Oxidative Conversion of 6-Nitrocatecholamines to Nitrosating Products

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Oxidative Conversion of 6-Nitrocatecholamines to Nitrosating Products: A Possible Contributory Factor in Nitric Oxide and Catecholamine Neurotoxicity Associated with Oxidative Stress and Acidosis Anna Palumbo,† Alessandra Napolitano,‡ Antonio Carraturo,§ Gian Luigi Russo,§ and Marco d’Ischia*,‡ Laboratory of Biochemistry, Zoological Station “Anton Dohrn”, Villa Comunale 80121 Naples, Italy, Department of Organic Chemistry and Biochemistry, University of Naples “Federico II”, Via Cinthia 4, I-80126 Naples, Italy, and Institute of Food Science and Technology, National Research Council, 83100 Avellino, Italy Received May 7, 2001

Oxidation of 6-nitrodopamine (1) and 6-nitronorepinephrine (2), as well as of the model compounds 4-nitrocatechol and 4-methyl-5-nitrocatechol, with horseradish peroxidase (HRP)/ H2O2, lactoperoxidase (LPO)/H2O2, Fe2+/H2O2, Fe2+-EDTA/H2O2 (Fenton reagent), HRP or Fe2+/ EDTA in combination with D-glucose-glucose oxidase, or Fe2+/O2, resulted in the smooth formation of yellowish-brown pigments positive to the Griess assay. In the case of 1, formation of the Griess positive pigment (GPP-1) promoted by HRP/H2O2 proceeded through the intermediacy of two main dimeric species that could be isolated and identified as 3 and the isomer 4, featuring the 4-nitro-6,7-dihydroxyindole system linked to a unit of 1 through ether bonds. Spectroscopic (FAB-MS, 1H NMR) and chemical analysis of GPP-1 indicated a mixture of oligomeric species related to 3 and 4 in which oxidative modification of the nitrocatechol moiety of 1 led to the generation of reactive nitro groups supposedly linked to sp3 hybridized carbons. In the pH range 3-6, GPP-1 induced concentration- and pH-dependent nitrosation of 2,3-diaminonaphthalene, but very poor (up to 2%) nitration of 600 µM tyrosine. At pH 7.4, 1 exerted significant toxicity to PC12 cells, while GPP-1 proved virtually innocuous. By contrast, when assayed on Lactobacillus bulgaricus cells at pH 3.5, 1 was inactive whereas GGP-1 caused about 70% inhibition of cell growth. Overall, these results hint at novel pH-dependent mechanisms of nitrocatecholamine-induced cytotoxicity of possible relevance to ischemia- or inflammation-induced catecholaminergic neuron damage.

Introduction Originally discovered in normal mammalian brain (1), and more recently detected also in the rat spinal cord (2), 6-nitrocatecholamines, e.g., 6-nitrodopamine (1) and 6-nitronorepinephrine (2), attract increasing interest as potential indicators of biochemical interactions between catecholamine neurotransmitters and the nitric oxide (NO) signaling pathway. During inflammatory or neurodegenerative processes, or following ischemia reperfusion, high fluxes of NO generated by the various isoforms of NO synthase (NOS) may combine with reactive oxygen species to produce a range of nitrosating/nitrating agents which may target catecholamine neurotransmitters giving rise to 6-nitrocatecholamines. Although apparently associated with a pathophysiological alteration of the catecholaminergic system, the biological significance of 6-nitrocatecholamines remains elusive. In vitro studies highlighted several effects of 6-nitrocatecholamines, including the inhibition of presynaptic catecholamine reuptake (1) and COMT activity (1), antinociceptive effects (2), a moderate neurotoxicity (3) * To whom correspondence should be addressed. † Laboratory of Biochemistry. ‡ Department of Organic Chemistry and Biochemistry. § Institute of Food Science and Technology.

and inhibition of neuronal NOS (4). In most of these studies, however, the focus was mainly on 6-nitrocatecholamines per se, and little attention was paid to the notion that 6-nitrocatecholamine formation requires an oxidative environment. This notion stems from mechanistic analysis of the various putative routes to 6-nitrocatecholamines, viz., the direct reaction of catecholamines with NO in the presence of oxygen (5, 6); the reaction with nitrite in acidic media (7); and the oxidation with peroxidase/H2O2/NO2- (3, 7) or with Fe2+-EDTA/H2O2NO2- (3). In all these routes, an oxidative step precedes or is associated with the introduction of the nitro group onto the aromatic catechol ring, implying that the biological activity of 6-nitrocatecholamines may be related at least in part to their oxidative metabolism. In fact, although less oxidizable than the parent catecholamines, 6-nitrocatecholamines, e.g., 1, have been shown in preliminary studies to be susceptible to oxidation under physiologically relevant conditions to yield mixtures of oligomeric products, only one of which, dimer 3, could be isolated and characterized (3, 8). In the course of those studies, it was noticed that prolonged oxidation of 6-nitrocatecholamines, as well as of the model compounds 4-nitrocatechol and 4-methyl-5-nitrocatechol, resulted in the gradual development of products positive

10.1021/tx015525z CCC: $20.00 © 2001 American Chemical Society Published on Web 07/24/2001

Nitrosating Species from Nitrocatecholamines

to the Griess reagent, denoting potential nitrosating species in an acidic environment. This unexpected observation prompted us to undertake a detailed investigation of the oxidative behavior of 6-nitrocatecholamines and related nitrocatechols under physiologically relevant conditions. Specific aims were to elucidate the structural features of the nitrosating species and to assess their chemical and biological properties, in relation to their possible involvement in the mechanisms of neuronal damage and tissue injury.

Experimental Procedures Materials. Dopamine hydrochloride, (-)-norepinephrine, 4-nitrocatechol, 3-nitrotyrosine, D-glucose, potassium thiocyanate, EDTA disodium salt, ferrous sulfate heptahydrate, sodium hydrogensulfite (NaHSO3), 2,3-diaminonaphthalene, nonstabilized hydrogen peroxide (35% solution in water) were used as obtained. Horseradish peroxidase (HRP) (donor, H2O2 oxidoreductase, EC 1.11.1.7) type II (167 units/mg, RZ E430/E275 ) 2.0), lactoperoxidase (LPO) from bovine milk (80 units/mg RZ E412/E280 ) 0.76), glucose oxidase from Aspergillus Niger (β-Dglucose, oxygen 1-oxidoreductase EC 1.1.3.4) type II (20 000 units/mg), bovine serum albumin (BSA) fraction V from cold alcohol precipitation were used as obtained. 6-Nitrodopamine (1), 6-nitronorepinephrine (2), 4-methyl-5-nitrocatechol were prepared from the corresponding catechols by reaction with sodium nitrite/sulfuric acid as reported previously (5, 9). Pyrrole2,3-dicarboxylic acid (PDCA) was prepared as previously described (10). Melanin pigments from 5,6-dihydroxyindole or dopamine were obtained by tyrosinase/O2 oxidation as reported (10). Analytical Methods. FAB/MS spectra were obtained using nitrobenzyl alcohol or glycerol as the matrix. 1H (13C) NMR spectra were recorded at 400 (100) MHz. 1H,13C HETCOR and 1H,13C HMBC NMR experiments were run at 400 MHz using standard pulse programs. Analytical and preparative HPLC was carried out with an instrument equipped with a UV detector set at 254 or 280 nm. Octadecylsilane coated columns (4.6 × 250 mm, 5 µm particle size, or 22 × 250 mm, 10 µm particle size) were used for analytical or preparative runs at a flow rate of 1 or 15 mL/min, respectively. Elution conditions: 0.05 M sodium citrate/MeOH 85:15, pH 4 (eluant I); 0.1 M formic acid containing 10% acetonitrile (solution A), 0.1 M formic acid containing 40% acetonitrile (solution B) from 0 to 70% solution B gradient, 40 min (eluant II); 0.05 M formic acid/acetonitrile 90:10 (eluant III); 0.1 M sodium phosphate/MeOH 95:5, pH 4 (eluant IV), 50 mM potassium phosphate buffer, pH 3, containing 5% methanol (eluant V). Biochemical Assays. Production of H2O2 by the D-glucoseglucose oxidase system was monitored by the Fe(II)-thiocyanate assay involving quantitation of the Fe(III)-thiocyanate complex (11). Nitrite was determined spectrophotometrically at 538 nm

Chem. Res. Toxicol., Vol. 14, No. 9, 2001 1297 using the Griess reagent (1% sulfanylamide, 0.1% naphthylethylenediamine in 2.5% phosphoric acid) (12). Enzymatic Oxidation of Nitrocatecholamines. To solutions of 1 or 2 (100 µM) in 0.1 M phosphate buffer (pH 7.4) in a water bath thermostated at 37 °C, HRP (1 unit/mL final concentration) and 0.6% H2O2 (100 µM final concentration) were added in that order under vigorous stirring. Aliquots of the reaction mixture were periodically withdrawn, treated with a solution of NaHSO3 in water up to 100 µM final concentration to terminate oxidation, and analyzed by HPLC using eluant I or II. Similar experiments were performed in which H2O2 was generated in situ using D-glucose (0.28 mM) and glucose oxidase (0.02 units/mL, final concentration). In other experiments where mammalian LPO was used, the enzyme (0.053 units/mL final concentration) was added to the incubation mixture containing the nitrocatecholamine (100 µM) followed by H2O2 (100 µM). Iron-Promoted Oxidation of Nitrocatecholamines. Stirred solutions of 1 or 2 (100 µM) in 0.1 M phosphate buffer (pH 7.4) in a water bath thermostated at 37 °C were treated sequentially with H2O2 or glucose-glucose oxidase and Fe(II)/EDTA complex obtained by premixing (NH4)2Fe(SO4) and EDTA, both at a final concentration of 100 µM, to start the reaction. Aliquots of the reaction mixture were periodically withdrawn, treated with NaHSO3 and analyzed as above. In other experiments, EDTA was omitted, or the oxidation mixture contained dopamine in the case of 1 or norepinephrine in the case of 2 at a final concentration of 100 µM. Similar experiments were performed in which solutions of the nitrocatecholamines (100 µM) in 0.1 M acetate buffer, pH 4, were treated sequentially with sodium nitrite (100 µM) and H2O2 (100 µM). In the iron-catalyzed air oxidation, nitrocatecholamine solutions (100 µM) containing (NH4)2Fe(SO4) (10 µM) were allowed to stand at 37 °C for 18 h under vigorous stirring. Isolation of 5-[5-(2-Aminoethyl)-2-hydroxy-4-nitrophenoxy]-6,7-dihydroxy-4-nitro-2,3-dihydroindole (4). A solution of 1 hydrogensulfate (500 mg, 1.7 mmol) in 0.05 M phosphate buffer, pH 7.4 (1 L) was treated with HRP (11 units/ mL) and 3% H2O2 (2.6 mmol) and taken under stirring at room temperature. After 1 h the mixture was treated with excess NaHSO3, concentrated to a small volume, centrifuged to remove solid material and fractionated by preparative HPLC (eluant III) to give, in addition to 3 (25 mg, 8% yield) (8), pure 4 (15 mg, 5% yield) as yellow amorphous solid homogeneous to HPLC (tR 28 min, eluant II): λmax 352 nm (pH 7.0); 287, 340 nm (pH 3.0); νmax (CHCl3/DMSO) 3675, 3392, 1607, 1515, 1417, 1375 cm-1; δH (DMSO-d6) 2.94-3.06 (12H, m), 7.22 (1H, s), 7.56 (1H, s); δC (DMSO-d6) 26.5 (CH2), 30.43 (CH2), 40.47 (CH2), 114.0 (C), 114.2 (CH) 120.3 (C), 123.6 (C), 127.3 (CH), 134.1 (C), 139.0 (C), 145.4 (C), 150.2 (C), 152.4 (C), 153.5 (C), 155.4 (C). FABMS: 393 [M + H]+, 415 [M + Na]+; HRFAB-MS calcd for C16H17N4O8 [M + H]+, 393.1046; found, m/z 393.1060. Nitrosating Species from Nitrocatecholamines and Nitrocatechols. Oxidation of 1 or 2 was carried out with HRP or LPO in the presence of H2O2 or glucose-glucose oxidase as described above. In other experiments, oxidation of 1, 2 or 4-methyl-5-nitrocatechol was carried out with the Fenton system or with omission of EDTA as detailed above. When required, the oxidation mixture contained the parent catecholamine/ catechol at the same concentrations. Aliquots of the oxidation mixture were periodically withdrawn and mixed with two volumes of the Griess reagent; after 2 min, the absorbance at 538 nm of the solution was determined. The nitrosating properties of the oxidation products of nitrocatecholamines, of nitrocatechols, of dimers 3 and 4 as well as of the Griess Positive Pigments (GPPs) isolated as detailed below, were expressed in terms of nitrite equivalents. Nitrite equivalents were quantitatively estimated by interpolating calibration curves obtained from nitrite solutions in the concentration range 2.5-100 µM. Typically, 1 absorbance unit at 538 nm corresponded to 25 ( 3 µM nitrite. The concentration of GPP solutions was determined spectrophotometrically assuming the extinction coefficient equal to that of the starting nitrocatecholamine/nitrocatechol at 345

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nm. In other experiments, a solution of GPP from 1 (GPP-1) in water was added via a syringe to 0.1 M acetate buffer, pH 3, in a rubber-capped vial. The gas which developed was conveyed with a flux of argon into a solution of the Griess reagent in a rubber-capped vial. When required the solution containing GPP-1 was acidified to pH 0 by addition of 1% sulfuric acid. In control experiments, nitrite solution in water were introduced in the rubber-capped vial containing the buffer at pH 3. Isolation of Griess Positive Pigments (GPPs) from Nitrocatecholamines/Nitrocatechols. To a solution of the appropriate nitrocatechol (50 mg) in 0.05 M phosphate buffer, pH 7.4, HRP (24 units/mL) and 3% H2O2 (3 M equiv) were added sequentially under stirring at room temperature. After 1 h, the reaction mixture was concentrated to a small volume, centrifuged to remove solid material and fractionated on a Bio Gel P-2 column (100 × 2 cm) using water as the eluant. The elution course was monitored both spectrophotometrically and by the Griess assay. Griess-positive fractions were combined and taken to dryness to give yellowish brown solids (GPPs) in amounts ranging from 30 to 45 mg. 2,3-Diaminonaphthalene Assay. Quantitative determination of the nitrosating reaction was carried out by addition of varying amounts of solutions of GPPs from nitrocatecholamines/ nitrocatechols in water, up to 600 µM final concentration, to a mixture containing 0.2 mM 2,3-diaminonaphthalene in 0.05 M acetate buffer (pH 4, 1 mL) at room temperature. At 30 min reaction time, the pH of the mixture was raised by addition of 1 M phosphate buffer, pH 7.4 (200 µL), to stop the reaction. The nitrosation of 2,3-diaminonaphthalene to 2,3-naphthotriazole was quantified by measuring the fluorescence using an excitation wavelength of 375 nm and an emission wavelength of 450 nm (13). Chemical Analysis of GPPs. Degradation of GPPs from 1 and 2 as well as of synthetic melanin pigments was performed by a modification of the reported procedure involving treatment with alkaline hydrogen peroxide (10). Briefly, a suspension/ solution of the appropriate sample in 0.1 M potassium carbonate (1 mg/mL) was treated with 30% H2O2 (35 µL/mL) and kept under stirring at room temperature overnight. The reaction was stopped by addition of 5% NaHSO3 (50 µL/mL) and the mixture was acidified to around pH 2 with 1 M HCl. The mixture was then analyzed by HPLC using eluant IV as the mobile phase for PDCA determination. Tyrosine Nitration. A solution of tyrosine (600 µM) in 0.1 M sodium phosphate buffer (pH 3.0 or 4.4) was incubated at room temperature for 24 h in the presence of GPP from 1 (GPP1) (final concentration in the range 1-4 mM). In separate experiments, sodium nitrite (1-4 mM) was used in place of GPP1. 3-Nitrotyrosine formation was assessed by HPLC analysis using eluant V as the mobile phase (tR 16 min), with detector set at 274 nm. The detection limit was 5 µM at 274 nm. BSA Nitration. BSA (0.5 mg/mL) in 0.1 M sodium acetate buffer (pH 3.5, 1 mL) was incubated at room temperature overnight in the presence of GPP-1 (200 µL, 1.2 mM final concentration) or sodium nitrite (1.2 mM). Incubations were terminated by the addition of Laemmli buffer containing 0.125 M Tris/HCl (pH 6.8), 4% (w/v) SDS, 10% (v/v) 2-mercaptoethanol, 20% (w/v) glycerol, and 0.02% (w/v) bromophenol blue. BSA was examined by electrophoresis on 10% SDS polyacrylamide gel and transferred to Hybond-ECL nitrocellulose (Amersham Corp.). The blots, after saturation for 1h with 3% nonfat dry milk/PBS, were incubated overnight with affinity-purified rabbit anti-nitrotyrosine IgG (Upstate Biotechnology) at a final concentration of 1 µg/mL. After washings, bound antibodies were detected by enhanced chemiluminescence (Amersham) according to the manufacturer’s instructions. Control used included omission of the primary antibody. As positive control a sample of BSA nitrated with tetranitromethane was used. Cell Line, Bacterial Strain, and Media. PC12 cells were grown in RPMI medium containing 5% fetal calf serum, 10% horse serum, 100 units/mL penicillin, 100 µg/mL streptomycin, and 2 mM glutamine at 37 °C with 95% air-5% CO2. Cells were

Palumbo et al. commonly grown in 1 cm multiwell dishes and the medium was changed every 48 h (14). Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 was purchased from the American Type Culture Collection (ATCC, Rockville, MD) and was grown anaerobically in MRS medium (15) at 42 °C. The pH of the medium was adjusted to 3.5. Antibacterial Activity of GPP-1 versus 1 and NO2-. Various volumes of samples, corresponding to predetermined amounts of NO2-, were added to a final volume of 1.5 mL of MRS broth containing 106 cfu/mL of an overnight culture of L. bulgaricus. The control experiments consisted of 1.5 mL of MRS broth containing the same bacterial inoculum. Bacterial growth was assessed by conventional plate count method (16). Briefly, decimal dilutions from overnight cultures were prepared, and 0.1 mL of each dilution was spread onto MRS agar plates, following anaerobical incubation at 42 °C for 3 days. Plates showing between 30 and 300 colonies were counted. Effect of 1 and GPP-1 on Cell Growth. To assay the effect of title compounds on cell growth, cells [(2-3) × 105/well] were seeded on a 12-well plate and incubated with medium containing 1 (0.5 mM) and GPP-1 at the concentration corresponding to 0.5 mM of free NO2- for 16 h. Subsequently, cell viability was determined by crystal violet assay (17).

Results Kinetic Studies. The oxidative behavior of 6-nitrocatecholamines (100 µM unless otherwise stated) was investigated in 0.1 M phosphate buffer, pH 7.4, at 37 °C using a variety of enzymatic and nonenzymatic systems of potential relevance to oxidative stress. Enzymatic systems included horseradish peroxidase (HRP)-hydrogen peroxide, lactoperoxidase (LPO)-hydrogen peroxide, and tandem HRP-glucose-glucose oxidase, the latter ensuring low constant fluxes of hydrogen peroxide. Nonenzymatic oxidizing systems were largely based on iron ions as activators of hydrogen peroxide and comprise Fe2+/EDTA/ hydrogen peroxide (Fenton reagent), Fe3+/EDTA/hydrogen peroxide, Fe2+/hydrogen peroxide, and Fe2+/EDTAglucose-glucose oxidase. The Fe2+-promoted autoxidation as well as reactions with nitrite or hydrogen peroxidenitrite in mildly acidic media (pH 3-5) were also included in the list. In virtually all cases examined, the oxidation was found to proceed with a gradual broadening and flattening of the nitrocatecholamine chromophore (λmax 420, pH 7) and was accompanied by the slow accumulation of yellow pigments positive to the Griess assay. Typical kinetic profiles of the oxidation of 1 and 2 with HRP-hydrogen peroxide (A) and HRP-glucose-glucose oxidase (B) as monitored by substrate decay and development of Griess positive material, are shown in Figure 1. These data show that HRP in combination with hydrogen peroxide, either added as a bolus or slowly produced by the glucose-glucose oxidase system, can effectively promote oxidation of 1 and 2. Griess positive material was expressed in terms of nitrite equivalents, that is, the amount of nitrite giving the same response to Griess reagent. Typically, in the case of 1, nitrite equivalents of the oxidation mixture by the HRP/H2O2 system after 2 h reaction time accounted for ca. 15% of the starting substrate on a molar basis. In other words, ca. 15% of 1 was converted on oxidation to species bearing a reactive nitro group responsible for nitrosation of Griess reagent. The mammalian peroxidase LPO proved likewise effective in inducing 6-nitrocatecholamine oxidation in the presence of hydrogen peroxide, the reaction kinetics being comparable to those of the HRP-promoted reaction (not shown).


Figure 1. Time course of nitrocatecholamine oxidation and GPP formation promoted by the peroxidase/H2O2 system. 1 or 2 (100 µM) was incubated with horseradish peroxidase (1 unit/ mL) and H2O2 (100 µM) or glucose (0.28 mM) and glucose oxidase (0.02 units/mL) as detailed in the Experimental Procedures. Data are means of three experiments, and SD < 5%. Plot A: left axis (solid line), consumption of 1 (9) or 2 (b); and right axis (dashed line), GPP-1 (9) and GPP-2 (b) formation by the peroxidase/ H2O2 system. Plot B: as in plot A with generation of H2O2 by the glucose-glucose oxidase system.

Figure 2 shows the kinetics of substrate decay and formation of Griess positive material for the oxidation of 1 and 2 by Fe2+-hydrogen peroxide in the presence and in the absence of EDTA. The results indicate that the Fenton reagent is also effective in inducing nitrocatecholamine oxidation and formation of Griess positive material. Omission of EDTA in the incubation mixture resulted in a marked decrease in the reaction rate, possibly on account of decreased iron recycling and stabilization of the ferric form following binding to the nitrocatechol moiety of 1 and 2. Consistent with this interpretation, replacement of Fe2+ with Fe3+ resulted in little or no substrate oxidation under the same experimental conditions. Significant formation of Griess positive material was observed with the Fe2+-EDTA/ glucose-glucose oxidase system. Though much slower than Fenton-type reactions, autoxidation of 1 and 2 induced by Fe2+ (10 µM) at 37 °C proceeded to an appreciable extent after 24 h (substrate consumption: 28 ( 3% in the case of 1 and 26 ( 4% in the case of 2) and was likewise associated with the formation of Griess positive material (ca. 8% nitrite equivalents on a molar basis). When 6-nitrocatecholamines were incubated with nitrite in phosphate buffer, pH 3-5, in the presence or in the absence of hydrogen peroxide, i.e., under conditions favoring formation of the potential oxidants peroxynitrous acid or nitrous acid, respectively, no significant substrate oxidation was observed. Since in vivo 6-nitrocatecholamines are likely to be formed in small amounts within a catecholamine pool, and are therefore expected to be exposed to oxidative environments in the presence of their parent catecholamines, in subsequent experiments 1 and 2 were oxidized

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Figure 2. Time course of nitrocatecholamine oxidation and GPP formation promoted by Fe2+-H2O2 system. 1 or 2 (100 µM) was incubated with Fe2+ or Fe2+/EDTA (100 µM) and H2O2 (100 µM) as detailed in the Experimental Procedures. Data are means of three experiments, and SD < 5%. Plot A: left axis (solid line), consumption of 1 (9) or 2 (b); and right axis (dashed line), GPP-1 (9) and GPP-2 (b) formation by the Fe2+/EDTAH2O2 system. Plot B: as in plot A using Fe2+-H2O2 as the oxidant.

in the presence of equimolar amounts of dopamine and norepinephrine, respectively, using the Fenton reagent as the oxidizing system. The data in Figure 3 show kinetic profiles for nitrocatecholamine decay and formation of Griess-positive material that match fairly well with those observed in the absence of the parent catecholamines. Initial rates of oxidation of 1 and 2 in the presence of the parent catecholamines were 1.4 ( 0.2 and 1.3 ( 0.2 µM/min, respectively, versus values of 1.7 ( 0.2 and 1.8 ( 0.3 µM/min for 1 and 2 determined from Figure 2. Structural Characterization of Nitrosating Products from Oxidation of 1. With this background, in subsequent experiments the attention was focused to the chemical nature of the products responsible for the Griess reaction during nitrocatecholamine oxidation. As representative nitrocatecholamine, 1 was chosen because of its better known oxidation behavior (8). HRP/hydrogen peroxide was preferred as the oxidizing system in view of its effectiveness in inducing nitrocatecholamine oxidation (3, 8). HPLC analysis of the oxidation of 1 revealed different chromatographic profiles depending on the amount of oxidant. At hydrogen peroxide/1 ratios of 3:1 the product mixture consisted largely of ill-defined materials giving no defined peaks on HPLC. However, at lower oxidant/ substrate ratios, e.g., 0.8:1, a few peaks could be detected (Figure 4). Peak I proved the dimer 3 (8). The compound eluted under peak II was isolated by preparative HPLC. The FAB-MS spectrum (m/z 393, M + H+) was virtually identical to that obtained from 3 suggesting an isomeric dimer. The 1H NMR spectrum was also similar to that of 3, displaying two 1H singlets at δ 7.22 and 7.56 correlating with carbon resonances at δ 114.2 and 127.3, respectively. In the 1H,13C HMBC spectrum, both proton

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Figure 5. Bio Gel P-2 elution profile of the products obtained from 1 by HRP/H2O2 oxidation. Absorbance at 280 nm (9); and 538 nm (O) (Griess assay).

Figure 3. Time course of nitrocatecholamine oxidation and GPP formation promoted by Fe2+/EDTA-H2O2 system in the presence of the parent catecholamines. Solutions of 1 or 2 (100 µM) containing dopamine and norepinephrine (100 µM), respectively, were incubated with Fe2+/EDTA (100 µM) and H2O2 (100 µM) as detailed in the Experimental Procedures. Data are means of three experiments, and SD < 5%. Plot A: left axis (solid line), consumption of 1 (9) and dopamine ([); and right axis (dashed line), GPP-1 formation by the Fe2+/EDTA-H2O2 system. Plot B: as in plot A consumption of 2 (b) and norepinephrine ([); and right axis (dashed line) formation of GPP-2 (b).

Figure 4. HPLC elution profile of the oxidation mixture of 1 by HRP/H2O2. 1 (100 µM) in 0.1 M phosphate buffer (pH 7.4) was oxidized with 1 unit/mL HRP and H2O2 (80 µM). The elutogram was registered after 20 min by stopping the oxidation with sodium hydrogensulfite.

signals displayed cross-peaks with two carbon resonances at δ 145.4 and 153.5, indicating a nitrocatechol unit unsubstituted on the nuclear positions. Further scrutiny of the NMR spectra in comparison with those of 3 and 1 led eventually to formulate the product as 5-[5-(2aminoethyl)-2-hydroxy-4-nitrophenoxy]-6,7-dihydroxy-4nitro-2,3-dihydroindole (4).

Figure 6. Low field region of the 1H NMR spectrum of GPP-1.

As expected, dimers 3 and 4 proved negative to the Griess reagent, in analogy with 1. However, on oxidation, they were converted to Griess-positive materials, suggesting that they were intermediates in the generation of the nitrosating species from 1. To gain an insight into the nitrosating species produced by oxidation of 1 the crude reaction mixture was subjected to chromatographic analysis. Silica gel, polyamide, cellulose, and octadecylsilane (HPLC) proved of little value. However, a satisfactory fractionation was achieved by Bio Gel P2 column chromatography using water as the eluant. As apparent from the typical chromatographic profile shown in Figure 5, most of the reactivity toward the Griess reagent was carried by a yellow pigment (Griess-positive pigment from 1, GPP-1) eluting after 150 mL as a well defined band. The other minor yellowcolored fractions were negative to the Griess reagent. Fractions containing GPP-1 were thus pooled and taken to dryness to afford a yellowish-brown glassy oil virtually insoluble in water, poorly soluble in methanol and relatively more soluble in DMSO. Figure 6 shows the low field region of the 1H NMR spectrum of GPP-1 displaying a number of signals comprised between δ 5 and 8, denoting a mixture of oligomeric species. Unfortunately, both the low solubility and the chemical heterogeneity of GPP-1 prevented acquisition of informative 13C NMR and heteronuclear 2D-NMR spectra. Mass spectrometric analysis (FAB-MS) showed distinct peaks at m/z 977, 783 (+Na+, 805), 589 and 392 (+Na+, 414), suggesting oligomeric products up to the pentamer stage. Peaks were also apparent at m/z 880, 803, 717, 676, 671, 663 (+Na+, 685), 567, 490, 482, 349 (+Na+, 371), and 307 (+Na+, 329), due probably to extensive breakdown of oligomer precursors with losses of CO, NO, NO2, and other fragments. Whether these degradation products were components of GPP-1 or were generated



Table 1. Chemical Analysis of GPPs from Nitrocatecholamines and Reference Melanin Pigments

a

compd

PDCA yield (µg/mg)a

GPP-1 GPP-2 1 dopamine melanin 5,6-dihydroxyindole melanin

2.0 1.5 2.8 4.7

Mean of three experiments, SD e 7%.

in the ionization chamber of the mass spectrometer could not be determined on the basis of available data. Treatment of the isolated fraction with NaBH4 resulted in a marked decrease in absorbance throughout the UV and visible regions, with appreciable loss of color. It should be pointed out, in this regard, that NaBH4 does not reduce the nitro group of nitrocatecholamines, so any spectral change with this reagent must be ascribed to reduction of carbonyls, quinones and other hydridereducible functionalities. Similar GPPs were obtained by Bio-Gel P2 chromatography from the oxidation of 2 (GPP-2) as well as of the model compounds 4-nitrocatechol and 4-methyl-5nitrocatechol. These species proved intractable and could be analyzed only by 1H NMR spectroscopy. The spectra showed in all cases several proton signals spread in the low field region. In the spectrum of the GPP from 4-nitrocatechol, distinct groups of signals were also observed in the region between δ 3 and 4.5, denoting protons linked to sp3 carbons (not shown). Unfortunately, FAB-MS analysis proved much less informative than in the case of the oxidation products of 1, and the fractions could not be investigated further. Since structures 3 and 4 featured a 2,3-dihydroindole moiety derived from intramolecular cyclization of the aminoethyl side chain ortho to the OH group, the possible presence of indole rings in the Griess positive material from 1 was investigated. To this aim, an analytical procedure specifically suited for the analysis of melanins and related insoluble pigments from catecholamine metabolism (10, 18) was adopted. This involves alkaline hydrogen peroxide degradation of the material followed by HPLC quantitation of 2,3,5-pyrroletricarboxylic acid (PTCA) and 2,3-pyrroledicarboxylic acid (PDCA), diagnostic markers of 2-substituted and unsubstituted hydroxylated indole units, respectively. The results indicated a substantial formation of PDCA but no PTCA. The yields of PDCA obtained from GPP-1 and GPP-2, reported on a weight-by-weight basis, are shown in Table 1 in comparison with those from synthetic melanin pigments obtained by tyrosinase oxidation of dopamine and 5,6dihydroxyindole. Under the same degradation conditions, no PDCA was detected from 1, indicating that the PDCA obtained from GPPs derived exclusively from cyclized nitrocatecholamine units. Nitrosating Properties and Toxicity of 6-Nitrocatecholamine Oxidation Products. The observed positivity to the Griess assay by 6-nitrocatecholamine oxidation products denoted nitrosating properties in strongly acidic media. This raised two critical issues, concerning (a) the nitrosating properties under mildly acidic conditions of closer physiological relevance, and (b) the mechanism of nitrosation. To address these issues, the nitrosating properties of GPPs from nitrocatecholamines and nitrocatechols were

Figure 7. N-nitrosation of 2,3-diaminonaphthalene by nitrocatecholamine/nitrocatechol GPPs measured as fluorescence emission at 450 nm of 2,3-naphthotriazole. Data are means of three experiments, and SD < 7%. Plot A: GPP from 1 (b), 2 (×), 4-nitrocatechol (9), or 4-nitro-5-methylcatechol (4) versus concentration. Plot B: GPP-1 (open bars) in comparison with nitrite ions (black bars) at different pHs.

tested by monitoring the reaction with 2,3-diaminonaphthalene at pH 4, giving a strongly fluorescent 1Hnaphthotriazole. Figure 7 shows that GPP-1 exhibits significant concentration-dependent nitrosating properties, comparable to those of GPP-2 and GPPs from the model nitrocatechols (Figure 7A). The nitrosating properties of GPP-1 were then compared with those of nitrite at comparable concentrations in the pH range from 4 to 6. The results, shown in Figure 7B, indicated appreciable nitrosation even at pH 6, with the extent of fluorophore formation approximating onethird of that caused by nitrite at all pH values examined. Nitrosation reactions of GPP may in principle involve two distinct routes, namely acid-promoted release of nitrite/nitrous acid in the medium or direct interaction with the nitrosyl-accepting amine substrate. To distinguish between these routes, GPP-1 was incubated in acidic media in a septum-capped vial purged with argon, and the outlet gas was collected in a solution of the Griess reagent. At pH as low as 3, no detectable evolution of nitrogen oxides occurred after sufficiently prolonged periods of time. In control experiments, carried out under the same conditions at pH 3, but with nitrite in place of GPP-1, a positive Griess test was observed. However, when the pH of the medium containing GPP-1 was drastically lowered by the addition of acid, a marked Griess reaction was observed, suggesting substantial degradation of the pigments with formation of nitrous acid and subsequent decomposition to volatile nitrogen oxides. When incubated at 37 °C at pH 3, GPP-1 proved fairly stable, as judged spectrophotometrically at various intervals of time. To investigate whether GPPs could cause tyrosine nitration under mildly acidic conditions (19, 20), 600 µM L-tyrosine was incubated at 37 °C for 24 h with GPP-1 (from 1 to 4 mM, for the definition of GPP concentration see experimental) or nitrite at the same concentration

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Palumbo et al. Scheme 1. Comparative Views of the Oxidation Pathways of Dopamine and 1 Leading to Pigment Products

Figure 8. Effect of 1 and GPP-1 on bacterial cell growth. 1, GPP-1, and NO2-, at concentration of 0.25 mM or in the case of GPP-1 corresponding to 0.25 mM of free NO2- were added to L. bulgaricus cultures as reported in the Experimental Procedures. Incubation was performed anaerobically for 6 h and growth inhibition determined by colony count. A similar pattern of growth inhibition was observed after 24 h of incubation (data not reported). The data are representative of two independent experiments in duplicate. Bars indicate standard error. ANOVA: P < 0.005.

Figure 9. Effect of 1 and GPP-1 on cell viability. PC12 cells were grown as reported in the Experimental Procedures and treated for 16 h with a concentration of 1 (0.5 mM) and GPP-1 corresponding to 0.5 mM of free NO2-. Cell viability was assayed by crystal violet method, and data were expressed as percentage respect to untreated cells. The data are representative of two independent experiments in duplicate. Bars represent standard error. ANOVA: P < 0.01.

at pH values ranging from 3 to 7.4. HPLC analysis showed detectable formation of 3-nitrotyrosine (10 µM) only in the presence of 4 mM GPP-1 at pH 3. With 4 mM nitrite, 3-nitrotyrosine yields were 85 µM at pH 3 and 20 µM at pH 4.4. The lack of substantial tyrosine nitration was confirmed in similar experiments carried out on bovine serum albumin (BSA) using polyclonal antibodies against nitrated proteins. As positive control, detectable BSA nitration was observed with nitrite at comparable concentrations (not shown). Finally, a preliminary insight into the biological properties of GPPs was gained by assaying the effects of GPP-1 on a dopaminergic Pheochromocytoma cell line (PC12) at pH 7.4 as well as on a Lactobacillus cell culture at pH 3.5, using 1 as control. The results showed that GPP-1 can inhibit the growth of Lactobacillus cells cultured in acidic medium (Figure 8), but has no effect on PC12 (Figure 9). A similar pattern of growth inhibition was observed after 24 h of incubation (data not reported). No significant toxicity was exerted by nitrite at concentrations selected to match the nitrosating efficacy of GPP1. Interestingly, in contrast to GPP-1, 1 was toxic to PC12 cells (Figure 9) but did not affect Lactobacillus cell growth. In all cases, 1 was fairly stable under the incubation conditions. Whereas the activity of GPP-1 was possibly due to an arrest or delay in bacterial cell cycle progression, since a

comparison between growth inhibition at 6 and 24 h of treatment showed similar values of viability without bacterial cell death, the effect of 1 on PC12 cells was clearly associated to cytoxicity.

Discussion Oxidation of catecholamine neurotransmitters is a peculiar metabolic feature of certain populations of pigmented neurons, e.g., in the substantia nigra pars compacta and in the locus coeruleus. Within nigral neurons, the process is apparently confined to lysosomeassociated organelles and is purportedly triggered by an oxidative stress condition associated with excess levels of cytosolic catecholamines overwhelming the normal capacity of vesicular monoamine transporter to ensure accumulation into synaptic vesicles (21). Although the nature of the oxidizing systems involved in catecholamine oxidation remains elusive, there is growing consensus that reactive oxygen species, chiefly hydrogen peroxide derived from both enzymatic and nonenzymatic sources, play a critical role along with Fe3+ ions. On the basis of the results of the present as well as of previous studies, it appears that a similar biochemical scenario may also account for 6-nitrocatecholamine generation and oxidation. Indeed, our data indicate that 1 and 2 can be efficiently oxidized not only by peroxidases, e.g., HRP and a mammalian enzyme, LPO, but also by a variety of Fe2+dependent systems, typically the Fenton reagent, with H2O2 either added as a bolus or generated at physiologically relevant fluxes by the glucose-glucose oxidase system. Of particular relevance, in this frame, is the finding that the rates of oxidation of 1 and 2 are not significantly slackened in the presence of the parent catecholamines. This finding is intriguing because, judging from the greater oxidizability of dopamine and norepinephrine compared to 1 and 2 observed in separate kinetic experiments (3), a faster oxidation of catecholamines preceding the onset of nitrocatecholamine decay could be predicted. Nonetheless, it provides a most convincing evidence of the biological relevance of the chemistry described in this paper since 6-nitrocatecholamine formation in vivo must occur within a catecholamine pool. Oxidation of catecholamines typically proceeds to yield black to dark brown pigments by way of transient o-quinones, which polymerize via unstable aminochromes and 5,6-dihydroxyindole intermediates (Scheme 1) (2224). Operation of a similar mechanism is, however, precluded in the case of 6-nitrocatecholamines in which the electron-withdrawing nitro group on the 6-position of the catechol ring hinders intramolecular cyclization of the putative nitroquinone intermediates to 5,6-dihydroxyindole species. In accord with this expectation, oxidation

Nitrosating Species from Nitrocatecholamines Scheme 2. Suggested Origin of Nitrosating Products by Oxidation of Dimer 3 and Possible Mechanism of Reaction with 2,3-Diaminonaphthalene as Representative Amine Substratea

a The generation of a highly resonance-stabilized cationic species driving nitrite transfer is highlighted.

of 6-nitrocatecholamines was found to afford yellowish brown materials quite different from the black-to-brown melanin pigments produced by oxidation of dopamine or norepinephrine. The lighter color of the oxidation products of 6-nitrocatecholamines probably reflects the presence of 4-nitro-6,7-dihydroxyindole-derived units (Scheme 1) linked through C-O-C bonds as in dimers 3 and 4, a feature precluding highly extended π electron delocalization. The formation of small amounts of PDCA by oxidative degradation of the GPP-1 corroborates the presence of indole rings or pyrrole units thereof (10). On the other hand, the lack of formation of PTCA, a diagnostic marker of 2-substituted hydroxyindole derivatives (25), would rule out any significant mechanism of dimerization/ polymerization of 3 or 4 through the pyrrole moiety of the indole ring. A critical mechanistic issue, in this scheme, concerns the origin of the nitrosating moieties. FAB-MS analysis of GPP-1 was not much revealing in this regard, offering, however, support to oligomeric structures derived from dimers 3 and 4. More insightful was the 1H NMR spectrum of GPP-1 as well as of the GPPs from model nitrocatechols, which indicated profound modifications of the nitrocatechol system, on account of a partial loss of the aromatic character. This might be due to inter- or intramolecular attacks of the hydroxyl groups onto the nitro-bearing positions of quinone intermediates, as exemplified in Scheme 2 for the oxidation of dimer 3. This scheme, leading to products with the marks of reactive aliphatic nitrocompounds, would well account for the observed positivity toward the Griess reagent. The use of this reagent has certain limitations regarding the identity of the nitrosating species, as it may be an indicator both of nitroso functionalities and of reactive nitro groups that can release nitrite under acidic conditions. However, the nature of the substrate and chemical arguments would restrict the range of candidate nitrosating agents to labile nitro groups. In the postulated scheme, the driving force for the acid-promoted loss/ transfer of nitrite would be provided by restoration of the aromatic/quinonoid system, assisted by the stabilizing


effects on the developing cationic center of at least three groups bearing lone pairs. This structural arrangement would favor heterolytic cleavage of the C-NO2 bond even in mildly acidic media, e.g., at pH 5. Similar oxidation pathways could be invoked in the case of 2, whereas the oxidation of the model nitrocatechols appears to proceed mainly through coupling of the nuclear positions via carbon-carbon bonds (8). This chemistry is unprecedented and no information is available concerning the generation of nitrosating species by oxidation of other nitrophenols, e.g., 3-nitrotyrosine. Another relevant issue concerns the mechanism of the acid-dependent nitrosation by GPPs. At mildly acidic pH the GPPs appeared to be stable. Accordingly, they should nitrosate target amine compounds presumably by direct interaction, without release of free nitrite and/or nitrous acid as mediators. This view would be supported by the different ability of GPP-1 and nitrite to effect protein nitration, as well as by the lack of nitrogen oxides in the purging gas from mildly acidic solutions of GPP-1. The comparable trends of 2,3-diaminonaphthalene nitrosation by GPP-1 and nitrite with changing pH indicate a similar dependence of the nitrosation reactions on acids. It can be noted, in this connection, that nitrite formation is a characteristic feature of 4-nitrocatechol degradation by microorganisms, e.g., Burkholderia c. and Arthrobacter p., and by sensitized photocatalysis in TiO2 slurries (26, 27). The hydrogen peroxide-dependent processes described in the present study, therefore, seem to involve milder oxidation mechanisms. That nitrite is not responsible for the nitrosating properties of GPPs in acidic media can also be inferred by the different responses elicited by nitrite ions and GPP-1 on Lactobacillus cells. This finding however must be interpreted with caution. In fact, what can be safely argued from those data is that nitrite ions are not the determinants of the toxic effects caused by GPP-1, although the apparent pH-dependent nature of the effects seems to indicate an acid-induced mechanism of cytotoxicity related to nitrosation. Of course, caution must be exercised in comparing effects on quite different types of cells; nonetheless, within these limitations, it is worthy of note that GPP-1 affect only cells growing in acidic media. It is also of interest that 1 exerts significant toxicity toward PC12 cells at pH 7.4. This observation lends further support to the proposed involvement of this putative metabolite in the mechanisms of catecholaminedependent neurotoxicity and neuronal degeneration (3, 5). Clearly, more data are necessary to definitively support the generation and role of 1 and 2 in catecholaminergic neurons and to demonstrate the actual formation and pathophysiological relevance of the oxidation products described in this paper. Although 6-nitrocatecholamine concentrations in mammalian brain and spinal cord are low, their levels are expected to increase dramatically under pathological conditions as happens, e.g., for nitrite levels. So, it can be speculated that they can give rise to significant amounts of oxidation products which may play a contributory role in neuronal degeneration and cell damaging processes associated with temporary pH drops (28). Tissue acidosis is a prominent feature of severe inflammatory and ischemic conditions, and is associated with a marked decrease in extracellular and/or intracellular pH. In the brain, ischemic neurons deprived of oxygen and glucose lose ATP and become depolarized,

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leading to synaptic release of the excitotoxic neurotransmitter glutamate and the electrogenic transport of glutamate from depolarized astrocytes. The resulting build up of extracellular glutamate may lead to several NMDA-receptor mediated neurotoxic events, including a drop in intracellular pH (average values of ca. 5.86.2). This more or less prolonged condition of acidity is thought to contribute to cellular injury by diverse, partially interrelated mechanisms currently under intense scrutiny as potential therapeutic targets (29-31). Critical factors supposed to contribute to post-ischemic injury to catecholaminergic neurons include lactic acid accumulation (32), enhanced production of free radicals and reactive oxygen species (during reperfusion) (33), overactivation/up-regulation of neuronal NOS (34, 35, 36) and, notably, catecholamine oxidation (37). Thus, at sites of CNS trauma, ischemia or inflammation, catecholaminergic neurons may experience redox and pH conditions suitable to sustain both 6-nitrocatecholamine oxidation and the potentially cytotoxic N-nitrosation processes mediated by the resulting products. With the two distinct types of reactivity (oxidation and acid-induced nitrosyl transfer) underlying formation of nitrosating products from 6-nitrocatecholamines, timing and location of the relevant steps may be critical to the biological implications in disease states. The present study indicates that 6-nitrocatecholamine oxidation is favored in nonacidic media, implying that this step should precede the pH drop required to induce the nitrosation reaction. Such a situation may be compatible, for example, with ischemia-induced nigrostriatal neurodegeneration. In this setting, a constant oxidation of the dopamine pool would result in the gradual accumulation of polymeric species incorporating nitrocatecholaminederived products which, when temporarily exposed to low pH conditions, may elicit nitrosation of surrounding targets and cytotoxicity. The possible location of the proposed oxidative events is also a critical issue. Catecholamine oxidation to neuromelanin is an essentially intracellular process. On the other hand, 6-nitrocatecholamine formation appears to reflect oxidative stress and excitotoxic responses with attendant overproduction of NO. Such a condition can be associated with enhanced release of catecholamines from, and decreased reuptake into, presynaptic nerve terminals (38), which hints at a possible extracellular setting for catecholamine nitration. These views are not mutually exclusive and their verification requires a more detailed knowledge of the sites and mechanisms of formation of 6-nitrocatecholamines. Several open questions obscure the latter issue at present, e.g., the nature of the nitrating species and the relevant inhibitory mechanisms, and raise interesting points concerning, e.g., the role of the one-electron reduction product of NO, nitroxyl anion, and the possible alteration of dopamine nitration pathways by neuromelanin, given the protective effects of dopamine melanin on peroxynitrite-induced tyrosine nitration (39, 40, 41).

Acknowledgment. This work was supported in part by grants from CNR (Rome). We thank the Servizio di Spettrometria di Massa del CNR e dell’Universita` di Napoli for mass spectra and the Centro Interdipartimentale di Metodologie Chimico-Fisiche of Naples University for NMR facilities. We thank Mr. Luigi De Martino for technical assistance.

Palumbo et al.

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Oxidative Conversion of 6-Nitrocatecholamines to Nitrosating Products

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