QUARTERLY

Peroxynitrite-mediated linoleic acid oxidation and tyrosine nitration were analysed in the presence of synthetic model neuromelanins: dopamine (DA) -melanin, cysteinyldopamine (CysDA) -melanin and various DA/CysDA copolymers. The presence of melanin significantly decreased the amount of 3-nitrotyrosine formed. This inhibitory effect depended on the type and concentration of melanin polymer. It was found that incorporation of CysDA-derived units into melanin attenuated its protective effect on tyrosine nitration induced by peroxynitrite. In the presence of bicarbonate, the melanins also inhibited 3-nitrotyrosine formation in a concentration dependent manner, although the extent of inhibition was lower than in the absence of bicarbonate. The tested melanins inhibited peroxynitrite-induced formation of linoleic acid hydroperoxides, both in the absence and in the presence of bicarbonate. In the presence of bicarbonate, among the oxidation products appeared 4-hydroxynonenal (HNE). CysDA-melanin inhibited the formation of HNE, while DA-melanin did not affect the aldehyde level. The results of the presented study suggest that neuromelanin can act as a natural scavenger of peroxynitrite.

3-Nitrotyrosine has been suggested as a marker of peroxynitrite-mediated damage in a variety of disease states, including neurodegenerative disorders such as Alzheimer's disease (Smith et al., 1997) and Parkinson's disease (Good et al., 1998;Ara et al., 1998).However, tyrosine nitration can also be caused by other biochemical pathways (Ischiropoulos, 1998;Pfeiffer et al., 2000).
Neuromelanin, a dark pigment that deposits with age in the cytoplasm of nigrostriatal dopaminergic neurons, originates from the oxidative pathway of dopamine (DA) metabolism in the human brain (Graham, 1978;Marsden, 1983).The synthesis of neuromelanin is initiated by the oxidation of DA to its o-quinone, which can undergo intramolecular cyclization and further oxidation to indoles (Graham, 1978;Costa et al., 1992), or can react with the thiol group of cysteine to give cysteinyl conjugates, mainly 5-S-cysteinyldopamine (5-S-CysDA) (Tse et al., 1976;Ito et al., 1986;Zhang & Dryhurst, 1995).These conjugates are easily oxidized (more easily than DA) to form benzothiazine derivatives (Shen & Dryhurst, 1996).Degradative studies of neuromelanin isolated from human substantia nigra have demonstrated that the pigment is a copolymer of eumelanin and phaeomelanin, and consists of indole-type and benzothiazinetype units (Carstam et al., 1991;Odh et al., 1994).It has been postulated that neuromelanin formation is an autoxidative process catalysed by iron and other transition metals (Rodgers & Curzon, 1975;Fornstedt et al., 1989; D'Ischia & Prota, 1997), although enzymes such as prostaglandin H synthase (Hastings, 1995), lipoxygenase (Rosei et al., 1994) and peroxidase (Okun, 1997) are capable of oxidizing DA to DA-quinone in vitro.The ability of peroxynitrite to promote DA oxidation has been also demonstrated (Kerry & Rice-Evans, 1999;LaVoie & Hastings, 1999).
Recently, we have shown that synthetic DA-melanin inhibited peroxynitrite-mediated nitration of free tyrosine, loss of tryptophan residues in bovine serum albumin and Ca 2+ -ATPase inactivation (Stêpieñ et al., 2000).In this work we investigated the effect of synthetic neuromelanins derived from DA and 5-S-CysDA on peroxynitrite-induced tyrosine nitration and linoleic acid oxidation.
Preparation of 5-S-CysDA and synthetic neuromelanins.5-S-Cysteinyldopamine was obtained by tyrosinase-catalysed oxidation of dopamine in the presence of cysteine, and purified by ion-exchange chromatography (Dowex 50Wx2) according to Ito et al. (1986).Synthetic models of neuromelanin were prepared by oxidative polymerization of dopamine (DA-melanin) or 5-S-cysteinyldopamine (CysDA-melanin), and by copolymerization of both precursors at molar ratios 3 : 1 (DA/CysDA(3 : 1)-melanin) and 1 : 1 (DA/ CysDA(1 : 1)-melanin).Solutions of the melanin precursors (total concentration 5 mM) in phosphate buffer (50 mM, pH 7.4) were incubated at 37°C for 48 h.The insoluble melanins formed during such oxidation were separated by centrifugation (3 000 ´g), washed with water and stored as aqueous suspensions of known concentrations.Elemental analysis of the melanin samples was performed by the Center for Molecular and Macromolecular Research of the Polish Academy of Sciences in £ódŸ (Poland).
Synthesis of peroxynitrite.Peroxynitrite was synthesised according to the method of Uppu and Pryor (1996), using a 2 M H 2 O 2 solution in NaOH (100 ml, pH 13) and isoamyl nitrite (0.2 mol).The aqueous phase that contained peroxynitrite was separated after centrifugation and washed with dichloromethane (6 ´100 ml) to remove the contaminating isoamyl alcohol and traces of isoamyl nitrite.The unreacted H 2 O 2 was removed by passing the aqueous phase through a column of MnO 2 (25 g) prewashed with water (20 ml) and 0.5 M NaOH (20 ml).A stock solution of peroxynitrite was stored frozen at -80°C.The peroxynitrite concentration was determined spectrophotometrically at 302 nm using a molar absorption coefficient of 1700 M -1 cm -1 (Bohle et al., 1996) before each experiment, and appropriate amounts of peroxynitrite solution were added to reaction mixtures.A drop of peroxynitrite was placed on the test tube wall and the samples were immediately vortexed for 30 s.In control experiments, decomposed peroxynitrite was used (Stêpieñ et al., 2000).
Tyrosine nitration.Peroxynitrite (final concentration 1 mM) was added to solutions of tyrosine (1 mM) in sodium phosphate buffer (0.1 M, pH 7.4) containing 0.1 mM diethylenetriaminepentaacetic acid (DTPA) and various amounts of model melanins (50-800 mg/ml).In a separate set of experiments, the reaction medium was supplemented with sodium bicarbonate (20 mM).Bicarbonate was introduced 2 min before the addition of peroxynitrite (pH of the solution increased to 7.6).Incubations were carried out for 30 min at room temperature.The formation of 3-nitrotyrosine was quantified by HPLC and expressed as a percent of the control incubated without melanin.
Linoleic acid oxidation.The reaction mixtures contained linoleic acid (1 mM) dispersed in sodium phosphate buffer (0.1 M, pH 7.4), DTPA (0.1 mM), various concentrations of DA-melanin or CysDA-melanin (10-200 mg/ ml) and 0 or 20 mM of sodium bicarbonate.The oxidation was initiated by adding peroxynitrite to a final concentration of 4 mM.After 2 h of incubation at room temperature, each sample was diluted with methanol (1 : 1, v/v) and directly analysed by HPLC.HPLC analysis.Reverse-phase HPLC was performed with a Hewlett-Packard model 1050 liquid chromatograph equipped with a HP 1100 diode array detector and interfaced to a HPLC ChemStation (HP).Each sample was filtered (filter Millex GV13, pore diameter 0.22 mm; Millipore) to remove melanin and then the reaction products were separated on an Eurospher 100 C18 column (particle size 5 mm, 250 ´4 mm; Knauer) at 35°C.3-Nitrotyrosine and tyrosine were eluted isocratically with 50 mM KH 2 PO 4 (pH 3) and methanol (92 : 8) and detected at 276 nm (tyrosine was eluted at 4.6 min and 3-nitrotyrosine at 12.4 min).Linoleic acid oxidation products were separated using water (A) and acetonitryle (B) containing 0.2% phosphoric acid and the following gradient programme: 0 min 50% A: 50% B, 2 min 50% A:50% B, 16 min 17% A: 83% B and 17 min 100% B. HNE, 13-HODE and four linoleic acid hydroperoxides isomers were identified by matching their retention times and UV-Vis spectra with respective standards.The observed retention times were 5.2 min, 19.1 min, 21.1 min, 21.9 min, 24.2 min and 25.5 min, respectively.The amounts of 3-nitrotyrosine, 13-HODE and HNE were calculated with respect to the appropriate calibration curves.Quantifications of linoleic acid hydroperoxides were made using 13-HPODE as a standard.
The results are expressed as mean values ± S.D. calculated from three or more independent experiments.
To determine the effect of synthetic neuromelanins on tyrosine nitration mediated by peroxynitrite, formation of 3-nitrotyrosine was measured by HPLC.In the absence of melanin, the yield of 3-nitrotyrosine was 95 ± 6 mM (n = 12) after the addition of 1 mM ONOO -.The presence of melanin in the reaction medium significantly decreased the amount of 3-nitrotyrosine formed.As shown in Fig. 1, this inhibitory effect depended on the type and concentration of melanin polymer.At low concentrations (50 and 100 mg/ ml), all the melanins tested had no effect on peroxynitrite-mediated nitration of tyrosine but a further increase in melanin concentrations caused a gradual decrease in the yield of 3-nitrotyrosine.DA-melanin was the most efficient inhibitor of 3-nitrotyrosine formation, while CysDA-melanin and DA/CysDA (1 : 1)melanin were the least effective and their inhibitory effects were virtually the same at any concentration employed.Addition of 20 mM bicarbonate, prior to peroxynitrite, enhanced the level of 3-nitrotyrosine in controls about 1.5-fold (132 ± 7 mM of 3-nitrotyrosine; n = 12).In the presence of bicarbonate, the melanins inhibited 3-nitrotyrosine formation in a concentration dependent manner, although the extent of inhibition was lower than in the absence of bicarbonate (Fig. 1).
The influence of DA-melanin and CysDAmelanin on the oxidation of linoleic acid by peroxynitrite was investigated.Treatment of linoleic acid with peroxynitrite led to the formation of isomeric hydroperoxides with cis, trans 13-HPODE and cis, trans 9-HPODE being the major products.The tested melanins inhibited formation of HPODEs, and the inhibition extent, similar for both melanins, depended on their concentration (Fig. 2A).When amounts of 13-HODE were analysed, only a little increase was observed for CysDA-melanin (maximally 1 ± 0.1 mM HODE for 200 mg/ml CysDA-melanin), while the effect of DA-melanin did not look significant.In the presence of bicarbonate, in control samples, the observed levels of linoleic acid hydroperoxides were about two times lower than in the absence of bicarbonate (Fig. 2).The addition of DA-melanin and CysDA-melanin caused a further decrease of HPODE concentration while the amounts of HODE remained almost unchanged.In experiments carried out with added bicarbonate, 4-hydroxy-2-nonenal, which was absent in the samples without bicarbonate, appeared among the analysed oxidation products.DA-melanin did not affect the HNE level at all, whereas CysDA-melanin showed an inhibiting effect (Fig. 3).

DISCUSSION
Recently, we have demonstrated the ability of DA-melanin to protect against nitrating action of peroxynitrite (Stêpieñ et al., 2000).In this study, the effect of model neuromelanins derived from CysDA and from DA/CysDA mixtures on peroxynitrite-mediated tyrosine nitration was investigated and compared to the effect of DA-melanin.It was found that all the melanins tested significantly inhibited the formation of 3-nitrotyrosine but DA-melanin was a more efficient inhibitor than CysDAmelanin and DA/CysDA-melanins.This indicates that incorporation of the phaeomelanin component into the eumelanin polymer attenuates its protective effect on tyrosine nitration induced by peroxynitrite.In the presence of 20 mM bicarbonate (which is in equilibrium with approximately 1 mM CO 2 ) stimulation of tyrosine nitration in the control was observed; this is in agreement with the data of other authors (Lymar et al., 1996;Lehnig, 1999).The observed stimulation depends only on the formation of the highly reactive peroxynitrite-CO 2 adduct and does not depend on the pH change caused by bicarbonate addition.As shown by Lymar et al. (1996), the CO 2 -catalysed 3-nitrotyrosine formation is not influenced by pH in the range of 5-8.Addition of the melanins diminished the levels of 3-nitrotyrosine formed, indicating their ability to protect against the nitrating action of peroxynitrite-CO 2 adduct.
Vol. 47 Peroxynitrite and synthetic neuromelanins 935  Study of the effect of the melanins on peroxynitrite-induced oxidation of linoleic acid demonstrated that both DA-melanin and CysDA-melanin significantly decreased the yield of linoleic acid hydroperoxides formed.As melanins are capable of reducing linoleic acid hydroperoxides to its hydroxyl derivatives (Stêpieñ et al., 1998;Wilczok et al., 1999), the formation of 13-HODE was analysed simultaneously with HPODEs after treatment of linoleic acid with peroxynitrite in the presence of the melanins.A little increase in the amount of 13-HODE formed was observed only for CysDA-melanin at the highest concentration applied.These experiments showed that DA-melanin and CysDA-melanin indeed inhibited linoleic acid oxidation induced by peroxynitrite.When the peroxynitrite-mediated oxidation of linoleic acid was carried out in the presence of bicarbonate, the levels of HPODEs were attenuated and HNE was detected among the oxidation products.This aldehyde was not found after the reaction of linoleic acid with peroxynitrite in the absence of bicarbonate.HNE is the secondary oxidation product derived from omega-6-polyunsaturated fatty acids such as 20:4 and 18:2 (Comporti, 1998).This suggests that the peroxynitrite-CO 2 adduct is able to decompose linoleic acid hydroperoxides.CysDA-melanin inhibited the formation of HNE, while DA-melanin did not affect the aldehyde level.The results indicate that CysDA-melanin is a more effective inhibitor of peroxynitrite/CO 2 -mediated linoleic acid oxidation than DA-melanin.
O' Donnell et al. (1999a;1999b) have shown that treatment of linoleic acid with peroxynitrite leads to the formation of nitrated linoleate species (LNO 2 /LONO), in addition to HPODE and HODE.In our study, we did not detect any linoleic acid nitration products.In our experiment, peroxynitrite was introduced to linoleic acid by bolus addition and products of the reaction were analysed without extraction, which requires acidification of samples and may cause secondary reactions.In the studies described by O'Donnell et al. (1999a; 1999b), the diluted solution of peroxynitrite in 0.1 M NaOH was infused at a constant rate over 15 min into the linoleate emulsion and an equal volume of 0.1 M HCl was simultaneously infused to keep a constant pH value.These authors noted that "relative yields of oxidized vs nitrated products are likely to vary depending on the administration method chosen".
The peroxynitrite anion is relatively unreactive, however, protonation or addition of CO 2 generates a reactive species that can either interact with target compounds or undergo isomerization to NO  one-electron oxidation of tyrosine, leading to tyrosyl radical (Zhang et al., 1997;Lehnig 1999).
Melanins are known to be able to scavenge the hydroxyl radicals (Sarna et al., 1986).Interactions of synthetic eu-and phaeo-melanins derived from 3,4-dihydroxyphenylalanine with strong oxidising radicals such as NO 2 × were described (Ró¿anowska et al., 1999).The observed inhibition of 3-nitrotyrosine formation by all the melanins tested, both in the absence and presence of added bi-carbonate, suggests that the model neuromelanins are capable of scavenging NO 2 × radicals.
The results of the presented study suggest that neuromelanin can act as natural scavenger of peroxynitrite.As peroxynitrite is proposed to be a mediator of neurotoxic processes associated with Parkinson's disease, a protective effect of neuromelanin against peroxynitrite may be of physiological importance.

Figure 1 .
Figure 1.Effect of melanins on peroxynitritemediated 3-nitrotyrosine formation.Peroxynitrite (final concentration 1 mM) was added to solutions of tyrosine (1 mM) in phosphate buffer (100 mM, pH 7.4) containing indicated concentrations of melanins in the absence (A) or in the presence (B) of 20 mM sodium bicarbonate.

Figure 2 .
Figure 2. Effect of melanins on peroxynitritemediated linoleic acid hydroperoxides formation.Peroxynitrite (final concentration 4 mM) was added to solutions of linoleic acid (1 mM) in phosphate buffer (100 mM, pH 7.4) containing indicated concentrations of melanins in the absence (A) or in the presence (B) of 20 mM sodium bicarbonate.

Figure 3 .
Figure 3. Decrease of 4-hydroxynonenal induced by melanins during peroxynitrite/CO 2 -mediated linoleic acid oxidation.Peroxynitrite (final concentration 4 mM) was added to solutions of linoleic acid (1 mM) in phosphate buffer (100 mM, pH 7.4) containing indicated concentration of melanins in the presence of 20 mM sodium bicarbonate.