Toxicity Of Peroxynitrite Research Articles

Peroxynitrite: A tale of two radicals

The published syntheses of peroxynitrite from azide, nitrite, amylnitrite, hydroxylamine, nitrogen monoxide, and ammonia are discussed. With one exception, all of these syntheses yield peroxynitrite contaminated with nitrate and nitrite as well as reactants. The rate constant for the reaction of nitrogen monoxide with superoxide has been determined by pulse radiolysis and flash photolysis. In pulse radiolysis studies, the formation of the reactants may be rate-limiting and could lead to underestimation of the second-order rate constant. The conditions of flash photolysis experiments can be chosen to minimize conflict between reactant formation and the reaction half-life, thus the rate constant of 1.6 × 1010 M−1 s−1 determined by flash photolysis is preferred. The toxicity of peroxynitrite can be attributed mainly to its rapid reaction with carbon dioxide to yield the oxidizing trioxidocarbonate(•1−) and nitrogen dioxide radicals.

Uric Acid: A Translational Journey in Cerebroprotection That Spanned Preclinical and Human Data.

Uric acid (UA) is a strong endogenous antioxidant that neutralizes the toxicity of peroxynitrite and other reactive species on the neurovascular unit generated during and after acute brain ischemia. The realization that a rapid reduction of UA levels during an acute ischemic stroke was associated with a worse stroke outcome paved the way to investigate the value of exogenous UA supplementation to counteract the progression of redox-mediated ischemic brain damage. The long translational journey for UA supplementation recently reached a critical milestone when the results of the multicenter NIH stroke preclinical assessment network (SPAN) were reported. In a novel preclinical paradigm, 6 treatment candidates including UA supplementation were selected and tested in 6 independent laboratories following predefined criteria and strict methodological rigor. UA supplementation was the only intervention in SPAN that exceeded the prespecified efficacy boundary with male and female animals, young mice, young rats, aging mice, obese mice, and spontaneously hypertensive rats. This unprecedented achievement will allow UA to undergo clinical testing in a pivotal clinical trial through a NIH StrokeNet thrombectomy endovascular platform created to assess new treatment strategies in patients treated with mechanical thrombectomy. UA is a particularly appealing adjuvant intervention for mechanical thrombectomy because it targets the microcirculatory hypoperfusion and oxidative stress that limits the efficacy of this therapy. This descriptive review aims to summarize the translational development of UA supplementation, highlighting those aspects that likely contributed to its success. It includes having a well-defined target and mechanism of action, and an approach that simultaneously integrated rigorous preclinical assessment, with epidemiologic and preliminary human intervention studies. Validation of the clinical value of UA supplementation in a pivotal trial would confirm the translational value of the SPAN paradigm in preclinical research.

Solvent Cage Concept for the Homolytic Fragmentation of the Peroxynitrite-CO2 Adduct, ONOOCO2.

The toxicity of peroxynitrite, ONOO-, is directed by carbon dioxide via the formation of the corresponding adduct, ONOOCO2-. Entity ONOOCO2- is believed to be a highly unstable compound that primarily decomposes to nitrate and carbon dioxide, but it also undergoes fractional homolysis to generate carbonate radical anion, CO3•-, and nitrogen dioxide, NO2•, in a so-called solvent (radical) cage reaction. Recently, Koppenol et al. reviewed their proposal that ONOOCO2- is a relatively long-lived intermediate, arguing that "the solvent cage as proposed is physically not realistic". To further address whether ONOOCO2- could be a long-lived species, bond dissociation enthalpies (BDE) were calculated by the composite reference method (SMD)W1BD. Anion ONOOCO2- can exist in two conformers, s-cis-gauche and s-trans-gauche with predicted gas-phase O-O BDEs of about 10.8 and 9.5 kcal mol-1, respectively. Therefore, both conformers should have very short lifetimes. The (SMD)W1BD method was also used to evaluate the thermodynamic parameters of interest, revealing that the homolytic decomposition of ONOOCO2- is the most reasonable pathway. Moreover, previously reported experimental chemically induced dynamic nuclear polarization data also support the intermediacy of the radical cage and the formation of products CO2 and NO3- at a total yield of about 70%. Because the solvent radical cage concept for the decay of ONOO- in the presence of CO2 is supported by a variety of spectrometric methods as well as by quantum chemical calculations at high levels of theory, it provides strong evidence against the "out-of-cage" construct. For clarification of the nature of the transient UV/vis absorption(s) between 600 and 700 nm, as observed by Koppenol et al., several experimental approaches are suggested.

Potential ghrelin-mediated benefits and risks of hydrogen water

Molecular hydrogen (H2) can scavenge hydroxyl radical and diminish the toxicity of peroxynitrite; hence, it has interesting potential for antioxidant protection. Recently, a number of studies have explored the utility of inhaled hydrogen gas, or of hydrogen-saturated water, administered parenterally or orally, in rodent models of pathology and in clinical trials, oftentimes with very positive outcomes. The efficacy of orally ingested hydrogen-rich water (HW) has been particularly surprising, given that only transient and rather small increments in plasma hydrogen can be achieved by this method. A recent study in mice has discovered that orally administered HW provokes increased gastric production of the orexic hormone ghrelin, and that this ghrelin mediates the favorable impact of HW on a mouse model of Parkinson’s disease. The possibility that most of the benefits observed with HW in experimental studies are mediated by ghrelin merits consideration. Ghrelin is well known to function as an appetite stimulant and secretagogue for growth hormone, but it influences physiological function throughout the body via interaction with the widely express GHS-R1a receptor. Rodent and, to a more limited extent, clinical studies establish that ghrelin has versatile neuroprotective and cognitive enhancing activity, favorably impacts vascular health, exerts anti-inflammatory activity useful in autoimmune disorders, and is markedly hepatoprotective. The stimulatory impact of ghrelin on GH-IGF-I activity, while potentially beneficial in sarcopenia or cachectic disorders, does raise concerns regarding the long-term impact of ghrelin up-regulation on cancer risk. The impact of ingesting HW water on ghrelin production in humans needs to be evaluated; if HW does up-regulate ghrelin in humans, it may have versatile potential for prevention and control of a number of health disorders.

Toxicity of endogenous peroxynitrite and effects of puerarin on transplanted retinal pigment epithelial sheets in the subretinal space in mice.

To evaluate the toxicity of endogeneous peroxynitrite on transplanted retinal pigment epithelial (RPE) sheets and the effect of puerarin on their survival in the C57BL/6 mice after RPE sheets have been transplanted into SD rats' subretinal space . C57BL/6 mice eyes were used to culture RPE cells. Ninety-six SD rats were involved in the experiment. They were divided into control (block control), streptozotocin (STZ, negative control), untransplanted RPE (positive control) and transplanted RPE groups respectively. Diabetes was induced in SD rats by intra-peritoneal STZ injection in the latter three groups. Saline was injected into the subretinal space of 24 SD rats in the untransplanted RPE group and primary RPE sheets were injected into the subretinal space of 24 SD rats in the transplanted RPE group. Puerarin (45mg/kg) was administrated into both untransplanted RPE and transplanted RPE groups of diabetic rats through intra-peritoneal injection route after RPE sheets transplantation. At 20, 40, 60 days after surgery, Western blotting analysis, DNA ladder and RT-PCR were used for determining the differences in expression of nitrotyrosine (NT, the foot print of peroxynitrite ), apoptosis and iNOS mRNA in the control, STZ, untransplanted RPE and transplanted RPE groups respectively. HE staining was used for determining the RPE survival in the subretinal space of the transplanted RPE group. Apoptosis and expression of NT and iNOS mRNA were observed in STZ, untransplanted RPE and transplanted RPE groups, but were delayed in untransplanted RPE and transplanted RPE groups in a time-dependent manner compared with control and STZ groups (P<0.01). There were no differences between the two groups (P>0.01). NT, DNA ladder, iNOS mRNA were down-regulated, which were associated with the decrease of expression of peroxynitrite. Numerous pigmented cells emerged and increased in number in the subretinal space during the 60-day observation period after transplantation. On day 20, heavily pigmented cells were visible at the transplant site; On day 40, monolayer and multilayered transplant was visible in the subretinal space; On day 60, heavily pigmented monolayer and multilayered transplants with round apical profile were present along Bruch's membrane. Puerarin increased the 60-day survival of C57BL/6 mice RPE xenografts in the SD rats' subretinal space, which may be related to its direct inhibition of apoptosis of RPE cells and antagnism of damage of peroxynitrite to RPE cells.

Etoposide Reduces Peroxynitrite-Induced Cytotoxicity via Direct Scavenging Effect

Previously, we reported that glucose-deprived astrocytes are more vulnerable to the cytotoxicity of peroxynitrite, the reaction product of nitric oxide and superoxide anion. The augmented vulnerability of glucose-deprived astrocytes to peroxynitrite cytotoxicity was dependent on their proliferation rate. Inhibition of cell cycle progression has been shown to inhibit the apoptotic cell death occurring in cerebral ischemia-reperfusion. In the present study, we demonstrate that the increased death of glucose-deprived astrocytes by peroxynitrte was largely blocked by the cell cycle phase G2/M transition blocker etoposide. However, the cytoprotective effect of etoposide was not associated with its inhibition of cell cycle progression. Instead, etoposide effectively scavenged peroxynitrite. However, etoposide did not scavenge individual nitric oxide and superoxide anion and it did not prevent the hydrogen peroxide-induced cytotoxicity. The present results indicate that etoposide prevents the toxicity of peroxynitrite in astrocytes by directly scavenging peroxynitrite, not by inhibiting cell cycle progression.

Peroxynitrite is a major trigger of cardiomyocyte apoptosis in vitro and in vivo

Recent evidence indicates that peroxynitrite represents a major cytotoxic effector in heart diseases, but its mechanisms of action are still not known exactly. Notably, the ability of peroxynitrite to trigger cardiomyocyte apoptosis, a crucial mode of cell death in many cardiac conditions, remains poorly defined. We evaluated apoptotic and necrotic cell death in cultured H9C2 cardiomyocytes, following a brief (20 min) exposure to peroxynitrite (50–500 μM). Peroxynitrite-dependent myocardial toxicity was then investigated in a rat model of myocardial ischemia-reperfusion (MIR), where the effects of peroxynitrite were blocked by the superoxide dismutase mimetics and peroxynitrite scavenger Mn(III)-tetrakis(4-benzoic acid) porphyrin (MnTBAP). In vitro, peroxynitrite killed cardiomyocytes mostly through apoptosis (DNA fragmentation, apoptotic nuclear alterations, caspase-3 activation, and PARP cleavage), but not necrosis (propidium iodide staining and LDH release). In vivo, MIR triggered myocardial oxidative stress (malondialdehyde generation), nitrotyrosine formation, neutrophil accumulation, and the cleavage of caspase-3 and PARP, indicating ongoing myocardial apoptosis. MnTBAP suppressed these alterations, allowing a considerable reduction of myocardial injury. Thus, peroxynitrite triggers apoptosis in cardiomyocytes in vitro and in the myocardium in vivo, through a pathway involving caspase-3 activation and the cleavage of PARP. These results provide important novel information on the mechanisms of myocardial toxicity of peroxynitrite.

Intracellular Zinc Release and ERK Phosphorylation Are Required Upstream of 12-Lipoxygenase Activation in Peroxynitrite Toxicity to Mature Rat Oligodendrocytes

Peroxynitrite toxicity has been implicated in the pathogenesis of white matter injury. The mechanisms of peroxynitrite toxicity to oligodendrocytes (OLs), the major cell type of the white matter, are unknown. Using primary cultures of mature OLs that express myelin basic protein, we found that 3-morpholinosydnonimine, a peroxynitrite generator, caused toxicity to OLs. N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine, a zinc chelator, completely blocked peroxynitrite-induced toxicity. Use of FluoZin-3, a specific fluorescence zinc indicator, demonstrated the liberation of zinc from intracellular stores by peroxynitrite. Peroxynitrite caused the sequential activation of extracellular signal-regulated kinase 42/44 (ERK42/44), 12-lipoxygenase, and generation of reactive oxygen species, which were all dependent upon the intracellular release of zinc. The same cell death pathway was also activated when exogenous zinc was used. These results suggest that in addition to preventing the formation of peroxynitrite, useful strategies in preventing disease progression in pathologies in which peroxynitrite toxicity plays a critical role might include maintaining intracellular zinc homeostasis, blocking phosphorylation of ERK42/44, inhibiting activation of 12-lipoxygenase, and eliminating the accumulation of reactive oxygen species.

Green Tea Polyphenol Epigallocatechin-3-gallate Protects Cells against Peroxynitrite-Induced Cytotoxicity: Modulatory Effect of Cellular G6PD Status

Glucose-6-phosphate dehydrogenase (G6PD) plays important roles in the maintenance of cellular redox balance. It was not until recently that the importance of G6PD in regulation of cellular growth and apoptosis emerged. In the present study, we found that G6PD-deficient fibroblasts were more susceptible to peroxynitrite-induced cytotoxicity. Treatment with peroxynitrite generator 3-morpholinosydnonimine (SIN-1) hydrochloride caused apoptosis in human fibroblast in a dose-dependent manner. This was preceded by a decrease in the intracellular level of glutathione (GSH) as well as accumulation of p53. The extent of apoptosis and glutathione depletion were greater in G6PD-deficient fibroblasts than in the normal counterpart. Pretreatment with green tea polyphenol epigallocatechin-3-gallate (EGCG) effectively blocked peroxynitrite-induced glutathione depletion, p53 accumulation, and apoptosis in both normal and G6PD-deficient cells. EGCG, administered to cells alone or as pretreatment, caused activation of Akt. The protective effect was abolished by phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin, and LY294002. Our findings suggest that G6PD deficiency enhances the toxicity of peroxynitrite and that EGCG initiates cell survival signaling via the PI3K/akt pathway.

Toxicity of nitric oxide and peroxynitrite to Photobacterium damselae subsp. piscicida

Virulent strains of Photobacterium damselae subsp. piscicida ( Pdp) were grown in media with or without glucose supplementation (to enhance polysaccharide capsule formation) and the bactericidal action of nitric oxide (NO) and peroxynitrites was evaluated in a cell-free assay. Treatment with the NO-donor S-nitroso-acetyl-penicillamine (SNAP) induced a dose-and time-dependent decrease in Pdp survival. This effect was greater for strains grown without glucose supplementation (C forms) than for their counterparts grown with glucose supplementation (C+ forms). Addition of superoxide anion (O 2 −) generating systems (Xanthine/Xanthine oxidase, glucose/glucose oxidase) to the culture media further enhanced the bactericidal effect of NO. A similar bactericidal effect, with the same pattern of sensitivity, was observed when C+ and C forms of the bacteria were treated with 3-morpholino-sydonimide hydrochloride (SIN-1), a compound which simultaneously generates NO and O 2 −. Addition of superoxide dismutase (SOD) or SOD plus catalase (CAT) did not fully reverse the toxic action of SIN-1 and the bactericidal effect was similar for both C and C+ forms suggesting that while NO alone is sufficient to cause damage in all strains of the pathogen tested, growth in glucose supplemented medium enhanced protection to reactive oxygen intermediates rather than NO.

The essential nutrient pyrroloquinoline quinone may act as a neuroprotectant by suppressing peroxynitrite formation.

Pyrroloquinoline quinone (PQQ) is a redox active essential nutrient that can generate or scavenge superoxide depending on its microenvironment. PQQ has been shown previously to be neuroprotective in a rodent stroke model. Here we test whether PQQ interacts with reactive nitrogen species, known to be involved in the pathogenesis of stroke. Using rat forebrain neurons in culture, we determined that the toxicity of SIN-1 was mediated by peroxynitrite and that PQQ could block this toxic action. However, PQQ could not block the toxicity of peroxynitrite itself. Both SIN-1 and peroxynitrite caused ATP depletion, but only SIN-1 evoked ATP depletion was blocked by PQQ. In a cell-free system, PQQ blocked nitration of bovine serum albumin produced by SIN-1, but potentiated peroxynitrite-induced nitration. PQQ was unable to block ATP depletion and cell death induced by NO. donors (DEA/NO, DPT/NO and DETA/NO), indicating that it does not directly interact with nitric oxide, and suggesting that it acts as a superoxide scavenger. PQQ significantly potentiated cGMP accumulation evoked by SIN-1, similar to the effect of superoxide dismutase (SOD). However, unlike SOD, which potentiated neurotoxicity induced by SIN-1, PQQ blocked its toxicity, arguing against the possibility that PQQ functions simply as a SOD mimetic. Indeed, substantially less H2O2 was produced by the incubation of SIN-1 with PQQ, when compared to SOD. These results suggest that PQQ scavenges superoxide without forming toxic levels of H2O2. Therefore, the protective effect of PQQ on stroke might be due, at least in part, to the suppression of peroxynitrite formation.

Production of Nitric Oxide by the Middle Ear Epithelium and Subsequent Inhibition of Sodium Transport

The production of nitric oxide (NO) within the middle ear has not previously been characterized. The presence of NO synthase (NOS) transcripts was demonstrated using RNA amplification by reverse transcriptase-polymerase chain reaction in rat middle ear mucosa and in rat primary cultured middle ear epithelial cells. The expression of NOS was indirectly assessed by nitrite measurement in the supernatant of primary cultured cells. The effect of NO on ion transport was investigated in a previously described middle ear epithelial cell line using the short-circuit current (Isc) technique. NO per se had no effect on Isc. However, previous work has shown that sodium transport is stimulated by reactive oxygen species (ROS). NO blunted this stimulation, an inhibition probably related to the toxicity of peroxynitrite, ONOO-, a highly reactive compound. These results suggest that NO is produced by middle ear epithelial cells and that, in the presence of ROS, NO may be responsible for an inhibition of ion transport via peroxynitrite formation.

Toxicity of nitric oxide and peroxynitrite to bacterial pathogens of fish.

The inhibitory effect of the nitric oxide (NO) donor S-nitroso-acetyl-penicillamine (SNAP) and the NO and O2- donor 3-morpholino-sydnonimine hydrochloride (SIN-1) was tested in a cell-free assay. Strains of the bacterial fish pathogens Aeromonas salmonicida, Renibacterium salmoninarum and Yersinia ruckeri were exposed to different concentrations of the NO donors for 24 h. The results showed that NO possesses inhibitory properties, while peroxynitrite had no effect. However, when SIN-1 was used in combination with superoxide dismutase (SOD) alone or with catalase, an inhibitory effect comparable to that caused by SNAP was seen. The implications of these results are discussed.

The neuronal toxicity of sulfite plus peroxynitrite is enhancede by glutathione depletion: implications for Parkinson’s disease

In Parkinson’s disease (PD) and incidental Lewy body disease glutathione levels in the substantia nigra are decreased by 40–50%. Both peroxynitrite (ONOO −) and alterations in the metabolism of sulfur-containing amino acids have been implicated in PD and we have previously shown that sulfite and ONOO − exert synergistic toxicity to a neuronal cell line. This article presents data to show that this synergistic toxicity of sulfite and ONOO − is greatly enhanced by 50% depletion of cellular glutathione levels. The toxicity of sulfite is also slightly enhanced. Neurones with decreased glutathione may be at increased risk from sulfite and especially from the synergistic damaging effects of ONOO − and sulfite. Because sulfite is present normally in the brain as a product of cysteine metabolism, and because increased ONOO − formation has been reported in PD, these events might contribute to neuronal cell death.

Nitric oxide and peroxynitrite. The ugly, the uglier and the not so good

Nitric oxide, a gaseous free radical, is poorly reactive with most biomolecules but highly reactive with other free radicals. Its ability to scavenge peroxyl and other damaging radicals may make it an important antioxidant in vivo, particular in the cardiovascular system, although this ability has been somewhat eclipsed in the literature by a focus on the toxicity of peroxynitrite, generated by reaction of O·-2 with NO· (or of NO- with O2). On balance, experimental and theoretical data support the view that ONOO- can lead to hydroxyl radical (OH·) generation at pH 7.4, but it seems unlikely that OH· contributes much to the cytotoxicity of ONOO-. The cytotoxicity of ONOO- may have been over-emphasized: its formation and rapid reaction with antioxidants may provide a mechanism of using NO· to dispose of excess O·-2, or even of using O·-2 to dispose of excess NO·, in order to maintain the correct balance between these radicals in vivo. Injection or instillation of “bolus” ONOO- into animals has produced tissue injury, however, although more experiments generating ONOO- at steady rates in vivo are required. The presence of 3-nitrotyrosine in tissues is still frequently taken as evidence of ONOO- generation in vivo, but abundant evidence now exists to support the view that it is a biomarker of several “reactive nitrogen species”. Another under-addressed problem is the reliability of assays used to detect and measure 3-nitrotyrosine in tissues and body fluids: immunostaining results vary between laboratories and simple HPLC methods are susceptible to artefacts. Exposure of biological material to low pH (e.g. during acidic hydrolysis to liberate nitrotyrosine from proteins) or to H2O2 might cause artefactual generation of nitrotyrosine from NO-2 in the samples. This may be the origin of some of the very large values for tissue nitrotyrosine levels quoted in the literature. Nitrous acid causes not only tyrosine nitration but also DNA base deamination at low pH: these events are relevant to the human stomach since saliva and many foods are rich in nitrite. Several plant phenolics inhibit nitration and deamination in vitro, an effect that could conceivably contribute to their protective effects against gastric cancer development.

The Effect of Bicarbonate on Oxidation by Peroxynitrite: Implication for Its Biological Activity

The effect of bicarbonate on the oxidation rates and yields of the reaction of peroxynitrite with NiII(cyclam), ferrocyanide, and ABTS was studied using the stopped-flow technique. In the absence of bicarbonate, NiII(cyclam) is oxidized directly by peroxynitrous acid whereas ferrocyanide and ABTS are oxidized by intermediates that are formed during the self-decomposition of peroxynitrous acid. In the presence of sufficient concentrations of bicarbonate, the rate-determining step of these oxidation processes is the reaction of ONOO- with CO2. The latter reaction results in the formation of an adduct ONOOCO2-, which is incapable of oxidizing directly these substrates. It is shown that about 33% of this adduct decomposes into highly oxidizing species, most probably into •NO2 and CO3•- via the homolysis of the peroxo O−O bond. Under physiological conditions, where the most abundant species is ONOO- and the concentration of CO2 exceeds 1 mM, the half-life of peroxynitrite is reduced, and consequently its diffusion distance is shortened. Therefore, indirect oxidation by peroxynitrite through intermediates that are formed during its self-decomposition will not take place in vivo, whereas direct oxidation by peroxynitrite may take place provided that the rate of this reaction competes efficiently with that of peroxynitrite with CO2. We conclude that the toxicity of peroxynitrite in vivo is most probably governed by ONOOCO2-, which has a reactivity different from that of peroxynitrite.

Inactivation of Dehydratase [4Fe-4S] Clusters and Disruption of Iron Homeostasis upon Cell Exposure to Peroxynitrite

Phagocytes produce both nitric oxide and superoxide as components of the oxidative defense against pathogens. Neither molecule is likely at physiological concentrations to kill cells. However, two of their reaction products, hydrogen peroxide and peroxynitrite, are strong oxidants, cell-permeant, and toxic. Hydrogen peroxide generates oxidative DNA damage, while the primary mechanism of toxicity of peroxynitrite has not yet been determined. Recent in vitro studies indicated that peroxynitrite is capable of oxidizing the [4Fe-4S] clusters of a family of dehydratases (Hausladen, A., and Fridovich, I. (1994) J. Biol. Chem. 269, 29405-29408; Castro, L., Rodriguez, M., and Radi, R. (1994) J. Biol. Chem. 269, 29409-29415). We demonstrate here that peroxynitrite at 1% of its lethal dose almost fully inactivated the labile dehydratases in Escherichia coli. The rate at which peroxynitrite inactivated the clusters substantially exceeded the rate at which it oxidized thiols or spontaneously decomposed. These results suggest that these dehydratases may be primary targets of peroxynitrite in vivo. Another consequence of the cluster damage was the release of 100 microM iron into the cytosol. During phagocytosis, this intracellular free iron could increase lethal DNA damage by hydrogen peroxide or protein modification by additional peroxynitrite. In response to peroxynitrite challenges, E. coli rapidly sequestered the intracellular free iron using an undefined scavenging system. The iron-sulfur clusters were more gradually repaired by a process that drew iron from its iron-storage proteins. These are likely to be critical events in the struggle between phagocyte and pathogen.

Oxidation of cholesterol in synaptosomes and mitochondria isolated from rat brains.

Cholesterol and alpha-tocopherol oxidations were studied in brain subcellular fractions isolated from cerebral hemispheres of 4-month-old, male Fischer 344 rats. The fractions were suspended in buffered media (pH 7.4, 37 degrees C0 and oxidized by adding (i) ferrous iron (Fe2+) with or without ascorbate or (ii) peroxynitrite (an endogenous oxidant produced by the reaction of superoxide and nitric oxide). Treatment of subcellular fractions with Fe2+ in the presence or absence of ascorbate produced primarily 7-keto- and 7-hydroxy-cholesterols and small amounts of 5 alpha, 6 alpha-epoxycholesterol. Since brain contains high levels of ascorbate, and release of iron could result in oxysterol formation. Peroxynitrite oxidized alpha-tocopherol but not cholesterol. Hence, the toxicity of peroxynitrite or nitric oxide could not be due to cytotoxic oxysterols. When synaptosomes were incubated for 5 min in the presence of 0.5 to 2 microM Fe2+ and ascorbate, alpha-tocopherol was oxidized while cholesterol remained unchanged. Thus, alpha-tocopherol is functioning as an antioxidant, protecting cholesterol. Diethylenetriaminepentaacetic acid blocked production of oxysterols, whereas citrate, ADP and EDTA did not. A significant percentage of mitochondrial cholesterol was oxidized by treatment with Fe2+ and ascorbate. Hence, mitochondrial membrane properties dependent on cholesterol could be particularly susceptible to oxidation. The oxysterols formed were retained within the membranes of synaptosomes and mitochondria. The 7-oxysterols produced are known to be inhibitors of membrane enzymes and also can modify membrane permeability. Hence, oxysterols may plan an important role in brain tissue damage during oxidative stress.

Toxicity of peroxynitrite and related reactive nitrogen species toward Escherichia coli.

The toxicity of peroxynitrite toward Escherichia coli (expressed as LD50, the concentration required to kill 50% of the bacteria) was found to be independent of bacterial cell densities over a wide experimental range, spanning 10(6)-10(10) colony-forming units/mL; the magnitude of LD50 was also pH-independent over the range pH 5.9-8.3. This highly unusual behavior can be quantitatively reproduced by a dynamical model in which (i) ONO2H is identified as the toxic form of the oxidant and (ii) the bulk of the added peroxynitrite decays to nitrate ion under these conditions. From the model, one estimates that 10(6)-10(7) ONO2H molecules are required to kill a bacterium, indicating a very high intrinsic toxicity (cf. HOCl, for which LD50 = 10(7)-10(8) molecules/cell of E. coli). Nearly complete protection was observed when bicarbonate ion was added to the buffer, even when concentrations of peroxynitrite exceeded 50 times the LD50 measured in the absence of bicarbonate. Consistent with previous reports, combinations of H2O2 and NO and, in weakly acidic media, H2O2 and NO2- were found to exhibit enhanced toxicities relative to the individual reactants. Protection by bicarbonate was utilized to assess the potential role of intermediary formation of ONO2H in bacterial killing in these systems. Approximately 25% protection by bicarbonate was observed for media containing H2O2 and NO2-, consistent with a minor contribution to killing by ONO2H under the experimental conditions. No protection was observed for media containing H2O2 and *NO in both anaerobic and aerobic environments, excluding extracellularly generated ONO2H as a participant in these bactericidal reactions.

Protein Nitration by Peroxynitrite: A Method for Monitoring Nitric Oxide Neurotoxicity

During ischemia, peroxynitrite may be a toxic intermediate which formsin vivowhen nitric oxide condenses with superoxide. Alone, peroxynitrite appears to directly react with aromatic and sulfhydryl nucleophiles. At physiological pH, peroxynitrite rapidly decomposes to species with·OH and NO·2character. These reactive species are shown to initiate lipid peroxidation, hydroxylate aromatic residues, and nitrate aromatic residues. Recently, peroxynitrite was found to inactivate the polyclonal antibody to cAMP. This functional loss of activity was also correlated to a loss of structural integrity as verified by capillary electrophoresis. Here, the dose-dependent effects of peroxynitrite on the electrophoretic migration of a monoclonal antibody (monoclonal to plasminogen activator inhibitor-1) were studied. Results showed that the kinetics of migrational change with peroxynitrite could be described by two mathematical models. The first model described high affinity, first-order reactivity at one site, and lower affinity, first-order reactivity at a second site. The second model described high affinity, first-order reactivity at one site, and nonspecific, linear reactivity at multiple sites. Amino acid analysis of the antibody protein that was exposed to increasing concentrations of peroxynitrite indicated that the second model was correct. Only tyrosine residues were lost at the lowest concentration of peroxynitrite, which is consistent with high affinity, first-order reactivity at a single site. At higher concentrations of peroxynitrite, several other primarily polar amino acids were lost at a more or less linear rate. This was consistent with the second component of the model. If tyrosine residues were lost with high affinity, then perhaps a known product of tyrosine nitration by peroxynitrite, namely nitrotyrosine, might be found. A reductive/oxidative electrochemical detection scheme of nitrotyrosine was found to be 50–100 times more sensitive than standard spectrophotometric detection. This redox HPLC assay was used to measure nitrotyrosine in hydrolyzed antibody protein samples that had been previously exposed to various concentrations of peroxynitrite dose-dependently. The results show that the formation of nitrotyrosine from peroxynitrite roughly parallels the loss of tyrosine. Previously, we have shown peroxynitrite to be toxic to cerebellar granule cells and mouse spinal neurons in cell culture. These results indicate that proteins may be important targets related to the toxicity of peroxynitrite. This data suggest that nitrotyrosine may be an important marker of peroxynitrite reactivityin vivo.