The discovery that mammalian cells have the ability to synthesize the free radical nitric oxide (NO) has stimulated an extraordinary impetus for scientific research in all the fields of biology and medicine. Since its early description as an endothelial-derived relaxing factor, NO has emerged as a fundamental signaling device regulating virtually every critical cellular function, as well as a potent mediator of cellular damage in a wide range of conditions. Recent evidence indicates that most of the cytotoxicity attributed to NO is rather due to peroxynitrite, produced from the diffusion-controlled reaction between NO and another free radical, the superoxide anion. Peroxynitrite interacts with lipids, DNA, and proteins via direct oxidative reactions or via indirect, radical-mediated mechanisms. These reactions trigger cellular responses ranging from subtle modulations of cell signaling to overwhelming oxidative injury, committing cells to necrosis or apoptosis. In vivo, peroxynitrite generation represents a crucial pathogenic mechanism in conditions such as stroke, myocardial infarction, chronic heart failure, diabetes, circulatory shock, chronic inflammatory diseases, cancer, and neurodegenerative disorders. Hence, novel pharmacological strategies aimed at removing peroxynitrite might represent powerful therapeutic tools in the future. Evidence supporting these novel roles of NO and peroxynitrite is presented in detail in this review.

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Nitric Oxide and Peroxynitrite in Health and Disease

Publisher Summary This chapter discusses the role of free radicals and catalytic metal ions in human disease. The importance of transition metal ions in mediating oxidant damage naturally leads to the question as to what forms of such ions might be available to catalyze radical reactions in vivo . The chapter discusses the metabolism of transition metals, such as iron and copper. It also discusses the chelation therapy that is an approach to site-specific antioxidant protection. The detection and measurement of lipid peroxidation is the evidence most frequently cited to support the involvement of free radical reactions in toxicology and in human disease. A wide range of techniques is available to measure the rate of this process, but none is applicable to all circumstances. The two most popular are the measurement of diene conjugation and the thiobarbituric acid (TBA) test, but they are both subject to pitfalls, especially when applied to human samples. The chapter also discusses the essential principles of the peroxidation process. When discussing lipid peroxidation, it is essential to use clear terminology for the sequence of events involved; an imprecise use of terms such as initiation has caused considerable confusion in the literature. In a completely peroxide-free lipid system, first chain initiation of a peroxidation sequence in a membrane or polyunsaturated fatty acid refers to the attack of any species that has sufficient reactivity to abstract a hydrogen atom from a methylene group.

Role of free radicals and catalytic metal ions in human disease: an overview.

O2- oxidizes the [4Fe-4S] clusters of dehydratases, such as aconitase, causing-inactivation and release of Fe(II), which may then reduce H2O2 to OH- +OH.. SODs inhibit such HO. production by scavengingO2-, but Cu, ZnSODs, by virtue of a nonspecific peroxidase activity, may peroxidize spin trapping agents and thus give the appearance of catalyzing OH. production from H2O2. There is a glycosylated, tetrameric Cu, ZnSOD in the extracellular space that binds to acidic glycosamino-glycans. It minimizes the reaction of O2- with NO. E. coli, and other gram negative microorganisms, contain a periplasmic Cu, ZnSOD that may serve to protect against extracellular O2-. Mn(III) complexes of multidentate macrocyclic nitrogenous ligands catalyze the dismutation of O2- and are being explored as potential pharmaceutical agents. SOD-null mutants have been prepared to reveal the biological effects of O2-. SodA, sodB E. coli exhibit dioxygen-dependent auxotrophies and enhanced mutagenesis, reflecting O2(-)-sensitive biosynthetic pathways and DNA damage. Yeast, lacking either Cu, ZnSOD or MnSOD, are oxygen intolerant, and the double mutant was hypermutable and defective in sporulation and exhibited requirements for methionine and lysine. A Cu, ZnSOD-null Drosophila exhibited a shortened lifespan.

Superoxide Radical and Superoxide Dismutases

Denitrification is a distinct means of energy conservation, making use of N oxides as terminal electron acceptors for cellular bioenergetics under anaerobic, microaerophilic, and occasionally aerobic conditions. The process is an essential branch of the global N cycle, reversing dinitrogen fixation, and is associated with chemolithotrophic, phototrophic, diazotrophic, or organotrophic metabolism but generally not with obligately anaerobic life. Discovered more than a century ago and believed to be exclusively a bacterial trait, denitrification has now been found in halophilic and hyperthermophilic archaea and in the mitochondria of fungi, raising evolutionarily intriguing vistas. Important advances in the biochemical characterization of denitrification and the underlying genetics have been achieved with Pseudomonas stutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans, Ralstonia eutropha, and Rhodobacter sphaeroides. Pseudomonads represent one of the largest assemblies of the denitrifying bacteria within a single genus, favoring their use as model organisms. Around 50 genes are required within a single bacterium to encode the core structures of the denitrification apparatus. Much of the denitrification process of gram-negative bacteria has been found confined to the periplasm, whereas the topology and enzymology of the gram-positive bacteria are less well established. The activation and enzymatic transformation of N oxides is based on the redox chemistry of Fe, Cu, and Mo. Biochemical breakthroughs have included the X-ray structures of the two types of respiratory nitrite reductases and the isolation of the novel enzymes nitric oxide reductase and nitrous oxide reductase, as well as their structural characterization by indirect spectroscopic means. This revealed unexpected relationships among denitrification enzymes and respiratory oxygen reductases. Denitrification is intimately related to fundamental cellular processes that include primary and secondary transport, protein translocation, cytochrome c biogenesis, anaerobic gene regulation, metalloprotein assembly, and the biosynthesis of the cofactors molybdopterin and heme D1. An important class of regulators for the anaerobic expression of the denitrification apparatus are transcription factors of the greater FNR family. Nitrate and nitric oxide, in addition to being respiratory substrates, have been identified as signaling molecules for the induction of distinct N oxide-metabolizing enzymes.

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Cell biology and molecular basis of denitrification.

Fenton chemistry encompasses reactions of hydrogen peroxide in the presence of iron to generate highly reactive species such as the hydroxyl radical and possibly others. In this review, the complex mechanisms of Fenton and Fenton-like reactions and the important factors influencing these reactions, from both a fundamental and practical perspective, in applications to water and soil treatment, are discussed. The review covers modified versions including the photoassisted Fenton reaction, use of chelated iron, electro-Fenton reactions, and Fenton reactions using heterogeneous catalysts. Sections are devoted to nonclassical pathways, by-products, kinetics and process modeling, experimental design methodology, soil and aquifer treatment, use of Fenton in combination with other advanced oxidation processes or biodegradation, economic comparison with other advanced oxidation processes, and case studies.

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Advanced Oxidation Processes for Organic Contaminant Destruction Based on the Fenton Reaction and Related Chemistry

The Fenton reagents.

The reaction of NO. with O2.- and HO2.: a pulse radiolysis study.

The toxicity of ascorbate towards phage lambda and the phages T2–T7 has been investigated. At room temperature the T-odd and lambda bacteriophages are highly susceptible to ascorbate-induced damage, whereas the T-even phages are practically resistant. The toxicity of ascorbate is dependent on the presence of copper (or iron) and oxygen, although oxygen is not required in the presence of H2O2. Hydrogen peroxide is essential for the ascorbate-induced phage inactivation and the damage is prevented by catalase. At the concentrations used, most of the copper ions are bound to the phage particles. Chelating agents such as EDTA or histidine fully protect the phages, whereas salicylate only reduces the rate of phage inactivation. OH scavengers such as sucrose, formate, mannitol, tert-butyl alcohol or poly(ethylene glycol) have no protective effect.



Experiments with DNA labeled phages indicate that both phage adsorption and DNA injection are impaired as a result of the exposure to ascorbate and copper. The failure to express the viral genetic information as a result of single and double-strand breaks in the DNA, probably also contribute to the loss of the plaque-forming ability of the phages.



The results are interpreted in terms of a ‘site-specific’ Fenton mechanism according to which the binding of the transition metal ions to the biological target is a prerequisite for the production of damage. The bound metal ion is reduced either by O−2, ascorbate or other reductants and is subsequently reoxidized by H2O2 yielding OH˙ radicals. This cyclic redox reaction of the metal generates OH˙ radicals which react with vital macromolecules with a high probability of causing ‘multi-hit’ damage. This ‘site-specific’ formation of OH˙ radicals, which takes place near the target molecules, accounts both for the high damaging efficiency and for the failure of OH˙ scavengers to protect against it.

Gidon Czapski

Papers

The Fenton reagents.

The reaction of NO. with O2.- and HO2.: a pulse radiolysis study.

On the cytotoxicity of vitamin C and metal ions. A site-specific Fenton mechanism.

The one-electron transfer redox potentials of free radicals. I. The oxygen/superoxide system.

Kinetics of Nitric Oxide Autoxidation in Aqueous Solution in the Absence and Presence of Various Reductants. The Nature of the Oxidizing Intermediates