Giuseppe L. Squadrito

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 Alzheimers disease, rheumatoid arthritis, atherosclerosis, lung injury, amyotrophic lateral sclerosis, and other diseases.

Hydrogen sulfide mediates the vasoactivity of garlic

The consumption of garlic is inversely correlated with the progression of cardiovascular disease, although the responsible mechanisms remain unclear. Here we show that human RBCs convert garlic-derived organic polysulfides into hydrogen sulfide (H2S), an endogenous cardioprotective vascular cell signaling molecule. This H2S production, measured in real time by a novel polarographic H2S sensor, is supported by glucose-maintained cytosolic glutathione levels and is to a large extent reliant on reduced thiols in or on the RBC membrane. H2S production from organic polysulfides is facilitated by allyl substituents and by increasing numbers of tethering sulfur atoms. Allyl-substituted polysulfides undergo nucleophilic substitution at the α carbon of the allyl substituent, thereby forming a hydropolysulfide (RSnH), a key intermediate during the formation of H2S. Organic polysulfides (R-Sn-R′; n > 2) also undergo nucleophilic substitution at a sulfur atom, yielding RSnH and H2S. Intact aorta rings, under physiologically relevant oxygen levels, also metabolize garlic-derived organic polysulfides to liberate H2S. The vasoactivity of garlic compounds is synchronous with H2S production, and their potency to mediate relaxation increases with H2S yield, strongly supporting our hypothesis that H2S mediates the vasoactivity of garlic. Our results also suggest that the capacity to produce H2S can be used to standardize garlic dietary supplements.

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.

The cascade mechanism to explain ozone toxicity: the role of lipid ozonation products.

Ozone is so reactive that it can be predicted to be entirely consumed as it passes through the first layer of tissue it contacts at the lung/air interface. This layer includes the lung lining fluid (tracheobronchial surface fluid and alveolar and small airway lining fluid) and, where the lung lining fluid is thin or absent, the membranes of the epithelial cells that line the airways. Therefore, the biochemical changes that follow the inhalation of ozone must be relayed into deeper tissue strat by a cascade of ozonation products. Lipid ozonation products (LOP) are suggested to be the most likely species to act as signal transduction molecules. This is because unsaturated fatty acids are present in the lipids in both the lung lining fluid and in pulmonary cell bilayers, and ozone reacts with unsaturated fatty acids to produce ozone-specific products. Further, lipid ozonation products are finite in number, have structures that are predictable from the Criegee ozonation mechanism, and are small, diffusible, stable (or metastable) molecules. Preliminary data show that individual LOP cause the activation of specific lipases, which trigger the release of endogenous mediators of inflammation.

A practical method for preparing peroxynitrite solutions of low ionic strength and free of hydrogen peroxide.

The reaction of ozone (approximately 5% in oxygen) with sodium azide (0.02-0.2 M in water) at pH 12 and 0-4 degrees C is shown to yield concentrated, stable peroxynitrite solutions of up to 80 mM. The product of this reaction is identified based on a broad absorption spectrum with a maximum around 302 nm and by its first-order rate of decomposition (k = 0.40 +/- 0.01 s-1 at pH 7.05 and 25 degrees C). These peroxynitrite solutions can be obtained essentially free of hydrogen peroxide (detection limit 1 microM) and only traces of azide (detection limit 0.1 mM). They are low in ionic strength and have a pH of about 12 but without buffering capacity; therefore, they can be adjusted to any pH by addition of buffer. These preparations of peroxynitrite frozen at -20 degrees C show negligible decomposition for about 3 weeks of storage and follow a first-order decomposition with a halflife of about 7 days at refrigerator temperatures (approximately 5 degrees C). These preparations give reactions that are characteristic of peroxynitrite. For example, at pH 7.0, they react with L-tyrosine to give a 7.3 mol % yield of nitrotyrosine(s), and with dimethyl sulfoxide to give a 8.2 mol % yield of formaldehyde, based on starting peroxynitrite concentration.

The formation of peroxynitrite in vivo from nitric oxide and superoxide

Peroxynitrite is predicted to be formed in vivo from the reaction of nitric oxide and superoxide. Nitric oxide at concentrations as low as 3 nM is expected to compete efficiently for superoxide near the surface of endothelial cells, based on competition kinetics.

Direct and indirect oxidations by peroxynitrite, neither involving the hydroxyl radical.

A new mechanism (Mechanism III) that combines features of mechanisms suggested earlier (Goldstein and Czapski, Inorg. Chem. 34:4041-4048; 1995; Pryor, Jin, and Squadrito Proc. Natl. Acad. Sci. USA 91:11173-11177; 1994) is proposed for oxidations by peroxynitrite. In Mechanism III, oxidations by peroxynitrite can take place either directly by ground-state peroxynitrous acid, ONOOH, or indirectly by ONOOH*, where ONOOH* is an activated form of peroxynitrous acid. In the direct oxidation pathway the reaction is first order in peroxynitrite and first order in substrate, and the oxidation yield approaches 100%. In the indirect oxidation pathway the reaction is first order in peroxynitrite and zero order in substrate. In the presence of sufficient concentrations of a substrate that reacts by the indirect oxidation pathway, about 50-60% of the ONOOH directly isomerizes to nitric acid, and about 40-50% of the ONOOH is converted into ONOOH*. Thus, the oxidation yields by the indirect pathway will not exceed 40-50%, and there will always be a residual yield of nitrate even in the presence of very high concentrations of the substrate. Competitive inhibition studies with various free radical scavengers showed that in some cases these scavengers have no effect on oxidation yields. In others, only partial inhibition was observed, far less than that predicted from to the known rate constants for the reactions of these scavengers with the hydroxyl radical. There are some cases where the extent of inhibition correlates well with the known rate constants of the reactions of these scavengers with hydroxyl radical; nevertheless, even in these cases, the involvement of hydroxyl radicals in indirect oxidations by peroxynitrite is ruled our on the basis of kinetics and oxidation yields. Thus, direct oxidations by peroxynitrite are explained in terms of ONOOH, and indirect oxidations in terms of ONOOH*, and substrates can react by one or both of these pathways.

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.

A new mechanism for the toxicity of ozone.

Ozone, with its high reactivity, is entirely consumed as it passes through the first layer of tissue it contacts at the lung/air interface. This layer includes the epithelial cell lining fluid (ELF) and, where the ELF is thin or absent, the membranes of the epithelial cells that line the airways. Thus the biochemical changes that follow the inhalation of ozone must be relayed into deeper tissue strata by a cascade of ozonation products. Lipid ozonation products (LOP) are suggested to be the most likely relay molecules of ozones signal. This is because unsaturated fatty acids are present in relatively high concentrations in both the ELF and in pulmonary cell bilayers, and ozone reacts with unsaturated fatty acids to produce ozone-specific products. Further, LOP are finite in number, have structures that are predictable from the Criegee ozonation mechanism, and are small, diffusible, stable (or meta-stable) molecules, similar to other lipid-derived signal transduction species. Preliminary data show that individual LOP cause the activation of specific lipases, which trigger the release of endogenous mediators of inflammation.

Mitigation of chlorine-induced lung injury by low-molecular-weight antioxidants

Chlorine (Cl(2)) is a highly reactive oxidant gas used extensively in a number of industrial processes. Exposure to high concentrations of Cl(2) results in acute lung injury that may either resolve spontaneously or progress to acute respiratory failure. Presently, the pathophysiological sequelae associated with Cl(2)-induced acute lung injury in conscious animals, as well as the cellular and biochemical mechanisms involved, have not been elucidated. We exposed conscious Sprague-Dawley rats to Cl(2) gas (184 or 400 ppm) for 30 min in environmental chambers and then returned them to room air. At 1 h after exposure, rats showed evidence of arterial hypoxemia, respiratory acidosis, increased levels of albumin, IgG, and IgM in bronchoalveolar lavage fluid (BALF), increased BALF surfactant surface tension, and significant histological injury to airway and alveolar epithelia. These changes were more pronounced in the 400-ppm-exposed rats. Concomitant decreases of ascorbate (AA) and reduced glutathione (GSH) were also detected in both BALF and lung tissues. In contrast, heart tissue AA and GSH content remained unchanged. These abnormalities persisted 24 h after exposure in rats exposed to 400 ppm Cl(2). Rats injected systemically with a mixture of AA, deferoxamine, and N-acetyl-L-cysteine before exposure to 184 ppm Cl(2) had normal levels of AA, lower levels of BALF albumin and normal arterial Po(2) and Pco(2) values. These findings suggest that Cl(2) inhalation damages both airway and alveolar epithelial tissues and that resulting effects were ameliorated by prophylactic administration of low-molecular-weight antioxidants.