Quinoid redox cycling as a mechanism for sustained free radical generation by inhaled airborne particulate matter.

The health effects of airborne fine particles are the subject of government regulation and scientific debate. The aerodynamics of airborne particulate matter, the deposition patterns in the human lung, and the available experimental and epidemiological data on health effects lead us to focus on airborne particulate matter with an aerodynamic mean diameter less than 2.5 microm (PM(2.5)) as the fraction of the particles with the largest impact in health. In this article we present a novel hypothesis to explain the continuous production of reactive oxygen species produced by PM(2.5) when it is deposited in the lung. We find PM(2.5) contains abundant persistent free radicals, typically 10(16) to 10(17) unpaired spins/gram, and that these radicals are stable for several months. These radicals are consistent with the stability and electron paramagnetic resonance spectral characteristics of semiquinone radicals. Catalytic redox cycling by semiquinone radicals is well documented in the literature and we had studied in detail its role on the health effects of cigarette smoke particulate matter. We believe that we have for the first time shown that the same, or similar radicals, are not confined to cigarette smoke particulate matter but are also present in PM(2.5). We hypothesize that these semiquinone radicals undergo redox cycling, thereby reducing oxygen and generating reactive oxygen species while consuming tissue-reducing equivalents, such as NAD(P)H and ascorbate. These reactive oxygen species generated by particles cause oxidative stress at sites of deposition and produce deleterious effects observed in the lung.

Role of free radicals in the toxicity of airborne fine particulate matter.

Exposure to airborne fine particles (PM2.5) is implicated in excess of 50 000 yearly deaths in the USA as well as a number of chronic respiratory illnesses. Despite intense interest in the toxicity of PM2.5, the mechanisms by which it causes illnesses are poorly understood. Since the principal source of airborne fine particles is combustion and combustion sources generate free radicals, we suspected that PM2.5 may contain radicals. Using electron paramagnetic resonance (EPR), we examined samples of PM2.5 and found large quantities of radicals with characteristics similar to semiquinone radicals. Semiquinone radicals are known to undergo redox cycling and ultimately produce biologically damaging hydroxyl radicals. Aqueous extracts of PM2.5 samples induced damage to DNA in human cells and supercoiled phage DNA. PM2.5-mediated DNA damage was abolished by superoxide dismutase, catalase, and deferoxamine, implicating superoxide radical, hydrogen peroxide, and the hydroxyl radical in the reactions inducing DNA damage.

Reaction of uric acid with peroxynitrite and implications for the mechanism of neuroprotection by uric acid.

Peroxynitrite, a biological oxidant formed from the reaction of nitric oxide with the superoxide radical, is associated with many pathologies, including neurodegenerative diseases, such as multiple sclerosis (MS). Gout (hyperuricemic) and MS are almost mutually exclusive, and uric acid has therapeutic effects in mice with experimental allergic encephalomyelitis, an animal disease that models MS. This evidence suggests that uric acid may scavenge peroxynitrite and/or peroxynitrite-derived reactive species. Therefore, we studied the kinetics of the reactions of peroxynitrite with uric acid from pH 6.9 to 8.0. The data indicate that peroxynitrous acid (HOONO) reacts with the uric acid monoanion with k = 155 M(-1) s(-1) (T = 37 degrees C, pH 7.4) giving a pseudo-first-order rate constant in blood plasma k(U(rate))(/plasma) = 0.05 s(-1) (T = 37 degrees C, pH 7.4; assuming [uric acid](plasma) = 0.3 mM). Among the biological molecules in human plasma whose rates of reaction with peroxynitrite have been reported, CO(2) is one of the fastest with a pseudo-first-order rate constant k(CO(2))(/plasma) = 46 s(-1) (T = 37 degrees C, pH 7.4; assuming [CO(2)](plasma) = 1 mM). Thus peroxynitrite reacts with CO(2) in human blood plasma nearly 920 times faster than with uric acid. Therefore, uric acid does not directly scavenge peroxynitrite because uric acid can not compete for peroxynitrite with CO(2). The therapeutic effects of uric acid may be related to the scavenging of the radicals CO(*-)(3) and NO(*)(2) that are formed from the reaction of peroxynitrite with CO(2). We suggest that trapping secondary radicals that result from the fast reaction of peroxynitrite with CO(2) may represent a new and viable approach for ameliorating the adverse effects associated with peroxynitrite in many diseases.

Induction of inflammatory mediators in human airway epithelial cells by lipid ozonation products.

We have proposed that exposure of epithelial cell membrane lipids in the lung (mainly phospholipids) to ozone will generate lipid ozonation products (LOP), which could be responsible for the proinflammatory effects of ozone. The ozonation of phosphocholine, the principal membrane phospholipid, produces a limited number of LOP, including hydroxyhydroperoxides and aldehydes. We now report that exposure of cultured human bronchial epithelial cells to the ozonized 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) product, 1-palmitoyl-2-(9-oxononanoyl)-sn-glycero-3-phosphocholine (PC-ALD), a phospholipase A(2) (PLA(2))-stimulatory LOP, resulted in a 113 +/- 11% increase in the amounts of tritiated platelet-activating factor ((3)H-PAF) released apically. (3)H-PAF release was also induced by 1-hydroxy-1-hydroperoxynonane of ozonized POPC (HHP-C9), a phospholipase C (PLC)- stimulatory LOP (134 +/- 40% increase in (3)H-PAF). PC-ALD at 10 microM, but not HHP-C9, induced a 127 +/- 24% increase in prostaglandin E(2) (PGE(2)) release (n = 6, p < 0.05). In contrast, HHP-C9, but not PC-ALD, induced interleukin (IL)-6 release (178 +/- 23% increase, n = 6, p < 0.05) and IL-8 release (101 +/- 23% increase, n = 8, p < 0. 05). These results suggest that LOP-dependent release of proinflammatory mediators may play an important role in the early inflammatory response seen during exposure to ozone.

Reaction of peroxynitrite with melatonin: A mechanistic study.

The pH profile of the peroxynitrite/melatonin reaction suggests that both peroxynitrous acid (ONOOH) and its anion (ONOO-) are reactive toward melatonin, but at physiological pH most of the reaction with melatonin involves ONOOH and the activated form of peroxynitrous acid (ONOOH). The formation of hydroxylated products (mainly 6-hydroxymelatonin) suggests that melatonin also reacts with ONOOH. The overall peroxynitrite/melatonin reaction is first-order in melatonin and first-order in peroxynitrite, but the hydroxylation of melatonin is presumed to be zero-order in melatonin. Melatonin is metabolized in the liver, mainly to 6-hydroxymelatonin, so we do not think this metabolite is a useful biomarker for melatonin's antioxidant activity; however, 6-hydroxymelatonin is a better chain-breaking antioxidant than melatonin and may contribute to the beneficial effects of melatonin in vivo. As is now well-known, CO2 modulates the reactions of peroxynitrite. The reaction of peroxynitrite with melatonin in the absence of added bicarbonate produces mainly 6-hydroxymelatonin and 1,2,3,3a,8, 8a-hexahydro-1-acetyl-5-methoxy-8a-hydroxypyrrolo[2,3-b]indole, with some isomeric 1,2,3,3a,8, 8a-hexahydro-1-acetyl-5-methoxy-3a-hydroxypyrrolo[2,3-b]indole. In the presence of added bicarbonate, product yields decrease and 6-hydroxymelatonin is not formed. These facts suggest that melatonin scavenges reactive species (such as CO3*- and *NO2) that are produced from the peroxynitrite/CO2 reaction. The spectrum of the melatoninyl radical cation is observed both in the absence and in the presence of added bicarbonate, suggesting that the melatoninyl radical cation is the initial product and the hydroxypyrrolo[2, 3-b]indole products are derived from it. Unlike tyrosine, where both nitrated and hydroxylated products can be isolated, nitromelatonin is not found in the final products from the melatonin/peroxynitrite reaction in either the absence or presence of added bicarbonate. However, we suggest that 2-hydroxy-3-nitro- and/or 2-hydroxy-3-peroxynitro-2,3-dihydromelatonin are formed as intermediates and subsequently decompose to give 1,2,3,3a,8, 8a-hexahydro-1-acetyl-5-methoxy-8a-hydroxypyrrolo[2,3-b]indole. Since peroxynitrite/CO2 governs the reactions of peroxynitrite in vivo, we suggest that the hydroxypyrrolo[2,3-b]indole products are the main products from the oxidation of melatonin by peroxynitrite-derived species in vivo, and that these products may serve as indexes for melatonin's antioxidant activity.

Direct and simultaneous ultraviolet second-derivative spectrophotometric determination of nitrite and nitrate in preparations of peroxynitrite.

We have determined the initial concentrations of nitrite and nitrate for three different methods of synthesizing peroxynitrite using an ultraviolet second-derivative spectroscopy method (Fig. 3). As expected, the net nitrogen balance in these preparations (Fig. 4) and the yields of nitrite and nitrate (Table II) indicate that, at pH 6.0, peroxynitrite decomposes to give essentially NO3-. Stock solutions of peroxynitrite prepared using method I (ozonation of azide) consistently contain more NO2- and NO3- than method II (isoamyl nitrite with hydrogen peroxide) and method III (hydrogen peroxide with nitrous acid). Method II gives the least amount of NO2- contaminants, and NO3- impurities are the lowest in method III (Table I).

The reaction of melatonin with peroxynitrite: formation of melatonin radical cation and absence of stable nitrated products.

Peroxynitrite is capable of hydroxylating and nitrating aromatic species. However, nitromelatonin is not found as a final product when melatonin was allowed to react with peroxynitrite either in the presence or absence of added bicarbonate. In the absence of bicarbonate, the two major products formed are 6-hydroxymelatonin and 5-methoxy-2-hydro-pyrroloindole, and the latter is the only major product with excess bicarbonate. A transient purple intermediate with a maximum absorbance at about 520 nm is observed upon mixing solutions containing peroxynitrite and melatonin. These observations indicate that the melatoninyl radical cation is formed in the peroxynitrite/melatonin reaction, providing a direct evidence for the one-electron oxidation ability of peroxynitrite. The melatoninyl radical cation also is observed with excess bicarbonate.

Nitrosation by peroxynitrite: use of phenol as a probe.

Nitrosation is an important pathway in the metabolism of nitric oxide, producing S-nitrosothiols that may be critical signal transduction species. The reaction of peroxynitrite with aromatic compounds in the pH range of 5 to 8 has long been known to produce hydroxylated and nitrated products. However, we here present evidence that peroxynitrite also can promote the nitrosation of nucleophiles. We chose phenol as a substrate because the nitrosation reaction was first recognized during a study of the CO2-modulation of the patterns of hydroxylation and nitration of phenol by peroxynitrite (Lemercier et al., Arch. Biochem. Biophys. 345, 160-170, 1997). 4-Nitrosophenol, the principal nitrosation product, is detected at pH 7.0, along with 2- and 4-nitrophenols; 4-nitrosophenol becomes the dominant product at pH >/= 8.0. The yield of 4-nitrosophenol continues to increase even after pH 11.1, 1. 2 units above the pKa of phenol, suggesting that the phenolate ion, and not phenol, is involved in the reaction. Hydrogen peroxide is not formed as a by-product. The nitrosation reaction is zero-order in phenol and first-order in peroxynitrite, suggesting the phenolate ion reacts with an activated nitrosating species derived from peroxynitrite, and not with peroxynitrite itself. Under optimal conditions, the yields of 4-nitrosophenol are comparable to those of 2- and 4-nitrophenols, indicating that the nitrosation reaction is as significant as the nitration of phenolic compounds by peroxynitrite. Low concentrations of CO2 facilitate the nitrosation reaction, but excess CO2 dramatically reduces the yield of 4-nitrosophenol. The dual effects of CO2 can be rationalized if O=N-OO- reacts with the peroxynitrite anion-CO2 adduct (O=N-OOCO-2) or secondary intermediates derived from it, including the nitrocarbonate anion (O2N-OCO-2), the carbonate radical (CO*-3), and *NO2. The product resulting from these reactions can be envisioned as an activated intermediate X-N=O (where X is -OONO2, -NO2, or -CO-3) that could transfer a nitrosyl cation (NO+) to the phenolate ion. An alternative mechanism for the nitrosation of phenol involves the one-electron oxidation of the phenolate ion by CO*-3 to give the phenoxyl radical and the oxidation of O=N-OO- by CO*-3 to give a nitrosyldioxyl radical (O=N-OO*), which decomposes to give *NO and O2; the *NO then reacts with the phenoxyl radical giving nitrosophenol. Both mechanisms are consistent with the high yields of NO-2 and O2 during the alkaline decomposition of peroxynitrite and the potent inhibitory effect of N-3 on the nitrosation of phenol by peroxynitrite and peroxynitrite/CO2 adducts. The biological significance of the peroxynitrite-mediated nitrosations is discussed.

Oxidative chemistry of nitric oxide: the roles of superoxide, peroxynitrite, and carbon dioxide.

The roles of superoxide (O2.-), peroxynitrite, and carbon dioxide in the oxidative chemistry of nitric oxide (.NO) are reviewed. The formation of peroxynitrite from .NO and O2.- is controlled by superoxide dismutase (SOD), which can lower the concentration of superoxide ions. The concentration of CO2 in vivo is high (ca. 1 mM), and the rate constant for reaction of CO2 with -OONO is large (pH-independent k = 5.8 x 10(4) M(-l)s(-1)). Consequently, the rate of reaction of peroxynitrite with CO2 is so fast that most commonly used scavengers would need to be present at very high, near toxic levels in order to compete with peroxynitrite for CO2. Therefore, in the presence of physiological levels of bicarbonate, only a limited number of biotargets react directly with peroxynitrite. These include heme-containing proteins such as hemoglobin, peroxidases such as myeloperoxidase, seleno-proteins such as glutathione peroxidase, proteins containing zinc-thiolate centers such as the DNA-binding transcription factors, and the synthetic antioxidant ebselen. The mechanism of the reaction of CO2 with OONO produces metastable nitrating, nitrosating, and oxidizing species as intermediates. An analysis of the lifetimes of the possible intermediates and of the catalysis of peroxynitrite decompositions suggests that the reactive intermediates responsible for reactions with a variety of substrates may be the free radicals .NO2 and CO3.-. Biologically important reactions of these free radicals are, for example, the nitration of tyrosine residues. These nitrations can be pathological, but they also may play a signal transduction role, because nitration of tyrosine can modulate phosphorylation and thus control enzymatic activity. In principle, it might be possible to block the biological effects of peroxynitrite by scavenging the free radicals .NO2 and CO3.-. Because it is difficult to directly scavenge peroxynitrite because of its fast reaction with CO2, scavenging of intermediates from the peroxynitrite/CO2 reaction would provide an additional way of preventing peroxynitrite-mediated cellular effects. The biological effects of peroxynitrite also can be prevented by limiting the formation of peroxynitrite from .NO by lowering the concentration of O2.- using SOD or SOD mimics. Increased formation of peroxynitrite has been linked to Alzheimer's disease, rheumatoid arthritis, atherosclerosis, lung injury, amyotrophic lateral sclerosis, and other diseases.

Lipid ozonation products activate phospholipases A2, C, and D.

Ozone exposure, in vitro, has been shown to activate phospholipases A2 (PLA2), C (PLC), and D (PLD) in airway epithelial cells. However, because of its high reactivity, ozone cannot penetrate far into the air/lung tissue interface. It has been proposed that ozone reacts with unsaturated fatty acids (UFA) in the epithelial lining fluid (ELF) and cell membranes to generate a cascade of lipid ozonation products (LOP) that mediate ozone-induced toxicity. To test this hypothesis, we exposed cultured human bronchial epithelial cells (BEAS-2B) to LOP (1-100 microM) produced from the ozonation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and measured the activity of PLA2, PLC, and PLD. The PLA2 isoform responsible for arachidonic acid release (AA) in stimulated cultures was also characterized. Activation of PLA2, PLC, and PLD by three oxidants, hydrogen peroxide (H2O2), tert-butyl hydroperoxide (t-BOOH) and 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) also was measured and compared to that of LOP. The derivatives of ozonized POPC at the sn-2 residue, 9-oxononanoyl (PC-ALD), 9-hydroxy-9-hydroperoxynonanoyl (PC-HHP), and 8-(-5-octyl-1,2,4-trioxolan-3-yl-) octanoyl (POPC-OZ) selectively activated PLA2 in a dose-dependent fashion. Cytosolic PLA2 (cPLA2) measured in the cytosolic fraction of stimulated cell lysates was found to be the predominant isoform responsible for AA release. PLC activation was exclusively induced by the hydroxyhydroperoxide derivatives. PC-HHP and the 9-carbon hydroxyhydroperoxide (HHP-C9) increased PLC activity. PLD activity also was induced by LOP generated from POPC. Incubation of cultures with H2O2 alone did not stimulate PLC; however, in the presence of the aldehyde, nonanal, a 62 +/- 2% increase in PLC activity was found, suggesting that the increase in activity was due to the formation of the intermediate HHP-C9. t-BOOH, and AAPH also failed to induce PLA2 activation, but did activate PLC, under conditions of exposure identical to that of LOP. Only t-BOOH activated PLD. These results suggest that biologically relevant concentrations of LOP activate PLA2, PLC, and PLD in the airway epithelial cell, a primary target to ozone exposure. The activation of these phospholipases may play a role in the development of lung inflammation during ozone exposure.

Inactivation of glutathione peroxidase by peroxynitrite.

Glutathione peroxidase (GSH-Px) is inactivated on exposure to peroxynitrite under physiologically relevant conditions. Stopped-flow kinetic studies show that the reaction between peroxynitrite and GSH-Px is first-order in each of the reactants, with an apparent second-order rate constant of 4.5 +/- 0.2 x 10(4) M-1 s-1 per monomer unit of enzyme. In good agreement with this value, GSH-Px inactivation experiments afford an apparent second-order rate constant of 1.8 +/- 0.1 x 10(4) M-1 s-1 per monomer unit of enzyme. The hydroxyl radical scavengers mannitol, DMSO, and benzoate (at 100 mM) afford only 8-12% protection of the enzyme, while addition of 25 mM bicarbonate results in 55% protection. The minimal protection by hydroxyl radical scavengers indicates, as expected, that hydroxyl radicals are not involved in the inactivation. Protection by bicarbonate occurs because peroxynitrite is rapidly trapped by CO2 to form the adduct nitrosoperoxycarbonate (ONOOCO2-), and/or other reactive species that preferentially decompose to nitrate rather than react with GSH-Px. The close agreement between the rate constants obtained from enzyme inactivation and from stopped-flow kinetics experiments suggests that the mechanism of the reaction between peroxynitrite and GSH-Px involves the oxidation of the ionized selenol of the selenocysteine residue in the enzyme's active site (E-Se-) by peroxynitrite. This reaction does not simply involve formation of the selenenic acid, E-SeOH, because E-SeOH is an intermediate in the catalytic cycle of the enzyme, and thus its formation cannot explain the inactivation we observe. Thus, the ionized selenol in the active site is transformed into a form of selenium that cannot easily be reduced back to the selenol.

Carbon dioxide modulation of hydroxylation and nitration of phenol by peroxynitrite.

We have examined the formation of hydroxyphenols, nitrophenols, and the minor products 4-nitrosophenol, benzoquinone, 2,2'-biphenol, and 4,4'-biphenol from the reaction of peroxynitrite with phenol in the presence and absence of added carbonate. In the absence of added carbonate, the product yields of nitrophenols and hydroxyphenols have different pH profiles. The rates of nitration and hydroxylation also have different pH profiles and match the trends observed for the product yields. At a given pH, the sum of the rate constants for nitration and hydroxylation is nearly identical to the rate constant for the spontaneous decomposition of peroxynitrite. The reaction of peroxynitrite with phenol is zero-order in phenol, both in the presence and absence of added carbonate. In the presence of added carbonate, hydroxylation is inhibited, whereas the rate of formation and yield of nitrophenols increase. The combined maximum yield of o- and p-nitrophenols is 20 mol% (based on the initial concentration of peroxynitrite) and is about fourfold higher than the maximal yield obtained in the absence of added carbonate. The o/p ratio of nitrophenols is the same in the presence and absence of added carbonate. These results demonstrate that hydroxylation and nitration occur via two different intermediates. We suggest that the activated intermediate formed in the isomerization of peroxynitrous acid to nitrate, ONOOH*, is the hydroxylating species. We propose that intermediate 1, O=N-OO-CO2-, or secondary products derived from it, is (are) responsible for the nitration of phenol. The possible mechanisms responsible for nitration are discussed.

The mechanism of the peroxynitrite-carbon dioxide reaction probed using tyrosine.

Peroxynitrite (ONOO-), which is produced in vivo by the reaction of nitric oxide (.NO) with superoxide (O2-.), is a selective oxidant. Peroxynitrite decomposes to form nitrate at neutral pH and the decomposition is accelerated by CO2, which is abundant in biological systems, but the mechanism of this CO2-assisted decomposition of peroxynitrite is not fully understood. Previously we showed that CO2 recycles and is a true catalyst in the reaction. In this work, we probed the reaction of peroxynitrite with a limiting amount of CO2 at pH 7.4 and 25 degrees C in the presence of tyrosine. Tyrosine does not react directly with peroxynitrite, but it is able to trap intermediates that arise from the CO2-peroxynitrite reaction. The addition of tyrosine to the CO2-peroxynitrite system results in a reduced rate of decay of peroxynitrite. This effect is easier to identify in later stages of the reaction, and therefore we have used the lifetime of peroxynitrite (the time it takes for the concentration of peroxynitrite to fall to 1/e of its initial value) to characterize the reaction. The values of the lifetime of peroxynitrite rapidly reach a plateau with increasing concentrations of tyrosine. However, even at the plateau value, the lifetime of peroxynitrite still is much shorter than that observed in the absence of added CO2. To rationalize these effects, we suggest that tyrosine partially traps intermediates that arise from the CO2-peroxynitrite reaction, causing the fraction of the CO2 that recycles to decrease and the lifetime of peroxynitrite to increase. Possible intermediates involved in the CO2-peroxynitrite reaction that could be trapped by tyrosine are discussed.

Inhibition of peroxynitrite-mediated oxidation of glutathione by carbon dioxide.

Peroxynitrite reacts with CO2 to from an adduct containing a weak O--O bond that can undergo homolytic and/or heterolytic cleavage to give other reactive intermediates. Because the peroxynitrite/CO2 reaction is fast and physiological concentrations of CO2 are relatively high, peroxynitrite-mediated oxidations of biological species probably involve the peroxynitrite-CO2 adduct and its subsequent reactive intermediates. We have examined the reaction of glutathione with peroxynitrite in the presence and absence of added bicarbonate. In the presence of added bicarbonate, CO2 competes with glutathione for peroxynitrite, resulting in a markedly decreased consumption of glutathione compared with that observed in the absence of added bicarbonate. However, the consumption of glutathione still is much higher than predicted from the assumption that the glutathione-peroxynitrite reaction is the only reaction that can consume glutathione in this system. These results suggest that glutathione partially, but not completely, traps intermediate(s) derived from the peroxynitrite and CO2 reaction. Some rate constants for the trapping of the intermediates are estimated by simulating the reactions, and possible mechanisms for the reaction of peroxynitrite with glutathione in the presence of added bicarbonate are discussed.

The catalytic role of carbon dioxide in the decomposition of peroxynitrite.

The fast reaction of peroxynitrite with CO2 and the high concentration of dissolved CO2 in vivo (ca. 1 mM) suggest that CO2 modulates most of the reactions of peroxynitrite in biological systems. The addition of peroxynitrite to CO2 produces of the adduct ONOO-CO2- (1). The production of 1 greatly accelerates the decomposition of peroxynitrite to give nitrate. We now show that the formation of 1 is followed by reformation of CO2 (rather than another carbonate species such as CO3 = or HCO3-). To show this, it is necessary to study systems with limiting concentrations of CO2. (When CO2 is present in excess, its concentration remains nearly constant during the decomposition of peroxynitrite, and the recycling of CO2, although it occurs, can not be detected kinetically). We find that CO2 is a true catalyst of the decomposition of peroxynitrite, and this fundamental insight into its action must be rationalized by any in vivo or in vitro reaction mechanism that is proposed. When the concentration of CO2 is lower than that of peroxynitrite, the reformation of CO2 amplifies the fraction of peroxynitrite that reacts with CO2. Even low concentrations of CO2 that result from the dissolution of ambient CO2 can have pronounced catalytic effects. These effects can cause deviations from predicted kinetic behavior in studies of peroxynitrite in noncarbonate buffers in vitro, and since 1 and other intermediates derived from it are oxidants and/or nitrating agents, some of the reactions attributed to peroxynitrite may depend on the availability of CO2.

Peroxynitrite-mediated oxidation of D,L-selenomethionine: kinetics, mechanism and the role of carbon dioxide.

Peroxynitrite oxidizes D,L-selenomethionine (MetSe) by two competing mechanisms, a one-electron oxidation that leads to ethylene and a two-electron oxidation that gives methionine selenoxide (MetSeO). Kinetic modeling of the experimental data suggests that both peroxynitrous acid and the peroxynitrite anion react with MetSe to form MetSeO with rate constants of 20,460 +/- 440 M-1 s-1 and 200 +/- 170 M-1 s-1, respectively at 25 degrees C. The enthalpy (delta H++) and entropy (delta S++) of activation for the reaction of peroxynitrous acid with MetSe at pH 4.6 are 2.55 +/- 0.08 kcal mol-1 and -30.5 +/- 0.3 cal mol-1 K-1, respectively. With increasing concentrations of MetSe at pH 7.4, the yield of ethylene decreases and that of MetSeO increases, suggesting, as with methionine, the reactions leading to ethylene and MetSeO have different kinetic orders. We propose that the activated form of peroxynitrous acid, HOONO*, is the one-electron oxidant and ground-state peroxynitrite is the two-electron oxidant in the reaction of peroxynitrite with MetSe. The peroxynitrite anion rapidly adds to CO2 to form an adduct, O = N-OO-CO2- (1), capable of generating potent reactive species, and we therefore examined the role of CO2 in the peroxynitrite/MetSe system. In presence of added bicarbonate, the yield of ethylene obtained from the reaction of 0.4 mM peroxynitrite with 1.0 mM MetSe increases slightly with an increase in the concentration of bicarbonate from 0 to 5.0 mM and remains constant with a further increase of bicarbonate up to 20 mM. The yield of MetSeO, from the reaction of 10 mM peroxynitrite with 10 mM MetSe, decreases by 35% with an increase in the concentration of bicarbonate from 0 to 25 mM. Kinetic simulations show that the decrease in the yield of MetSeO is due to reaction of the peroxynitrite anion with CO2. These results suggest that CO2 partially protects MetSe from peroxynitrite-mediated oxidation and that 1 or its derivatives do not mediate the oxidation of MetSe to MetSeO.

Detection of aldehydes in bronchoalveolar lavage of rats exposed to ozone.

We report the detection of hexanal, heptanal, and nonanal in the bronchoalveolar lavage (BAL) of rats exposed to 0.5 to 10 ppm ozone with or without simultaneous 5% CO2. These three aldehydes primarily result from the Criegee ozonation of specific mono- or polyunsaturated fatty acids that are present in significant amounts in the rat lung; e.g., palmitoleic acid gives heptanal, oleic gives nonanal, and linoleic and arachidonic can give hexanal. Hexanal also is produced in the ozone-initiated autoxidation of any n-6 polyunsaturated fatty acid, and thus is a measure of generalized oxidative stress. (Monounsaturated fatty acids do not undergo appreciable autoxidation.) This detection and quantitation of aldehydes directly demonstrates for the first time that unsaturated fatty acids undergo Criegee ozonation in the lung when ozone is inhaled. Exposure to ozone alone produced smaller apparent yields of the three aldehydes than did exposure to ozone plus 5% CO2. Hexanal, heptanal, and nonanal can be detected in BAL of rats 5 hr after the end of the ozone exposure, but after more than 5 hr only hexanal can be found, probably from ozone-induced autoxidation of n-6 PUFA that continues after ozone exposure. The measured amounts of aldehydes are low, and that, coupled with inherent biovariability, suggests that aldehydes may not be useful as quantitative dosimeters. However, they can be useful biomarkers, since some of these aldehydes (e.g., nonanal) are produced in ozone-specific pathways and aldehydes are the most easily detected among the lipid ozonation products (LOP). Furthermore, our identification of these aldehydes by BAL, coupled with our recognition that ozone itself cannot penetrate far enough into the lung to cause many of the effects associated with the inhalation of ozone, suggests that these aldehydes, as well as other types of LOP (such as hydroxyhydroperoxides and Criegee ozonides), may act as signal transduction molecules, activating lipases and causing the release of inflammatory molecules by a variety of pathways not yet entirely elucidated.

Acceleration of peroxynitrite oxidations by carbon dioxide.

Stopped-flow kinetic studies of the isomerization of peroxynitrite to give nitrate have been performed in carbonate-enriched buffers using pH jump and carbonic anhydrase as probes. The data are consistent with the reaction of CO2 and the peroxynitrite anion rapidly forming an unstable nitrosoperoxy-carbonate anion adduct, O=N-OOCO2- (1). The CO2 catalysis of the isomerization of peroxynitrite is not accompanied by the formation of nitrite, hydrogen peroxide, or other hydroperoxidic material like peroxycarbonate. The reaction proceeds via the transient formation of an oxidant or oxidants that is (are) capable of promoting electrophilic nitration reactions. We propose that O=N-OOCO2- rearranges to give a nitrocarbonate anion, O2N-OCO2- (2) which in turn, may serve as the proximal oxidant in biological systems that produce peroxynitrite. At least four different mechanistic classes of reactions that have been ascribed to peroxynitrite can be envisioned to involve 2: (a) hydrolysis to nitrate, (b) one-electron or (c) two-electron oxidations, and (d) electrophilic nitration. Given the fast reaction of peroxynitrite with carbon dioxide and the ubiquitous presence of the latter, the role of CO2 cannot be neglected in complex peroxynitrite reactions in vitro and in vivo.