8-Nitro-2'-deoxyguanosine, a specific marker of oxidation by reactive nitrogen species, is generated by the myeloperoxidase-hydrogen peroxide-nitrite system of activated human phagocytes

Reactive intermediates generated by phagocytes damage DNA and may contribute to the link between chronic inflammation and cancer. Myeloperoxidase, a heme protein secreted by activated phagocytes, is a potential catalyst for such reactions. Recent studies demonstrate that this enzyme uses hydrogen peroxide (H2O2) and nitrite (NO2-) to generate reactive nitrogen species which convert tyrosine to 3-nitrotyrosine. We now report that activated human neutrophils use myeloperoxidase, H2O2, and NO2- to nitrate 2'-deoxyguanosine, one of the nucleosides of DNA. Through HPLC, UV/vis spectroscopy, and mass spectrometry, the two major products of this reaction were identified as 8-nitroguanine and 8-nitro-2'-deoxyguanosine. Nitration required each component of the complete enzymatic system and was inhibited by catalase and heme poisons. However, it was independent of chloride ion and little affected by scavengers of hypochlorous acid, suggesting that the reactive agent is a nitrogen dioxide-like species that results from the one-electron oxidation of NO2- by myeloperoxidase. Alternatively, 2'-deoxyguanosine might be oxidized directly by the enzyme to yield a radical species which subsequently reacts with NO2- or NO2* to generate the observed products. Human neutrophils stimulated with phorbol ester also generated 8-nitroguanine and 8-nitro-2'-deoxyguanosine. The reaction required NO2- and was inhibited by catalase and heme poisons, implicating myeloperoxidase in the cell-mediated pathway. These results indicate that human neutrophils use the myeloperoxidase-H2O2-NO2- system to generate reactive species that can nitrate the C-8 position of 2'-deoxyguanosine. Our observations raise the possibility that reactive nitrogen species generated by myeloperoxidase and other peroxidases contribute to nucleobase oxidation and tissue injury at sites of inflammation.     

Alternative oxidase modulates leaf mitochondrial concentrations of superoxide and nitric oxide

? The nonenergy-conserving alternative oxidase (AOX) has been hypothesized to modulate the amount of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in plant mitochondria but there is sparse direct in planta evidence to support this. ? Laser scanning fluorescent confocal microscopy and biochemical methods were used to directly estimate in planta leaf concentrations of superoxide (O2(-)), nitric oxide (NO), peroxynitrite (ONOO(-)) and hydrogen peroxide (H(2)O(2)) in wildtype (Wt) tobacco (Nicotiana tabacum) and transgenic tobacco with altered amounts of AOX. ? We found that plants lacking AOX have increased concentrations of leaf mitochondrial-localized O2(-) and leaf NO in comparison to the Wt, while leaf concentrations of H(2)O(2) were similar or lower in the AOX-suppressed plants. ? Based on our results, we suggest that AOX respiration acts to reduce the generation of ROS and RNS in plant mitochondria by dampening the leak of single electrons from the electron transport chain to O(2) or nitrite. This may represent a universal role for AOX in plants. More work is now needed to establish the functional implications of this role, such as during abiotic and biotic stress.     

Reactions of peroxynitrite with globin proteins and their possible physiological role

It is now widely accepted that, besides their well-established function in O(2) transport, hemoglobin and myoglobin also undergo several redox reactions aimed to scavenge toxic free radicals and reactive oxygen and nitrogen species. At least some of these reactions are believed to play an important physiological role in the defense against oxidative stress. This aspect is exemplified by the recently discovered neuroglobin, a globin expressed in the brain. Rather than being considerably involved in reversible O(2) binding, neuroglobin is likely to undergo redox reactions to protect neurons against oxidative and potentially pathogenic pathways, as those operating after episodes of tissue hypoxia or ischemia. A major part of the cellular damage occurring under such conditions has been ascribed to formation of peroxynitrite, that originates from the reaction between two biologically important free radicals, nitric oxide (NO ) and superoxide. Here we review the current knowledge of the reactions of different forms of hemoglobin, myoglobin, and neuroglobin with peroxynitrite and discuss their physiological role on the basis of measured rate constants and on the probability of occurrence of these reactions in vivo.     

Cell signalling following plant/pathogen interactions involves the generation of reactive oxygen and reactive nitrogen species

It is now clear that reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂), and reactive nitrogen species, such as nitric oxide (NO), are produced by plant cells in response to a variety of stresses, including pathogen challenge. Such molecules may be involved in direct defence mechanisms, such as cross-linking of plant cell walls, or as antimicrobial agents. However, it is also apparent that cells generate such reactive species as signalling molecules, produced at controlled levels, and leading to defined responses. Signalling responses to ROS and NO include the activation of mitogen-activated protein kinases, and the up- and down-regulation of gene expression, often leading to localised programmed cell death, characteristic of the hypersensitive response. Therefore, ROS and NO are key molecules which may help to orchestrate events following pathogen challenge. Here we review the generation and role of both reactive oxygen and reactive nitrogen species in plant cells.     

Evidence that covalent complex formation between BCNU-treated oligonucleotides and E. coli alkyltransferases requires the O6-alkylguanine function.

Chloroethylnitrosoureas (CENUs) are thought to induce cytotoxic DNA interstrand cross-links via an initial reaction at O6-position of guanine, yielding a rearranged intermediate, O6,N1-ethanoguanine. Repair of these adducts by mammalian and bacterial DNA alkyltransferases blocks the formation of cross-links. Human alkyltransferase can form a covalent complex with DNA containing BCNU-induced cross-link precursors, but the nature of the DNA-protein linkage remains unknown. Using E. coli alkyltransferases expressed by the ada and ogt genes, we now demonstrate that both enzymes can form such complexes with CENU-treated DNA. We attribute this reaction to the O6-alkylguanine repair function, because an N-terminal fragment of the ada protein, which has only alkylphosphotriester repair activity, failed to form a similar complex. This result is consistent with the idea that complex formation requires an alkyltransferase reaction with a guanine adduct, such as O6,N1-ethanoguanine. It tends to exclude the possibility that such reactions simply involve alkylation of the enzyme by reactive DNA adducts such as chloroethylphosphate or chloroethylguanine.     

A tale of two controversies: defining both the role of peroxidases in nitrotyrosine formation in vivo using eosinophil peroxidase and myeloperoxidase-deficient mice, and the nature of peroxidase-generated reactive nitrogen species

Nitrotyrosine is widely used as a marker of post-translational modification by the nitric oxide ((.)NO, nitrogen monoxide)-derived oxidant peroxynitrite (ONOO(-)). However, since the discovery that myeloperoxidase (MPO) and eosinophil peroxidase (EPO) can generate nitrotyrosine via oxidation of nitrite (NO(2)(-)), several questions have arisen. First, the relative contribution of peroxidases to nitrotyrosine formation in vivo is unknown. Further, although evidence suggests that the one-electron oxidation product, nitrogen dioxide ((*)NO(2)), is the primary species formed, neither a direct demonstration that peroxidases form this gas nor studies designed to test for the possible concomitant formation of the two-electron oxidation product, ONOO(-), have been reported. Using multiple distinct models of acute inflammation with EPO- and MPO-knockout mice, we now demonstrate that leukocyte peroxidases participate in nitrotyrosine formation in vivo. In some models, MPO and EPO played a dominant role, accounting for the majority of nitrotyrosine formed. However, in other leukocyte-rich acute inflammatory models, no contribution for either MPO or EPO to nitrotyrosine formation could be demonstrated. Head-space gas analysis of helium-swept reaction mixtures provides direct evidence that leukocyte peroxidases catalytically generate (*)NO(2) formation using H(2)O(2) and NO(2)(-) as substrates. However, formation of an additional oxidant was suggested since both enzymes promote NO(2)(-)-dependent hydroxylation of targets under acidic conditions, a chemical reactivity shared with ONOO(-) but not (*)NO(2). Collectively, our results demonstrate that: 1) MPO and EPO contribute to tyrosine nitration in vivo; 2) the major reactive nitrogen species formed by leukocyte peroxidase-catalyzed oxidation of NO(2)(-) is the one-electron oxidation product, (*)NO(2); 3) as a minor reaction, peroxidases may also catalyze the two-electron oxidation of NO(2)(-), producing a ONOO(-)-like product. We speculate that the latter reaction generates a labile Fe-ONOO complex, which may be released following protonation under acidic conditions such as might exist at sites of inflammation.     

Oxygen and nitrogen are pro-carcinogens. Damage to DNA by reactive oxygen, chlorine and nitrogen species: measurement, mechanism and the effects of nutrition.

Humans are exposed to many carcinogens, but the most significant may be the reactive species derived from metabolism of oxygen and nitrogen. Nitric oxide seems unlikely to damage DNA directly, but nitrous acid produces deamination and peroxynitrite leads to both deamination and nitration. Scavenging of reactive nitrogen species generated in the stomach may be an important role of flavonoids, flavonoids and other plant-derived phenolic compounds. Different reactive oxygen species produce different patterns of damage to DNA bases, e.g., such patterns have been used to implicate hydroxyl radical as the ultimate agent in H(2)O(2)-induced DNA damage. Levels of steady-state DNA damage in vivo are consistent with the concept that such damage is a major contributor to the age-related development of cancer and so such damage can be used as a biomarker to study the effects of diet or dietary supplements on risk of cancer development, provided that reliable assays are available. Methodological questions addressed in this article include the validity of measuring 8-hydroxydeoxyguanosine (8OHdG) in cellular DNA or in urine as a biomarker of DNA damage, the extent of artifact formation during analysis of oxidative DNA damage by gas chromatography-mass spectrometry and the levels of oxidative damage in mitochondrial DNA.     

Hypothesis: the role of reactive sulfur species in oxidative stress.

Oxidative stress arises from an imbalance in the metabolism of redox-active species promoting the formation of oxidizing agents. At present, these species are thought to include reactive oxygen, reactive nitrogen, and reactive nitrogen oxygen species (ROS, RNS, and RNOS, respectively). Reactive species have their origin in enzymatic synthesis, environmental induction, or by the further chemical reaction of an active species with other endogenous molecules to generate a second-generation reactive species. These second-generation species possess a different spectrum of activity to the parent species, with different redox reactions and biological targets. We now propose that an additional group of redox active molecules termed "reactive sulfur species" (RSS) are formed in vivo under conditions of oxidative stress. RSS are likely to include disulfide-S-oxides, sulfenic acids, and thiyl radicals, and are predicted to modulate the redox status of biological thiols and disulfides.     

Free radical biology and medicine: it's a gas, man!

We review gases that can affect oxidative stress and that themselves may be radicals. We discuss O(2) toxicity, invoking superoxide, hydrogen peroxide, and the hydroxyl radical. We also discuss superoxide dismutase (SOD) and both ground-state, triplet oxygen ((3)O(2)), and the more energetic, reactive singlet oxygen ((1)O(2)). Nitric oxide ((*)NO) is a free radical with cell signaling functions. Besides its role as a vasorelaxant, (*)NO and related species have other functions. Other endogenously produced gases include carbon monoxide (CO), carbon dioxide (CO(2)), and hydrogen sulfide (H(2)S). Like (*)NO, these species impact free radical biochemistry. The coordinated regulation of these species suggests that they all are used in cell signaling. Nitric oxide, nitrogen dioxide, and the carbonate radical (CO(3)(*-)) react selectively at moderate rates with nonradicals, but react fast with a second radical. These reactions establish "cross talk" between reactive oxygen (ROS) and reactive nitrogen species (RNS). Some of these species can react to produce nitrated proteins and nitrolipids. It has been suggested that ozone is formed in vivo. However, the biomarkers that were used to probe for ozone reactions may be formed by non-ozone-dependent reactions. We discuss this fascinating problem in the section on ozone. Very low levels of ROS or RNS may be mitogenic, but very high levels cause an oxidative stress that can result in growth arrest (transient or permanent), apoptosis, or necrosis. Between these extremes, many of the gasses discussed in this review will induce transient adaptive responses in gene expression that enable cells and tissues to survive. Such adaptive mechanisms are thought to be of evolutionary importance.     

Critical O2 and NO concentrations in NO-induced cell death in a rat liver sinusoidal endothelial cell line

Nitric oxide (NO) plus oxygen (O2) are known to cause cell damage via formation of reactive nitrogen species. NO itself directly inhibits cytochrome oxidase of the mitochondrial respiratory chain in competition with O2, thus inducing a hypoxic-like injury. To assess the critical NO and O2 concentrations for both mechanisms of NO-induced cell injury, cells of a rat liver sinusoidal endothelial cell line were incubated in the presence of the NO donor spermineNONOate at different O2 concentrations, and their loss of viability was determined by the release of lactate dehydrogenase. Protection by ascorbic acid was used as indication for the involvement of reactive nitrogen species, whereas a hypoxic-like injury was indicated by the protective effects of glycine and glucose and the increase in NAD(P)H fluorescence. High concentrations of NO (approx. 10 microM NO) and O2 (21% O2) were required to induce endothelial cell death mediated by formation of reactive nitrogen species. On the other hand, pathophysiologically relevant NO concentrations at low but physiological O2 concentrations (ca. 2 microM NO at 5% O2 and about 1 microM NO at 2% O2) induced hypoxic-like cell death in the endothelial cells that was prevented by the presence of glucose.     

Metal ions and oxygen radical reactions in human inflammatory joint disease

Activated phagocytic cells produce superoxide (O2-) and hydrogen peroxide (H2O2); their production is important in bacterial killing by neutrophils and has been implicated in tissue damage by activated phagocytes. H2O2 and O2- are poorly reactive in aqueous solution and their damaging actions may be related to formation of more reactive species from them. One such species is hydroxyl radical (OH.), formed from H2O2 in the presence of iron- or copper-ion catalysts. A major determinant of the cytotoxicity of O2- and H2O2 is thus the availability and location of metal-ion catalysts of OH. formation. Hydroxyl radical is an initiator of lipid peroxidation. Iron promoters of OH. production present in vivo include ferritin, and loosely bound iron complexes detectable by the 'bleomycin assay'. The chelating agent Desferal (desferrioxamine B methanesulphonate) prevents iron-dependent formation of OH. and protects against phagocyte-dependent tissue injury in several animal models of human disease. The use of Desferal for human treatment should be approached with caution, because preliminary results upon human rheumatoid patients have revealed side effects. It is proposed that OH. radical is a major damaging agent in the inflamed rheumatoid joint and that its formation is facilitated by the release of iron from transferrin, which can be achieved at the low pH present in the micro-environment created by adherent activated phagocytic cells. It is further proposed that one function of lactoferrin is to protect against iron-dependent radical reactions rather than to act as a catalyst of OH. production.     

Formation of 8-nitroguanosine in cellular RNA as a biomarker of exposure to reactive nitrogen species

Reactive nitrogen species, such as peroxynitrite, nitrogen oxides and nitryl chloride, have been implicated as a cause of diverse pathophysiological conditions, including inflammation, neurodegenerative and cardiovascular diseases and cancer. We previously reported that 8-nitroguanine is formed by reactions of guanine or calf-thymus DNA with peroxynitrite in vitro. In the present study, we have studied the formation of 8-nitroguanosine and 8-oxo-7,8-dihydroguanosine in reactions of calf-liver RNA with various reactive nitrogen species. 8-Nitroguanosine in RNA was found to be much more stable than 8-nitro-2' -deoxyguanosine in DNA, which rapidly depurinates to release 8-nitroguanine. Both 8-nitroguanosine and 8-oxo-7,8-dihydroguanosine were formed in calf-liver RNA following exposure to various reactive nitrogen species, such as synthetic peroxynitrite. They were also formed in RNA by reactive species formed from nitric oxide and superoxide anion generated concomitantly from 3-morpholino-sydnonimine (SIN-1) and those formed with myeloperoxidase or horseradish peroxidase in the presence of nitrite and hydrogen peroxide. 8-Nitroguanosine was detected by HPLC with an electrochemical detector in enzymatic hydrolyzates of RNA isolated from human lung carcinoma cells incubated with synthetic peroxynitrite. Our results indicate that 8-nitroguanosine in cellular RNA could be measured as a marker of damage caused by endogenous reactive nitrogen species in tissues and cells.     

Redox imbalance

Substantial evidence implies that redox imbalance attributable to an overproduction of reactive oxygen species or reactive nitrogen species that overwhelm the protective defense mechanism of cells contributes to all forms of Parkinsons disease. Factors such as dopamine, neuromelanin, and transition metals may, under certain circumstances, contribute to the formation of oxygen species such as H₂O₂, superoxide radicals, and hydroxyl radicals and react with reactive nitrogen species such as nitric oxide or peroxinitrite. Mitochodrial dysfunction and excitotoxicity may be a cause and a result of oxidative stress. Consequences of this redox imbalance are lipid peroxidation, oxidation of proteins, DNA damage, and interference of reactive oxygen species with signal transduction pathways. These consequences become even more harmful when genetic variations impair the normal degradation of altered proteins. Therefore, therapeutic strategies must aim at reducing free-radical formation and scavenging free-radicals.     

Sphingolipid signaling and redox regulation

Sphingolipids including ceramide and its derivatives such as ceramide-1-phosphate, glycosyl-ceramide, and sphinogosine (-1-phosphate) are now recognized as novel intracellular signal mediators for regulation of inflammation, apoptosis, proliferation, and differentiation. One of the important and regulated steps in these events is the generation of these sphingolipids via hydrolysis of sphingomyelin through the action of sphingomyelinases (SMase). Several lines of evidence suggest that reactive oxygen species (ROS; O2-, H2O2, and OH-,) and reactive nitrogen species (RNS; NO, and ONOO-) and cellular redox potential, which is mainly regulated by cellular glutathione (GSH), are tightly linked to the regulation of SMase activation. On the other hand, sphingolipids are also known to play an important role in maintaining cellular redox homeostasis through regulation of NADPH oxidase, mitochondrial integrity, and antioxidant enzymes. Therefore, this paper reviews the relationship between cellular redox and sphingolipid metabolism and its biological significance.     

Generation of oxygen free radicals in thyroid cells and inhibition of thyroid peroxidase

We examined whether superoxide (O(2)(-)) is produced as a precursor of hydrogen peroxide (H(2)O(2)) in cultured thyroid cells using the cytochrome c method and the electron paramagnetic resonance (EPR) method. No O(2)(-) or its related radicals was detected in thyroid cells under the physiological condition. The presence of quinone, 2,3-dimethoxy-l-naphthoquinone (DMNQ), or 2-methyl-1, 4-naphthoquinone (menadione), in the medium produced O(2)(-) and hydroxyl radicals (OH*); the amount of H(2)O(2) generation was also increased. Incubation of follicles with DMNQ or menadione inhibited iodine organification (a step of thyroid hormone formation) and its catalytic enzyme, thyroid peroxidase (TPO). This inhibition should be caused by reactive oxygen species because the two quinones, particularly DMNQ, exert their effect through the generation of reactive oxygen species. It is speculated that the site-specific inactivation of TPO might have occurred at the heme-linked histidine residue of the TPO molecule, a critical amino acid for enzyme activity because OH* (vicious free radicals) can be formed at the iron-linked amino acid. TPO mRNA level and electrophoretic mobility of TPO were not inhibited by quinones. Our study suggests that thyroid H(2)O(2) is produced by divalent reduction of oxygen without O(2)(-) generation. If thyroid cells happen to be exposed to significant amount of reactive oxygen species, TPO and subsequent thyroid hormone formation are inhibited.     

Nitration and nitrosation of N-acetyl-L-tryptophan and tryptophan residues in proteins by various reactive nitrogen species

Proteins are targets of reactive nitrogen species such as peroxynitrite and nitrogen dioxide. Among the various amino acids in proteins, tryptophan residues are especially susceptible to attack by reactive nitrogen species. We carried out experiments on the reactions of peroxynitrite and other reactive nitrogen species with N-acetyl-L-tryptophan under various conditions. Four major products were identified as 1-nitroso-N-acetyl-L-tryptophan, 1-nitro-N-acetyl-L-tryptophan, 6-nitro-N-acetyl-L-tryptophan, and N-acetyl-N'-formyl-L-kynurenine on the basis of their mass and UV spectra. The reactions with SIN-1 (a peroxynitrite generator), Angeli's salt (a nitroxyl donor), and spermine NONOate (a nitric oxide donor) generated the nitroso derivative but not the nitro derivatives. A myeloperoxidase-H(2)O(2)-NO(2)(-) system generated the nitro derivatives but not the nitroso derivative. Under physiological conditions 6-nitro-N-acetyl-L-tryptophan was stable, whereas the 1-nitroso and 1-nitro derivatives decomposed with half-lives of 1.5 and 18 h, respectively. After treatment with various reactive nitrogen species, bovine serum albumin was enzymatically hydrolyzed and analyzed for 6-nitro-L-tryptophan and 3-nitro-L-tyrosine by HPLC with electrochemical detection. Levels of 6-nitro-L-tryptophan and 3-nitro-L-tyrosine were similar in the nitrated protein. 6-Nitro-L-tryptophan in proteins can be measured as an additional biomarker of protein nitration.     

Body nitrosation potential measured by a novel 15N breath test

Oxygenated nitrogen species, for example, the protonated form of nitrous acid (H2ONO+), dinitrogentrioxide (N2O3), dinitrogentetroxide (N2O4), or peroxynitrite (ONOO-), can react with amines to form molecular nitrogen. These reactions can occur spontaneously with primary aliphatic amines or via cytochrome P450 catalysed reactions with secondary amines. In principle measurements of the excretion of the molecular nitrogen generated by these reactions could be used as an index of the levels of oxygenated nitrogen compounds acting as nitrosating agents. To test this idea, [15N2]urea (3 mmol) was administered orally to five patients infected with Helicobacter pylori (as diagnosed by the [13C]urea breath test) and to four healthy volunteers. All participants ingested 3-mmol sodium nitrate as a precursor for NA 5 min before the ingestion of the nitrogen tracer. During the test the participants breathed 100% oxygen to increase the sensitivity of detection of endogenous molecular nitrogen. After the administration of [15N2]urea, the patients with H. pylori showed significantly increased 15N enrichments of exhaled N2, expressed as delta value (per 1000), compared with healthy volunteers (patients: 3.5 +/- 0.9 vs. volunteers: 1.3 +/- 0.4; p < .05). We speculate that the endogenous production of molecular nitrogen is a protective process controlling the body NO and nitrite levels. The 15N breath technique allows the noninvasive estimation of the body nitrosation and could indicate the health risk, possibly the oxidative stress status, caused by highly reactive oxygenated nitrogen species and carbenium ion intermediates.     

Cells with dysfunctional telomeres are susceptible to reactive oxygen species hydrogen peroxide via generation of multichromosomal fusions and chromosomal fragments bearing telomeres

During genotoxic stress, reactive oxygen species hydrogen peroxide (H(2)O(2)) is a prime mediator of the DNA damage response. Telomeres function both to assist in DNA damage repair and to inhibit chromosomal end-to-end fusion. Here, we show that telomere dysfunction renders cells susceptible to H(2)O(2), via generation of multichromosomal fusion and chromosomal fragments. H(2)O(2) caused formation of multichromosomal end-to-end fusions involving more than three chromosomes, preferentially when telomeres were erosive. Interestingly, extensive chromosomal fragmentation (yielding small-sized fragments) occurred only in cells exhibiting such multichromosomal fusions. Telomeres were absent from fusion points, being rather present in the small fragments, indicating that H(2)O(2) cleaves chromosomal regions adjacent to telomeres. Restoration of telomere function or addition of the antioxidant N-acetylcysteine prevented development of chromosomal aberrations and rescued the observed hypersensitivity to H(2)O(2). Thus, chromosomal regions adjacent to telomeres become sensitive to reactive oxygen species hydrogen peroxide when telomeres are dysfunctional, and are cleaved to produce multichromosomal fusions and small chromosomal fragments bearing the telomeres.     

Unique oxidative mechanisms for the reactive nitrogen oxide species, nitroxyl anion

The nitroxyl anion (NO-) is a highly reactive molecule that may be involved in pathophysiological actions associated with increased formation of reactive nitrogen oxide species. Angeli's salt (Na2N2O3; AS) is a NO- donor that has been shown to exert marked cytotoxicity. However, its decomposition intermediates have not been well characterized. In this study, the chemical reactivity of AS was examined and compared with that of peroxynitrite (ONOO-) and NO/N2O3. Under aerobic conditions, AS and ONOO- exhibited similar and considerably higher affinities for dihydrorhodamine (DHR) than NO/N2O3. Quenching of DHR oxidation by azide and nitrosation of diaminonaphthalene were exclusively observed with NO/N2O3. Additional comparison of ONOO- and AS chemistry demonstrated that ONOO- was a far more potent one-electron oxidant and nitrating agent of hydroxyphenylacetic acid than was AS. However, AS was more effective at hydroxylating benzoic acid than was ONOO-. Taken together, these data indicate that neither NO/N2O3 nor ONOO- is an intermediate of AS decomposition. Evaluation of the stoichiometry of AS decomposition and O2 consumption revealed a 1:1 molar ratio. Indeed, oxidation of DHR mediated by AS proved to be oxygen-dependent. Analysis of the end products of AS decomposition demonstrated formation of NO2- and NO3- in approximately stoichiometric ratios. Several mechanisms are proposed for O2 adduct formation followed by decomposition to NO3- or by oxidation of an HN2O3- molecule to form NO2-. Given that the cytotoxicity of AS is far greater than that of either NO/N2O3 or NO + O2, this study provides important new insights into the implications of the potential endogenous formation of NO- under inflammatory conditions in vivo.     

Vasoactive intestinal peptide, a singlet oxygen quencher

The neuropeptide vasoactive intestinal peptide (VIP), a highly basic 28-amino acid peptide, has a widespread distribution in the body. The functional specificity of this peptide not only includes its potent vasodilatory activity, but also its role in protecting lungs against acute injury, in preventing T-lymphocyte proliferation and in modulating immune function. We have investigated the possible antioxidant properties of VIP and found that VIP does not have significant O2-, OH., or H2O2 scavenging ability. However, VIP was found to inhibit, in a dose-dependent manner, the 1O2-dependent 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO) formation. 1O2 was produced in photosensitizing systems using rose bengal or methylene blue as sensitizers and was detected as TEMP-1O2 product (TEMPO) by electron paramagnetic resonance (EPR) spectroscopic techniques. The formation of TEMPO signal was strongly inhibited by known singlet quenchers, e.g. beta-carotene, histidine as well as azide, but not by catalase (20 micrograms/ml) which removes H2O2 and mannitol (6 mM) or ethanol (5.9 mM) which remove OH.. Superoxide dismutase (2.5 micrograms/ml) inhibited the photoreaction up to 20% by removing O2- and most probably by blocking the secondary charge transfer pathway of 1O2 formation. These results suggest that the formation of nitroxide radical by 1O2 attack on TEMP may be used as a simple and specific assay for 1O2, and VIP can serve as an effective 1O2 scavenger/quencher, thus it may modulate the oxidative tissue injury caused by this reactive species of oxygen.