Endogenous antioxidant changes in the myocardium in response to acute and chronic stress conditions

Oxygen is a diradical and because of its unique electronic configuration, it has the potential to form strong oxidants (e.g. superoxide radical, hydrogen peroxide and hydroxyl radical) called oxygen free radicals or partially reduced forms of oxygen (PRFO). These highly reactive oxygen species can cause cellular injury by oxidizing lipids and proteins as well as by causing strand breaks in nucleic acids. PRFO are produced in the cell during normal redox reactions including respiration and there are various antioxidants in the cell which scavenge these radicals. Thus in order to maintain a normal cell structure and function, a proper balance between free radical production and antioxidant levels is absolutely essential. Production of PRFO in the myocardium is increased during variousin vivo as well asin vitro pathological conditions and these toxic radicals are responsible for causing functional, biochemical and ultrastructural changes in cardiac myocytes. Indirect evidence of free radical involvement in myocardial injury is provided by studies in which protection against these alterations is seen in the presence of exogenous administration of antioxidants. Endogenous myocardial antioxidants have also been reported to change under various physiological as well as pathophysiological conditions. It appears that endogenous antioxidants respond and adjust to different stress conditions and failure of these compensatory changes may also contribute in cardiac dysfunction. Thus endogenous and/or exogenous increase in antioxidants might have a therapeutic potential in various pathological conditions which result from increased free radical production.     

Kinetic analysis-based quantitation of free radical generation in EPR spin trapping

Because short-lived reactive oxygen radicals such as superoxide have been implicated in a variety of disease processes, methods to measure their production quantitatively in biological systems are critical for understanding disease pathophysiology. Electron paramagnetic resonance (EPR) spin trapping is a direct and sensitive technique that has been used to study radical formation in biological systems. Short-lived oxygen free radicals react with the spin trap and produce paramagnetic adducts with much higher stability than that of the free radicals. In many cases, the quantity of the measured adduct is considered to be an adequate measure of the amount of the free radical generated. Although the intensity of the EPR signal reflects the magnitude of free radical generation, the actual quantity of radicals produced may be different due to modulation of the spin adduct kinetics caused by a variety of factors. Because the kinetics of spin trapping in biochemical and cellular systems is a complex process that is altered by the biochemical and cellular environment, it is not always possible to define all of the reactions that occur and the related kinetic parameters of the spin-trapping process. We present a method based on a combination of measured kinetic data for the formation and decay of the spin adduct alone with the parameters that control the kinetics of spin trapping and radical generation. The method is applied to quantitate superoxide trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO). In principle, this method is broadly applicable to enable spin trapping-based quantitative determination of free radical generation in complex biological systems.     

Reperfusion injury

Several lines of evidence point to a major role of oxygen free radicals in the pathogenesis of cell death or dysfunction in a variety of disease processes. Recent studies from this as well as other laboratories have demonstrated that oxygen free radicals play a major role in the pathogenesis of post-ischemic reperfusion injury in the heart. We have recently developed methods for direct measurement of radical species and/or specific byproducts of radical injury. Timely administration of oxygen radical scavengers reduced the quantity of free radicals generated following reperfusion and in addition improved recovery of post-ischemic ventricular function and metabolism. In a regionally ischemic model the free radical scavenger recombinant human superoxide dismutase also administered at the time of reflow was shown to limit infarct size. In this article we review the biophysical and molecular mechanisms of oxygen free radical generation that are viewed as contributing to post-ischemic reperfusion injury. We also discuss the mechanisms by which the body defends against free radical attack and the interrelationships of free radical injury to other mechanisms of tissue injury.     

Hydroxyl radical in living systems and its separation methods

It has recently been shown that hydroxyl radicals are generated under physiological and pathological conditions and that they seem to be closely linked to various models of pathology putatively implying oxidative stress. It is now recognized that the hydroxyl radical is well-regulated to help maintain homeostasis on the cellular level in normal, healthy tissues. Conversely, it is also known that virtually every disease state involves free radicals, particularly the most reactive hydroxyl radical. However, when hydroxyl radicals are generated in excess or the cellular antioxidant defense is deficient, they can stimulate free radical chain reactions by interacting with proteins, lipids, and nucleic acids causing cellular damage and even diseases. Therefore, a confident analytical approach is needed to ascertain the importance of hydroxyl radicals in biological systems. In this paper, we provide information on hydroxyl radical trapping and detection methods, including liquid chromatography with electrochemical detection and mass spectrometry, gas chromatography with mass spectrometry, capillary electrophoresis, electron spin resonance and chemiluminescence. In addition, the relationships between diseases and the hydroxyl radical in living systems, as well as novel separation methods for the hydroxyl radical are discussed in this paper.     

Electron paramagnetic resonance evidence that cellular oxygen toxicity is caused by the generation of superoxide and hydroxyl free radicals

Cells require molecular oxygen for the generation of energy through mitochondrial oxidative phosphorylation; however, high concentrations of oxygen are toxic and can cause cell death. A number of different mechanisms have been proposed to cause cellular oxygen toxicity. One hypothesis is that reactive oxygen free radicals may be generated; however free radical generation in hyperoxic cells has never been directly measured and the mechanism of this radical generation is unknown. In order to determine if cellular oxygen toxicity is free radical mediated, we applied electron paramagnetic resonance, EPR, spectroscopy using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide, DMPO, to measure free radical generation in hyperoxic pulmonary endothelial cells. Cells in air did not give rise to any detectable signal. However, cells exposed to 100% O2 for 30 min exhibited a prominent signal of trapped hydroxyl radical, DMPO-OH, while cell free buffer did not give rise to any detectable radical generation. This cellular radical generation was demonstrated to be derived from the superoxide radical since the observed signal was totally quenched by superoxide dismutase, but not by equal concentrations of the denatured enzyme. It was confirmed that the hydroxyl radical was generated since in the presence of ethanol the CH3 CH(OH) radical was formed. Loss of cell viability as measured by uptake of trypan blue dye was observed paralleling the measured free radical generation. Thus, superoxide and hydroxyl radicals are generated in hyperoxic pulmonary endothelial cells and this appears to be an important mechanism of cellular oxygen toxicity.     

巨噬细胞产生NO.和O_2~-自由基的分子机理

建立了用顺磁共振（ＥＳＲ）和化学发光技术测定巨噬细胞产生ＮＯ和氧自由基的方法．捕捉到了巨噬细胞受佛波酯刺激产生的ＮＯ．和Ｏ－２自由基．测定了在不同浓度Ｌ－精氨酸存在时佛波酯刺激后巨噬细胞产生的ＮＯ自由基．研究了巨噬细胞产生的ＮＯ和氧自由基的分子机理．结果表明巨噬细胞不仅产生氧自由基而且产生ＮＯ自由基．ＮＡＤＰＨ氧化酶产生氧自由基的部位位于巨噬细胞膜的外侧．ＮＯ合成酶活化产生ＮＯ自由基比ＮＡＤＰＨ氧化酶活化产生氧自由基晚几分钟．     

The activity of recombinant human neuroglobin as an antioxidant and free radical scavenger

Free radicals are by-products of metabolism and exist in a homeostasis between generation and scavenging in vivo. Excessive free radicals cause various diseases, including nervous system diseases. Neuroglobin (Ngb), a nervous system-specific oxygen-binding protein, has been suggested to be a potential free radical scavenger in the nervous system in vivo; however, its underlying mechanism remains unclear. In this study, we investigated the antioxidant potential and free radical scavenging properties of recombinant human Ngb (rhNgb) in vitro. Interestingly, we found that the rhNgb protein itself has a direct and distinct antioxidant capacity and can efficiently scavenge a variety of free radicals, including the [2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid)] (ABTS) cation, superoxide anion, hydrogen peroxide, and hydroxyl radical. The capacity of rhNgb to scavenge the superoxide anion and hydrogen peroxide was even comparable to that of vitamin C. In addition, rhNgb had Fe(2+) chelating activity but hemoglobin did not. In conclusion, our results indicated that the rhNgb protein itself has antioxidant and free radical scavenging activities, providing fundamental evidence for the neuroprotective function of Ngb. These data provide key information for the origin of the neuroprotective and physiological role of Ngb and will promote the treatment of reactive oxygen species (ROS)-related diseases using this novel oxygen-binding globin.     

Central nervous system trauma and stroke. I. Biochemical considerations for oxygen radical formation and lipid peroxidation

The generation of oxygen radicals and the process of lipid peroxidation have become a focus of attention for investigators in the fields of central nervous system (CNS) trauma and stroke (e.g., ischemia). Considering our level of understanding of free radical and lipid peroxidation chemistry, absolute proof for their involvement in the pathophysiology of traumatic and ischemic damage to the CNS has been meager. While direct, unequivocal evidence for the participation of free radicals and lipid peroxidation as primary contributors to the death of neuronal tissue waits to be established, numerous recent studies have provided considerable support for the occurrence of free radical and lipid peroxidation reactions in the injured or ischemic CNS. In addition, the pharmacological use of antioxidants and free radical scavengers in the treatment of experimental CNS trauma and ischemia has provided convincing, although indirect evidence, for the involvement of oxygen radicals and lipid peroxidation in these conditions. The intent of this and its companion paper is to review: 1) the biochemical processes which may give rise to free radical reactions in the CNS, 2) the environment of the ischemic cell as it may affect the generation of oxygen radicals and the catalysis of lipid peroxidation reactions, 3) the evidence for the involvement of free radical mechanisms in CNS trauma and ischemia, and 4) the pathophysiological consequences of these phenomena.     

Antioxidant properties of bile salt micelles evaluated with different chemiluminescent assays: a possible physiological role

The antioxidant activity of a representative series of free, glycine- and taurine-conjugated bile acids was evaluated by two different chemiluminescent assays: (a) the enhanced chemiluminescence system based on horseradish peroxidase and luminol/oxidant/enhancer reagent, and (b) the hypoxanthine/xanthine oxidase/Fe²⁺-EDTA/luminol system. Bile acids were studied at final concentrations ranging from 1 to 28 mmol/L. All of the bile acids studied inhibited the steady-state chemiluminescent reaction and the extent of inhibition depended upon the structure of the bile acids, whereas the duration was related to bile acid concentration. The mechanism of the light inhibition is probably due to trapping of oxygen free radicals generated in the chemiluminescent reactions, within bile acid micelles. The free radicals trapped into micelles reduced the formation of luminol radicals and consequently the light output; when the micelles were saturated, the oxygen free radicals in solution again produced luminol radicals. The micelle interaction with reactive oxygen species could be a physiological mechanism of defence against the toxicity of those species in the intestinal content. On the other hand, alterations in bile acid organ distribution, concentration and composition leads to a membrane damage caused by their detergent-like properties, which could be associated to oxygen free radical production. © 1998 John Wiley & Sons, Ltd.     

Carnivorous pitcher plant uses free radicals in the digestion of prey

Abstract

A study of the involvement of free oxygen radicals in trapping and digestion of insects by carnivorous plants was the main goal of the present investigation. We showed that the generation of oxygen free radicals by pitcher fluid of Nepenthes is the first step of the digestion process, as seen by EPR spin trapping assay and gel-electrophoresis. The EPR spectrum of N. gracilis fluid in the presence of DMPO spin trap showed the superposition of the hydroxyl radical spin adduct signal and of the ascorbyl radical signal. Catalase addition decreased the generation of hydroxyl radicals showing that hydroxyl radicals are generated from hydrogen peroxide, which can be derived from superoxide radicals. Gel-electrophoresis data showed that myosin, an abundant protein component of insects, can be rapidly broken down by free radicals and protease inhibitors do not inhibit this process. Addition of myoglobin to the pitcher plant fluid decreased the concentration of detectable radicals. Based on these observations, we conclude that oxygen free radicals produced by the pitcher plant aid in the digestion of the insect prey.     

In situ measurement of superoxide and hydroxyl radicals by frequency mixing detection technique

Frequency mixing magnetic detection (FMMD) was used to detect superoxide from hypoxanthine and xanthine reaction and to detect hydroxyl radical from the Fenton reaction. FMMD was also applied to measure the reactive oxygen species (ROS) level released from microglial cells. We could assess the formation and extinction of the free radicals without a spin trap reagent. The FMMD signal amplitude scaled with the concentration of the radicals. It was verified that no signals are obtained from the substrates and reagents. Based on the observations and on previous research, we suggest that the FMMD signals originate from superoxide and hydroxyl radicals, indicating that FMMD can be used to detect O-centered radicals. Subsequent analysis of free radicals generated from living microglial cells showed that there were significant differences between the activated microglial cells and resting ones. The results of this research are promising regarding the applications of FMMD for in situ measurement of free radicals from various sources, including the cell.     

Production, detection, and adaptive responses to free radicals in exercise

Free radicals (particularly oxygen- and nitrogen-centered radicals), and related reactive oxygen and nitrogen species, are generated in cells and tissues during exercise. Mitochondria (actually, 'leakage' of electrons from ubisemiquinone and other electron transport chain components), xanthine oxidase, and phagocytes such as neutrophils may all contribute to free radical production. In this article we review mechanisms of free radical production during exercise and methods for detecting free radicals and related reactive species, during, or immediately following exercise. The evidence presented strongly suggests that free radicals generated during mild to moderate endurance-type exercise actually form part of the mechanism of exercise adaptation that includes extensive biogenesis of muscle mitochondria, increased muscle blood supply, and altered fuel consumption patterns. We suggest, as originally proposed [1], that (at moderately increased levels) free radicals actually act as intracellular signaling molecules to initiate exercise adaptation. In contrast, endurance exercise of extreme duration and extreme intensity appears to generate much higher levels of free radicals that overwhelm cellular antioxidant defenses, and cause tissue damage. Such free radical damage requires effective protein, lipid, and DNA repair systems, and sufficient recuperation, before exercise adaptation can recommence.     

Adventitious chemistry at reduced water activities: free radicals and polyhydroxy agents

Free radicals have been associated with loss of viability of lyophilized bacteria exposed to oxygen. Free radical concentration was proportional to the log of the oxygen pressure in the sample. Sugars, such as lactose or sucrose, preserved viability and inhibited free radical production. Lyophilized tissue, particularly liver and spleen, also reacted with oxygen to produce free radicals, which appear to be associated with ascorbic acid in the tissues. Pure ascorbic acid in air does not produce free radicals, but when mixed with protein before lyophilization it reacts with oxygen in air. When a mixture of sodium ascorbate and phenylalanine or tryptophan is lyophilized, free radicals identical to those observed in tissue are obtained. Propyl gallate and di- or trihydroxybenzoates also react with oxygen when lyophilized with phenylalanine, but the g value of the free radical is significantly less than that obtained with ascorbate. A number of amino acids and similar nitrogenous compounds act as catalysts to form propyl gallate free radicals. As with the bacterial or tissue preparations, various sugars or similar carbohydrates inhibited free radical production by either ascorbate or gallate. In the absence of water the free radicals produced by the action of oxygen on lyophilized samples are stable for years. The rate of free radical production is increased by small amounts of moisture (exposure to moist air), but at humidities over 30% rh the radicals are unstable.     

Cell damage by oxygen free radicals

The exposure of isolated and cultured cells to oxygen free radicals generated extracellularly or intracellularly during the metabolism of foreing compounds results in the development of damage that eventually lead to cell death. Multiple mechanisms are involved in these cytopathological processes, including direct attack of free radicals to macromolecules essential for cell life, as well as indirect activation of catabolic processes such as proteases, endonucleases and phospholipases. A key role in triggering these indirect events is played by Ca²⁺ whose cytosolic concentration during oxidative stress raises well above the physiological limits.     

Mechanism of calcium potentiation of oxygen free radical injury to renal mitochondria. A model for post-ischemic and toxic mitochondrial damage

With a variety of forms of ischemic and toxic tissue injury, cellular accumulation of Ca2+ and generation of oxygen free radicals may have adverse effects upon cellular and, in particular, mitochondrial membranes. Damage to mitochondria, resulting in impaired ATP synthesis and diminished activity of cellular energy-dependent processes, could contribute to cell death. In order to model, in vitro, conditions present post-ischemia or during toxin exposure, the interactions between Ca2+ and oxygen free radicals on isolated renal mitochondria were characterized. The oxygen free radicals were generated by hypoxanthine and xanthine oxidase to simulate in vitro one of the sources of oxygen free radicals in the early post-ischemic period in vivo. With site I substrates, pyruvate and malate, Ca2+ pretreatment, followed by exposure to oxygen free radicals, resulted in an inhibition of electron transport chain function and complete uncoupling of oxidative phosphorylation. These effects were partially mitigated by dibucaine, a phospholipase A2 inhibitor. With the site II substrate, succinate, the electron transport chain defect was not manifest and respiration remained partially coupled. The electron transport chain defect produced by Ca2+ and oxygen free radicals was localized to NADH CoQ reductase. Calcium and oxygen free radicals reduced mitochondrial ATPase activity by 55% and adenine nucleotide translocase activity by 65%. By contrast oxygen free radicals alone reduced ATPase activity by 32% and had no deleterious effects on translocase activity. Dibucaine partially prevented the Ca2+-dependent reduction in ATPase activity and totally prevented the Ca2+-dependent translocase damage observed in the presence of oxygen free radicals. These findings indicate that calcium potentiates oxygen free radical injury to mitochondria. The Ca2+-induced potentiation of oxygen free radical injury likely is due in part to activation of phospholipase A2. This detrimental interaction associated with Ca2+ uptake by mitochondria and exposure of the mitochondria to oxygen free radicals may explain the enhanced cellular injury observed during post-ischemic reperfusion.     

Free radicals and anticancer drug resistance: Oxygen free radicals in the mechanisms of drug cytotoxicity and resistance by certain tumors

Certain anticancer agents form free radical intermediates during enzymatic activation. Recent studies have indicated that free radicals generated from adriamycin and mitomycin C may play a critical role in their toxicity to human tumor cells. Furthermore, it is becoming increasingly apparent that reduced drug activation and or enhanced detoxification of reactive oxygen species may be related to the resistance to these anticancer agents by certain tumor cell lines. The purposes of this review are to summarize the evidence pointing toward the significance of free radicals formation in drug toxicity and to evaluate the role of decreased free radical formation and enhanced free radical scavenging and detoxification in the development of anticancer drug resistance by a spectrum of tumor cell types. Studies failing to support the participation of oxyradicals in the cytotoxicity and resistance of adriamycin are also discussed.     

Measurement of superoxide-derived free radicals in the reperfused heart. Evidence for a free radical mechanism of reperfusion injury

There has been considerable controversy regarding the role of oxygen free radicals as important mediators of cell damage in reperfused myocardium. This controversy regards whether superoxide and hydroxyl free radicals are generated on reperfusion and if these radicals actually cause impaired contractile function. In this study, EPR studies using the spin trap 5,5-dimethyl-1-pyroline-n-oxide (DMPO) demonstrate the formation of .OH and R. free radicals in the reperfused heart. EPR signals of DMPO-OH, aN = aH = 14.9 G, and DMPO-R aN = 15.8 G aH = 22.8 G are observed, with peak concentrations during the first minute of reperfusion. It is demonstrated that these radicals are derived from .O2- since reperfusion in the presence of enzymatically active recombinant human superoxide dismutase markedly reduced the formation of these signals while inactive recombinant human superoxide dismutase had no effect. On reperfusion with perfusate pretreated to remove adventitial iron, the concentration of the DMPO-OH signal was increased 2-fold and a 4-fold decrease in the DMPO-R signal was observed demonstrating that iron-mediated Fenton chemistry occurs. Hearts reperfused with recombinant human superoxide dismutase exhibited improved contractile function in parallel with the marked reduction in measured free radicals. In order to determine if the reperfusion free radical burst results in impaired contractile function, simultaneous measurements of free radical generation and contractile function were performed. A direct relationship between free radical generation and subsequent impaired contractile function was observed. These studies suggest that superoxide derived .OH and R. free radicals are generated in the reperfused heart via Fenton chemistry. These radicals appear to be key mediators of myocardial reperfusion injury.     

Mechanism of horseradish peroxidase-catalyzed oxidation of malonaldehyde

The mechanism of malonaldehyde oxidation by horseradish peroxidase in the presence of manganese(II) and acetate was investigated. Our results show that an apparent oxygenase behavior demonstrated by peroxidase in this system can be explained in terms of normal peroxidase activity. A free radical is generated from the reaction of malonaldehyde with compounds I and II of peroxidase; this radical is scavenged by dissolved molecular oxygen to give the appearance of peroxidase acting as an oxygenase. Oxygen consumption, absorbance spectra, and kinetic results show that the reaction is initiated by autoxidation of malonaldehyde to give a free radical. The radical reacts with oxygen and through the action of manganese(II), a peroxide is generated. This peroxide drives the peroxidase cycle to generate more free radicals which propagate the oxygen consumption reaction.     

Free radical scavenging efficiency of Nano-Se in vitro

In this study, we showed that smaller size particles of Nano-Se have better scavenging effects on the following free radicals: carbon-centered free radicals (R*) generated from 2,2'-azo-bis-(2-amidinopropane) hydrochloride (AAPH), the relatively stable free radical 1,1-diphenyl-2-picryhydrazyl (DPPH), the superoxide anion (O2*-) generated from the xanthine/xanthine oxidase (X/XO) system, singlet oxygen (1O2) generated by irradiated hemoporphyrin. Furthermore, the three sizes of Nano-Se studied also show protective effects against the oxidation of DNA. The three samples all have potential size-dependent characteristics on scavenging the free radicals. Although in this study we regarded Nano-Se as a whole without considering interactions between BSA and the red selenium nano-particles, there is the possibility that the apparent free radical scavenging effects may be partially contributed by such interactions.     

Direct measurement of free radicals in the brain cortex and the blood serum after nociceptive stimulation in rats

The concentrations of ROS were measured in samples of the sensorimotor brain cortex and in the rat blood. We measured the following parameters: The six lines spectra, nitroxide radical, free hydroxyl radical and singleton oxygen. Their concentration was measured under physiological conditions, after the nociceptive stimulation and after the application of melatonin, both in normal and stimulated animals. In the brain cortex only the singleton oxygen decreased after the nociceptive stimulation, whereas the nitroxide radicals and six lines spectra increased. The free hydroxyl radicals did not change significantly. In the blood serum the six lines spectra and nitroxide radical increased, the concentration of the free hydroxyl radicals did not change. Melatonin increased both the hydroxyl and nitroxide radicals. There was a non-significant decrease in the six lines spectra. The estimation of ROS can be used as a tool for detecting metabolic changes and the consequences of different environmental influences, in our case the influence of nociception and melatonin.