Radical–radical reactions of superoxide: a potential route to toxicity (original) (raw)
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How superoxide radical damages the cell
Protoplasma, 2001
Superoxide is considered to be poorly reactive, and cell damage has been attributed to HO· generated via the Haber-Weiss reaction. The function of O2− in this reaction is only to reduce Fe3+ to Fe2+. In vivo, however, superoxide could not out-compete cellular reductants such as glutathione, NADPH, and ascorbate, which makes the observed O2− toxicity rather puzzling. Little attention has been paid to the idea that, irrespective of its poor chemical reactivity, superoxide might be capable of interacting directly with specific intracellular targets; and that even the Haber-Weiss reaction might be a consequence of such direct interactions. This paper summarizes latest data that support the concept of such a mechanism.
Free Radical Biology and Medicine, 2014
Tyrosine (Tyr) residues are major sites of radical generation during protein oxidation. We used insulin as a model to study the kinetics, mechanisms, and products of the reactions of radiation-induced or enzyme-generated protein-tyrosyl radicals with superoxide to demonstrate the feasibility of these reactions under oxidative stress conditions. We found that insulin-tyrosyl radicals combined to form dimers, mostly via the tyrosine at position 14 on the α chain (Tyr14). However, in the presence of superoxide, dimerization was largely outcompeted by the reaction of superoxide with insulin-tyrosyl radicals. Using pulse radiolysis, we measured a second-order rate constant for the latter reaction of (6 71) Â 10 8 M À 1 s À 1 at pH 7.3, representing the first measured rate constant for a protein-tyrosyl radical with superoxide. Mass-spectrometry-based product analyses revealed the addition of superoxide to the insulin-Tyr14 radical to form the hydroperoxide. Glutathione efficiently reduced the hydroperoxide to the corresponding monoxide and also subsequently underwent Michael addition to the monoxide to give a diglutathionylated protein adduct. Although much slower, conjugation of the backbone amide group can form a bicyclic Tyr-monoxide derivative, allowing the addition of only one glutathione molecule. These findings suggest that Tyr-hydroperoxides should readily form on proteins under oxidative stress conditions where protein radicals and superoxide are both generated and that these should form addition products with thiol compounds such as glutathione.
Chemistry, physiology and pathology of free radicals
Life Sciences, 1999
The superoxide anion radical and other reactive oxygen species (ROS) are formed in all aerobic organisms by enzymatic and nonenzymatic reactions. ROS arise in both physiological and pathological processes, but efficient mechanisms have evolved for their detoxification, Similarly, reactive nitrogen intermediates (RNI) have physiological activity, but can also react with different types of molecules, including superoxide, to form toxic products. ROS and RN1 participate in the destruction of microorganisms by phagocytes, as in the formation of a myeloperoxidase-hydrogen peroxidechloride/iodide complex which can destroy many cells, including bacteria. It is known that the cellular production of ROS and RN1 is controlled by different mechanisms. These free radicals can react with key cellular structures and molecules, thus altering their biological function. An imbalance between the systems producing and removing ROS and RN1 may result in pathological consequences.
2000
This review covers recent literature on the use of EPR techniques to investigate the formation and reactions of radicals in biochemical, biological, and medical systems during the period 1994 (when this area was last reviewed') to early 1998. It covers both direct EPR spectroscopy and spin trapping studies as well as related techniques; it does not cover recent developments in the synthesis and chemistry of spin traps, the formation and reactions of radicals in enzymes and metalloproteins, DNA damage, or spin labelling studies; these topics are covered elsewhere in this volume. Food irradiation, and its detection by EPR spectroscopy, has recently been extensively and thoroughly reviewed,2 and is also therefore not covered. Owing to the increasing interest in, and use of, EPR in the biomedical field, this review cannot be all encompassing and complete due to space limitations; we have, however, endeavoured to cover major advances that have occurred during this time period, and apologise for any omissions. Emphasis has been placed on novel discoveries and processes, and hence we have deliberately omitted the majority of (the very large number of) studies where EPR spin trapping has been employed in the assessment of putative antioxidant / scavenging compounds, in which (typically) the trapping of HO* or 0 2 ' -by DMPO* (to give the well-characterised DMPO-OH or DMPO-OOH adducts) has been employed purely as a competitive 'clock' reaction. The very large volume of literature that has developed over the last few years on the trapping on nitric oxide (NO.), which has a wide variety of important biological functions, is reviewed briefly, with particular emphasis on EPR methods.
Biochemistry of Free Radicals and Antioxidants
The biochemistry of reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide, hydroxyl radicals, and singlet oxygen is important in aerobic metabolism of the cell mostly reactive nitrogen species are well recognised for playing dual function as both dangerous and beneficial species. Overproduction of ROS from mitochondrial electron transport chain leakage or excessive stimulation of xanthine oxidase and other oxidative enzymes results in oxidative stress, a process that can be an important mediator of damage to cell structure and function, lipids, proteins, carbohydrates and DNA. In contrast, beneficial effects of ROS/RNS occur at very low concentrations and involve physiological roles in cellular responses in defence against infectious agents, gene expression, cellular growth, in the function of a number of cellular signalling pathways, hypoxia and respiratory burst. In the past and present years, progress has been made in the recognition and understanding of the roles of reactive oxygen species in many diseases. The body protects itself from the potential damages of reactive oxygen species, by utilizing antioxidant enzymes and non-antioxidant enzymes e.g superoxide dismutases, glutathione peroxidases, glutathione reductase and catalase. Scientists have indicated that antioxidant obtained from daily diets such as non-enzymatic antioxidants vitamin E, vitamin C, carotenoids and polyphenols can scavenge the reactive oxygen species. These compounds may also be required as cofactors for antioxidant enzymes or be used by cells for up-regulating enzymatic antioxidants.
Intracellular production of superoxide radical and of hydrogen peroxide by redox active compounds
Archives of Biochemistry and Biophysics, 1979
Several compounds have been found capable of diverting the electron flow in Escherichia coli and thus causing increased intracellular production of 0; and H,O,. One indication of this electron-shunting action was increased cyanide-resistant respiration and one cellular response was increased biosynthesis of the manganese-containing superoxide dismutase and of catalase. Blocking cytochrome oxidase with cyanide or azide increased the electron flow available for reduction of paraquat and presumably of the other exogenous compounds tested and thus increased their biological effects. Paraquat, pyocyanine, phenazine methosulfate, streptonigrin, juglone, menadione, plumbagin, methylene blue, and azure C were all effective in elevating intracellular production of 0; and H202. The effect of alloxan appeared paradoxical in that it increased cyanide-resistant respiration without significantly increasing the cell content of the manganese-superoxide dismutase and with only a small effect on the level of catalase. The alloxan effect on cyanide-resistant respiration was artifactual and was due to an oxygen-consuming reaction between alloxan and cyanide, rather than to a diversion of the intracellular electron flow. With paraquat as a representative electron-shunting compound, the increase in biosynthesis of the manganese-superoxide dismutase was prevented by inhibitors of transcription or of translation, but not by an inhibitor of replication. The increase in this enzyme activity, caused by paraquat and presumably by the other compounds, was thus due to de novo enzyme synthesis activated or derepressed at the level of transcription.
Pharmacology & Therapeutics, 1988
Free-radical-mediated oxidative damage, formerly considered to be a typical pathogenetic event in the development of the biological effects of ionizing radiation, has been recognized during the last 15 to 20 years to occur in a variety of other pathological conditions . These include aging, inflammation, carcinogenesis and toxicity of drugs and chemicals. As with ionizing radiation where its role in the lethal response of cells has long been established, the hydroxyl radical has been suggested to be mainly responsible for reactions leading to oxidative damage in critical bio-molecules. Investigations of free-radical reactivities with appropriate model compounds are therefore relevant to understanding the molecular mechanism in the development of both radiation injury and the above mentioned noxious conditions. By the same token, studies on possible interactions of oxygen-free radicals with antiagents, such as radioprotectors, antioxidants, and anti-inflammatory drugs, may be informative as to the mechanisms available to prevent or minimize harmful biological consequences. Particular attention has been paid to the reaction of the thiyl radical RS. with oxygen . The most prominent sulfur-centered radical intermediate resulting from defensive processes involving thiols against free-radical attack in cell systems, i.e. RS., adds oxygen with a second order rate constant of approximately 109dm 3.mol-l.s-l for glutathione, cysteine and penicillamine.
Detection of drug-induced, superoxide-mediated cell damage and its prevention by antioxidants
Free Radical Biology and Medicine, 2001
The mode of the cytotoxic activity of three benzo(c)fluorene derivatives was characterized. The observed morphological changes of lysosomes or variations of mitochondrial activity are assumed to be the consequence of cell protection against oxidative damage and/or the part of the damage process. To establish the relationship between the quantity of superoxide (O 2 •Ϫ) generated and the degree of damage resulting from O 2 •Ϫ , a simple system based on measurement of 3-(4-iodophenyl)-2-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT) reductase activity in the presence of superoxide dismutase (SOD) was used. The functionality of the chosen battery of in vitro tests was proved using several known superoxide inducers: cyclosporin A (CsA) and benzo(a)pyrene (BP), as well as noninducers: citrinin (CT) and cycloheximide (CH). From the results followed that the cell growth tests are much better indices of toxicity than the other tests. The model system for the evaluation of the protective capacity of antioxidants against superoxideinduced cytotoxicity included simultaneous exposure of HeLa cells to cytotoxic drugs and to quercetin (Qe), an antioxidant of plant origin. The complete abolishment of the inhibition of cell proliferation and clonogenic survival was concluded to be due to the protective effect of the antioxidant. These observations correlated with the decrease of superoxide content as estimated by the INT-reductase assay in the presence of SOD using the same model system, as well as with the increase of intracellular SOD content and its activity.