Characterization of sulfur-centered radical intermediates formed during the oxidation of thiols and sulfite by peroxynitrite. ESR-spin trapping and oxygen uptake studies.

Using a novel phosphorylated spin trap, 5-diethoxy-phosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), an analog of the commonly used trap 5,5'-dimethyl-1-pyrroline N-oxide (DMPO), we have investigated the reactions of sulfur-centered radicals produced from the oxidation of thiols and sulfite by peroxynitrite. The predominant species trapped in all cases are the corresponding sulfur-centered radicals, i.e. glutathionyl radical (GS) from glutathione (GSH), N-acetyl-DL-penicillamine thiyl radical (S-NAP) from N-acetyl-DL-penicillamine (NAP) and sulfate anion radical (SO3-) from sulfite. These radicals consume molecular oxygen forming either peroxyl or superoxide anion radicals. GS, S-NAP, and (SO3-)-derived radicals react with ammonium formate to form the carbon dioxide anion radical (CO2-). Further support of spin adduct assignments and radical reactions are obtained from photolysis of S-nitrosoglutathione and S-nitroso-N-acetyl-DL-penicillamine. We conclude that the direct reaction of peroxynitrite with thiols and sulfate forms thiyl and sulfate anion radicals, respectively, by a hydroxyl radical-independent mechanism. Pathological implications of thiyl radical formation and subsequent oxyradical-mediated chain reactions are discussed. Oxygen activation by thiyl radicals formed during peroxynitrite-mediated oxidation of glutathione may limit the effectiveness of GSH against peroxynitrite-mediated toxicity in cellular systems.

The reaction between nitric oxide ( ⅐ NO) and superoxide anion (O 2 . ) generates peroxynitrite 1  This reaction has been implicated in many pathological conditions including reperfusion injury (3), atherosclerosis (4), and neurodegenerative diseases (5). There are many potential targets for peroxynitrite in biological systems including thiols, lipids, and DNA (6 -8). Although free radical intermediates have been proposed in peroxynitrite-mediated oxidation reactions, they have been directly detected in only a few cases (9 -13). Because of its role in pathophysiology, further understanding of the free radical reactions of peroxynitrite with potential biological targets is essential. The chemistry of decomposition of peroxynitrite is complex (14). ONOO Ϫ is stable in alkaline solutions. However, upon protonation (pK a ϭ 6.9), ONOOH decays rapidly (t1 ⁄2 Ϸ 1 s at pH 7.4) (15). The decay of ONOOH has been shown to have some free radical character as evidenced by malondialdehyde formation from deoxyribose by a process inhibitable by hydroxyl radical scavengers and aromatic hydroxylation of sodium benzoate (15). This activity has been shown to have many similarities to the oxidant produced in the Fenton reaction and is often referred to as a "hydroxyl radical"-like species. Koppenol et al. (14) have shown that homolytic decomposition of ONOOH is unlikely and that the oxidant is probably an active conformer produced during internal rearrangement to nitric acid. The oxidative effects of peroxynitrite are not confined to this species, however, as ONOO Ϫ and ONOOH can act as both oneelectron and two-electron oxidants (6,10,16). For example, ONOO Ϫ will oxidize glutathione (GSH) to glutathione disulfide (GSSG) (6).
In this study we have attempted to characterize the reactions of sulfur-centered radicals formed from the one-electron oxidation of thiols by peroxynitrite using the spin-trapping technique. Previous electron spin resonance (ESR) studies of the decomposition of peroxynitrite at physiological pH have used 5,5Ј-dimethyl-1-pyrroline N-oxide (DMPO) 2 as the spin trap and the results are controversial. Shi et al. (13) reported the formation of 5,5Ј-dimethyl-1-pyrrolidin-2-one-1-oxy, a further oxidation product of DMPO, at high concentration of peroxynitrite and concluded that free hydroxyl radical is not released. However, Augusto et al. (9) reported the formation of DMPO/ ⅐ OH from DMPO using low peroxynitrite concentrations and a strong and persistent DMPO/ ⅐ OH signal at higher concentrations only in the presence of GSH. Recently, Pou et al. (11) reported that the yield of formation of HO ⅐ from peroxynitrite decomposition was about 1 to 4% based on the methane sulfinic acid assay. A more recent study from Lemercier et al. (12) claimed that DMPO/ ⅐ OH adduct detected during the reaction between DMPO and ONOO Ϫ is formed from a molecule-induced homolysis mechanism and not from trapping of HO ⅐ .
The use of DMPO does not allow easy discrimination be-* This research was supported by National Institutes of Health Grants RR01008, HL 45058, and HL 47250 from the National Heart, Lung and Blood Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
tween DMPO/ ⅐ OH and DMPO/ ⅐ SG, the radical adduct formed from trapping of the glutathionyl radical (GS ⅐ ). To obviate this problem, we have used a novel spin trap, 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) (17)(18)(19)45), to examine the oxidative reactions of peroxynitrite. The phosphorus nucleus (I ϭ 1/2) in DEPMPO spin adducts has a large hyperfine splitting (a P between 45 and 53 G) that is sensitive to the nature of the radical (R ⅐ ) trapped and to the nitroxide spin adduct conformation (Scheme 1) (18). For the DEPMPO/ ⅐ R spectrum, the extra hyperfine coupling to phosphorus (I ϭ 1/2) results, in most cases, in a spectrum that resembles a juxtaposition of two DMPO/ ⅐ R spin adduct spectra. In this paper, using ESR, gas chromatography, and oxygen uptake studies, we have characterized the reactions of sulfurcentered radicals formed from the reaction of peroxynitrite with sodium bisulfite and several thiols including glutathione. Biological implications of thiyl radical production from peroxynitrite in cellular systems are discussed.
Gas Chromatography-CO 2 analysis was performed using a Varian 3700 Gas Chromatograph equipped with a 6 feet ϫ 1/8 inch Porapak Q column and thermal conductivity detector operating at 30°C with a flow rate of 5 ml/min. Samples were prepared in a sealed vial and peroxynitrite was injected through the seal immediately prior to measurement. Headspace gas (100 l) was injected into the gas chromatograph with an airtight Hamilton syringe. The CO 2 retention time was 2.1-2.3 min.
Oxygen Uptake-Oxygen uptake experiments were performed using a YSI Oxygen electrode (Yellow Springs Instruments, Columbus, OH) at 37°C. The electrode was calibrated using air-saturated water (240 M oxygen) and sodium dithionite-treated water (0 M oxygen). All experiments were performed using phosphate buffer (200 mM, pH 7.4) with a final volume of 5 ml. Data are given as mean Ϯ S.D. (n ϭ 3). Fig. 1, a and b, shows the ESR spectra of authentic DEPMPO/ ⅐ OH (a N ϭ 14.0 G, a H ϭ 13.2 G, a H ␥ ϭ 0.27 G ( 3 H), and a P ϭ 47.3 G) and DEPMPO/ ⅐ SG (a N ϭ 14.1 G, a H ϭ 14.9 G, and a P ϭ 45.8 G) adducts generated by a Fenton system and by photolysis of GSNO, respectively. There is a clear difference between these two spin adduct spectra that is mainly due to the variations in phosphorus hyperfine splittings. A simulated spectrum of a mixture of DEPMPO/ ⅐ OH (20%) and DEPMPO/ ⅐ SG (80%) is shown in Fig. 1c. The majority of the spectral lines overlap and cannot be used as diagnostic indicators of either radical. However, the central portion of the spectrum clearly shows the contributions of DEPMPO/ ⅐ OH and DEPMPO/ ⅐ SG to the signal.

Spin-trapping of Radicals Formed from the Reaction between
Peroxynitrite, Glutathione, and DEPMPO-Incubation of DEPMPO with peroxynitrite in phosphate buffer (200 mM, pH 7.4) did not result in any ESR detectable spin adduct (Fig. 2a). This is in contrast to previous studies with DMPO wherein the investigators reported formation of DMPO/ ⅐ OH during the reaction between DMPO and peroxynitrite (9,11,12). The addition of HO ⅐ scavengers (ethanol, 1.8 M; Me 2 SO, 1.4 M; ammonium formate, 1 M; mannitol, 1 M, or t-butanol, 1.1 M) also gave no ESR detectable adduct. In the presence of GSH (1 mM), the spectrum shown in Fig. 2b was observed. This signal is complex and consists of several spin adducts. The predominant species is DEPMPO/ ⅐ SG which corresponds to 55 to 60% of the signal. This represents a concentration of 12 to 14 M DEPMPO/ ⅐ SG indicating that the efficiency of the one-electron oxidation of GSH to GS ⅐ by peroxynitrite is about 1-2%. The high performance liquid chromatography data indicate that DEPMPO does not affect the yield of GSSG during ONOO Ϫ -dependent oxidation suggesting that GS ⅐ is a minor product from the reaction between GSH and ONOO Ϫ . 3 The origin of the remaining signal in Fig. 2b is uncertain, but the whole spectrum can be simulated (Fig. 2b, dotted line) by assuming the presence of two radical adducts with hyperfine splitting constants characteristic of carbon-centered adducts (17) (a N ϭ 14.5 G, a H ϭ 21.5 G, and a P ϭ 45.9 G; a N ϭ 14.8 G, a H ϭ 20.7 G, and a P ϭ 47.8 G) and a species (from 10 to 19%) with parameters similar to DEPMPO/ ⅐ OH (a N ϭ 14.0 G, a H ϭ 13.4 G, a H ␥ ϭ 0.6 G (3H), and a P ϭ 47.5 G). Both DEPMPO/ ⅐ SG and DEPMPO/ ⅐ OH adducts decayed with time leaving only the signal assigned to the carbon-centered radical adducts (Fig. 2c). This spectrum can be simulated (Fig. 2c, dotted line) using the same ESR parameters for the carbon-centered radicals shown in Fig. 2b. We also observed a similar behavior after the decay of the DEPMPO/ ⅐ SG adduct obtained during photolysis of GSNO (data not shown). Augusto et al. (9) also reported the detection of an unidentified carbon-centered radical during oxidation of GSH by peroxynitrite in the presence of DMPO. The source of these carbon-centered radical is unknown but Grierson et al. (22) and Zhao et al. (23) have reported formation of a carbon-centered radical from an intramolecular rearrangement of the glutathionyl radical.
In order to examine whether the DEPMPO/ ⅐ OH spin adduct in Fig. 2b is formed from trapping of HO ⅐ , the effects of HO ⅐ scavengers were investigated. An alternate route by which DEPMPO/ ⅐ OH can be formed is via decomposition of DEPMPO/ ⅐ OOH to DEPMPO/ ⅐ OH. However, unlike DMPO/ ⅐ OOH, the DEPMPO/ ⅐ OOH adduct does not decay spontaneously to DEPMPO/ ⅐ OH (18, 24). It is conceivable that this conversion is facilitated by high concentrations of GSH. This reaction is similar to the GSH-dependent reduction of hydroperoxide to alcohol (Equation 2). In support of this, GSH was shown to facilitate the conversion of chemically or enzymatically synthesized DEPMPO/ ⅐ OOH to DEPMPO/ ⅐ OH (data not shown).
To further verify that DEPMPO/ ⅐ OH is actually formed from DEPMPO/ ⅐ OOH, we investigated the effect of superoxide dismutase mimic in peroxynitrite/GSH system. 4 The spectrum obtained in the absence of superoxide dismutase mimic was simulated assuming the presence of DEPMPO/ ⅐ SG (44%), DEPMPO/ ⅐ OH (19%), and two carbon-centered adducts (37%) (Fig. 4a). No ESR spectrum was obtained in the absence of peroxynitrite. The spectrum obtained in the presence of superoxide dismutase mimic (1 mM) shows no evidence for the presence of the DEPMPO/ ⅐ OH adduct (Fig. 4b) as determined by simulation using DEPMPO/ ⅐ SG (88%) and one carbon-centered adduct (12%) (Fig. 4b, dotted line). The spectral intensity of DEPMPO/ ⅐ SG was enhanced in the presence of superoxide dismutase mimic (Fig. 4b), perhaps indicating a superoxide-mediated destruction of the DEPMPO/ ⅐ SG adduct. This is consistent with a previous report where it was shown that addition of superoxide dismutase enhanced the signal intensity of DMPO/ ⅐ SG adduct formed during horseradish peroxidase/H 2 O 2 -catalyzed oxidation of GSH (25). Effect of Ammonium Formate (HCO 2 NH 4 ) on Spin Adduct Formation during the Reaction between Peroxynitrite, GSH, and DEPMPO-As mentioned above, the reaction between peroxynitrite (0.8 mM) and HCO 2 NH 4 (1 M) in the presence of DEPMPO (20, 50, and 100 mM) generated no ESR signal. However, in the presence of GSH (1 mM), DEPMPO/ ⅐ CO 2 Ϫ was observed ( Fig. 5, a-c). Lower concentrations of HCO 2 NH 4 (100 mM) gave an ESR spectrum that consisted of a mixture of DEPMPO/ ⅐ SG (marked E) and DEPMPO/ ⅐ CO 2 Ϫ (marked q) ad-ducts ( Fig. 5a). At higher concentrations of HCO 2 NH 4 (0.4 and 1 M), the DEPMPO/ ⅐ CO 2 Ϫ adduct dominated the spectrum (Fig.  5, b and c, respectively). The dotted line signal represents the computer simulation of the spectrum 5c which is a mixture of DEPMPO/ ⅐ CO 2 Ϫ (80%) and DEPMPO/ ⅐ SG (20%). The authentic DEPMPO/ ⅐ CO 2 Ϫ spin adduct (a N ϭ 14.5 G, a H ϭ 17.3 G, and a P ϭ 51.6 G) was generated by the Fenton reaction in the presence of DEPMPO (20 mM) and HCO 2 NH 4 (1 M) (Fig. 5d). The generation of GS ⅐ from photolysis of GSNO (1 mM) in the presence of DEPMPO (20 mM) and HCO 2 NH 4 (400 mM) led to a time-dependent formation of DEPMPO/ ⅐ CO 2 Ϫ (Fig. 6, a-e). These results indicate that GS ⅐ generated from reaction of peroxynitrite with GSH reacts with HCO 2 NH 4 to form the CO 2 . radical (Equations 3 and 4). formed from the reaction between peroxynitrite and HCO 2 NH 4 . However, in the presence of GSH, CO 2 formation was observed (Fig. 7). DEPMPO (30 mM) completely inhibited CO 2 formation in this reaction system ( fig. 7), suggesting that GS ⅐ is respon-  (Fig. 8a). The spectrum shown in Fig. 8a was simulated by assuming the presence of two diastereoisomeric DEPMPO/ ⅐ S-NAP adducts with slightly different hyperfine splitting constants (a N ϭ 14.3 G, a H ϭ 16.5 G, and a P ϭ 44.9 G (32%); a N ϭ 14.4 G, a H ϭ 15.2 G, and a P ϭ 46.2 G (38%)) and a carbon-centered radical adduct (a N ϭ 14.8 G, a H ϭ 20.8 G, and a P ϭ 48.0 G (30%)) (Fig. 8a, dotted line).
The incubation of NAP (10 mM) with DEPMPO (50 mM) and peroxynitrite (0.8 mM) in phosphate buffer led to the formation of a spectrum (Fig. 8b) which was essentially identical to the spectrum obtained by photolysis of SNAP (Fig. 8a). No evidence for DEPMPO/ ⅐ OH adduct was observed and the addition of HO ⅐ scavengers such as ethanol (1.8 M) or Me 2 SO (1.4 M) had no effect on the signal. DEPMPO/ ⅐ OH was also not observed when N-acetyl-DL-cysteine was used (data not shown). Addition of peroxynitrite to a mixture containing DEPMPO (50 mM), NAP (10 mM), and ammonium formate produced an ESR spectrum (Fig. 8c) consisting of both DEPMPO/ ⅐ S-NAP and DEPMPO/ ⅐ CO 2 Ϫ adducts. The spectrum was simulated by assuming the presence of two diastereoisomers of DEPMPO/ ⅐ S-NAP (33%) and DEPMPO/ ⅐ CO 2 Ϫ (67%) (Fig. 8c, dotted line). These results indicate that ⅐ S-NAP also reacts with ammonium formate to form CO 2 . . group which is lost upon conversion to ⅐ S-NAP. In ⅐ S-NAP the " ⅐ S" indicates the presence of a thiyl radical. Thiyl Radical-mediated Oxygen Uptake-Decomposition of peroxynitrite results in the evolution of O 2 (7). The concentration of oxygen formed was an approximately linear function of peroxynitrite concentration up to 1 mM (10% yield) and was unaffected by DEPMPO (20 mM). However, in the presence of GSH (1 mM), rapid consumption of O 2 was observed (Fig. 9). The addition of DEPMPO (20 mM) inhibited O 2 consumption by 62 Ϯ 2% (Fig. 9, inset), indicating the possible involvement of thiyl radicals.
The O 2 uptake during the reaction between peroxynitrite and NAP in phosphate buffer (200 mM, pH 7.4) at 37°C is shown in Fig. 10. The O 2 uptake increased as a function of NAP concentration (Fig. 10, inset). Under similar experimental conditions, the O 2 uptake with NAP was much higher than with GSH, suggesting differences in the reaction mechanism of the corresponding thiyl radicals.
Spin-trapping of Sulfite Radical Anion Formed during the Reaction between Sodium Bisulfite and Peroxynitrite-The reaction between peroxynitrite and NaHSO 3 in the presence of DEPMPO resulted in the formation of DEPMPO/ ⅐ SO 3 Ϫ adduct (a N ϭ 13.3 G, a H ϭ 14.9 G, and a P ϭ 48.9 G) (Fig. 11a). For comparison, the DEPMPO/ ⅐ SO 3 Ϫ adduct formed from HO ⅐ and NaHSO 3 is shown in Fig. 11c. Hydroxyl radical scavengers (ethanol, 1.8 M, and Me 2 SO 1.4 M) had only a slight effect (5 to 10%) on the ESR signal intensity shown in Fig. 11a indicating that SO 3 . probably arises from a direct reaction between HSO 3 Ϫ and peroxynitrite. The signal intensity of DEPMPO/ ⅐ SO 3 . adduct was diminished in oxygen saturated buffer. This supports trapping of free SO 3 . by DEPMPO to form the DEPMPO/ ⅐ SO 3 Ϫ adduct, as SO 3 . radical reacts rapidly with oxygen (27).
Sulfite Radical Anion-mediated Oxygen Uptake-Addition of peroxynitrite to various concentrations of NaHSO 3 (from 0.1 to 1 mM) caused a concentration-dependent increase in O 2 consumption (Fig. 12). During the reaction between 200 M NaHSO 3 and 300 M peroxynitrite, DEPMPO (20 mM) inhibited oxygen consumption by 69 Ϯ 6% (Fig. 12, inset). The inhibition in O 2 uptake observed in the presence of DEPMPO is probably due to effective trapping of SO 3 . by DEPMPO.

DEPMPO/ ⅐ CO 2
Ϫ adduct. This is likely to be due to the more effective trapping of SO 3 . under these conditions. It is likely that SO 4 . formed from SO 3 . and O 2 could also oxidize formate anion to the CO 2 . radical.

DISCUSSION
Peroxynitrite-mediated Oxidation of DEPMPO and DMPO-Our data show that incubating DEPMPO with peroxynitrite does not form the DEPMPO/ ⅐ OH adduct (Fig. 2a). This is in contrast to previous findings using DMPO (9,11,12). Several investigators have detected DMPO/ ⅐ OH adduct during the reaction between DMPO and peroxynitrite (9,11,12). Proposed mechanisms of formation of DMPO/ ⅐ OH include oxidation of DMPO to DMPO radical cation followed by hydrolysis, as shown below (12): The inability of peroxynitrite to oxidize DEPMPO is of interest. The oxidation potentials of DMPO and DEPMPO were measured to be about 1.87 V (versus NHE) and 2.24 V (versus NHE), respectively. 6 It is likely that the 400 mV difference in oxidation potential could account for the lack of oxidation of DEPMPO by peroxynitrite. The present data show that the DEPMPO/ ⅐ OH adduct that is formed from the reaction between peroxynitrite and DEPMPO is likely to be derived from GSHdependent decomposition of DEPMPO/ ⅐ OOH. Peroxynitrite-dependent Sulfur Radical Formation-Thiyl radical formation in peroxynitrite-mediated oxidation of thiols has been previously detected using DMPO (9,12,13). This observation is now confirmed using DEPMPO, as thiyl radical adducts of GSH, cysteine, and NAP were observed. The formation of thiyl adducts was independent of the hydroxyl radicallike reactivity of peroxynitrite, as hydroxyl radical scavengers did not affect the DEPMPO-thiyl radical spectra. It has been previously reported that ONOO Ϫ reacts with thiols to generate thiol disulfide (6). It is not clear whether one-electron oxidation of GSH by peroxynitrite is an intermediate step in disulfide formation or represents an independent pathway of oxidation by one of the conformers of ONOOH. As discussed earlier, the DEPMPO-dependent inhibition of O 2 uptake observed during peroxynitrite-mediated oxidation of GSH and NAP is due to Peroxynitrite oxidized sulfite by an one-electron mechanism to generate the radical. This again appears to be a direct reaction as hydroxyl radical scavengers did not affect the ESR spectral intensity of DEPMPO/ ⅐ SO 3 Ϫ spectrum and the signal was diminished in the presence of 100% oxygen.
Formation of DEPMPO/Carbon-centered Adducts during Peroxynitrite-mediated Oxidation of GSH and NAP-Our results indicate that both DEPMPO/ ⅐ SR and DEPMPO/ ⅐ R (DEPMPO-carbon centered adduct) were detected during oxidation of thiols by peroxynitrite (Figs. 2b and 7a). Evidence for formation of ␣-amino acid carbon-centered radicals from glutathionyl and other sulfur-centered radicals had been obtained using low-temperature (28) and fast-flow ESR spectroscopy (22). This mechanism involves an intramolecular abstraction of an ␣-hydrogen atom of the glutamyl residue by the thiyl radical center. A diagrammatic representation of this process is shown in Scheme 2.
It is possible that the thiyl radical, once formed, undergoes a conformational change which brings the thiyl radical center closer to the ␣-hydrogen atom. The equilibrium between GS ⅐ and ⅐ GSH is dependent on the pH and is shifted to the right side at higher pH (22,23). However, a considerable fraction of GS ⅐ presumably exists as the ␣-amino acid radical at physiological pH. A similar reaction mechanism can also be proposed for formation of the NAP-derived carbon-centered radical from ⅐ S-NAP. The ␣-amino acid carbon-centered radical has an asymmetric carbon center and the resulting DEPMPO-carboncentered adduct will exist as diastereoisomers. Further ESR structural characterization of these DEPMPO-carbon-centered adducts must await syntheses of carbon-13 labeled GSNO and SNAP.
Peroxynitrite-dependent Oxidation of Formate-No spin adducts were observed from the reaction between peroxynitrite and formate in the absence of GSH. However, in the presence of GSH, the formation of CO 2 . was observed. Augusto et al. (9) reported similar results using DMPO and indicated that for-mate was acting as a typical hydroxyl radical scavenger. The observation that GS ⅐ , generated from the photolysis of GSNO, is also able to abstract a hydrogen atom from formate to form CO 2 . indicates another mechanism whereby formate can affect spin adduct formation (30 NAP elicited a greater consumption of oxygen than GSH for the same concentration of thiol and peroxynitrite. It is likely that the additional steric hindrance at the sulfur atom of NAP hinders dimerization of the corresponding thiyl radical (i.e. ⅐ S-NAP). Whereas rapid dimerization of GS ⅐ will reduce the probability of a reaction between GS ⅐ and oxygen. Oxygen consumption during the reaction between sulfite and peroxynitrite can be explained by the following reactions (27). SO 3 . formed from the reaction between peroxynitrite and Superoxide is also formed as a minor product of reaction between SO 3 . and O 2 (Equation 21): Biological Implications of Peroxynitrite-mediated Oxidation of Thiols-It has been suggested that nitric oxide-induced cytotoxicity is amplified by thiol depletion (33). Nitric oxidemediated neurotoxicity has been thought to be due to peroxynitrite formation (34). Although the critical biological target of toxicity for peroxynitrite is not known, it has been suggested that thiols and protein sulfhydryls are major targets for peroxynitrite in neurons (35). It was recently shown that intact neurons are more susceptible than astrocytes to peroxynitritemediated toxicity. This differential sensitivity was attributed to higher concentrations of GSH and other antioxidants/antioxidant enzymes in astrocytes (35). Although the one-electron mechanism of oxidation of GSH by peroxynitrite probably occurs to a minor extent (36), the glutathionyl radical can initiate self-sustaining free radical chain reactions (Equations 11-15) limiting the effectiveness of GSH as an antioxidant (31,32). Peroxynitrite, which is initially formed by the reaction between ⅐ NO and O 2 . , can regenerate O 2 . as a result of scavenging by GSH (Scheme 3). Thus, superoxide dismutase is required to prevent the chain oxidation of GSH (32). The formation of thiyl radicals may contribute to the neurotoxicity associated with peroxynitrite and nitric oxide (Scheme 3). Thiyl radicals have been shown to initiate lipid peroxidation (37). Studies from Stadtman's laboratory (38) have shown that thiyl radicals in bacterial systems are detoxified by a thiol-specific antioxidant enzyme. However, it is not clear whether a similar enzymatic machinery exists in mammalian cells to detoxify thiyl radicals. It has also been reported that the reaction between peroxynitrite and GSH results in low yields (Ͻ1%) of GSNO (36,39). The mechanism for this is as yet unclear but may involve the intermediacy of thiyl radicals (40). GSH clearly protects the cell against oxidative damage by a number of mechanisms. Our data show that the reaction between peroxynitrite and GSH cannot simply be regarded as a 1:1 annihilation of the oxidant as radical species are generated that may propagate the oxidative insult. This propagation is clearly of low efficiency and, at normal GSH levels, can be easily controlled by the cell. However, if GSH levels are compromised by peroxynitrite-dependent oxidation or by some independent route, the formation of intracellular thiyl radicals may represent a mechanism for the propagation of peroxynitrite-dependent oxidation.
Peroxynitrite Interaction with Sulfite: Biological Implications-Recently it was reported that sulfites could counteract in a concentration-dependent manner the ability of ⅐ NO to inhibit platelet aggregation (41). This effect was attributed to a facile reaction between ⅐ NO and sulfite anion. In biological buffers, sulfite anion undergoes rapid autoxidation to produce the superoxide anion (42,43). It is likely that peroxynitrite is formed during the addition of ⅐ NO or NO-donors to solutions containing sulfite in the absence of metal-chelators such as DTPA. Since peroxynitrite has been shown to cause platelet aggregation (36), an alternative explanation for the effect of sulfite and ⅐ NO on platelet aggregation could be due to the oxidative chain reaction that occurs after the reaction between peroxynitrite and sulfite. Peroxynitrite-mediated formation of sulfite anion radicals could also be involved in environmental carcinogenesis induced by polycyclic aromatic hydrocarbons (44).
Conclusion-We have shown using a novel phosphorylated spin trap that peroxynitrite oxidizes a variety of thiols and sulfite forming the corresponding thiyl and sulfite anion radicals. These reactions are not mediated by hydroxyl radical. In contrast to DMPO, DEPMPO does not undergo direct oxidation by peroxynitrite to form the corresponding hydroxyl adduct. Peroxynitrite-dependent oxidation of formate in the presence of thiols is mediated by thiyl radicals. The oxidation of thiols by peroxynitrite is accompanied by oxyradical formation. It is possible that self-sustaining free radical reactions triggered by peroxynitrite contribute to cellular toxicity and limit the antioxidant potential of GSH. . can be generated enzymatically during inflammation. The reaction between ONOO Ϫ and GSH can amplify the oxidative damage via formation of O 2 . and may limit the antioxidant potential of GSH.