Here we report the structural characterization of the product formed from the reaction between hydroethidine (HE) and superoxide ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}-}\end{equation*}\end{document} ). By using mass spectral and NMR techniques, the chemical structure of this product was determined as 2-hydroxyethidium (2-OH-E + ). By using an authentic standard, we developed an HPLC approach to detect and quantitate the reaction product of HE and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}-}\end{equation*}\end{document} formed in bovine aortic endothelial cells after treatment with menadione or antimycin A to induce intracellular reactive oxygen species. Concomitantly, we used a spin trap, 5- tert -butoxycarbonyl-5-methyl-1-pyrroline N -oxide (BMPO), to detect and identify the structure of reactive oxygen species formed. BMPO trapped the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}-}\end{equation*}\end{document} that formed extracellularly and was detected as the BMPO-OH adduct during use of the EPR technique. BMPO, being cell-permeable, inhibited the intracellular formation of 2-OH-E + . However, the intracellular BMPO spin adduct was not detected. The definitive characterization of the reaction product of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}-}\end{equation*}\end{document} with HE described here forms the basis of an unambiguous assay for intracellular detection and quantitation of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}-}\end{equation*}\end{document} . Analysis of the fluorescence characteristics of ethidium (E + ) and 2-OH-E + strongly suggests that the currently available fluorescence methodology is not suitable for quantitating intracellular \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}-}\end{equation*}\end{document} . We conclude that the HPLC/fluorescence assay using HE as a probe is more suitable reactive oxygen species for detecting intracellular \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}-}\end{equation*}\end{document} .

Adriamycin (or doxorubicin) is an active and broad spectrum chemotherapeutic agent. Unfortunately, its clinical use is severely restricted by a dose-limiting cardiotoxicity which has been linked to the formation of superoxide. Enzymatic one-electron reduction of adriamycin forms adriamycin semiquinone radical, which rapidly reacts with oxygen to form superoxide and adriamycin. In this way, adriamycin provides a kinetic mechanism for the one-electron reduction of oxygen by flavoenzymes such as NADPH-cytochrome P450 reductase and mitochondrial NADH dehydrogenase. We demonstrate here that the endothelial isoform of nitric oxide synthase (eNOS) reduces adriamycin to the semiquinone radical. As a consequence, superoxide formation is enhanced and nitric oxide production is decreased. Adriamycin binds to eNOS with a Km of approximately 5 microM, as calculated from both eNOS-dependent NADPH consumption and superoxide generation. Adriamycin stimulated superoxide formation is not affected by calcium/calmodulin and is abolished by the flavoenzyme inhibitor, diphenyleneiodonium. This strongly suggests that adriamycin undergoes reduction at the reductase domain of eNOS. A consequence of eNOS-mediated reductive activation of adriamycin is the disruption of the balance between nitric oxide and superoxide. This may lead eNOS to generate peroxynitrite and hydrogen peroxide, potent oxidants implicated in several vascular pathologies.

1؉ cluster increased with increasing additions of superoxide to m-aconitase. This reaction was reversible, as >90% of the initial aconitase activity was recovered upon treatment with glutathione and iron(II). This mechanism presents a scenario in which ⅐ OH may be continuously generated in the mitochondria.There is much debate in the literature on the relative importance of hydroxyl radical ( ⅐ OH) and peroxynitrite in free radical pathology (1). Clarification of the mechanism centered on this subject is of considerable importance, especially in mitochondria, cellular organelles that are constantly exposed to low levels of superoxide anion (2, 3). Several neurodegenerative diseases (e.g. Alzheimer's disease, Parkinson's disease, and Lou Gehrig's disease or amyotrophic lateral sclerosis) and aging have been linked to mitochondrial oxidative damage that results in decreased mitochondrial function (3, 4). However, in biological systems, it is nearly impossible to associate a specific damage to a single oxidant. For example, superoxide and nitric oxide ( ⅐ NO) co-generated at very low levels (Ϸ10 Ϫ8 M) in cells will form peroxynitrite (ONOO Ϫ ) via a nearly diffusion-controlled reaction (5, 6). The toxicological significance of these species is clearly dependent on cell type, the biological targets, and their relationship to one another. One of the sensitive biological targets in oxidative damage to mitochondria is aconitase, an iron-sulfur protein that catalyzes the stereospecific dehydration-hydration of citrate to isocitrate in the Krebs cycle (7). Aconitase activity in mitochondria has been reported to be a sensitive redox sensor of reactive oxygen and nitrogen species in cells (8 -11). Aconitase contains a cubane-type [4Fe-4S] 2ϩ cluster in its active site with three iron atoms bound to cysteinyl groups and inorganic sulfur atoms and a fourth labile iron atom (Fe-␣). This Fe-␣ is unique in that it is not bound to a protein cysteine, but rather to a hydroxyl group of substrate and water (7 (13, 14). However, the reaction between aconitase and peroxynitrite is strongly inhibited by the addition of substrate that binds to the enzyme with high affinity (14).It was recently proposed that the reaction between mitochondrial aconitase (m-aconitase) 1 and superoxide plays a major role in mitochondrial oxidative damage (15)(16)(17). During this reaction, it has been proposed that iron is released from maconitase as iron(II) with the concomitant generation of hydrogen peroxide. This facilitates the formation of "free" hydroxyl radical in mitochondria. In the presence of intracellular reducing agents (e.g. glutathione, ascorbate, and NADPH), iron(II) is reincorporated into the inactive form of m-aconitase to regenerate the active form. According to this proposal, hydroxyl radical should be continuously generated in mitochondria as a result of the reaction between superoxide and aconitase. However, the experimental verification of this intriguing mechanism has so far been lacking.The objective of this study is to provide e...

The effects of nitric oxide ('NO) and nitrovasodilators on the oxidation of low-density lipoprotein (LDL) have been studied. S-Nitroso-Nacetylpenicillamine (SNAP) and sodium nitroprusside (SNP) inhibited Cu 2÷-and 2,2'-azobis-2-amidinopropane hydrochloride-dependent oxidation of LDL as monitored by oxygen consumption and the formation of thiobarbituric acid-reactive substances, conjugated dienes, and lipid hydroperoxides. In the case of SNP, inhibition of LDL oxidation occurred only when the incubation mixture was irradiated with visible light. SNAP, however, exerted a dose-dependent inhibition of CuZ÷-catalyzed oxidation of LDL even in the dark. Addition of "NO dissolved in deoxygenated buffer also inhibited the progression of LDL oxidation. Mouse peritoneal macrophages were less able to degrade LDL that had been oxidized in the presence of SNAR Using an "NO electrode, it was estimated that a continuous production of'NO (~< 760 nM/min) could retard the progression of LDL oxidation. We propose that "NO can inhibit LDL oxidation by acting as a chain-breaking antioxidant that is capable of scavenging carbon-centered and peroxyl radicals. Biological implications of this novel °NO antioxidant property are discussed in relation to atherogenesis and contrasted to the prooxidant property of "NO when generated in the presence of superoxide.