Reperfusion-induced arrhythmias: do free radicals play a critical role?

This article assesses whether oxygen-derived free radicals are one of the molecular causes of life-threatening arrhythmias that arise upon reperfusion of the ischemic myocardium. Evidence supporting this proposition has been obtained from studies of the effects of free radical scavengers and antioxidants, free radical generating systems, inhibition of various sources of free radicals and studies investigating the formation of free radicals and their products during early reperfusion. It has been hypothesized that free radical formation causes localised membrane damage to the sarcolemma that results in focal alterations in transmembrane ionic fluxes, particularly potassium. These changes in ionic fluxes may then lead to electrophysiological abnormalities that culminate in ventricular arrhythmias.     

Artemisinin: mechanisms of action,resistance and toxicity

Artemisinin and its derivatives are widely used throughout the world. The mechanism of action of these compounds appears to involve the heme-mediated decomposition of the endoperoxide bridge to produce carbon-centred free radicals. The involvement of heme explains why the drugs are selectively toxic to malaria parasites. The resulting carbon-centred free radicals are alkylate heme and proteins, one of which is the translationally controlled tumour protein. Clinically relevant artemisinin resistance has not been demonstrated, but it is likely to occur since artemisinin resistance has been obtained in laboratory models. At high doses, artemisinin can be neurotoxic but toxicity has not been found in clinical studies. The mechanism of neurotoxicity may be similar to the mechanism of action.     

The generation of free radicals by nitric oxide synthase

Nitric oxide synthase (NOS) is an example of a family of heme-containing monooxygenases that, under the restricted control of a specific substrate, can generate free radicals. While the generation of nitric oxide (NO*) depends solely on the binding of L-arginine, NOS produces superoxide (O(2)*(-)) and hydrogen peroxide (H(2)O(2)) when the concentration of the substrate is low. Not surprisingly, effort has been put forth to understand the pathway by which NOS generates NO*, O(2)*(-) and H(2)O(2), including the role of substrate binding in determining the pathways by which free radicals are generated. By binding within the distal heme pocket near the sixth coordination position of the NOS heme iron, L-arginine alters the coordination properties of the heme iron that promotes formation of the perferryl complex NOS-[Fe(5+)=O](3+). This reactive iron intermediate has been shown to abstract a hydrogen atom from a carbon alpha to a heteroatom and generate carbon-centered free radicals. The ability of NOS to produce free radicals during enzymic cycling demonstrates that NOS-[Fe(5+)=O](3+) behaves like an analogous iron-oxo complex of cytochrome P-450 during aliphatic hydroxylation. The present review discusses the mechanism(s) by which NOS generates secondary free radicals that may initiate pathological events, along with the cell signaling properties of NO*, O(2)*(-) and H(2)O(2).     

Using anti-5,5-dimethyl-1-pyrroline N-oxide (anti-DMPO) to detect protein radicals in time and space with immuno-spin trapping

The detection of protein free radicals using the specific free radical reactivity of nitrone spin traps in conjunction with nitrone-antibody sensitivity and specificity greatly expands the utility of the spin trapping technique, which is no longer dependent on the quantum mechanical electron spin resonance (ESR). The specificity of the reactions of nitrone spin traps with free radicals has already made spin trapping with ESR detection the most universal, specific tool for the detection of free radicals in biological systems. Now the development of an immunoassay for the nitrone adducts of protein radicals brings the power of immunological techniques to bear on free radical biology. Polyclonal antibodies have now been developed that bind to protein adducts of the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). In initial studies, anti-DMPO was used to detect DMPO protein adducts produced on myoglobin and hemoglobin resulting from self-peroxidation by H2O2. These investigations demonstrated that myoglobin forms the predominant detectable protein radical in rat heart supernatant, and hemoglobin radicals form inside red blood cells. In time, all of the immunological techniques based on antibody-nitrone binding should become available for free radical detection in a wide variety of biological systems.     

胶原蛋白的电子活性与矽肺纤维化Ⅱ正常大鼠与实验性矽肺大鼠肺Ⅰ型胶原稳定自由基的研究

由正常大鼠肺撮取的酸溶性Ⅰ型胶原出现一个明显的ESR信号(g=2.006),而由矽肺大鼠提取的酸溶性Ⅰ型胶原较少或缺乏该ESR信号,该信号不是顺磁过渡元素产生,而是稳定自由基的反映.肺胶原可能存在两种稳定的自由基,一种易受紫外线辐照的激发并且在电场极化下自旋耦合而减弱,另一种自由基则相对稳定,对紫外线辐照和电场极化不敏感.矽肺胶原缺乏活泼性较高的稳定自由基.     

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.     

Applications of electron spin resonance spectroscopy to the identification of radicals produced during lipid peroxidation

Electron spin resonance (ESR) spectroscopy, which is the only commonly available method for directly detecting free radicals in biological systems, has now been quite extensively used to study radicals produced by metabolism of xenobiotic chemicals and the interaction of such species with lipid molecules. This review examines a variety of different xenobiotic systems and tissues and summarises the information obtained from these studies, with particular reference to the elucidation of the nature of the radicals involved in the initiation and propagation of lipid peroxidation.     

ESR and optical absorption evidence for free radical involvement in the photosensitizing action of furocoumarin derivatives and for their singlet oxygen production.

Frozen aqueous solutions of thymine and its derivatives were irradiated with visible light (lambda greater than 320 nm) in the presence of various furocoumarins. ESR analysis revealed the induction of hydrogen adduct free radicals at C-6 position of thymine, only with those furocoumarin derivatives which show a skin-photosensitizing ability. It has been shown, moreover, that the photocycloaddition of psoralen to thymine, which is responsible for the biological effects of this dye, is inhibited when the induction of free radicals in thymine moiety has been prevented by electron scavengers. It is suggested that the free radicals observed could be involved in the biological photosensitization. The mechanism of free radical generation and singlet oxygen production by furoccoumarins were also investigated.     

Spin trapping in biological systems. Oxidation of the spin trap 5,5-dimethyl-1-pyrroline-1-oxide by a hydroperoxide-hematin-system.

We have used the spin trap 5,5-dimethyl-1-pyrroline-1-oxide to determine if primary free radicals are involved in the hematin-cumene hydroperoxide system which has been shown to oxidize N-hydroxy-2-acetylaminofluorene into the nitroxyl free radical form of this carcinogen. We have found that the spin trap was oxidized itself rather than trapping either primary free radicals or carcinogen free radicals.     

Free radicals in the 1900's: from in vitro to in vivo

Remarkable progress has been achieved in the past 100 years in the field of free radical chemistry, biology and medicine since the discovery of free radicals in 1900. Free radical-mediated processes play a major role in the present industrial chemistry, but they also cause deleterious effects on rubber, plastics, oil products and foods. The importance of free radicals in vivo has been recognized increasingly from both positive and negative sides. Free radicals play an important role in phagocytosis, the production of some biologically essential compounds and possibly cell signaling. At the same time, they may cause oxidative modification of biological molecules, which leads to oxidative damage and eventually to various diseases, cancer and aging. The role and beneficial effects of antioxidants against such oxidative stress support this view. Furthermore, novel issues have been continuously found in this fascinating and yet controversial field of free radicals in biology. In this short article, the past work, present problems and future perspectives of free radicals in life science will be briefly discussed.     

Amyloid precursor protein-mediated free radicals and oxidative damage: implications for the development and progression of Alzheimer's disease

Alzheimer's disease (AD) is a late-onset dementia that is characterized by the loss of memory and an impairment of multiple cognitive functions. Advancements in molecular, cellular, and animal model studies have revealed that the formation of amyloid beta (Abeta) and other derivatives of the amyloid precursor protein (APP) are key factors in cellular changes in the AD brain, including the generation of free radicals, oxidative damage, and inflammation. Recent molecular, cellular, and gene expression studies have revealed that Abeta enters mitochondria, induces the generation of free radicals, and leads to oxidative damage in post-mortem brain neurons from AD patients and in brain neurons from cell models and transgenic mouse models of AD. In the last three decades, tremendous progress has been made in mitochondrial research and has provided significant findings to link mitochondrial oxidative damage and neurodegenerative diseases such as AD. Researchers in the AD field are beginning to recognize the possible involvement of a mutant APP and its derivatives in causing mitochondrial oxidative damage in AD. This article summarizes the latest research findings on the generation of free radicals in mitochondria and provides a possible model that links Abeta proteins, the generation of free radicals, and oxidative damage in AD development and progression.     

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.     

Free radicals in the 1900's: from in vitro to in vivo

Remarkable progress has been achieved in the past 100 years in the field of free radical chemistry, biology and medicine since the discovery of free radicals in 1900. Free radical-mediated processes play a major role in the present industrial chemistry, but they also cause deleterious effects on rubber, plastics, oil products and foods. The importance of free radicals in vivo has been recognized increasingly from both positive and negative sides. Free radicals play an important role in phagocytosis, the production of some biologically essential compounds and possibly cell signaling. At the same time, they may cause oxidative modification of biological molecules, which leads to oxidative damage and eventually to various diseases, cancer and aging. The role and beneficial effects of antioxidants against such oxidative stress support this view. Furthermore, novel issues have been continuously found in this fascinating and yet controversial field of free radicals in biology. In this short article, the past work, present problems and future perspectives of free radicals in life science will be briefly discussed.     

Superoxide Dismutase Therapy for Myocardial Ischemia

Oxygen derived free radicals have been shown to be generated during reperfusion of ischemic myocardium by a variety of approaches including spin trap probes. Three levels of injury have been described for the reperfused heart. Periods of ischemia of only several minutes can trigger lethal arrhythmias on reperfusion. Anti-oxidants including SOD and or catalase, as well as iron chelators reduce the incidence of these arrhythmias in both dog and rat. Xanthine oxidase inhibitors arc equipotent with SOD in this model suggesting that xanthine oxidase is the source of the radicals. Periods of occlusion lasting 10–15 minutes produce a recoverable defect in contractility termed “stunning”. SOD plus catalase has been shown to reduce the incidence of stunning in a variety of models including the xanthine oxidase deficient rabbit. Neither agent on its own seemed to be effective against stunning in either the rabbit or the dog. Stunning is more difficult to demonstrate in the rabbit heart, presumably due to its lack of xanthine oxidase. Periods of ischemia in excess of 20 minutes will result in some irreversible cell death (infarction) with reperfusion. While studies using histochemical methods suggested that SOD plus Catalase given at the time of reper-fusion could limit necrosis in the dog model, histological studies reveal that infarct size was not modified but rather, SOD appears to interfere with the ability of tetrazoliurn to histochemically discriminate between living and dead cells. While PEG SOD with its extended plasma half life was reported to reduce infarct size in the dog. it was unable to protect the reprefused rabbit heart. To date. none of the scavengers have been proven to limit infarction suggesting that free radicals contribute to arrythmias and stunning, but do not. kill cells in the reperfused heart.     

Free radical scavenging reactions of sulfasalazine, 5-aminosalicylic acid and sulfapyridine: mechanistic aspects and antioxidant activity

Reactions of sulfasalazine (SAZ) and its metabolites, 5-aminosalicylic acid (5-ASA) and sulfapyridine (SP), with various oxidizing and reducing free radicals (hydroxyl, haloperoxyl, one-electron oxidizing, lipid peroxyl, glutathiyl, superoxide, tryptophanyl, etc.) have been studied to understand the mechanistic aspects of its action against free radicals produced during inflammation. Nanosecond pulse radiolysis technique coupled with transient spectrophotometry has been used for in situ generation of free radicals and to follow their reaction pathways. The transients produced in these reactions have been assigned and radical scavenging rate constants have been measured. In addition to scavenging of various primary and secondary free radicals by SAZ, 5-ASA and SP, 5-ASA has also been observed to efficiently scavenge radicals of biomolecules. 5-ASA has been found to be the active moiety of SAZ involved in the scavenging of oxidizing free radicals whereas reduction of SAZ produced molecular radical anion. The study suggests that free radical scavenging activity of 5-ASA may be a major path of pharmacological action of SAZ against inflammatory bowel diseases (IBD).     

不仅是"益母"草:益母草的心脏保护作用

益母草作为一种传统的妇科中药,近年来的研究表明其作用是多方面的。在心血管方面,益母草能改善心肌缺血、增加冠状动脉血流、提高心功能,其机制主要是在氧化应激状态下通过清除氧自由基、抑制活性氧簇生成发挥抗氧化作用。益母草心脏保护作用的另一机制是促进血管发生。临床试验也表明,益母草能抑制冠心病人的血小板聚集,起抗凝、抗血栓形成作用,从而改善血流变学参数。本文根据目前研究进展,对益母草的心脏保护作用简要综述。     

A protocol for detecting and scavenging gas-phase free radicals in mainstream cigarette smoke

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking (5,6,7,12). Although tobacco tars and related products in the particle phase of cigarette smoke are major causes of carcinogenic and mutagenic related diseases, cigarette smoke contains significant amounts of free radicals that are also considered as an important group of carcinogens(9,10). Free radicals attack cell constituents by damaging protein structure, lipids and DNA sequences and increase the risks of developing various types of cancers. Inhaled radicals produce adducts that contribute to many of the negative health effects of tobacco smoke in the lung(3). Studies have been conducted to reduce free radicals in cigarette smoke to decrease risks of the smoking-induced damage. It has been reported that haemoglobin and heme-containing compounds could partially scavenge nitric oxide, reactive oxidants and carcinogenic volatile nitrosocompounds of cigarette smoke(4). A 'bio-filter' consisted of haemoglobin and activated carbon was used to scavenge the free radicals and to remove up to 90% of the free radicals from cigarette smoke(14). However, due to the cost-ineffectiveness, it has not been successfully commercialized. Another study showed good scavenging efficiency of shikonin, a component of Chinese herbal medicine(8). In the present study, we report a protocol for introducing common natural antioxidant extracts into the cigarette filter for scavenging gas phase free radicals in cigarette smoke and measurement of the scavenge effect on gas phase free radicals in mainstream cigarette smoke (MCS) using spin-trapping Electron Spin Resonance (ESR) Spectroscopy(1,2,14). We showed high scavenging capacity of lycopene and grape seed extract which could point to their future application in cigarette filters. An important advantage of these prospective scavengers is that they can be obtained in large quantities from byproducts of tomato or wine industry respectively(11,13).     

Protective effects of lazaroids against oxygen-free radicals induced lysosomal damage

Lipid peroxidation of membranes by oxygen free radicals has been implicated in various disease states. Different antioxidants and iron chelators have been used to reduce lipid peroxidation. Lazaroids have been used for the acute treatment of central nervous system disorders such as trauma and ischemia wherein lipid peroxidative processes take place.In this study we evaluated the effect of lazaroids (U-785 18F and U-74389F) on the release of acid phosphatase activity and formation of malondialdehyde (MDA) in rat liver lyosomes subjected to exogenously generated oxygen free radicals. There was a significant increase in the acid phosphatase release and MDA formation in the presence of oxygen free radicals. This was prevented by both the lazaroids. In a separate study the effect of lazaroid U-74389F was seen on the zymosan-stimulated polymorphonuclear (PMN) leukocyte-derived chemiluminescence. The PMN leukocyte chemiluminescent activity was attenuated by the lazaroid in a dose-dependent manner. These studies suggest that lazaroids may inhibit lipid peroxidation and stabilize the membrane.     

Inflammation and free radicals in tumor development and progression

Inflammation is thought to be one of the major contributors to carcinogenesis. Accumulated studies in this field revealed that free radicals produced by inflammatory cells not only cause direct damage to DNA but also exert indirect effects such as de-regulation of cell proliferation and apoptosis, stimulation of angiogenesis, and modification of gene/protein expressions and protein activities, all of which are a critical step toward carcinogenesis. Free radicals have also been reported to act as both initiator and promoter of carcinogenic process. Recent evidence shows that free radicals convert benign tumors to more malignant ones (i.e. tumor progression) leading to the final stage of carcinogenesis. This article reviews the current findings linking inflammation and cancer, and shed light on inflammatory cell-derived free radicals as major endogenous reactive substances for tumor development and progression.     

氢气生物学作用的生物酶基础

氢气具有广泛的生物学功能,近年来逐渐引起广泛关注。但是氢气发挥生物学作用的机理一直都有争论,制约了氢生物学的进一步发展。现在被广泛接受的是氢气选择性与毒性自由基反应的理论,但是生理条件下氢气与自由基直接反应的证据并不充分,多数属于间接证据,无法区分氢气是与自由基直接反应还是影响了自由基的产生。氢气具有抗氧化作用,本团队研究表明,氢气不是在自由基产生之后去清除,而是减少自由基的产生,类似于在自由基产生之初就关上“开关”;氢气可以提高包括线粒体复合物Ⅰ、乙酰胆碱酯酶、HRP在内的生物酶的活性,可以影响线粒体膜电位和调节神经细胞膜电位,细胞膜的氧化还原酶类及离子通道等都受到氢气的调节,这表明氢气的作用可能是多靶点的主要基于酶学反应的过程,高等生物具有产生和利用氢气的氢化酶活性。主要探讨了氢气和自由基的关系以及氢气作用的生物酶学基础,以期为揭示氢气发挥生物学作用的机理提供参考。