Reaction of Peroxynitrite with Reduced Nicotinamide Nucleotides, the Formation of Hydrogen Peroxide*

NAD(P)H acts as a two-electron reductant in physiological, enzyme-controlled processes. Under nonenzymatic conditions, a couple of one-electron oxidants easily oxidize NADH to the NAD⋅ radical. This radical reduces molecular oxygen to the superoxide radical (O⨪2) at a near to the diffusion-controlled rate, thereby subsequently forming hydrogen peroxide (H2O2). Because peroxynitrite can act as a one-electron oxidant, the reaction of NAD(P)H with both authentic peroxynitrite and the nitric oxide (⋅NO) and O⨪2 releasing compound 3-morpholinosydnonimineN-ethylcarbamide (SIN-1) was studied. Authentic peroxynitrite oxidized NADH with an efficiency of ∼25 and 8% in the absence and presence of bicarbonate/carbon dioxide (HCO3 −/CO2), respectively. NADH reacted 5–100 times faster with peroxynitrite than do the known peroxynitrite scavengers glutathione, cysteine, and tryptophan. Furthermore, NADH was found to be highly effective in suppressing peroxynitrite-mediated nitration reactions even in the presence of HCO3 −/CO2. Reaction of NADH with authentic peroxynitrite resulted in the formation of NAD+ and O⨪2 and, thus, of H2O2 with yields of about 3 and 10% relative to the added amounts of peroxynitrite and NADH, respectively. Peroxynitrite generated in situ from SIN-1 gave virtually the same results; however, two remarkable exceptions were recognized. First, the efficiency of NADH oxidation increased to 60–90% regardless of the presence of HCO3 −/CO2, along with an increase of H2O2 formation to about 23 and 35% relative to the amounts of added SIN-1 and NADH. Second, and more interesting, the peroxynitrite scavenger glutathione (GSH) was needed in a 75-fold surplus to inhibit the SIN-1-dependent oxidation of NADH half-maximal in the presence of HCO3 −/CO2. Similar results were obtained with NADPH. Hence, peroxynitrite or radicals derived from it (such as, e.g. the bicarbonate radical or nitrogen dioxide) indeed oxidize NADH, leading to the formation of NAD+ and, via O⨪2, of H2O2. When peroxynitrite is generated in situ in the presence of HCO3 −/CO2,i.e. under conditions mimicking the in vivosituation, NAD(P)H effectively competes with other known scavengers of peroxynitrite.

erated in situ from SIN-1 has been shown to attack many biological targets (e.g. low density lipoproteins (3)) in nearly the same manner as bolus addition of authentic peroxynitrite.
We have recently reported (8) that both authentic and in situ generated peroxynitrite oxidizes HEPES and related tertiary amines in a one-electron step with initial formation of an amine radical cation. Subsequent ␣-deprotonation generates an ␣-aminoalkyl radical that rapidly reduces molecular oxygen to O 2 . , from which H 2 O 2 is further produced. This reaction appears to be so effective that, in the presence of HEPES, SIN-1-induced cytotoxicity to L929 cells was mainly conveyed by H 2 O 2 but not by ONOO Ϫ /ONOOH (9). As HEPES and related tertiary amines are widely used to maintain the pH in biological systems, numerous studies of the (patho)physiological effects of peroxynitrite must be expected to be obscured by this mechanism, that is H 2 O 2 -driven pathways instead of peroxynitrite-mediated effects may have been observed. Consequently, the question arises whether the reaction mechanism elucidated for the reaction of peroxynitrite with HEPES also applies to biologically relevant tertiary amines. One very likely candidate for such an in vivo relevant tertiary amine is NADH as indicated by the following findings: (i) Kobayashi et al. (10) have found that peroxynitrite reacts with NADH with a second-order rate constant of 4 ϫ 10 3 M Ϫ1 s Ϫ1 at pH 7.4, and Ewing et al. (11) observed a high efficiency of SIN-1 to oxidize NADH to NAD ϩ in the absence of HCO 3 Ϫ / CO 2 . However, both groups did not provide any further information on the resulting products and on the underlying mechanism. (ii) NADH is very sensitive toward one-electron oxidants (12). (iii) The Br 2 . radical, which has a standard reduction potential close to that of peroxynitrous acid [E°(Br 2 . /2 Br Ϫ ) ϭ 1.62 V (13), E°(ONOOH/NO 2 ⅐ (aq) ) ϭ 1.6 V (14)], has been reported to attack NADH via one-electron transfer (Reaction 1) (15). Subsequent deprotonation leads to an alkyl radical (NAD ⅐ ) that rapidly (k ϭ 2 ϫ 10 9 M Ϫ1 s Ϫ1 ) reduces oxygen to superoxide radicals (15) from which H 2 O 2 is formed (Reactions 2-4).
* 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.
Solutions-Care was taken to exclude possible contamination by bicarbonate/carbon dioxide. Double-distilled water was bubbled (2 l/min) with synthetic air at room temperature for 20 min. This water was used for synthesis of oxoperoxonitrate(1Ϫ), NaOH (0.01-0.5 N) and for all other solutions. Potassium phosphate buffer (50 mM) containing DTPA (0.1 mM) was prepared freshly each day. The pH was adjusted to 7.5 at 37°C, and the solution was again bubbled (2 l/min) with synthetic air or with the CO 2 mixture for 20 min. In the case of bubbling with the CO 2 mixture, the pH had to be readjusted to 7.5. SIN-1 solutions were prepared as 100ϫ stock solutions at 4°C in 50 mM KH 2 PO 4 and used within 15 min.
Experimental Conditions-SIN-1 was added to 1 ml of phosphate buffer and incubated in 12-well cell culture plates (volume of each well was 7 ml, Falcon, Heidelberg, Germany). For the detection of H 2 O 2 with the catalase assay, SIN-1 was added to 10 ml of buffer and incubated in tissue culture dishes (75 ml, Falcon, Heidelberg, Germany). Under HCO 3 Ϫ /CO 2 -free conditions, these plates/dishes were placed in an airtight vessel (10 l). During the first 15 min of each experiment, these vessels were flushed (5 l/min) with synthetic air in a warming incubator (Heraeus, Hanau, Germany). In the presence of HCO 3 Ϫ /CO 2 , the plates/ dishes were placed in an incubator for cell culture (37°C, humidified atmosphere of 95% authentic air and 5% CO 2 , Labotect, Göttingen, Germany). The experiments with authentic peroxynitrite (2 l of 0.35-0.035 M ONOO Ϫ in 0.5 N NaOH was added to 1 ml of reaction solution) were performed in reaction tubes (1.4 ml, Eppendorf, Hamburg, Germany) by using the drop-tube Vortex mixer technique as described previously (8) (19). Various concentrations of peroxynitrite (50 -2000 M) were vortexed to the reaction solution in the presence of NADH (500 M) and cytochrome c 3ϩ (20 M) or cytochrome c 3ϩ plus SOD (625 nM, i.e. 100 units/ml). NADH was added in surplus amounts to prevent reaction of (residual) peroxynitrite with SOD and cytochrome c 2ϩ formed. The resulting mixture was stored for 2 min at 37°C. Cytochrome c 2ϩ formation was determined by reading its absorbance at 550 nm (⌬⑀ 550 ϭ 21000 M Ϫ1 cm Ϫ1 ) (20). The difference in cytochrome c reduction in the presence and absence of SOD was used to calculate the amount of trapped O 2 . .
Determination of SIN-1 and SIN-1C-SIN-1 and SIN-1C were quantified by capillary zone electrophoresis on a Beckman P/ACE 5000 apparatus as described previously (8).
Determination of NAD ϩ , NADH, and NADPH-NAD ϩ was determined by capillary zone electrophoresis under the following conditions: fused silica capillary (50-cm effective length, 75-m internal diameter), hydrodynamic injection for 5 s, 30°C temperature, 15 kV voltage, reversed polarity, UV detection at 254 nm. 50 mM sodium phosphate, 1 mM EDTA, 100 mM dodecyltrimethylammonium bromide (pH 7.0) was used as electrolyte system. To each sample, 0.25 mM 4-aminobenzamide was added as internal standard. Alternatively, NAD ϩ was determined by an enzymatic method using yeast alcohol dehydrogenase, ethanol, and semicarbazide (alcohol dehydrogenase assay) as described by Klingenberg (21). After treatment with authentic peroxynitrite or with SIN-1, residual NAD(P)H was quantified by reading its fluorescence with excitation at 339 nm and emission at 460 nm (21). Standard calibration curves were prepared from known amounts of NAD(P)H. Additionally, the oxidation of NAD(P)H was also followed photometrically at 340 nm using ⌬⑀ 340 ϭ 6200 M Ϫ1 cm Ϫ1 (21). Both methods gave identical results; therefore, only one parameter, decrease of fluorescence, will be shown.
Because HCO 3 Ϫ /CO 2 is believed to act as peroxynitrite scavenger, the influence of other known peroxynitrite scavengers, namely cysteine, glutathione, methionine, and tryptophan, on peroxynitrite-dependent consumption of NADH was studied (Table I). Rather high concentrations of these scavengers, i.e.
[scavenger] Ͼ5 ϫ [NADH], were needed to inhibit peroxynitritedependent consumption of NADH half-maximally. Interestingly, the HO ⅐ radical scavenger Me 2 SO (up to 50 mM), which convincingly has been shown to scavenge HO ⅐ radicals derived from peroxynitrite (25), did not exhibit any protective effect on peroxynitrite (0.25 mM)-mediated oxidation of NADH (200 M, data not shown). In further experiments, the chemical reactivity of NADPH toward peroxynitrite was compared with that of NADH. Virtually the same results were obtained. Thus, after addition of 0.25 mM peroxynitrite to 200 M NADPH in the absence of HCO 3 Ϫ /CO 2 133.8 Ϯ 4.7 M, residual NADPH was detected, and the concentration of GSH necessary to protect NADPH half-maximal was also 1.1 Ϯ 0.1 mM.
Based on the data of Table I, we suspected that NADH inhibited peroxynitrite-mediated nitration reactions (Table II). Interactions of peroxynitrite (1 mM) with the target compounds p-HPA, tyrosine, and tryptophan (each 1 mM) in the absence and in the presence of HCO 3 Ϫ /CO 2 yielded amounts of nitrated products that were virtually identical to those reported previously (8,22,26). The addition of NADH very strongly decreased the peroxynitrite-mediated nitration of the selected compounds under both conditions. Concentrations of NADH in the range of 100 -200 M reduced the formation of nitroaromatics by 50% in the HCO 3 Ϫ /CO 2 -free situation, in the presence of HCO 3 Ϫ /CO 2 the concentrations of NADH had to be increased to 480 -580 M to inhibit the formation of the nitrated products half-maximally.
Formation of NAD ϩ -The reaction of peroxynitrite with NADH yielded exclusively NAD ϩ at concentrations of peroxynitrite lower or comparable with those of NADH, irrespective of the presence of HCO 3 Ϫ /CO 2 (capillary zone electrophoresis and alcohol dehydrogenase assay, data not shown). The situation somehow changed when excess concentrations of peroxynitrite were employed. Whereas in the presence of HCO 3 Ϫ /CO 2 , NADH was still exclusively oxidized to NAD ϩ (up to 4 mM peroxynitrite, 200 M NADH), in the absence of HCO 3 Ϫ /CO 2 , only 167 Ϯ 7 M NAD ϩ were formed upon addition of 2 mM peroxynitrite to 200 M NADH (alcohol dehydrogenase assay). Now ADP-ribose was detected as additional reaction product (capillary zone electrophoresis, data not shown).
Formation of O 2 . -The most commonly used assay for O 2 .
comprises the reduction of cytochrome c 3ϩ in the absence and presence of SOD (19). Cytochrome c 2ϩ and SOD, however, react rapidly with peroxynitrite at reaction rate constants of 2.3 ϫ 10 5 M Ϫ1 s Ϫ1 (27)  were found upon addition of ONOO Ϫ to mere phosphate buffer (Fig. 3), reflecting the detectable hydrogen peroxide base level of 0.29 mol % of our peroxynitrite stock solution rather than H 2 O 2 formation from peroxynitrite. In the presence of NADH, H 2 O 2 was produced, and its concentration increased linearly with the concentration of peroxynitrite. The yield of H 2 O 2 accounted for ϳ2.7% of the amount of added peroxynitrite and about 10.8% of the consumed NADH. At peroxynitrite concentrations higher than 700 M, i.e. when all of the NADH was consumed (see Fig. 1   Ϫ /CO 2 (20 mM, 5%), NADH (0 -1000 M) in 50 mM potassium phosphate buffer (0.1 mM DTPA, 37°C, pH 7.5). After a 5-min incubation at 37°C, the formation of nitrated products (3-(NO 2 )-4-HPA, 3-nitrotyrosine, 5-nitrotryptophan) were determined by reading the absorbance at maximum (400 -430 nm). Each value represents the mean Ϯ S.D. of four experiments performed in duplicate.

NAD(P)H as a Target for Peroxynitrite-In the absence of SIN-1, about 95% (85%) of the initial NADH (NADPH) concentration (200 M) could still be detected after 4 h of incubation, regardless of the presence of HCO 3
Ϫ /CO 2 (20 mM, 5%) (Fig. 4). In the absence of HCO 3 Ϫ /CO 2 , SIN-1 oxidized NADH with an efficiency decreasing from 90 to 60% with increasing SIN-1 concentration. Surprisingly, and in sharp contrast to the experiments performed with authentic peroxynitrite in the presence of HCO 3 Ϫ /CO 2 , a slightly higher efficiency of SIN-1 to degrade NADH was observed. The nonlinear concentration dependences (Fig. 4) implied that the decomposition product of SIN-1, SIN-1C, might interfere with the NADH oxidation. SIN-1C (0 -400 M), however, did not have any effect on NADH oxidation both in the absence and presence of HCO 3 Ϫ /CO 2 (data not shown). SIN-1 (0.2 mM) also effectively oxidized NADPH (0.2 mM). In the absence and presence of HCO 3 Ϫ /CO 2 , only 56.2 Ϯ 1.2 M and 35.9 Ϯ 1.8 M residual NADPH, respectively, were found after 4 h of incubation. Thus, peroxynitrite in situ generated from SIN-1 was remarkably more effective than authentic peroxynitrite added as a bolus. This is especially true in the presence of HCO 3 Ϫ /CO 2 . To compare the reactivity of peroxynitrite generated from SIN-1 with that of authentic peroxynitrite, the effects of peroxynitrite scavengers on SIN-1-mediated NADH oxidation were analyzed in the absence of HCO 3 Ϫ /CO 2 (Table III). In line with the experiments performed with authentic peroxynitrite (Table I), concentrations of scavengers severalfold higher than those of NADH were necessary to inhibit the SIN-1-mediated oxidation of NADH half-maximally. The apparent reaction rate constants (ϳ1 ϫ 10 4 M Ϫ1 s Ϫ1 , Table III) are in good agreement with the values found for authentic peroxynitrite (Table I) studied in the presence of HCO 3 Ϫ /CO 2 (Fig. 5, A and B). Under that condition, roughly 65 M residual NAD(P)H could be detected after 4 h of incubation in the absence of GSH. Between 0.4 and 5 mM GSH, SIN-1-mediated oxidation of NAD(P)H was somewhat reduced. The residual amount of NAD(P)H increased to values in the range of 80 -100 M and was found to be largely independent of the concentration of GSH. A further increase of the GSH concentration led to an approximate exponential increase of the residual concentration of NAD(P)H. A limiting value of 200 M of residual NAD(P)H was extrapolated at ϳ36 mM GSH. The effect of GSH on SIN-1-dependent oxidation of NAD(P)H was half-maximal at 15.6 Ϯ 0.1 mM GSH (for NADH) and at 14.8 Ϯ 0.1 mM GSH (for NADPH), respectively. As deduced from the data in Table III, GSH protected NAD(P)H effectively in the absence of HCO 3 Ϫ /CO 2 (Fig. 5, A and  B). Noticeably, now the "plateau" at 80 -100 M NAD(P)H in the 0.4 -5 mM GSH region could not be detected, and full protection of NAD(P)H oxidation was already achieved at ϳ10 mM GSH. Thus, the addition of HCO 3 Ϫ /CO 2 strongly diminished the inhibitory effect of GSH on SIN-1-mediated oxidation of NAD(P)H. Cysteine inhibited SIN-1 (0.15 mM)-mediated oxidation of NAD(P)H (0.2 mM) in the presence of HCO 3 Ϫ /CO 2 more effectively, with half-maximal concentrations of 2.0 Ϯ 0.1 mM (for NADH) and 3.0 Ϯ 0.1 mM (for NADPH), respectively. These experiments with thiols strongly suggest that an oxidant(s) other than peroxynitrite itself is mainly responsible for the oxidation of NAD(P)H in the presence of HCO 3 Ϫ /CO 2 . To demonstrate that the NADH-oxidizing species indeed derives from peroxynitrite, Cu, Zn-, and Mn-superoxide dismutases (100 units/ml each) were tested for their abilities to suppress SIN-1 (150 M)-mediated oxidation of NADH (200 M). In the absence and in the presence of HCO 3 Ϫ /CO 2 , SIN-1-dependent oxidation of NADH was inhibited by SOD by about 50 and 35%, respectively, virtually independent of the type of SOD applied (three experiments performed in duplicate).
Formation of NAD ϩ -In line with the results reported for authentic peroxynitrite, SIN-1-mediated oxidation of NADH yielded exclusively NAD ϩ (alcohol dehydrogenase assay, data not shown), provided that the concentration of SIN-1 was less than twice the concentration of NADH. Ϫ /CO 2 and 69.6 Ϯ 3.7 M H 2 O 2 in its presence (catalase assay). Thus, SIN-1 dependent production of H 2 O 2 from NAD(P)H is highly effective, corresponding to about 30 and 35% of the amount of decomposed NAD(P)H in the absence and in the presence of HCO 3 Ϫ /CO 2 , respectively.

TABLE III
Effects of peroxynitrite scavengers on SIN-1-mediated oxidation of NADH SIN-1 (0.15 mM) was mixed to NADH (0.2 mM) in the presence of various scavengers (0 -10 mM) in 50 mM potassium phosphate buffer (0.1 mM DTPA, 37°C, pH 7.5). To find the scavenger concentration necessary to inhibit SIN-1-mediated oxidation of NADH half-maximal, the concentration of the scavengers was increased stepwise (⌬[scavenger], 125 M) from 0 to 1.25 mM (Cys, GSH, thiourea). In the case of both methionine and tryptophan, concentration was increased by 1 mM steps to reach a final concentration of 10 mM. After the addition of SIN-1, the samples were stored for 4 h in a warming incubator. NADH was quantified by reading the fluorescence with excitation at 339 nm and emission at 460 nm. Data are means Ϯ S.D. of three experiments performed in duplicate.

DISCUSSION
Oxidizing Species-Various oxidants and radicals can be formed from peroxynitrite at physiological pH values (Fig. 7). Under HCO 3 Ϫ /CO 2 -free conditions, peroxynitrous acid (ONOOH) has been reported to directly attack a variety of substrates such as tryptophan (22). Alternatively, it may dissociate with a yield of roughly 10% (25) into the highly reactive free radicals HO ⅐ and NO 2 ⅐ (29). In the presence of HCO 3 Ϫ /CO 2 , however, ONOO Ϫ rapidly reacts with CO 2 (30) to give the free radicals CO 3 . (31) and NO 2 ⅐ (32) via the putative intermediate nitrosoperoxycarbonate (ONOOCO 2 Ϫ ). Because of the facts that (i) NADH is readily oxidized by one-electron oxidants (12) to the NAD ⅐ radical (15), (ii) ONOOH can act as an one-electron oxidant (33), and (iii) HO ⅐ , NO 2 ⅐ and CO 3 . radicals are also one-electron oxidants, there can be little doubt that reaction of peroxynitrite with NADH will initially produce the NAD ⅐ radical. This radical readily reduces O 2 to O 2 . , and both products of this reaction, namely NAD ϩ and O 2 . , were indeed found from reaction of authentic peroxynitrite with NADH. As O 2 . dismutates spontaneously to H 2 O 2 , the latter was likewise detected after reaction of NAD(P)H with both authentic peroxynitrite and peroxynitrite generated in situ from SIN-1. As expected, both reduced nicotinamides, NADH and NADPH, react in virtually the same manner. Because most of the above free radicals can react with each other, the yields of the various recombination products cannot be estimated. For example, NO 2 ⅐ reacts at pH 7.5 about 2 ϫ 10 5 times faster with O 2 . than O 2 . reacts with itself (34,35). Therefore, H 2 O 2 is unlikely to be formed in roughly stoichiometric amounts.
In the absence of HCO 3 Ϫ /CO 2 , ONOOH appears to oxidize NADH directly. This follows from the fact that the HO ⅐ radical scavenger Me 2 SO was unable to protect NADH against the attack of peroxynitrite and also because reaction of HO ⅐ with NADH should lead to significant (ϳ40%) formation of products other than NAD ϩ (12). In the presence of HCO 3 Ϫ /CO 2 , peroxynitrite scavengers but not superoxide dismutases were largely ineffective in protecting NADH against the attack of SIN-1.
Hence, ONOOH appears not to be the major oxidant in such a situation. The experiments performed with GSH and cysteine as additive indicate that the CO 3 . radical is the presumed attacking species. Compared with cysteine, a 7.6-fold higher concentration of GSH was necessary to inhibit SIN-1-mediated oxidation of NADH half-maximal, in good agreement with the fact that the CO 3 . radical reacts about 8.7 times faster with cysteine than with GSH (36). The reactivity of CO 3 . on NADH can be estimated by making use of the fact that the rate constants of the reaction of NADH with a variety of radicals correlate with the reduction potentials of the oxidizing radicals. 2 Thus, CO 3 . is expected to react with NADH with a rate constant of 7 ϫ 10 8 M Ϫ1 s Ϫ1 . Because of its lower reduction potential, NO 2 ⅐ should react about seven-fold slower with NADH .
Authentic Versus in Situ-generated Peroxynitrite-In the absence of HCO 3 Ϫ /CO 2 , the steady-state concentration of ONOOH generated in situ from SIN-1 should be much less than upon bolus addition of authentic peroxynitrite, as the release of ⅐ NO and O 2 . from SIN-1 is a slow process at pH 7.5 (t1 ⁄2 ϭ 40 min (9)).
In the presence of HCO 3 Ϫ /CO 2 , in situ generated peroxynitrite from SIN-1 was about 10-fold more effective in oxidizing NADH than authentic peroxynitrite. This surprisingly different capability is hard to explain with the current knowledge about the chemical characteristics of authentic and in situ generated peroxynitrite.
Putative (Patho)Physiological Significance-GSH is considered to be a major scavenger of intracellularly operating peroxynitrite (5), and, indeed, in the absence of HCO 3 Ϫ /CO 2 GSH prevented effectively the (physiologically uncommon) one-electron oxidation of NAD(P)H by in situ generated peroxynitrite. In the presence of HCO 3 Ϫ /CO 2 , however, peroxynitrite predominantly reacts with CO 2 (39) to yield CO 3 . and NO 2 ⅐ (see above). GSH and other thiols readily react with NO 2 ⅐ (40, 41), but GSH reacts about 100-fold slower with the CO 3 . radical (36). Therefore, the GSH/NAD(P)H ratio must be high to protect NAD(P)H effectively against the attack of peroxynitrite-derived CO 3 . radicals. For instance, the total amount of GSH in rat liver typically lies in the range of 5-10 mol/g wet weight (e.g. Ref. 42).
To the contrary, the total amount of NAD(P)H in rat liver cytosol was found in the range of 90 nmol/g wet weight (43), whereas the bulk of NAD(P)H is located in the mitochondria with a total amount up to ϳ0.4 mol/g wet weight (43), i.e. ϳ20 times lower than the total amount of GSH. On the other hand, as GSH in a 75 ϫ surplus was necessary to inhibit the SIN-1mediated oxidation of NAD(P)H half-maximal in the presence of HCO 3 Ϫ /CO 2 , GSH should be rather ineffective in inhibiting the attack of in vivo generated peroxynitrite on NAD(P)H. This implies that the reaction products O 2 . /H 2 O 2 may be the dominating factor decisively contributing to the (patho)physiological effects ascribed originally attributed to peroxynitrite. In line with this notion, H 2 O 2 and nitrotyrosine (the latter is believed to be a marker for peroxynitrite) have been reported to be elevated in patients with adult respiratory distress syndrome (44,45). Hydrogen peroxide induces the activation of NF-B (46,47), and this transcription factor has been implicated in atherosclerosis (48) and rheumatoid arthritis (49), i.e. in diseases where formation of nitrotyrosine has also been detected (50,51). Furthermore, both peroxides can induce DNA single strand breaks (52)(53)(54)(55), thus activating poly(ADP-ribose) synthetase (52,56). Accordingly, authentic peroxynitrite-dependent DNA cleavage and subsequent apoptosis in HL-60 cells in phosphate-buffered saline has been reported to be mediated mainly by hydrogen peroxide (57).