Synthesis of peroxynitrite by azide-ozone reaction.
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Azides↗
Biomedical subjects
R Cueto
Publications and source records attributed to R Cueto.
What does ozone react with at the air/lung interface? Model studies using human red blood cell membranes.
We exposed human red blood cell (RBC) membranes to low levels of ozone and measured the oxidative damage that occurs to the proteins and the unsaturated lipids that are present. Oxidative damage to proteins causes significant decreases in the content of thiol groups, the fluorescence of protein-tryptophan residues, and the activity of membrane-bound acetylcholinesterase. Oxidative damage to lipids causes changes in some of the unsaturated fatty acids (UFA) in the lipid fraction of these RBC membranes. Significant amounts of hexanal, heptanal, and nonanal are formed from the ozonation of UFA. Although no decrease in the amount of oleate is detected, it does undergo ozonation to yield nonanal; thus, as would be expected, product appearance is a more sensitive measure of ozonation than is substrate disappearance. These results imply that both proteins and unsaturated lipids undergo simultaneous and competitive ozonation in human RBC membranes when ozone is the limiting reactant. The ratios of reaction of ozone with different targets can be predicted in reasonably good agreement with the observed values using calculations (W. A. Pryor and R. M. Uppu (1993) J. Biol. Chem. 268, 3120-3126; R. M. Uppu and W. A. Pryor (1994) Chem. Res. Toxicol. 7, 47-55) that take into account the reactivities and relative amounts of protein and lipid functionalities present in the RBC membranes. Similar calculations are used to predict the reaction of ozone with unsaturated lipids and proteins at the air/lung interface, and both UFA and proteins are predicted to react with ozone in the lung, as in RBC membranes.
Acetylcholinesterase↗
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.
Azides↗
Effect of lipid ozonation products on liposomal membranes detected by Laurdan fluorescence.
We report here the influence of the lipid ozonation products, 1-palmitoyl-2-(9-oxononanoyl)-sn-glycero-3-phosphocholine (PC-aldehyde) and 1-palmitoyl-2[8-(5-octyl-1, 2, 4,-trioxolan-3-yl)- octanoyl]-sn-glycero-3-phosphocholine (PC-Criegee ozonide), on the phase domains of small unilamellar vesicles. (See Scheme 1 for structures.) 6-Lauroyl-2-dimethylaminonaphtalene (Laurdan) fluorescence excitation and emission spectra and generalized polarization measurements allowed us to study how lipid ozonation products affect the phase components of phospholipid membranes. A shift of excitation and emission spectra and a decrease in generalized polarization reveal the presence of a more polar environment surrounding the probe. We find that when either PC-aldehyde or PC-Criegee ozonide are incorporated into a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane, or when the POPC membrane is directly ozonated, a change in polarity of the phospholipid environment occurs that changes the properties of the bilayer. The introduction of more oxygenated and more polar phospholipids creates a more polar environment allowing the deeper penetration of water molecules into the membrane. Water penetration also is facilitated by the membrane disorder-producing effect of the ozonation products. The presence of an increased number of water molecules in the membrane effects the bilayer, altering packing order and cooperatively among fatty acyl chains as well as enhancing membrane fluidity.
Liposomes↗
Quantifying aldehydes and distinguishing aldehydic product profiles from autoxidation and ozonation of unsaturated fatty acids.
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Aldehydes↗
Oxygen consumption and oxyradical production from microsomal reduction of aqueous extracts of cigarette tar.
Aqueous extracts of cigarette tar (ACT) contain a quinone-hydroquinone-semiquinone system that undergoes autoxidation to produce superoxide and hydrogen peroxide. In this paper we ask whether the production of activated oxygen species from this quinone system can be enhanced via an NAD(P)H-dependent autoxidation in which electrons are donated from electron transport components of the microsomal system to electron acceptors in ACT such as quinones and/or metal chelates. Therefore, the ability of ACT to enhance the oxidation of NADPH, consumption of oxygen, and production of oxyradicals during microsomal electron transport has been investigated. Rates of NADPH oxidation and oxygen consumption are enhanced in a concentration-dependent manner by both aqueous and dimethyl sulfoxide (DMSO) extracts of tar over that observed in the presence of microsomes alone. Over the time course studied, neither NADPH oxidation nor oxygen consumption by tar extracts is observed in the absence of microsomes. Microsomal NADPH oxidation is enhanced by 1,6- and 1,3-benzo[a]pyrene quinones and by iron-EDTA; per unit weight, the enhancement by these compounds is similar to that of tar. NADPH oxidation in the presence of tar and absence of iron-EDTA is inhibited by about 15% by the addition of deferoxamine to the reaction mixture. Chrysene quinone enhances NADPH oxidation to a much greater extent than does either of the benzo[a]pyrene quinones or tar. Inhibition of tar-stimulated microsomal oxygen uptake by superoxide dismutase, stimulation of oxygen uptake by azide (an inhibitor of catalase), and the presence of spin-trappable superoxide anion radical all indicate the reduction of tar components to superoxide and hydrogen peroxide. The similar effects of tar on NADPH oxidation catalyzed either by microsomes or by purified NADPH-cytochrome P450 reductase suggest that cytochrome P450 reductase is an important locus of electron transfer from microsomes to components of cigarette tar extracts. These data suggest that the cytotoxic and mutagenic effects of smoking may be mediated by metabolic activation of quinones and, to a lesser extent, metal chelates in cigarette tar to oxyradicals.
Animals↗
Identification of heptanal and nonanal in bronchoalveolar lavage from rats exposed to low levels of ozone.
Heptanal and nonanal are identified from in vitro studies as potential biomarkers of exposure to ozone, the former resulting from ozonation of palmitoleic acid and the latter from oleic acid. An analytical method is developed based on derivatization using O-pentafluorobenzylhydroxylamine HCl and gas chromatography. These molecules also are present in the lung lavage of Sprague-Dawley rats exposed to 1.3 ppm ozone for 10 hr. These results suggest aldehydes may be useful dosimeters for ozone and indicate that unsaturated fatty acids in the lung lining fluid layer undergo ozonation in vivo.
Aldehydes↗
Production of the Criegee ozonide during the ozonation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes.
It is likely that Criegee ozonides are formed in small amounts in the lungs of animals breathing ozone-containing air. This makes these compounds potential candidates to act as secondary toxins which relay the toxic effects of ozone deeper into lung tissue than ozone itself could penetrate. Therefore, we have determined the yields of Criegee ozonides from unsaturated lipids in liposomal systems as a model of the types of yields of Criegee ozonides that might be expected both in the lung lining fluid layer and in biological membranes. Ozonation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes produced both cis- and trans-Criegee ozonides. These ozonides have been isolated by solid phase extraction and high-performance liquid chromatography of the ozonized lipid, and the products have been identified by two-dimensional 1H nuclear magnetic resonance. The combined yield of the cis- and trans-Criegee ozonides is 10.7 +/- 2.8% (avg. +/- SD, n = 7) with small unilamellar liposomes and 10.6 +/- 2.7% (n = 3) with large multilamellar liposomes. We had previously reported (Chem. Res. Toxicol. 5, 505-511, 1992) that ozonation of methyl oleate in sodium dodecylsulfate micelles also produces an 11% yield of the Criegee ozonides. Thus, ozonation in a variety of models gives about 11% of the Criegee ozonide, suggesting that these products also would be formed in small but significant amounts in the lungs of animals breathing polluted air. Further research on the pharmacokinetics and possible toxicity of the Criegee ozonides of fatty acids is suggested.
Liposomes↗