conditions.
It should be emphasized, in this context, that under
the oxidative stress conditions characterizing neuronal
degeneration in Parkinson’s disease and related disor-
ders, with more or less extensive inflammation and
gliosis, relatively high levels of NO may be produced by
overactivation of the calcium-dependent neuronal isoform
of nitric oxide synthase as well as by stimulation of the
inducible isoform found in astrocytes and microglial cells.
Three-dimensional diffusion of NO through membranes
-
(64) may lead to an accumulation of NO2 in virtually
Routes (ii) and (iii) would instead be more relevant to
iron-promoted, nonenzymatic reactions. Previous studies
with linoleic acid hydroperoxide have suggested that
hydroperoxides can directly react with the catechola-
mine-Fe3+ chelate causing an OH radical-independent
hydroxylation at the highly reactive 6-position of the
aromatic ring (20). We can speculate, for example, that
chelation of Fe3+ by the catechol functionality would
cause a redistribution of the charge density on the
aromatic ring, possibly enhancing the reactivity of the
all compartments of inflamed tissues and at levels locally
much greater than those measured in plasma and other
body fluids. When it is considered that, because of the
observed ease of degradation of 2 in an oxidizing medium,
the actual extent of dopamine nitration has been under-
estimated in the study presented here, we may speculate
that under conditions of enhanced NO synthase activity
and abnormal NO2 accumulation, a certain proportion
of the dopamine pool in the degenerating substantia nigra
may be converted to 2. That is, on the basis of the results
-

Dopamine Nitration and Hydroxylation
Chem. Res. Toxicol., Vol. 12, No. 12, 1999 1221
(7) Halliwell, B., and Gutteridge, J . M. C (1984). Oxygen toxicity,
oxygen radicals, transition metals and disease. Biochem. J . 219,
1-14.
(8) Smythies, J ., and Galzigna, L. (1998) The oxidative metabolism
of catecholamines in the brain: a review. Biochim. Biophys. Acta
1380, 159-162.
presented here, it seems that special conditions would
have to prevail if 2 is to be formed in vivo. Most likely,
dopamine nitration may take place in the cytosol of
degenerating neurons, and should prevail under condi-
tions of iron accumulation, after the primary toxic
processes are underway.
(9) Slivka, A., and Cohen, G. (1985) Hydroxyl radical attack on
dopamine. J . Biol. Chem. 260, 15466-15472.
From the preliminary biological data reported in this
paper, it appears that catecholamine 2 is less toxic than
1 and other endogenous dopamine metabolites, e.g.,
benzothiazines (12) and tetrahydroisoquinolines (65), but
can partially counteract the effects induced by the
apomorphine challenge. Experiments are currently being
carried out to assess on a quantitative basis the actual
neurotoxicity of 2 by HPLC analysis of striatal dopamine
levels after stereotaxic injection of 2 as well as by
systematic evaluation of the incidence and severity of
behavioral changes, in comparison with those of 1 and
dopamine itself. Waiting for those definitive data to
become available regarding the potential ability of 2 to
cause destruction of dopamine nerve terminals or loss of
dopamine receptors which are activated by apomorphine,
we find it possible here only to speculate that compound
2, being relatively more stable than 1, may persist
enough to contribute to the general NO-dependent im-
pairment of dopaminergic neurotransmission (66) be-
cause of its reduced ability to interact with D1-dopa-
minergic receptors compared to the parent catecholamine
(49).
Despite considerable efforts, 2 has not yet been identi-
fied in vivo. However, the analogous derivative of nor-
epinephrine, 6-nitronorepinephrine, has been detected in
mammalian brain at levels correlating with the degree
of NO synthase activity (50) and has been shown to have
inhibitory effects on catechol O-methyltransferase and
on norepinephrine activation. When tested on rat aorta,
6-nitronorepinephrine caused a dose-dependent contrac-
tion in both intact endothelium and denuded aorta, and
inhibited acetylcholine-induced relaxation, through a
H2O2-dependent mechanism (67). Further work is there-
fore warranted to identify 2 in vivo and to definitively
assess the actual significance of nitration routes in
catecholamine metabolism.
(10) Palumbo, A., d’Ischia, M., Misuraca, G., De Martino, L., and Prota,
G. (1995) Iron and peroxide-dependent conjugation of dopamine
with cysteine: oxidative routes to the novel brain metabolite 5-S-
cysteinyldopamine. Biochim. Biophys. Acta 1245, 255-261.
(11) Spencer, J . P., J enner, P., Daniel, S. E., Lees, A. J ., Marsden, D.
C., and Halliwell, B. (1998) Conjugates of catecholamines with
cysteine and GSH in Parkinson’s disease: possible mechanisms
of formation involving reactive oxygen species. J . Neurochem. 71,
2112-2122.
(12) Zhang, F., and Dryhurst, G. (1994) Effects of L-cysteine on the
oxidation chemistry of dopamine: new reaction pathways of
potential relevance to idiopathic Parkinson’s disease. J . Med.
Chem. 37, 1084-1098.
(13) Rabinovic, A. D., and Hastings, T. G. (1998) Role of endogenous
glutathione in the oxidation of dopamine. J . Neurochem. 71,
2071-2078.
(14) Hou, J . G., Cohen, G., and Mytilineou, C. (1997) Basic fibroblast
growth factor stimulation of glial cells protects dopamine neurons
from 6-hydroxydopamine toxicity: involvement of the glutathione
system. J . Neurochem. 69, 76-83.
(15) Cohen, G., Yakushin, S., and Dembiec-Cohen, D. (1998) Protein
L-dopa as an index of hydroxyl radical attack on protein tyrosine.
Anal. Biochem. 23, 232-239.
(16) Nappi, A. J ., Vass, E., Prota, G., and Memoli, S. (1995) The effects
of hydroxyl radical attack on dopa, dopamine, 6-hydroxydopa and
6-hydroxydopamine. Pigm. Cell Res. 8, 283-293.
(17) Napolitano, A., Crescenzi, O., Pezzella, A., and Prota, G. (1995)
Generation of the neurotoxin 6-hydroxydopamine by oxidation of
dopamine with peroxidase/H2O2. J . Med. Chem. 38, 917-922.
(18) Garner, C. D., and Nachtman, J . P. (1989) Manganese catalyzed
autoxidation of dopamine to 6-hydroxydopamine in vitro. Chem.-
Biol. Interact. 69, 345-351.
(19) Linert, W., Herlinger, E., J ameson, R. F., Kienzl, E., J ellinger,
K., and Youdim, M. B. H. (1986) Dopamine, 6-hydroxydopamine,
iron and dioxygen, their mutual interaction and possible implica-
tion in the development of Parkinson’s disease. Biochim. Biophys.
Acta 1316, 160-168.
(20) Pezzella, A., d’Ischia, M., Napolitano, A., Misuraca, G., and Prota,
G. (1997) Iron-mediated generation of the neurotoxin 6-hydroxy-
dopamine quinone by reaction of fatty acid hydroperoxides with
dopamine:
a possible contributory mechanism for neuronal
degeneration in Parkinson’s disease. J . Med. Chem. 40, 2211-
2216.
(21) Moncada, S., and Higgs, A. (1993) The L-arginine-nitric oxide
pathway. N. Engl. J . Med. 329, 2002-2012.
(22) Pfeiffer, S., Mayer, B., and Hemmens, B. (1999) Nitric oxide:
Chemical puzzles posed by a biological messenger. Angew. Chem.,
Int. Ed. 38, 1714-1731.
(23) Rubbo, H., Darley-Usmar, V., and Freeman, B. (1996) Nitric oxide
regulation of tissue free radical injury. Chem. Res. Toxicol. 9,
809-820.
(24) d’Ischia, M., and Costantini, C. (1995) Nitric oxide induced
nitration of catecholamine neurotransmitters: a key to neuronal
degeneration? Bioorg. Med. Chem. 3, 923-927.
Ack n ow led gm en t. This work has been supported in
part by grants from CNR. We thank Professor G. Prota
for helpful discussions and Mr. Luigi De Martino for
technical assistance.
Refer en ces
(25) de la Breteche, M. L., Servy, C., Lenfant, M., and Ducrocq, C.
(1994) Nitration of catecholamines with nitrogen oxides in mild
conditions. Tetrahedron Lett. 35, 7231-7232.
(26) Fukuto, J . M., and Ignarro, L. J . (1997) In vivo aspects of nitric
oxide (NO) chemistry: does peroxynitrite (-OONO) play a major
role in cytotoxicity? Acc. Chem. Res. 30, 149-152.
(27) Wink, D. A., Darbyshire, J . F., Nims, R. W., Saavedra, J . E., and
Ford, P. C. (1993) Reactions of the regulatory agent nitric oxide
in oxygenated aqueous media: determination of the kinetics for
oxidation and nitrosation by intermediates generated in the NO/
O2 reaction. Chem. Res. Toxicol. 6, 23-27.
(28) Beckman, J . S., and Koppenol, W. H. (1996) Nitric oxide, super-
oxide and peroxynitrite: the good, the bad and the ugly. Am. J .
Physiol. 271, C1424-C1437.
(1) Cohen, G. (1983) The pathobiology of Parkinson’s disease: Bio-
chemical aspects of dopamine neuron senescence. J . Neural
Transm., Suppl. 19, 89-103.
(2) Ebadi, M., Srinivasan, S. K., and Baxi, M. D. (1996) Oxidative
stress and antioxidant therapy in Parkinson’s Disease. Prog.
Neurobiol. 48, 1-9.
(3) Riederer, P., Sofic, E., Rausch, W. D., Schmidt, B., Reynolds, G.
P., J ellinger, K., and Youdim, M. B. H. (1989) Transition metals,
ferritin, glutathione and ascorbic acid in Parkinsonian brain. J .
Neurochem. 52, 515-520.
(4) Spina, M. B., and Cohen, G. (1989) Dopamine turnover and
glutathione oxidation: implications for Parkinson’s disease. Proc.
Natl. Acad. Sci. U.S.A. 86, 1398-1400.
(5) Sofic, E., Paulus, W., J ellinger, K., Riederer, P., and Youdim, M.
B. H. (1991) Selective increase of iron in substantia nigra zona
compacta of parkinsonian brains. J . Neurochem. 56, 978-982.
(6) Dexter, D. T., Carayon, A., Vidaihlet, M., Ruberg, M., Agid, F.,
Agid, Y., Lees, A. J ., Wells, F. R., and Marsden, C. D. (1990)
Decreased ferritin levels in brain in Parkinson’s disease. J .
Neurochem. 55, 16-20.
(29) van der Vliet, A., Eiserich, J . P., Halliwell, B., and Cross, C. E.
(1997) Formation of reactive nitrogen species during peroxidase-
catalyzed oxidation of nitrite. A potential additional mechanism
of nitric oxide-dependent toxicity. J . Biol. Chem. 272, 7617-7625.
(30) Nappi, A. J ., and Vass, E. (1998) Hydroxyl radical formation
resulting from the interaction of nitric oxide and hydrogen
peroxide. Biochim. Biophys. Acta 1380, 55-63.

1222 Chem. Res. Toxicol., Vol. 12, No. 12, 1999
Palumbo et al.
(31) Lewis, R. S., and Deen, W. M. (1994) Kinetics of the reaction of
nitric oxide with oxygen in aqueous solutions. Chem. Res. Toxicol.
7, 568-574.
(32) Prutz, W. A., Monig, H., Butler, J ., and Land, E. J . (1985)
Reactions of nitrogen dioxide in aqueous model systems: oxidation
of tyrosine units in peptides and proteins. Arch. Biochem. Biophys.
243, 125-134.
(33) Klebanoff, S. J . (1993) Reactive nitrogen intermediates and
antimicrobial activity: role of nitrite. Free Radical Biol. Med. 14,
351-360.
(34) Sampson, J . B., Ye, Y. Z., Rosen, H., and Beckman, J . S. (1998)
Myeloperoxidase and horseradish peroxidase catalyzed tyrosine
nitration in proteins from nitrite and hydrogen peroxide. Arch.
Biochem. Biophys. 356, 207-213.
(35) Eiserich, J . P., Hristova, M., Cross, C. E., J ones, A. D., Freeman,
B. A., Halliwell, B., and van der Vliet, A. (1998) Formation of
nitric oxide-derived inflammatory oxidants by myeloperoxidase
in neutrophils. Nature 391, 393-397.
(36) Eiserich, J . P., Cross, C. E., J ones, A. D., Halliwell, B., and van
der Vliet, A. (1996) Formation of nitrating and chlorinating
species by reaction of nitrite with hypochlorous acid. A novel
mechanism for nitric oxide-mediated protein modification. J . Biol.
Chem. 271, 19199-19208.
(37) Singh, R. J ., Goss, S. P. A., J oseph, J ., and Kalyanaraman, B.
(1998) Nitration of γ-tocopherol and oxidation of R-tocopherol by
copper-zinc superoxide dismutase/H2O2/NO2-: Role of nitrogen
dioxide free radical. Proc. Natl. Acad. Sci. U.S.A. 95, 12912-
12917.
(49) Daveu, C., Servy, C., Dendane, M., Marin, P., and Ducrocq, C.
(1997) Oxidation and nitration of catecholamines by nitrogen
oxides derived from nitric oxide. Nitric Oxide 1, 234-243.
(50) Shintani, F., Kinoshita, T., Kanba, S., Ishikawa, T., Suzuki, E.,
Sasakawa, N., Kato, R., Asai, M., and Nakaki, T. (1996) Bioactive
6-nitronorepinephrine identified in mammalian brain. J . Biol.
Chem. 271, 13561-13565.
(51) Koppenol, W. H., Moreno, J . J ., Pryor, W. A., Ischiropoulos, H.,
and Beckman, J . S. (1992) Peroxynitrite, a cloaked oxidant formed
by nitric oxide and superoxide. Chem. Res. Toxicol. 5, 834-842.
(52) Kalyanaraman, B., Felix, C. C., and Sealy, R. C. (1984) Peroxidatic
oxidation of catecholamines. A kinetic electron spin resonance
investigation using the spin stabilization approach. J . Biol. Chem.
259, 7584-7589.
(53) Roman, R., and Dunford, H. B. (1973) Studies on horseradish
peroxidase. XII. A kinetic study of the oxidation of sulfite and
nitrite by compounds I and II. Can. J . Chem. 51, 588-596.
(54) Kerry, N., and Rice-Evans, C. A. (1998) Peroxynitrite oxidises
catechols to o-quinones. FEBS Lett. 437, 167-171.
(55) Zhouen, Z., Side, Y., Weizhen, L., Wenfeg, W., Yizun, J ., and
Nianyun, L. (1998) Mechanism of reaction of nitrogen dioxide
radical with hydroxycinnamic acid derivatives: a pulse radiolysis
study. Free Radical Res. 29, 13-16.
(56) Pannala, A. S., Razaq, R., Halliwell, B., Singh, S., and Rice-Evans,
C. A. (1998) Inhibition of peroxynitrite dependent tyrosine nitra-
tion by hydroxycinnamates: nitration or electron donation? Free
Radical Biol. Med. 24, 594-606.
(57) Rosenblatt, D. H., Epstein, J ., and Levitch, M. (1953) Some
nuclearly substituted catechols and their acid dissociation con-
stants. J . Am. Chem. Soc. 75, 3277-3278.
(58) Marquez, L. A., and Dunford, B. H. (1995) Kinetics of oxidation
of tyrosine and dityrosine by myeoloperoxidase compounds I and
II. J . Biol. Chem. 270, 30434-30440.
(59) Lide, D. R., Ed. (1995) CRC Handbook of Chemistry and Physics,
75th ed., CRC Press, Boca Raton, FL.
(60) Sawyer, D. T., Kang, C., Llobet, A., and Redman, C. (1993) Fenton
reagents (1:1 FeIILx/HOOH) react via LxFeIIOOH(BH+) (1) as
hydroxylases (RH-ROH), not as generators of free hydroxyl
radicals (OH•). J . Am. Chem. Soc. 115, 5817-5818.
(61) Forni, L. G., Mora-Arellano, V. O., Packer, J . E., and Willson, R.
L. (1986) Nitrogen dioxide and related free radicals: electron-
transfer reactions with organic compounds in solutions containing
nitrite or nitrate. J . Chem. Soc., Perkin Trans. 2, 1-6.
(62) Richter, H. W., and Waddell, W. H. (1983) Mechanism of the
oxidation of dopamine by the hydroxyl radical in aqueous solution.
J . Am. Chem. Soc. 105, 5434-5440.
(63) Gerard, C., Chehhal, H., and Hugel, R. P. (1994) Complexes of
iron(III) with ligands of biological interest: dopamine and 8-hy-
droxyquinoline-5-sulphonic acid. Polyhedron 13, 591-597.
(64) Lancaster, J . R. (1994) Simulation of the diffusion and reaction
of endogenously produced nitric oxide. Proc. Natl. Acad. Sci.
U.S.A. 91, 8137-8141.
(65) Naoi, M., Maruyama, W., Dostert, P., Hashizume, Y., Nakahara,
D., Takahashi, T., and Ota, M. (1996) Dopamine-derived endog-
enous 1(R),2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroiso-
quinoline, N-methyl-(R)-salsolinol, induced parkinsonism in rat:
biochemical, pathological and behavioral studies. Brain Res. 709,
285-295.
(66) Macarthur, H., Mattammal, M. B., and Westfall, T. C. (1995) A
new perspective on the inhibitory role of nitric oxide in sympa-
thetic neurotransmission. Biochem. Biophys. Res. Commun. 216,
686-692.
(38) Zecca, L., Fariello, R. G., Galimberti, M., Racagni, G., and
Ambrosini, A. (1997) Changes in nitric oxide metabolite levels in
stimulated substantia nigra neurons. NeuroReport 8, 2121-2125.
(39) Milstien, S., Sakai, N., Brew, B. J ., Krieger, C., Vickers, J . H.,
Saito, K., and Heyes, M. P. (1994) Cerebrospinal fluid nitrite/
nitrate levels in neurologic diseases. J . Neurochem. 63, 1178-
1180.
(40) Qureshi, G. A., Baig, S., Bednar, I., Sodersten, P., Forsberg, G.,
and Siden, A. (1995) Increased cerebrospinal fluid concentration
of nitrite in Parkinson’s disease. NeuroReport 6, 1642-1644.
(41) Shergill, J . K., Cammack, R., Cooper, C. E., Cooper, J . M., Mann,
V. M., and Shapira, A. H. V. (1996) Detection of nitrosyl complexes
in human substantia nigra, in relation to Parkinson’s disease.
Biochem. Biophys. Res. Commun. 228, 298-305.
(42) NIH Guide for the Care and Use of Laboratory Animals (1985)
NIH Publication 85-23, National Institutes of Health, Bethesda,
MD.
(43) Pellegrino, L. J ., Pellegrino, A. S., and Cushman, A. (1979) A
stereotaxic atlas of the rat brain, Plenum, New York.
(44) Vallone, D., Pellecchia, M. T., Morelli, M., Verde, P., Di Chiara,
G., and Barone, P. (1997) Behavioural sensitization in 6-hydroxy-
dopamine-lesioned rats is related to compositional changes of the
AP-1 transcription factor: evidence for induction of FosB- and
J unD-related proteins Mol. Brain Res. 52, 307-317.
(45) J ourd’heuil, D., Miranda, K. M., Kim, S. M., Espey, M. G.,
Vodovotz, Y., Laroux, S., Mai, C. T., Miles, A. M., Grisham, M.
B., and Wink, D. A. (i999) The oxidative and nitrosative chemistry
of the nitric oxide/superoxide reaction in the presence of bicarbon-
ate. Arch. Biochem. Biophys. 365, 92-100.
(46) Lemercier, J .-N., Padmaja, S., Cueto, R., Squadrito, G. L., Uppu,
R. M., and Pryor, W. A. (1997) Carbon dioxide modulation of
hydroxylation and nitration of phenol by peroxynitrite. Arch.
Biochem. Biophys. 345, 160-170.
(47) Lerch, K. (1981) Copper monooxygenase:tyrosinase and dopamine
â-hydroxylase. In Metal Ions in Biological System (Sigel, H., Ed.)
Vol. 13, pp 143-186, Marcel Dekker, New York.
(48) Ungerstedt, U. (1971) Histochemical studies on the effect ofin-
tracerebral and intraventricular injections of 6-hydroxydopamine
on monoamine neurons in the rat brain. In 6-Hydroxydopamine
and catecholamine neurons (Malmfors, T., and Thoenen, H., Eds.)
pp 101-128, Elsevier, New York.
(67) Nakaki, T., Fujii, T., Suzuki, E., and Shintani, F. (1998) Endo-
thelium-independent and -dependent vasoactivity of 6-nitronor-
epinephrine. Eur. J . Pharmacol. 357, 193-197.
TX990121G