Rao M. Uppu

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 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.

SYNTHESIS OF PEROXYNITRITE BY AZIDE-OZONE REACTION

Publisher Summary This chapter discusses the synthesis of peroxynitrite by azide-ozone reaction. A simple method for preparing stable, concentrated solutions of peroxynitrite that are low in ionic strength, low in alkali, and free of H 2 O 2 . The method is based on the reaction of ozone with azide ions in water at pH 12. There are advantages to the use of these peroxynitrite solutions in chemical investigations, especially where alkali and H 2 O 2 contamination pose a problem. These solutions also are useful in biological studies, if the contamination by the unreacted residual azide is kept to a minimum. A simplification and improvement of the original azide–ozone method is described by using a Sander model 200 ozonator. The Sander ozonator is designed for use with aquariums, and is readily available, inexpensive, and easily used in any laboratory setup. It is possible to prepare peroxynitrite solutions containing an unreacted residual azide of ≤5μM. The residual azide in these peroxynitrite solutions does not interfere with the assay of several hemoprotein and nonhemoprotein enzymes.

SELECTING THE MOST APPROPRIATE SYNTHESIS OF PEROXYNITRITE

Publisher Summary This chapter discusses various methods and the selection criteria used for the synthesis of Peroxynitrite. Ozonation of azide ions is performed at 0–4° in a mildly alkaline solution. This method gives peroxynitrite solutions up to 80 mM that are low in alkali and free of H 2 O 2 , however may contain some unreacted azide. These peroxynitrite preparations are used in several chemical and biochemical reactions, including those mediated by nonhemoprotein and hemoprotein enzymes. The autooxidation reactions of hydroxylamine lead to the formation of peroxynitrite. The reaction of hydrogen peroxide with nitrous acid involves a rapid reaction of an acidified solution of H 2 O 2 with a solution of sodium nitrite followed by stabilization of the product, peroxynitrous acid, with strong alkali. The reaction of alkaline H 2 O 2 with an alkyl nitrite (RONO) can also produce peroxynitrite. Among the various solution-based methods, peroxynitrite prepared by the azide–ozone reaction contains low levels of alkali. All preparations of peroxynitrite contain nitrite and nitrate to a greater or lesser degree. Nitrite is produced when peroxynitrite undergoes decomposition in a transition metal ion-assisted reaction, and nitrate is produced in the acid-catalyzed decomposition of peroxynitrite.

A major ozonation product of cholesterol, 3β-hydroxy-5-oxo-5,6-secocholestan-6-al, induces apoptosis in H9c2 cardiomyoblasts

Cholesterol, a major neutral lipid component of biological membranes and the lung epithelial lining fluids, is susceptible to oxidation by reactive oxygen and nitrogen species including ozone. The oxidation by ozone in biological environments results in the formation of 3β‐hydroxy‐5‐oxo‐5,6‐secocholestan‐6‐al (cholesterol secoaldehyde or CSeco, major product) along with some other minor products. Recently, CSeco has been implicated in the pathogenesis of atherosclerosis and Alzheimers disease. In this communication, we report that CSeco induces cytotoxicity in H9c2 cardiomyoblasts with an IC50 of 8.9 ± 1.29 μM (n = 6). The observed effect of CSeco at low micromolar concentrations retained several key features of apoptosis, such as changes in nuclear morphology, phosphatidylserine externalization, DNA fragmentation, and caspase 3/7 activity. Treatment of cardiomyocytes with 5 μM CSeco for 24 h, for instance, resulted in 30.8 ± 3.28% apoptotic and 1.8 ± 1.11% of necrotic cells as against DMSO controls that only showed 1.3 ± 0.33% of apoptosis and 1.6 ± 0.67% of necrosis. In general, the loss of cellular viability paralleled the increased occurrence of apoptotic cells in various CSeco treatments. This study, for the first time, demonstrates the induction of apoptotic cell death in cardiomyocytes by a cholesterol ozonation product, implying a role for ozone in myocardial injury.

CARDIOVASCULAR EFFECTS OF PEROXYNITRITE

1 Peroxynitrite (PN) is formed in biological systems from the reaction of nitric oxide (·NO) with superoxide (·) and both exist as free radicals. By itself, PN is not a free radical, but it can generate nitrogen dioxide (·NO2) and carbonate radical (·) upon reaction with CO2. 2 The reaction of CO2 constitutes a major pathway for the disposition of PN produced in vivo and this is based on the rapid reaction of PN anion with CO2 and the availability of CO2 in both intra‐ and extracellular fluids. The free radicals ·NO2 and ·, in combination with ·NO, generated from nitric oxide synthase, can bring about oxidation of critical biological targets resulting in tissue injury. However, the reactions of ·NO2, · and ·NO with carbohydrates, protein and non‐protein thiols, phenols, indoles and uric acid could result in the formation of a number of nitration and nitrosation products in the vasculature. These products serve as long‐acting ·NO donors and, therefore, contribute to vasorelaxant properties, protective effects on the heart, inhibition of leucocyte–endothelial cell interactions and reduction of reperfusion injury. 3 Herein, we review the chemistry of PN, the observations that the effects of PN could be mediated by formation of an ·NO donor‐like substance and review the physiological and beneficial effects of PN.

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 bothcis- andtrans-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-dimensional1H nuclear magnetic resonance. The combined yield of thecis- andtrans-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.

Cytotoxic effects of oxysterols produced during ozonolysis of cholesterol in murine GT1-7 hypothalamic neurons

Ozone present in the photochemical smog or generated at the inflammatory sites is known to oxidize cholesterol and its 3-acyl esters. The oxidation results in the formation of multiple “ozone-specific” oxysterols, some of which are known to cause abnormalities in the metabolism of cholesterol and exert cytotoxicity. The ozone-specific oxysterols have been shown to favor the formation of atherosclerotic plaques and amyloid fibrils involving pro-oxidant processes. In the present communication, cultured murine GT1-7 hypothalamic neurons were studied in the context of cholesterol metabolism, formation of reactive oxygen species, intracellular Ca2 + levels and cytotoxicity using two most commonly occurring cholesterol ozonolysis products, 3β- hydroxy-5-oxo-5,6-secocholestan-6-al (ChSeco) and 5β, 6β-epoxy-cholesterol (ChEpo). It was found that ChSeco elicited cytotoxicity at lower concentration (IC50 = 21 ± 2.4 μM) than did ChEpo (IC50 = 43 ± 3.7 μM). When tested at their IC50 concentrations in GT1-7 cells, both ChSeco and ChEpo resulted in the generation of ROS, the magnitude of which was comparable. N-acetyl-l-cysteine and Trolox attenuated the cytotoxic effects of ChSeco and ChEpo. The intracellular Ca2 + levels were not altered by either ChSeco or ChEpo. Methyl-β-cyclodextrins, which cause depletion of cellular cholesterol, prevented ChSeco- but not ChEpo-induced cytotoxicity. The cell death caused by ChEpo, but not ChSeco, was prevented by exogenous cholesterol. Although oxidative stress plays a significant role, the results of the present study indicate differences in the pathways of cell death induced by ChSeco and ChEpo in murine GT1-7 hypothalamic neurons.

Prooxidant actions of bisphenol A (BPA) phenoxyl radicals: implications to BPA-related oxidative stress and toxicity

Abstract We investigated the prooxidant effects of bisphenol A (BPA) phenoxyl radicals in comparison with the phenoxyl radicals of 3-tert-butyl-4-hydroxyanisole (BHA), 2,6-di-tert-butyl-methylphenol (BHT) and 4-tert-butylphenol (TBP). The phenoxyl radicals, generated in situ by 1-electron oxidation of the corresponding phenol, were allowed to react with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and rifampicin. The antioxidant activity of various phenols was examined based on the reduction of 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH). It was found that the prooxidant activity of BPA phenoxyl radicals far exceeded those of BHA and BHT of phenoxyl radicals. Unlike Trolox, BPA showed minimal DPPH scavenging activity. The strong prooxidant properties of BPA phenoxyl radicals propelled us to study the markers of cellular oxidative stress in GT1-7 hypothalamic neurons exposed to BPA. It was observed that neuronal cells exposed to BPA had increased generation of intracellular peroxides and mitochondrial superoxide (). The formation of peroxides and were time- and dose-dependent and that co-incubation with N-acetyl-l-cysteine or Trolox greatly lowered their levels. The results of the present study are consistent with emerging evidence that human populations (non-institutionalized) having higher levels of urinary BPA also have increased levels of oxidative stress markers and are prone to higher risk of cardiovascular diseases, diabetes and abnormalities in hepatic enzymes.

Cholesterol secoaldehyde, an ozonation product of cholesterol, induces amyloid aggregation and apoptosis in murine GT1-7 hypothalamic neurons

Aldehydic products from ozonation of cholesterol and peroxidation of phospholipids have been shown to accelerate aggregation of amyloid-beta (Abeta) in vitro. Here, we show that 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al (ChSeco), an ozonation product of cholesterol, induces Abeta aggregation, generation of reactive oxygen species (ROS), and cytotoxicity in murine GT1-7 hypothalamic neurons. The formation of Abeta aggregates in situ was dose-dependent at ChSeco concentrations ranging from 1 to 20 microM. The increase in insoluble Abeta aggregates at increasing concentrations of ChSeco was accompanied by a decrease in soluble Abeta as evidenced by Western blot analysis. The formation of ROS in neuronal cells was found to be dose- and time-dependent with the magnitude being higher at 20 microM compared to 10 microM ChSeco or untreated controls. The increase in ROS was associated with depletion of GSH. The cytotoxicity induced by ChSeco involved changes in phosphatidylserine translocation, DNA fragmentation, and caspase 3/7 activity that are characteristic of apoptosis. Pretreatment of neuronal cells with Trolox, a water-soluble analog of alpha-tocopherol offered partial, but significant protection against ChSeco-induced cell death, whereas, N-acetyl-L-cysteine (NAC) completely prevented the cytotoxic effects of ChSeco. NAC and Trolox were without any effects on ChSeco-induced Abeta aggregation. Fibrillogenesis inhibitors, which inhibited Abeta aggregation, did not inhibit cell death induced by ChSeco, implying that ROS generation, and not Abeta aggregation, plays a major role in the observed cytotoxicity. However, since Alzheimers and other neurodegenerative diseases are slow and progressive, the formation of Abeta aggregates in vivo by ChSeco may have long-term pathological consequences.