Peroxynitrite: A Possible

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Succinylacetone Oxidation by Oxygen/Peroxynitrite: A Possible Source of Reactive Intermediates in Hereditary Tyrosinemia Type I Leandro O. Royer,† Fernanda S. Knudsen,† Marcone A. de Oliveira,‡ Marina F. M. Tavares,‡ and Etelvino J. H. Bechara*,† Departamento de Bioquı´mica and Departamento de Quı´mica Fundamental, Instituto de Quı´mica, Universidade de Sa˜ o Paulo, CP 26077, 05513-970, Sa˜ o Paulo, SP, Brazil Received December 4, 2003

Hereditary tyrosinemia type I (HT1) is an inborn metabolic error characterized by hepatorenal dysfunction. Affected patients excrete large quantities of succinylacetone (SA), a tyrosine catabolite believed to be involved in the pathogenesis of HT1. A growing body of evidence relates the oxidative stress observed in metabolic disorders to free radicals generated from accumulated metabolites. In this context, oxidation of SA by peroxynitrite or cytochrome c yielding reactive intermediates and products was investigated here. Both peroxynitrite and cytochrome c were able to initiate oxygen consumption by SA, which was followed by polarimetric and chemiluminescence measurements. The light emission arises from triplet carbonyls formed by the thermolysis of dioxetane intermediates, as indicated by energy transfer experiments. EPR spintrapping studies with 2-methyl-2-nitrosopropane revealed the intermediacy of two different carbon-centered radicals, one of them originating from cleavage of the triplet carbonyl product. The pH profiles obtained by oxygen consumption, chemiluminescence, and stopped-flow spectrophotometry point to the peroxynitrite anion as the initiator of SA aerobic oxidation. Overstoichiometric formation of organic acids based on added peroxynitrite confirms the occurrence of an oxygen-dependent chain reaction, here proposed to be initiated by one electron abstraction from the enolic form of SA. The results obtained may help shed light on the role of both SA and oxidative stress in the pathogenesis of HT1.

Introduction HT11 is an inborn error of the metabolism caused by an inherited deficiency of FAH (EC 3.7.1.2) activity, the enzyme involved in the last step of the catabolic pathway of tyrosine (1). It is characterized by tubular renal dysfunction (2), progressive liver disease, and a high incidence of hepatocellular carcinoma (3). Patients with FAH deficiency excrete large amounts of SA, a specific biochemical marker of this disease (1). SA has been shown to inhibit in vitro the overall DNAligase activity present in normal cell extracts (4) and to react nonenzymatically with proteins and free amino acids (5). SA is also able to generate glucosuria, proteinuria, and amino aciduria in adult rats (6). Moreover, it has recently been suggested that the renal dysfunction is, at least partly, related with SA (7). Also noteworthy is the fact that SA is a strong competitive inhibitor of ALA dehydratase, leading to reported accumulation of ALA in tissues and, consequently, triggering hepatic and neurological manifestations similar to those characteristic of acute intermittent porphyria (1). ALA overload in porphyric disorders may constitute a source of reactive * To whom correspondence should be addressed. Tel: 55-11-30913869. E-mail: ebechara@ iq.usp.br. † Departamento de Bioquı´mica. ‡ Departamento de Quı´mica Fundamental. 1 Abbreviations: ALA, δ-aminolevulinic acid; DOVA, 4,5-dioxovaleric acid; FAH, fumarylacetoacetate hydrolase; HT1, hereditary tyrosinemia type 1; HRP, horseradish peroxidase; MNP, 2-methyl-2-nitrosopropane; SA, succinylacetone.

oxygen species capable of damaging biomolecules, membranes, and other cell structures (8-10). Like any other β-diketone, SA is expected to undergo enolization in an aqueous medium, either directly or by phosphate catalysis. Previous oxidation studies with 3-methylacetoacetone, in the presence of peroxynitrite or HRP/H2O2, suggest that the enol form of these compounds undergoes one electron abstraction, followed by oxygen consumption and production of triplet excited carbonyls in high yields (11). Triplet species share chemical similarities with free radicals, being able to induce DNA strand breaks (12), promote lipid peroxidation (13), and damage isolated rat liver mitochondria (14). Different enzymes can catalyze the aerobic oxidation of enolizable carbonyls, including HRP, myeloperoxidase, myoglobin, hemoglobin, and cytochrome c (15-17). Peroxynitrite, a strong and versatile oxidant, can also promote the oxidation of these compounds (18). Formed in vivo by a diffusion-controlled reaction of nitric oxide with the superoxide anion radical [k ) (4.3-10) × 109 M-1 s-1] (19), peroxynitrite anion (ONOO-) and its conjugate acid, peroxynitrous acid (pKa ) 6.6 at 37 °C) (20), are oxidizing species capable of reacting with a wide variety of biomolecules, including DNA, lipids, thiols, and amino acids. Oxidative stress has been implicated in the pathogenesis of several inborn metabolic errors (21, 22), as is the case of HT1 (23), due to excessive free radical production (24) related with the accumulation of certain metabolites, including ALA (25). In this context, the present study

10.1021/tx0342520 CCC: $27.50 © 2004 American Chemical Society Published on Web 04/02/2004

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was undertaken to investigate possible production of free radicals and excited species in the oxidation of SA by two biological oxidants, cytochrome c and peroxynitrite. Although SA was measured in the plasma of HT1 patients in micromolar concentrations (6-43 µM) (26), millimolar concentrations of SA were used throughout this work to simulate an acute effect of this metabolite, as cell compartmentalization and tissue concentration effects might increase its pathological concentration by several orders of magnitude.

Materials and Methods Chemicals. Chelex-100, cytochrome c, hemoglobin, sodium nitrite, succinic acid, and SA were obtained from Sigma Chemical Co. (St. Louis, MO). Sodium acetate, sodium bicarbonate, and hydrogen peroxide were supplied by Merck (Darmstadt, Germany). MNP dimer was obtained from Aldrich (Milwaukee, WI). [Ru(bpy)3]Cl2‚6H2O was purchased from G. Frederick Smith Chemical Co. (Columbus, OH). Peroxynitrite was synthesized from sodium nitrite (0.6 M) and hydrogen peroxide (0.7 M) in a quenched-flow reactor and quantified spectrophotometrically at 302 nm ( ) 1670 M-1 cm-1) (27). Bovine oxyhemoglobin was prepared and assayed as described elsewhere (28). All of the solutions were prepared in distilled water treated in a Millipore Milli-Q system, and the buffers were pretreated with Chelex-100. Oxygen Consumption. Oxygen uptake studies were followed in an oxygen monitor (Yellow Spring Instruments model 53 at 37 °C). The saturating oxygen concentration at this temperature was taken to be 220 µM (29). Low-Level Chemiluminescence Measurements. Lowlevel chemiluminescence was measured either with a Hamamatsu TVC 767 photoncounter equipped with a red sensitive Thorn EMI (9658AM) photomultiplier cooled to -12 °C by a Thorn EMI (model Fackt MKIII) thermoelectric cooler or with a luminometer (Biorbit model 1251). Stopped-Flow Kinetics. The rate of peroxynitrite decomposition was monitored with a stopped-flow spectrophotometer (Applied Photophysics model SX-18V) at 340 nm to avoid interference due to light absorption by SA. Apparent rate constants for the reaction of SA with peroxynitrite, kobs (s-1), were determined by nonlinear least-squares fitting of stoppedflow data to a single-exponential function with a nonzero offset. The reported values are the average of at least 10 separate determinations. The temperature was maintained constant at 37 ( 0.2 °C, and the pH of the reaction mixtures was determined at the outlet. EPR Spin-Trapping Experiments. EPR spectra were recorded at room temperature using a Bruker EMX spectrometer. In the experiments with peroxynitrite, all of the spectra were recorded 1 min after the addition of the oxidant, while the pH was measured at the end of the experiment to detect changes caused by the addition of alkaline stock solutions of peroxynitrite. Computer simulation analyses of some spectra were performed using the software described by Duling (30). Capillary Electrophoresis. Organic ions were analyzed with an instrument (Beckman Instruments model P/ACE system 5510) equipped with a UV detector set at 254 nm. The column consisted of a fused-silica capillary (75 cm × 75 µm i.d.); the electrolytic solution was 10 mM 3,5-dinitrobenzoic acid and 0.2 mM cetyltrimethylammonium bromide at pH 4.43, and separation was performed at -20 kV. Samples were injected by the hydrodynamic method at 3.5 kPa for 1 s. Sodium acetate and succinate standards were used in the quantitative analyses.

Results Oxygen Consumption and Chemiluminescence. In the presence of peroxynitrite, SA consumes the dissolved oxygen within a few seconds in normally aerated

Figure 1. Total oxygen consumption (b) and chemiluminescence intensity ([) by 0.5 mM SA at different peroxynitrite concentrations in 500 mM phosphate buffer (pH 7.2) at 37 °C.

Figure 2. Oxygen uptake by 1.5 mM SA in the presence of 0.5 mM hydrogen peroxide (A) and 30 µM cytochrome c (B) or 20 µM hemoglobin (C) in 100 mM phosphate buffer (pH 7.2) at 37 °C.

Chelex-treated phosphate buffer. The extent of oxygen consumption by SA depends on both the peroxynitrite concentration (Figure 1) and the SA concentration (not shown), with depletion of the dissolved oxygen being attained with 500 µM SA. Oxygen consumption was also observed using cytochrome c/H2O2 or hemoglobin/H2O2 as oxidants (Figure 2). Direct chemiluminescence can be detected from SA oxidation by peroxynitrite and is dependent on peroxynitrite (Figure 1) and SA concentrations (not shown), as described for oxygen uptake. The excited species can be characterized using an adequate luminescent energy acceptor (31). The double-reciprocal plot of the sensitized chemiluminescence intensity vs the sensitizer concentration gives a straight line with a slope equal to kETτ, where τ is the excited species’ lifetime and kET is the rate constant of triplet-singlet energy transfer (31). Using [Ru(bpy)3]2+ as sensitizer (32), the slope obtained was 1.72 × 104 (Figure 3), from which the lifetime of the excited species was estimated to be 1.8 µs. This value is consistent with triplet carbonyl lifetimes reported in the literature: 2 µs for triplet acetone and 20 µs for biacetyl, both in normally aerated media (33, 34). The H2PO4- and other oxygenated anions are known to catalyze the enolization of carbonyl compounds in

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Figure 3. Double reciprocal plot of the effect of [Ru(bpy)3]2+ concentration upon the light intensity from the SA (5 mM)/ peroxynitrite (0.5 mM) system in 100 mM phosphate buffer (pH 7.2) at 37 °C.

Figure 4. Effect of phosphate buffer concentration on chemiluminescence ([) and oxygen consumption (b). SA (0.5 and 0.2 mM, respectively) was incubated with peroxynitrite (0.5 mM) in phosphate buffer (pH 7.2) at 37 °C.

aqueous media (35). Because the enol form is the actual substrate of the oxidation of carbonyls by one electron oxidants (36), we studied the effects of phosphate buffer concentration upon oxygen consumption and light emission. At pH 7.2, a 2-fold increase in total oxygen uptake was observed upon raising the phosphate concentration from 100 to 500 mM (Figure 4). A similar result was obtained for light emission, which was 50% more intense in 500 mM phosphate buffer. pH Profile. Aiming to identify the nature of the oxidant-peroxynitrite anion or peroxynitrous acid, we studied the pH dependence of SA oxidation by peroxynitrite. The pH profile of this reaction was obtained by three methods: total oxygen consumption, chemiluminescence, and stopped-flow spectrophotometry. The pH profiles of the SA oxygen consumption and light emission (Figure 5) roughly describe the peroxynitrous acid titration curve, pointing to peroxynitrite anion as the initiator of SA aerobic oxidation. A similar result was observed in stopped-flow spectrophotometry studies. The values of kobs shown in Figure 6 represent the pseudo-first-order rates for the peroxynitrite-initiated decomposition of SA. The values for the spontaneous decomposition of peroxynitrite varied with the pH and were subtracted from the respective rates

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Figure 5. pH dependence of chemiluminescence ([) and oxygen consumption (b) for the reaction of SA (0.5 and 0.2 mM, respectively) with peroxynitrite (0.5 mM) in 500 mM phosphate buffer at 37 °C.

Figure 6. pH profile of the observed pseudo-first-order rate constant (kobs) for the reaction of SA (10 mM) with peroxynitrite (0.5 mM) in 500 mM phosphate buffer at 25 °C.

obtained with SA. The higher reaction rates observed at alkaline pH values attest to peroxynitrite anion as the reaction initiator. EPR Spin-Trapping Studies. EPR spin-trapping studies with MNP were conducted to detect and possibly identify carbon-centered radical intermediates. A nine line spectrum [aN ) 1.63 mT; aH ) 0.99 mT (2H)] was observed upon exposure of SA to peroxynitrite at pH 7.2 in the presence of MNP (Figure 7). This spectrum can be assigned to a •CH2R radical, as predicted by the reaction mechanism shown in Scheme 1, where the nucleophilic attack of ONOO- on the SA γ-carbonyl group initiates the oxygen-consuming reaction (18 and references therein). Also present is the oxidation product of MNP, di-tert-butylnitroxide (aN ) 1.71 mT) (37, 38). Incubation of SA (10 mM) with cytochrome c (30 µM) in the presence of hydrogen peroxide (0.5 mM) and MNP (90 mM) in 0.1 M phosphate buffer (pH 7.2) led to the detection of the EPR spectrum shown in Figure 8. This spectrum is a composite of two radical adducts, both triplets. One triplet (aN ) 1.71 mT) can be assigned to the MNP oxidation product. The other one (aN ) 0.83 mT) can be attributed to an MNP adduct with cetyl radical. This radical may arise from the carbonyl-carbonyl cleavage of the electronically excited triplet products, either methylglyoxal or 4,5-dioxovalerate (Scheme 2), formed by a reaction initiated by electron abstraction


Figure 7. EPR spectra of MNP radical adducts obtained after incubation for 1 min in 100 mM phosphate buffer (pH 7.2) at room temperature of 60 mM MNP and 1 mM peroxynitrite in the presence (A) or absence (B) of 10 mM SA. Spectrum C is the computer simulation of panel A. Instrumental conditions were as follows: microwave power, 20 mW; modulation amplitude, 0.1 mT; time constant, 81.92 ms; scan rate, 0.1192 mT/s; and gain, 6.32 × 105.

from SA, as described for the oxidation of acetoacetone (38) and 3-methylacetoacetone (39) by HRP/H2O2 and peroxynitrite, respectively. Product Analysis. Both reaction pathways proposed in Schemes 1 and 2 predict the formation of organic acids as products. Therefore, their concentrations were determined in the spent reaction mixtures of air-equilibrated aqueous SA (10 mM) treated with peroxynitrite (final pH 7-8) in an attempt to determine their relative importance. In one set of experiments (set 1), the system was maintained closed after the addition of a bolus of 0.5 mM peroxynitrite. In another set (set 2), 1 mM peroxynitrite was added slowly and continuously and the system was kept open and vigorously stirred, allowing it to become reoxygenated. Using capillary electrophoresis, succinate was found at a concentration of 0.27 ( 0.04 and 0.56 ( 0.02 mM for sets 1 and 2, respectively. The concentrations obtained for acetate were 0.40 ( 0.02 and 11.00 ( 0.21 mM for sets 1 and 2, respectively. These results indicate


Figure 8. EPR spectra of MNP radical adducts obtained after incubation for 4 min in 100 mM phosphate buffer (pH 7.2) at room temperature of 90 mM MNP, 30 µM cytochrome c, and 0.5 mM hydrogen peroxide in the presence (A) or absence (B) of 10 mM SA. Instrumental conditions were as follows: microwave power, 20 mW; modulation amplitude, 0.1 mT; time constant, 327.7 ms; scan rate, 0.0298 mT/s; and gain, 1.0 × 106.

the concomitant operation of two reaction mechanisms, with acetate being formed by the oxygen-dependent chain reaction under continuous aeration. The formation of acetate in a 20-fold higher concentration than succinate points to a predominance of the oxygen-propagated reaction via dioxetane (Scheme 2i). In the presence of peroxynitrite, SA undergoes spectral changes (Figure 9), with the appearance of a weak absorption band in the region around 350 nm, along with a decrease in the SA absorption band at 280 nm. The increased absorbance in the 320-360 nm range observed in the reaction of peroxynitrite (2 mM) with ethyl acetoacetate (100 mM) was ascribed to the formation of nitrated products (40), although the highest yields of these compounds were observed only in the presence of 20 mM NaHCO3. The actual nitration reagent, •NO2, seems to be derived from nitrosoperoxycarbonate adduct (ONOO-CO2-) (41, 42). This led us initially to interpret the modest absorbance increase at 350 nm as due to minor SA nitration by the ONOO-CO2- adduct formed from contaminant HCO3- present in both the buffer and the peroxynitrite stock solutions. However, upon prior

Scheme 1. SA Oxidation by Oxygen Initiated by the Addition of Nucleophilic Peroxynitrite

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Scheme 2. Final Products from SA Oxidation by O2 upon the Addition of Peroxynitrite

addition of 15 mM NaHCO3 to the reaction mixture, the absorption band in the 320-360 nm range did not change significantly, while there was little decrease at 280 nm, suggesting that SA nitration under our experimental conditions is minor or nonexistent.

Discussion Carbonyl compounds such as aliphatic aldehydes (43, 44) and ketones (45, 46) have previously been shown to react with peroxynitrite, catalyzing its decomposition to nitrate and producing free radicals and organic acids at low yields. The reaction is initiated by the nucleophilic addition of peroxynitrite anion to the carbonyl group, forming an adduct analogous to the nitrosoperoxycarbonate anion (Scheme 1). This carbonyl-ONOO adduct is unstable (t1/2 ) 0.2-3.3 µs) (46) and undergoes O-O homolysis followed by β-scission, producing a carboncentered radical and an organic acid. Recently, it was demonstrated that peroxynitrite addition may also promote one electron oxidation of the enol form of carbonyls (18), initiating their aerobic oxidation to triplet carbonyls (Scheme 2). The former reaction pathway (Scheme 1),

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however, seems to initiate the oxidation of SA by peroxynitrite studied here. Accordingly, in addition to the detection of a •CH2R radical by EPR spin-trapping studies (Figure 7), succinate anion was found to be one of the major products. The •CH2R radical and succinate are the species expected from the β-scission of the intermediate radical formed by the homolysis of the SA-ONOO adduct (Scheme 1). Nevertheless, one electron abstraction of enol SA followed by oxygen insertion proposed in Scheme 2 also seems to occur, as indicated by the observed oxygen depletion (Figure 1). Poorly or nonenolizable carbonyls, which are expected to undergo only peroxynitrite nucleophilic addition, show little oxygen consumption when treated with peroxynitrite, suggesting that oxygen consumption in this case is probably not associated with the route shown in Scheme 2 but with the Russell reaction of the alkylperoxyl intermediates shown in Scheme 1. A product analysis of the SA/peroxynitrite/O2 system attested to the occurrence of both reaction pathways. Accordingly, the yield of succinate in reaction mixtures in which peroxynitrite (0.5 mM) was allowed to react with excess SA (10 mM) was found to be 0.56 mol/mol of peroxynitrite employed. The same yield was obtained when the peroxynitrite concentration was doubled and the experimental conditions favored reoxygenation, indicating that the formation of succinate is not limited by oxygen. A different pattern was observed for acetate yields under the same experimental conditions: 0.40 and 11.00 mol/mol peroxynitrite. This finding indicates that acetate is formed in an oxygen-dependent chain reaction, as predicted by the route shown in Scheme 2. The pH profile of the SA/peroxynitrite/O2 system obtained by plotting total oxygen consumption, chemiluminescence, and stopped-flow spectrophotometry points to peroxynitrite anion as the initiator of SA aerobic oxidation. However, there is no report in the literature on peroxynitrite anion acting as a one electron oxidant, which leads us to envisage a third reaction pathway (Scheme 3) that merges the two abovementioned routes. Scheme 3 indicates that the reaction is initiated by the addition of peroxynitrite nucleophilic to SA, forming the SA-ONOO adduct, which suffers homolysis, producing an oxyl radical (Scheme 3I) and the nitrogen dioxide radical (43-46). These radicals may (i) recombine, producing nitrate and the original compound, and (ii) act as one electron oxidants, initiating the chemiluminescent aerobic oxidation of SA. Accordingly, Jovanovic et al. have

Figure 9. Absorption spectrum of 0.5 mM SA before (A) and 5 min after the addition of 0.5 mM peroxynitrite in the presence (B) or absence (C) of 15 mM sodium bicarbonate in 500 mM phosphate buffer (pH 7.2) at room temperature.



Scheme 3. Suggested Mechanism for the Oxidation of SA by O2/Peroxynitrite

shown that both the methyl radical and the tert-butoxyl radical can promote the oxidation of the β-diketone curcumin, abstracting an H-atom from the central methylene group, whose reaction rate constants are 3.5 ( 0.3 × 109 and 7.5 ( 0.8 × 109 M-1 s-1, respectively (47). The aerobic oxidation of SA may also be promoted by cytochrome c/H2O2 or hemoglobin/H2O2 (Figures 2 and 8), indicating that different biological oxidants may trigger the one electron oxidation of SA to reactive intermediates and products. The acetyl radical detected in the cytochrome c-containing system probably arises from the carbonyl-carbonyl cleavage of the excited triplet carbonyl products shown in Scheme 2. In this case, high oxidative states of ferricytochrome c or ferrihemoglobin may respond for the one electron abstraction of hydrogen from enol SA, initiating its chain oxidation by dioxygen (48). The secondary radical predicted to be formed during the aerobic oxidation of SA (Scheme 2) was not detected here, possibly due to the unstable nature of the MNP adducts of these radicals (38). In conclusion, we have shown that peroxynitrite and hemeproteins/H2O2 can promote the aerobic oxidation of SA, producing free radicals as intermediates and triplet carbonyls as products. The latter species possess a reactivity pattern similar to that observed for alkoxyl radicals (15). Moreover, because of their intrinsically long lifetime, triplet species may participate in bimolecular processes, such as chemical reactions (isomerization, cycloaddition, and hydrogen abstraction) and energy transfer processes (e.g., singlet dioxygen production by collisional energy transfer) (36). Furthermore, the route (ii) in Scheme 2, if operative, would constitute an additional source of DOVA, an R-oxoaldehyde produced in vivo by (i) the metal-catalyzed oxidation of ALA (48), (ii) by an amino oxidase reaction 49, and (iii) by transamination (50). DOVA is an efficient alkylating agent of the guanine moieties in both nucleoside and isolated DNA (51, 52), being able to induce strand breaks in pBR322 DNA (53). These findings may not only help explain the observed oxidative stress in HT1 (23) but also shed light on the role of SA and DOVA in the pathogenesis of HT1.

Acknowledgment. This work was supported by grants from the Fundaç aõ de Amparo a` Pesquisa do Estado de Saõ Paulo (FAPESP), the Programa de Apoio a Nućleos de Exceleˆncia (PRONEX), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), and the von Humboldt Foundation. We are grateful to Dr. Omar El Seoud and Dr. Ana Campa for providing access to the stopped-flow spectrophotometer and luminometer, respectively.

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