Chem. Res. Toxicol. 1999, 12, 1213-1222
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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 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 July 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 NO2using 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 H2O2dependent 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.
Introduction Functional abnormalities of dopaminergic neuronal pathways, due to selective degenerative changes and the loss of pigmented nigrostriatal neurons, and a progressively severe depletion of dopamine constitute the primary 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 reactions (7). * 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:
[email protected]. † Zoological Station. ‡ Department of Organic and Biological Chemistry, University of Naples “Federico II”. § Department of Neurological Sciences, University of Naples “Federico II”.
Whether and by what modalities elevated fluxes of reactive oxygen species can affect the structural and functional integrity of dopamine and impair dopaminergic nigrostriatal connectivities has not yet been assessed in sufficient detail. Frequently cited theories advocate aberrant oxidation pathways of the labile catechol functionality as the leading cause of dopamine-mediated neurotoxicity (8). These pathways may proceed through either electron transfer steps, to give dopamine semiquinone and o-quinone via subsequent disproportionation, or direct electrophilic attack to the aromatic ring by a reactive oxygen species, e.g., the OH• radical (9). 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 traditionally occupied a prominent position because of its established ability to destroy dopaminergic neurons and
10.1021/tx990121g CCC: $18.00 © 1999 American Chemical Society Published on Web 11/18/1999
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cause Parkinsonism, possibly through oxygen radical production and alteration of zinc metallothionein (1, 2, 14). Extensive investigations aimed at identifying possible 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 dopaminerelated 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 (2629) produced by coupling of NO with O2-. An additional pathway of NO-dependent toxicity involves interaction with H2O2 promoting hydroxylation of aromatic substrates (30).
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 inflammatory 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 excessive 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 spectrometry.
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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 stressdependent neurotoxicity.
Experimental Procedures Materials. Dopamine hydrochloride, 2,4,5-trihydroxyphenylethylamine 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 (β-Dglucose:oxygen 1-oxidoreductase, EC 1.1.3.4) type II (20 000 units/mg), and mushroom tyrosinase (o-diphenol:O2 oxidoreductase, 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. Analytical Methods. 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/acetonitrile [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 reproducibilities 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-quadrupole mass spectrometer. Biochemical Assays. 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). Enzymatic Oxidation of Dopamine. To solutions of dopamine (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
Dopamine Nitration and Hydroxylation (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 products were carried out by comparing retention times and integrated peak areas with external calibration curves for authentic samples. Iron-Promoted Oxidation of Dopamine. 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. Product Identification or Isolation. 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 dopamine (20 mg) in the presence of NO2- (10 mM) was 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-SGlutathionyldopamine was identified by co-injection with a synthetic sample with eluants B and C. Preliminary Animal Experiments. 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 individual 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.
Results In a survey of the effects of NO2- on dopamine oxidation chemistry, the horseradish peroxidase (HRP)/ H2O2 system was initially chosen because of its established 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
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Figure 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 unreacted 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-dihydroxyindole, the rearrangement product of the aminochrome, as main detectable products (Figure 1). When the reaction was carried out in the presence of an excess of NO2-, 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 spectrometry (ES/MS) coupled with UV analysis. Compound 2 (pKa ) 6.2) is easily monitored by its characteristic pHdependent 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 resembling melanin. In control experiments, no detectable dopamine nitration was observed by incubating the substrate for at least 30 min with various NO2- concentrations 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 suggested reduction with sodium borohydride as the most advisable procedure. 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,
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Figure 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 withdrawn, 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-dihydroxyindole ([). Right axis (solid lines): 1 (b) and 2 (9).
Figure 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).
Figure 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
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, concentrations 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 comparative 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 experiments. 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 increased with increasing NO2- concentrations (Figure 4), whereas the level of generation of 1 was slightly decreased. 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 ascorbate under the same conditions caused significant inhibition. Notably, in the presence of 1.0 mM reduced glutathione, dopamine was converted to a major reaction product, which was identified as 5-S-glutathionyldopa-
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Table 1. Effect of Additives on Dopamine Hydroxylation and Nitration by HRP/H2O2/NO2- a yieldb (µM) additive
1
2
ascorbate (1 mM) HCO3- (25 mM) glutathione (1 mM) SOD (300 units/mL)
7.2 1.1 4.3 6.9
1.7 0.5 1.5 1.5
a Dopamine (1 mM) in 0.1 M phosphate buffer (pH 7.4) was 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%.
Figure 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).
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), induced 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 by HRP in the presence of 10 mM NO2- was achieved 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 concentration of 0.1 mM in the presence of 1 mM NO2-. 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, lactoperoxidase (LPO), proved to be likewise effective in converting 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 formation of detectable amounts of 2 from 1 mM dopamine with a NO2- concentration of 0.5 mM, conditions under which HRP proved to be virtually uneffective. Since thiocyanate (SCN) is a presumed major physiological 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
Table 2. Dopamine Hydroxylation and Nitration by LPO/H2O2/NO2- a [NO2-] (mM)
additive
1
2
unreacted dopamineb (mM)
0.5 1.0 2.0 5.0 10.0 10.0 10.0
SCN- (1 mM) SCN- (10 mM)
5.0 4.8 5.2 4.9 3.8 3.2 3.9 2.9
0.60 0.82 0.92 1.2 1.6 1.6 1.1
0.60 0.62 0.53 0.59 0.55 0.57 0.66 0.70
yieldb (µM)
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%.
Figure 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).
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 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 after 18 h, increasing linearly with NO2- concentration and exceeding that observed with the various peroxidase/ H2O2 systems (Figure 6). Addition of higher concentrations of Fe2+, however, considerably decreased the level of formation of 2 despite comparable or enhanced rates of dopamine consumption (not shown). In control experiments 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 dopamine in the presence of NO2- (data not shown). 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
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Figure 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 Udenfriend- and Fenton-type systems in the presence of ascorbate (9), was not detected. 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 inhibition by added mannitol. With a view to gaining an insight into the mechanism of catecholamine nitration, in further experiments dopamine was subjected to aerial oxidation in the presence of tyrosinase, a copper enzyme causing direct twoelectron oxidation of catecholic substrates to the corresponding 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 decreased 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. 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 episodes 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
Figure 8. Dopamine hydroxylation and nitration by tyrosinase/ O2/NO2- as a function of NO2- concentration. Dopamine (1 mM) in 0.1 M phosphate buffer (pH 7.4) was incubated with tyrosinase (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.
Discussion Increasing lines of evidence support the possible generation of 6-nitrocatecholamines by NO-dependent mechanisms 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 exclusive, which may have been overlooked in the general panorama of biologically relevant nitrations. A typical case in point is provided by the peroxidase/ H2O2/NO2--mediated nitration. Ample experimental evi-
Dopamine Nitration and Hydroxylation Scheme 1
dence indicates that HRP and LPO active forms are thermodynamically competent in bringing about oxidation of NO2- to NO2 (E° ) -0.99 V) (51), which, in the 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 compared to tyrosine would account for a faster and more efficient oxidation by peroxidase. As a consequence, dopamine would be expected to compete with NO2- for oxidation by peroxidase compounds I and II more efficiently 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). Furthermore, 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 peroxidase-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 peroxidase- and tyrosinase-catalyzed reactions in the presence of NO2-, a mechanistic scheme is proposed in which 1 and 2 arise mainly via nucleophilic attack of NO2- and H2O2 on dopamine o-quinone (4) generated by disproportionation of enzymatically produced semiquinone 3 (Scheme 1).
Chem. Res. Toxicol., Vol. 12, No. 12, 1999 1219
In this scheme, the quinone 4 partitions among three main competitive routes, the major involving intramolecular 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 evidence for the proposed ultimate involvement of NO2in 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 generation 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 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. 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+/H2O2induced oxidations. In the presence of EDTA, i.e., under conditions in which most of the Fe3+ produced by oxidation 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 Fenton-type reagents can bring about competitive attack on 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 reagent (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-
1220
Chem. Res. Toxicol., Vol. 12, No. 12, 1999 Scheme 2
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 precursor 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 oxygenating agent, namely, (i) the nucleophilic addition of H2O2 to dopamine quinone followed by carbonyl-forming cleavage 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 Fentontype species. Whenever sufficient concentrations of dopamine quinone are generated, e.g., by peroxidase/H2O2, tyrosinase, or ferricyanide or during autoxidation, formation of 1 should expectedly involve mainly nucleophilic attack of H2O2 as in route (i) (17). 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 catecholamine-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
Palumbo et al.
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) reported 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 necessary for obtaining detectable nitration of dopamine, compared to that of tyrosine (29). This reflects (a) the higher susceptibility of catechols to oxidation and polymerization 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 nitrocatechols compared to that of nitrophenols (data not shown), which accounts for significant product decomposition during the reaction. Indeed, although the electronwithdrawing 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 dopamine 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 overactivated under conditions of oxidative stress and severe antioxidant depletion, whereby dopamine is slowly oxidized in the presence of excess NO2-. Thus, in the case of dopamine and other catechols, NO2--dependent nitration 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 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 disorders, 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 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 underestimated 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
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 conditions of iron accumulation, after the primary toxic processes are underway. 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 impairment of dopaminergic neurotransmission (66) because of its reduced ability to interact with D1-dopaminergic receptors compared to the parent catecholamine (49). Despite considerable efforts, 2 has not yet been identified in vivo. However, the analogous derivative of norepinephrine, 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 contraction in both intact endothelium and denuded aorta, and inhibited acetylcholine-induced relaxation, through a H2O2-dependent mechanism (67). Further work is therefore warranted to identify 2 in vivo and to definitively assess the actual significance of nitration routes in catecholamine metabolism.
Acknowledgment. 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.
References (1) Cohen, G. (1983) The pathobiology of Parkinson’s disease: Biochemical 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., Jellinger, 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., Jellinger, 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.
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. (9) Slivka, A., and Cohen, G. (1985) Hydroxyl radical attack on dopamine. J. Biol. Chem. 260, 15466-15472. (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-Scysteinyldopamine. Biochim. Biophys. Acta 1245, 255-261. (11) Spencer, J. P., Jenner, 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., Jameson, R. F., Kienzl, E., Jellinger, K., and Youdim, M. B. H. (1986) Dopamine, 6-hydroxydopamine, iron and dioxygen, their mutual interaction and possible implication 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-hydroxydopamine quinone by reaction of fatty acid hydroperoxides with dopamine: a possible contributory mechanism for neuronal degeneration in Parkinson’s disease. J. Med. Chem. 40, 22112216. (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. (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, superoxide and peroxynitrite: the good, the bad and the ugly. Am. J. Physiol. 271, C1424-C1437. (29) van der Vliet, A., Eiserich, J. P., Halliwell, B., and Cross, C. E. (1997) Formation of reactive nitrogen species during peroxidasecatalyzed 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
(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., Jones, 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., Jones, 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., Joseph, 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, 1291212917. (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, 11781180. (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-hydroxydopamine-lesioned rats is related to compositional changes of the AP-1 transcription factor: evidence for induction of FosB- and JunD-related proteins Mol. Brain Res. 52, 307-317. (45) Jourd’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 bicarbonate. 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 β-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 ofintracerebral 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.
Palumbo et al. (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 nitration 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 constants. 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: electrontransfer 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-hydroxyquinoline-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 endogenous 1(R),2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, 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 sympathetic neurotransmission. Biochem. Biophys. Res. Commun. 216, 686-692. (67) Nakaki, T., Fujii, T., Suzuki, E., and Shintani, F. (1998) Endothelium-independent and -dependent vasoactivity of 6-nitronorepinephrine. Eur. J. Pharmacol. 357, 193-197.
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