Electron paramagnetic resonance spectroscopy with N-methyl-D-glucamine dithiocarbamate iron complexes distinguishes nitric oxide and nitroxyl anion in a redox-dependent manner: applications in identifying nitrogen monoxide products from nitric oxide synthase

Though a large number of studies indicate that nitric oxide synthase (NOS) is responsible for NO&z.rad; production in biological systems, controversy still remains concerning whether NOS directly produces NO&z.rad;. Schmidt et al. (PNAS 93:144492, 1996) proposed that NOS first synthesizes nitroxyl anion (NO(-)), which is then converted to NO&z.rad; by superoxide dismutase (SOD). With electron paramagnetic resonance spectroscopy using N-methyl-D-glucamine dithiocarbamate iron (Fe-MGD), we directly detected NO&z.rad; from purified NOS in the absence of SOD (Xia et al., PNAS 94:12705, 1997). We also showed that the requirement for SOD in the previous NO&z.rad; measurements appeared to be due to the high levels of exogenous superoxide production in their reaction system because of the presence of free FAD. However, it was recently questioned whether Fe-MGD can discriminate NO&z.rad; from NO(-) (Komarov et al., FRBM 28:739-742, 2000). In this study we examined the trapping specificity of different redox forms of Fe-MGD. With Fe(2+)-MGD, NO&z.rad; generated characteristic triplet NO&z.rad;-Fe(2+)-MGD signals (g = 2. 04, a(N) = 12.7 G), whereas NO(-) from Angeli's salt was EPR silent. Both NO&z.rad; and NO(-) gave rise to NO&z.rad;-Fe(2+)-MGD signals when Fe(3+)-MGD was used. Strong NO&z.rad; signals were measured from purified nNOS using the NO&z.rad; selective Fe(2+)-MGD and this was not affected by SOD. Thus, spin trapping with Fe-MGD can distinguish NO&z.rad; and NO(-) and this depends on the redox status of the iron. The detection of NO&z.rad; from purified NOS by Fe(2+)-MGD unambiguously reconfirms our previous report that NOS directly synthesizes NO&z.rad; but not NO(-).     

Further insights into the reaction of melatonin with hydroxyl radical

Recent interest has focused on the use of exogenous melatonin as an antioxidant, particularly to scavenge the highly cytotoxic hydroxyl radical (HO(z.rad;)) which may be generated in many pathological conditions. However, in vitro and in vivo studies aimed at assessing the antioxidant properties of melatonin have produced conflicting results. While it is known that HO(z.rad;) reacts with melatonin at a diffusion limited rate, very little is known about the products of this reaction. In this investigation it is shown that incubation of melatonin with a Fenton-type HO(z.rad;)-generating system at pH 7.4 forms a complex mixture of primary products. These include 2-hydroxymelatonin, which was isolated as its more stable oxindole tautomer, 4- and 6-hydroxymelatonin, N-acetyl-N(2)-formyl-5-methoxykynurenine and 7,7(')-bi-(5-methoxy-N-acetyltryptamine-4-one). Reaction pathways that might lead to these products are described. The differing biological effects of these products, while currently incompletely understood, might account for the controversy concerning the antioxidant properties of melatonin.     

Product formation and kinetic simulations in the pH range 1-14 account for a free-radical mechanism of peroxynitrite decomposition

The yields of nitrate and nitrite from decomposition of peroxynitrite in phosphate buffer at 37 degrees C were determined in the pH range 1-14. The NO(2)(-)/NO(3)(-) yields showed a stepwise variation with pH, with inflection points at approximately pH 3.1, 5.8, 6.8, 8.0, and 11.9. Nitrite formation increased strongly above pH 7 at the expense of nitrate, but above pH 12 nitrate again became the major product (80% at pH 14). At this pH, the Arrhenius parameters were E(a)=24.1+/-0.2kcal mol(-1) and A=(4.9+/-1.3)x10(12)s(-1). The yields of NO(2)(-), NO(3)(-), and O(2) measured at pH 5.8, 7.4, and 8.5 as a function of the initial peroxynitrite concentration (50-1000 microM) were linear only at pH 5.8. In the presence of carbon dioxide, oxygen production at pH 7.5 and pH 10 was found to be linear on the CO(2) concentration. The experimental observations were satisfactorily reproduced by kinetic simulations including principal component analyses. These data strongly suggest that the chemistry of peroxynitrite is exclusively mediated by z.rad;NO(2) and HO(z.rad;) radicals in the absence, and by z.rad;NO(2) and CO(3)(z.rad;-) radicals in the presence of CO(2).     

Simulated air dives induce superoxide,nitric oxide,peroxynitrite, and Ca 2+ alterations in endothelial cells

Human diving is known to induce endothelial dysfunction. The aim of this study was to decipher the mechanism of ROS production during diving through the measure of mitochondrial calcium concentration, peroxynitrite, NO°, and superoxide towards better understanding of dive-induced endothelial dysfunction. Air diving simulation using bovine arterial endothelial cells (compression rate 101 kPa/min to 808 kPa, time at depth 45 min) was performed in a system allowing real-time fluorescent measurement. During compression, the cells showed increased mitochondrial superoxide, peroxynitrite, and mitochondrial calcium, and decreased NO° concentration. MnTBAP (peroxynitrite scavenger) suppressed superoxide, recovered NO° production and promoted stronger calcium influx. Superoxide and peroxynitrite were inhibited by L-NIO (eNOS inhibitor), but were further increased by spermine-NONOate (NO° donor). L-NIO induced stronger calcium influx than spermine-NONOate or simple diving. The superoxide and peroxynitrite were also inhibited by ruthenium red (blocker of mitochondrial Ca2+ uniporter), but were increased by CGP (an inhibitor of mitochondrial Na+-Ca2+ exchange). Reactive oxygen and nitrogen species changes are associated, together with calcium mitochondrial storage, with endothelial cell dysfunction during simulated diving. Peroxynitrite is involved in NO° loss, possibly through the attenuation of eNOS and by increasing superoxide which combines with NO° and forms more peroxynitrite. In the field of diving physiology, this study is the first to unveil a part of the cellular mechanisms of ROS production during diving and confirms that diving-induced loss of NO° is linked to superoxide and peroxynitrite.     

Involvement of the mitogen-activated protein kinase cascade in peroxynitrite-mediated arachidonic acid release in vascular smooth muscle cells

Eicosanoid production is reduced when the nitric oxide (NO·) pathway is inhibited or when the inducible NO synthase gene is deleted, indicating that the NO· and arachidonic acid pathways are linked. We hypothesized that peroxynitrite, formed by the reaction of NO· and superoxide anion, may cause signaling events leading to arachidonic acid release and subsequent eicosanoid generation. Western blot analysis of rat arterial smooth muscle cells demonstrated that peroxynitrite (100–500 µM) and 3-morpholinosydnonimine (SIN-1; 200 µM) stimulate phosphorylation of extracellular signal-regulated kinase (ERK), p38, and cytosolic phospholipase A₂ (cPLA₂). We found that peroxynitrite-induced arachidonic acid release was completely abrogated by the mitogen-activated protein/ERK kinase (MEK) inhibitor U0126 and by calcium chelators. With the p38 inhibitor SB-20219, we demonstrated that peroxynitrite-induced p38 phosphorylation led to minor arachidonic acid release, whereas U0126 completely blocked p38 phosphorylation. Addition of arachidonic acid caused p38 phosphorylation, suggesting that arachidonic acid or its metabolites are responsible for p38 activation. KN-93, a specific inhibitor of Ca²⁺/calmodulin-dependent kinase II (CaMKII), revealed no role for this kinase in peroxynitrite-induced arachidonic acid release in our cell system. Together, these results show that in response to peroxynitrite the cell initiates the MEK/ERK cascade leading to cPLA₂ activation and arachidonic acid release. Thus studies investigating the role of the NO· pathway on eicosanoid production must consider the contribution of signaling pathways initiated by reactive nitrogen species. These findings may provide evidence for a new role of peroxynitrite as an important reactive nitrogen species in vascular disease. reactive nitrogen species; prostaglandin H₂ synthase; extracellular signal-regulated kinase; p38; cytosolic phospholipase A₂     

Chemical and biological evidence for base propenals as the major source of the endogenous M1dG adduct in cellular DNA

The endogenous DNA adduct, M(1)dG, has been shown to arise in vitro in reactions of dG with malondialdehyde (MDA), a product of both lipid peroxidation and 4'-oxidation of deoxyribose in DNA, and with base propenals also derived from deoxyribose 4'-oxidation. We now report the results of cellular studies consistent with base propenals, and not MDA, as the major source of M1dG under biological conditions. As a foundation for cellular studies, M1dG, base propenals, and MDA were quantified in purified DNA treated with oxidizing agents known to produce deoxyribose 4'-oxidation. The results revealed a consistent pattern; Fe2+-EDTA and gamma-radiation generated MDA but not base propenals or M1dG, whereas bleomycin and peroxynitrite (ONOO-) both produced M1dG as well as base propenals with no detectable MDA. These observations were then assessed in Escherichia coli with controlled membrane levels of polyunsaturated fatty acids (PUFA). ONOO- treatment (2 mm) of cells containing no PUFA (defined medium with 18:0/stearic acid) produced 6.5 M1dG/10(7) deoxynucleotides and no detectable lipid peroxidation products, including MDA, as compared with 3.8 M1dG/10(7) deoxynucleotides and 0.07 microg/ml lipid peroxidation products with control cells grown in a mixture of fatty acids (0.5% PUFA) mimicking Luria-Bertani medium. In cells grown with linoleic acid (18:2), the level of PUFA rose to 54% and the level of MDA rose to 0.14 microg/ml, whereas M1dG fell to 1.4/10(7) deoxynucleotides. Parallel studies with gamma-radiation revealed levels of MDA similar to those produced by ONOO- but no detectable M1dG. These results are consistent with base propenals as the major source of M1dG in this model cell system.     

beta-Carotene: interactions with alpha-tocopherol and ascorbic acid in microsomal lipid peroxidation

beta-Carotene, alpha-tocopherol, and ascorbic acid were tested for their ability to inhibit, enhance, or react synergistically with O(2) (15, 150, 760 torr) and, 2,2'-azobis (2-amidino-propane) dihydrochloride (AAPH) or 1,1'-azobis (cyclohexane-carbonitrile) (ACCN) in isolated rat liver microsomes. beta-Carotene did not protect against lipid peroxidation, i.e., malondialdehyde (MDA) formation, in microsomal samples incubated at 37 degrees C with aqueous soluble AAPH at all added beta-carotene concentrations and oxygen tensions. More MDA (16%, p < 0.001) was produced at 15 torr of O(2,) and 160 nmol/mg protein of beta-carotene compared to respective vehicle control. Individually, alpha-tocopherol and ascorbic acid exhibited antioxidant protection (ascorbic acid &z.Gt; alpha-tocopherol); however, a mixture of both compounds was no more protective than ascorbic acid alone. beta-Carotene demonstrated a concentration-dependent antioxidant affect at 15 torr O(2) (p < 0.01); but a prooxidant effect at higher O(2) at 150 and 760 torr (>57%, p < 0.001) by lipid-soluble ACCN. alpha-Tocopherol exhibited concentration-dependent inhibitory effects on microsomal MDA formation at all oxygen tensions, but was most effective under 150 torr. Ascorbic acid demonstrated a concentration-dependent antioxidant effect only at 150 torr. ACCN-induced lipid peroxidation was no greater for the combination of the three compounds than ascorbic acid added alone. Thus, antioxidant or prooxidant activities for beta-carotene, alpha-tocopherol, and ascorbic acid in microsomal suspensions are related to O(2) tension, solubility, antioxidant concentrations and are governed by complex interactions. Differences between AAPH- and ACCN-induced lipid peroxidation are related to differences in lipid solubility.     

Superoxide formed from cigarette smoke impairs polymorphonuclear leukocyte active oxygen generation activity.

Reactive free radicals contained in cigarette smoke (CS) and compromised phagocytic antimicrobial activities including those of polymorphonuclear leukocytes (PMNs) have been implicated in the pathogenesis of severe CS-related pulmonary disorders. In CS-exposed buffer solutions, O2-. was the predominant generated reactive oxygen species, as demonstrated by lucigenin-amplified chemiluminescence and electron spin resonance (ESR) spin-trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO). When PMNs were incubated in this buffer, phorbol 12-myristate 13-acetate (PMA)-stimulated active oxygen production and coupled O2 consumption were strongly impaired without appreciably affecting PMN viability (1-min exposure inhibited active oxygen production by 75%). Superoxide dismutase (SOD) totally protected and an iron chelator, diethylenetriaminepentaacetic acid (DETAPAC), also protected the CS-exposed PMNs, suggesting that generated O2-. was an initiating factor in the impairment and OH. generation was a subsequent injurious factor. Pretreatment of PMNs with antioxidants such as alpha-tocopherol and dihydrolipoic acid (DHLA) was partially protective. The results suggest that (i) O2-. is probably generated in the upper and lower respiratory tract lining fluid when they come in contact with CS; (ii) such generated O2-. can primarily impair PMN capabilities to generate reactive oxygen species; and (iii) since these effects may contribute to the pathogenesis of CS-related lung diseases, prior supplementation with antioxidants such as alpha-tocopherol or DHLA might be successful in preventing these deleterious effects.     

Radicals associated with the catalytic intermediates of bovine cytochrome c oxidase

Two radicals have been detected previously by electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies in bovine cytochrome oxidase after reaction with hydrogen peroxide, but no correlation could be made with predicted levels of optically detectable intermediates (P(M), F and F(z.rad;)) that are formed. This work has been extended by optical quantitation of intermediates in the EPR/ENDOR sample tubes, and by comparison with an analysis of intermediates formed by reaction with carbon monoxide in the presence of oxygen. The narrow radical, attributed previously to a porphyrin cation, is detectable at low levels even in untreated oxidase and increases with hydrogen peroxide treatments generally. It is presumed to arise from a side-reaction unrelated to the catalytic intermediates. The broad radical, attributed previously to a tryptophan radical, is observed only in samples with a significant level of F(z.rad;) but when F(z.rad;) is generated with hydrogen peroxide, is always accompanied by the narrow radical. When P(M) is produced at high pH with CO/O(2), no EPR-detectable radicals are formed. Conversion of the CO/O(2)-generated P(M) into F(z.rad;) when pH is lowered is accompanied by the appearance of a broad radical whose ENDOR spectrum corresponds to a tryptophan cation. Quantitation of its EPR intensity indicates that it is around 3% of the level of F(z.rad;) determined optically. It is concluded that low pH causes a change of protonation pattern in P(M) which induces partial electron redistribution and tryptophan cation radical formation in F(z.rad;). These protonation changes may mimic a key step of the proton translocation process.     

Effects of oxygen radicals on the conformation of sulfhydryl groups on human polymorphonuclear leukocyte membranes.

The healthy intact polymorphonuclear leukocytes (PMNs) were labeled with 4-maleimide-TEMPO spin labeling compound (MAL) to study the effects of oxygen radicals produced by phorbol myristate acetate (PMA)-stimulated PMNs on the conformation of sulfhydryl (SH) groups of PMN membrane proteins. The lipid peroxidation induced by PMA-stimulated PMNs was detected by evaluating the formation of malonaldehyde (MDA) with the thiobarbituric acid (TBA) test. From the experiments of luminol-dependent chemiluminescence (CL) and fluorometry, it was found that Chinese herbs schizandrin B (Sin B) and quercetin (Q) possessed scavenging properties for oxygen radicals produced during the PMN respiratory burst. These two herbs can also inhibit the conformation changes in SH binding sites on the PMN membrane proteins caused by oxygen radicals produced by the PMNs themselves. They also decreased the amount of MDA, which was a final product formed during lipid peroxidation.     

DELAYED URIC ACID ACCUMULATION IN PLASMA PROVIDES ADDITIONAL ANTI-OXIDANT PROTECTION AGAINST IRON-TRIGGERED OXIDATIVE STRESS AFTER A WINGATE TEST

Reactive oxygen species are produced during anaerobic exercise mostly by Fe ions released into plasma and endothelial/muscle xanthine oxidase activation that generates uric acid (UA) as the endpoint metabolite. Paradoxically, UA is considered a major antioxidant by virtue of being able to chelate pro-oxidative iron ions. This work aimed to evaluate the relationship between UA and plasma markers of oxidative stress following the exhaustive Wingate test. Plasma samples of 17 male undergraduate students were collected before, 5 and 60 min after maximal anaerobic effort for the measurement of total iron, haem iron, UA, ferric-reducing antioxidant activity in plasma (FRAP), and malondialdehyde (MDA, biomarker of lipoperoxidation). Iron and FRAP showed similar kinetics in plasma, demonstrating an adequate pro-/antioxidant balance immediately after exercise and during the recovery period (5–60 min). Slight variations of haem iron concentrations did not support a relevant contribution of rhabdomyolysis or haemolysis for iron overload following exercise. UA concentration did not vary immediately after exercise but rather increased 29% during the recovery period. Unaltered MDA levels were concomitantly measured. We propose that delayed UA accumulation in plasma is an auxiliary antioxidant response to post-exercise (iron-mediated) oxidative stress, and the high correlation between total UA and FRAP in plasma (R-Square = 0.636; p = 0.00582) supports this hypothesis.     

Hemiketal formation of dehydroascorbic acid drives ascorbyl radical anion disproportionation

In this paper the relationship between the ascorbate anion (AH(-)) and its oxidation products, ascorbyl radical anion (A&z.rad;(-)) and dehydroascorbic acid (DHA), are studied by means of theoretical calculations. Additional calculations are performed on alpha-hydroxytetronate, a model compound of ascorbate lacking the side chain. The method uses density functional theory with the B3LYP functional and a polarizable conductor dielectric model to compute solvation effects. Our results indicate that the model compound reacts with the alpha-tocopheroxyl radical to regenerate vitamin E with a free energy change of reaction (in water) of -7.4 kcal/mol. This reaction is 2.9 kcal/mol more exergonic than the corresponding reaction involving ascorbate, suggesting that the model compound may make a more effective antioxidant than ascorbate. However, the disproportionation of the ascorbyl radical anion, a reaction that regenerates AH(-), is found to be exergonic while the similar reaction involving the model compound is slightly endergonic. The reason for the difference is that the disproportionation of A&z. rad;(-) is found to be driven by the formation of the hemiketal structure of dehydroascorbic acid (DHA).     

Determination of salicylate hydroxylation products as an in vivo oxidative stress marker

The in vivo measurement of highly reactive free radicals, such as the z.rad OH radical, is very difficult. New specific markers, which are based on the ability of z.rad OH to attack the benzene rings of aromatic molecules, are currently under investigation. The produced hydroxylated compounds can be measured directly. In vivo, radical metabolism of salicylic acid produces two main hydroxylated derivatives (2,3- and 2,5-dihydroxybenzoic acids). The latter acid can be also produced by enzymatic pathways through the cytochrome P-450 system, while the former acid is reported to be solely formed by direct hydroxyl radical attack. Therefore, measurement of 2, 3-DHBA, following oral administration of the drug acetyl salicylate, could be proposed for assessment of oxidative stress in vivo. In this paper, a sensitive method for the identification and quantification of hydroxylation products from the reaction of z. rad OH with salicylate in vivo is presented. It employs a high performance liquid chromatography and electrochemical detection system. A detection limit of < 1 pmol for the hydroxylation products has been achieved with linear response over at least five orders of magnitude. Using this technique, we measured plasma levels of 2,3- and 2,5-DHBA dihydroxylated derivatives and salicylic acid and determined the ratios following administration of 1 g acetyl salicylate in 20 healthy subjects.     

In vitro studies of interactions of NO· donor drugs with superoxide and hydroxyl radicals

Nitric oxide (NO·) is a free radical characterized by a high spontaneous chemical reactivity with many other molecules including the superoxide radical (O₂·^–). This complex interaction may generate a peroxynitrite anion (ONOO^–), which behaves as an important mediator of oxidative stress in many pathological states. In the present study, in vitro experiments were performed to assess directly the O₂·^– and hydroxyl (·OH) radical scavenging effects of various NO· donor drugs, i.e. sodium nitroprusside (SNP), sodium nitrite (NaNO₂), molsidomine and SIN 1, at pH 7.4, 7 or 6. Concentrations of NO· in the incubation medium containing the different NO· donor drugs were measured by the assay based on the reaction of Fe-N-methyl-D-glucamine dithiocarbamate (MGD) with NO· that yields a stable spin-adduct measured by electron paramagnetic resonance (EPR). O₂·^– and ·OH generation was characterized by EPR spin trapping techniques, using the spin trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO). These free radicals were generated from the enzymatic system xanthine-xanthine oxidase, in phosphate buffer adjusted at pH 7.4, 7 and 6. Under these experimental conditions, SNP exhibited the strongest superoxide scavenging properties, characterized by IC₅₀ values expressed in the µmolar range, which decreased at low pH. Addition of SNP (800 µM) to solution containing MGD and Fe²⁺ (5:1) at pH 7 4 produced a three line EPR spectrum which is identified to [(MGD)₂-Fe²⁺-NO]. In control experiments no EPR signal was observed. We obtained the same results with NaNO₂ and an augmentation of the spin-adduct level was noted with the prolongation of the incubation period. In return, molsidomine (2 mM) did not produce, in our conditions, a detectable production of NO·. NaNO₂ displayed a significant superoxide scavenging effect only at pH 6, whilst neither molsidomine nor SIN 1 had any effect. Therefore, the superoxide scavenging properties of SNP, NaNO₂, and molsidomine appeared to be closely related to their potential for NO· release, which partially depends on the pH conditions. The behaviour of SIN 1 is more complicated, the speed of oxygen diffusion probably acting as a limiting factor in NO· formation in our conditions. The production of NO· was detected in presence of SIN 1. The intensity of the complex is comparable with the signal founded with NaNO₂. By contrast, all molecules exhibited hydroxyl radical scavenging properties, highlighting the capacity of ·OH to react with a wide range of molecules. In conclusion, considering the poor chemical reactivity of O₂·^–, the NO· donor drugs/O₂·^– interactions suggest a special relationship between these two radical species, which, in certain pathological states, could lead to the generation of cytotoxic end-products with strong oxidizing properties.     

The role of mitochondrial dysfunction and neuronal nitric oxide in animal models of neurodegenerative diseases

Excitotoxicity, mitochondrial dysfunction and free radical induced oxidative damage have been implicated in the pathogenesis of several different neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinson's disease (PD), Alzheimer's disease (AD), and Huntington's disease. Much of the interest in the association of neurodegeneration with mitochondrial dysfunction and oxidative damage emerged from animal studies using mitochondrial toxins. Within mitochondria 1-methyl-4-phenylpyridinium (MPP⁺), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), acts to inhibit NADH-coenzyme Q reductase (complex I) of the electron transport chain. MPTP produces Parkinsonism in humans, primates, and mice. Similarly, lesions produced by the reversible inhibitor of succinate dehydrogenase (complex II), malonate, and the irreversible inhibitor, 3-nitropropionic acid (3-NP), closely resemble the histologic, neurochemical and clinical features of HD in both rats and non-human primates. The interruption of oxidative phosphorylation results in decreased levels of ATP. A consequence is partial neuronal depolarization and secondary activation of voltage-dependent NMDA receptors, which may result in excitotoxic neuronal cell death (secondary excitotoxicity). The increase in intracellular Ca²⁺ concentration leads to an actiation of Ca²⁺ dependent enzymes, including the constitutive neuronal nitric oxide synthase (cnNOS) which produces NO·. NO· may react with the superoxide anion to form peroxynitrite. We show that systemic administration of 7-nitroindazole (7-NI), a relatively specific inhibitor of cnNOS in vivo. attenuates lesions produced by striatal malonate injections or systemic treatment with 3-NP or MPTP. Furthermore 7-NI attenuated increases in lactate production and hydroxyl radical and 3-nitrotyrosine generation in vivo, which may be a consequence of peroxynitrite formation. Our results suggest that neuronal nitric oxide synthase inhibitors may be useful in the treatment of neurologic diseases in which excitotoxic mechanisms play a role. (Mol Cell Biochem 174: 193–197, 1997)     

Nitric oxide production by rat bronchoalveolar macrophages or polymorphonuclear leukocytes following intratracheal instillation of lipopolysaccharide or silica

Exposure of the lung to lipopolysaccharide (LPS) or silica results in an activation of alveolar macrophages (AMs), recruitment of polymorphonuclear leukocytes (PMNs) into bronchoalveolar spaces, and the production of free radicals. Nitric oxide (NO) is one of the free radicals generated by bronchoalveolar lavage (BAL) cell populations following either LPS or silica exposure. The purpose of the present study was to assess the relative contributions of AMs and PMNs to the amounts of NO produced by BAL cells following intratracheal (IT) instillation of either LPS or silica. Male Sprague Dawley rats (265-340 g body wt.) were given LPS (10 mg/100 g body wt.) or silica (5 mg/100 g body wt.). BAL cells were harvested 18-24 h post-IT and enriched for AMs or PMNs using density gradient centrifugation. Media levels of nitrate and nitrite (NOx; the stable decomposition products of NO) were then measured 18 h after ex vivo culture of these cells. Following IT exposure to either LPS or silica, BAL cell populations were approximately 20% AMs and approximately 80% PMNs. After density gradient centrifugation of BAL cells from LPS- or silica-treated rats, cell fractions were obtained which were relatively enriched for AMs (approximately 60%) or PMNs (approximately 90%). The amounts of NOx produced by the AM-enriched fractions from LPS- or silica-treated rats were approximately 2-4-fold greater than that produced by the PMN-enriched fractions. Estimations of the relative contribution of AMs or PMNs to the NOx produced indicated that: (i) following LPS treatment, 75%-89% of the NOx was derived from AMs and 11%-25% from PMNs; and (ii) following silica treatment, 76%-100% of the NOx was derived from AMs and 0-24% from PMNs. Immunohistochemistry for inducible NO synthase on lung tissue sections supported these findings. We conclude that AMs are the major source of the NO produced by BAL cells during acute pulmonary inflammatory responses to LPS or silica.     

Inhibition of hypochlorous acid-induced oxidative reactions by nitrite: is nitrite an antioxidant?

Acute and chronic inflammation result in increased nitrogen monoxide (z.rad;NO) formation and the accumulation of nitrite (NO(2)(-)). Neutrophils stimulated by various inflammatory mediators release myeloperoxidase to produce the cytotoxic agent hypochlorous acid (HOCl). At physiologically attainable concentrations, we found that NO(2)(-) significantly inhibits HOCl-mediated DNA strand breakage and ascorbate depletion. HOCl-mediated inactivation of pure alpha(1)-antiproteinase or of the elastase inhibitory capacity of human plasma was inhibited by the addition of NO(2)(-). NO(2)(-) was more effective than ascorbate, GSH, and urate at inhibiting HOCl-mediated toxicity to human HepG2 cells in culture. These data suggest that NO(2)(-) may act in an antioxidant manner by removing HOCl at sites of inflammation where both HOCl and z.rad;NO are overproduced.     

The reactions of hydrogen peroxide with bovine cytochrome c oxidase

Oxidised cytochrome c oxidase is known to react with two molecules of hydrogen peroxide to form consecutively 607 nm 'Peroxy' and 580-nm 'Ferryl' species. These are widely used as model compounds for the equivalent P and F intermediates of the catalytic cycle. However, kinetic analysis of the reaction with H(2)O(2) in the pH range 6.0-9.0 reveals a more complex situation. In particular, as the pH is lowered, a 580-nm compound can be formed by reaction with a single H(2)O(2). This species, termed F(&z.rad;), is spectrally similar, but not identical, to F. The reactions are equivalent to those previously reported for the bo type quinol oxidase from Escherichia coli (T. Brittain, R.H. Little, C. Greenwood, N.J. Watmough, FEBS Lett. 399 (1996) 21-25) where it was proposed that F(&z.rad;) is produced directly from P. However, in the bovine oxidase F(&z.rad;) does not appear in samples of the 607-nm form, P(M), produced by CO/O(2) treatment, even at low pH, although this form is shown to be identical to the H(2)O(2)-derived P state, P(H), on the basis of spectral characteristics and kinetics of reaction with H(2)O(2). Furthermore, lowering the pH of a sample of P(M) or P(H) generated at high pH results in F(&z.rad;) formation only on a minutes time scale. It is concluded that P and F(&z.rad;) are not in a rapid, pH-dependent equilibrium, but instead are formed by distinct pathways and cannot interconvert in a simple manner, and that the crucial difference between them lies in their patterns of protonation.     

Reactions of Peroxynitrite with Uric Acid: Formation of Reactive Intermediates,Alkylated Products and Triuret,and In Vivo Production of Triuret Under Conditions of Oxidative Stress

Hyperuricemia is associated with hypertension, metabolic syndrome, preeclampsia, cardio-vascular disease and renal disease, all conditions associated with oxidative stress. We hypothesized that uric acid, a known antioxidant, might become prooxidative following its reaction with oxidants; and, thereby contribute to the pathogenesis of these diseases. Uric acid and 1,3-¹⁵N₂-uric acid were reacted with peroxynitrite in different buffers and in the presence of alcohols, antioxidants and in human plasma. The reaction products were identified using liquid chromatography-mass spectrometry (LC-MS) analyses. The reactions generate reactive intermediates that yielded triuret as their final product. We also found that the antioxidant, ascorbate, could partially prevent this reaction. Whereas triuret was preferentially generated by the reactions in aqueous buffers, when uric acid or 1,3-¹⁵N₂-uric acid was reacted with peroxynitrite in the presence of alcohols, it yielded alkylated alcohols as the final product. By extension, this reaction can alkylate other biomolecules containing OH groups and others containing labile hydrogens. Triuret was also found to be elevated in the urine of subjects with preeclampsia, a pregnancy-specific hypertensive syndrome that is associated with oxidative stress, whereas very little triuret is produced in normal healthy volunteers. We conclude that under conditions of oxidative stress, uric acid can form reactive intermediates, including potential alkylating species, by reacting with peroxynitrite. These reactive intermediates could possibly explain how uric acid contributes to the pathogenesis of diseases such as the metabolic syndrome and hypertension.