Pyridoxamine protects intestinal epithelium from ionizing radiation-induced apoptosis

Reactive oxygen species (ROS) and reactive carbonyl species (RCS) are the major causes of biological tissue damage during exposure to ionizing radiation (IR). The existing strategies to protect normal tissues from the detrimental effects of IR suffer from several shortcomings including highly toxic side effects, unfavorable administration routes, and low efficacy. These shortcomings emphasize a need for radioprotective treatments that combine effectiveness with safety and ease of use. In this paper, we demonstrate that pyridoxamine, a ROS and RCS scavenger with a very favorable safety profile, can inhibit IR-induced gastrointestinal epithelial apoptosis in cell culture and in an animal model. Pyridoxamine was more effective at protecting from radiation-induced apoptosis than amifostine, a synthetic thiol compound and the only FDA-approved radioprotector. We suggest that pyridoxamine has potential as an effective and safe radioprotective agent.     

Structural studies of an eukaryotic cambialistic superoxide dismutase purified from the mature seeds of camphor tree

Inflammation is one of the leading causes of the many pathological states associated with oxidative stress. A crucial role in the development of inflammation-induced oxidative stress is played by reactive oxidant species (ROS), which are very difficult to detect in vivo. One of the most sensitive and definitive methods in the detection of ROS is electron spin resonance, especially as used in conjunction with spin trapping. Unfortunately, the commonly used nitrone spin traps have a very low efficacy for trapping superoxide radicals, and their radical adducts are not stable. To address this deficiency, we have developed negatively charged cyclic hydroxylamines such as 1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidine (PP-H) for the detection of reactive oxidant species as a diagnostic tool for extracellular inflammation-induced oxidative stress. We used inflammation induced by a bacterial endotoxin lipopolysaccharide (LPS) as a model. ROS formation was tested in cultured macrophages, in blood and in vivo. PP-H reacts with reactive oxidant species generating the stable nitroxide radical 4-phosphonooxy-TEMPO. It was shown that a 5-h treatment of macrophages with LPS (1 microg/ml) leads to a threefold increase in superoxide formation as demonstrated using superoxide dismutase. Formation of reactive oxidant species 5 h after LPS (1 mg/kg) treatment of Fischer rats was analyzed in arterial blood; formation of reactive oxidant species in LPS-treated animals increased by a factor of 2.2 and was dependent upon the LPS dose. Diphenyleneiodonium (0.1 mM) inhibited formation of LPS-stimulated reactive oxidant species by 80%. We suggest that this test could be used as a noninvasive diagnostic tool for inflammation-induced oxidative stress.     

Reactive nitrogen species in the chemical biology of inflammation

The preponderance of epidemiological evidence now points to a strong association between chronic inflammation and cancers of several organs, including the gastrointestinal tract, liver, and lungs. The strongest evidence for a mechanistic link here involves the generation of reactive oxygen and nitrogen species by macrophages and neutrophils that respond to cytokines and other signaling processes arising at sites of inflammation. These reactive species cause oxidation, nitration, halogenation, and deamination of biomolecules of all types, including lipids, proteins, carbohydrates, and nucleic acids, with the formation of toxic and mutagenic products. This review, in honor of Bruce Ames, will focus on recent advances in our understanding of the protein and DNA damage caused by reactive nitrogen species produced by macrophages and neutrophils, with emphasis on nitric oxide, nitrous anhydride, peroxynitrite, and nitrogen dioxide radical.     

Actions of melatonin in the reduction of oxidative stress

Melatonin was discovered to be a direct free radical scavenger less than 10 years ago. Besides its ability to directly neutralize a number of free radicals and reactive oxygen and nitrogen species, it stimulates several antioxidative enzymes which increase its efficiency as an antioxidant. In terms of direct free radical scavenging, melatonin interacts with the highly toxic hydroxyl radical with a rate constant equivalent to that of other highly efficient hydroxyl radical scavengers. Additionally, melatonin reportedly neutralizes hydrogen peroxide, singlet oxygen, peroxynitrite anion, nitric oxide and hypochlorous acid. The following antioxidative enzymes are also stimulated by melatonin: superoxide dismutase, glutathione peroxidase and glutathione reductase. Melatonin has been widely used as a protective agent against a wide variety of processes and agents that damage tissues via free radical mechanisms.     

活性氧在创伤愈合中作用的研究

：创伤愈合是一个复杂的生物学过程,包括出血与凝血、炎症渗出、血管和肉芽组织的形成、再上皮化、纤维化和瘢痕改建等,在这一系列的生物学活动过程中都需要能量支持;高等动物使用氧气作为终端氧化剂,通过对碳水化合物的氧化作用为愈合过程中的各种生命活动提供能量,但该过程却可以产生大量的活性氧,这些活性氧在创伤愈合的过程中扮演着重要的角色,在低浓度情况下可以促进伤口的愈合,而在高浓度时会抑制伤口愈合,而活性量浓度的过高过低都会影响创口的正常愈合过程。     

What do we measure by a luminol-dependent chemiluminescence of phagocytes?

The review presents a survey of published findings concerning the mechanism of luminol-dependent chemiluminescence in biological systems. The potential of various oxygen species (superoxide anion, hydrogen peroxide, hydroxyl radical) to react with luminol is discussed. The ability of commonly used enzymes (superoxide dismutase, catalase), inhibitors, and oxygen radical scavengers to discriminate between individual oxygen species is assessed together with the potential of a variety of substances encountered in biological systems to interfere in luminol-dependent chemiluminescence reactions. It is concluded that luminol-dependent chemiluminescence gives at present very little ability to discriminate between individual oxygen or radical species. Furthermore, luminol-dependent chemiluminescence used in biological systems is extremely prone to many interferences, which are very difficult to control.     

Alcohol-induced oxidative stress

Alcohol-induced oxidative stress is linked to the metabolism of ethanol involving both microsomal and mitochondrial systems. Ethanol metabolism is directly involved in the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). These form an environment favourable to oxidative stress. Ethanol treatment results in the depletion of GSH levels and decreases antioxidant activity. It elevates malondialdehyde (MDA), hydroxyethyl radical (HER), and hydroxynonenal (HNE) protein adducts. These cause the modification of all biological structures and consequently result in serious malfunction of cells and tissues.     

Analysis of the antimicrobial effects of nonthermal plasma on fungal spores in ionic solutions

The antimicrobial efficiency of reactive species-based control strategies is significantly affected by the dynamics of reactive species in the biological environment. Atmospheric-pressure nonthermal plasma is an ionized gas in which various reactive species are produced. The various levels of antimicrobial activity may result from the dynamic interaction of the plasma-generated reactive species with the environment. However, the nature of the interaction between plasma and environments is poorly understood. In this study, we analyzed the influence of the ionic strength of surrounding solutions (environment) on the antimicrobial activity of plasma in relation to the plasma-generated reactive species using a model filamentous fungus, Neurospora crassa. Our data revealed that the presence of sodium chloride (NaCl) in the background solution attenuated the deleterious effects of plasma on germination, internal structure, and genomic DNA of fungal spores. The protective effects of NaCl were not explained exclusively by pH, osmotic stability, or the level of reactive species in the solution. These were strongly associated with the ionic strength of the background solution. The presence of ions reduced plasma toxicity, which might be due to a reduced access of reactive species to fungal spores, and fungal spores were inactivated by plasma in a background fluid of nonionic osmolytes despite the low level of reactive species. Our results suggest that the surrounding environment may affect the behavior of reactive species, which leads to different biological consequences regardless of their quantity. Moreover, the microbicidal effect of plasma can be synergistically regulated through control of the microenvironment.     

Primary antioxidant free radical scavenging and redox signaling pathways in higher plant cells

Antioxidants in plant cells mainly include glutathione, ascorbate, tocopherol, proline, betaine and others, which are also information-rich redox buffers and important redox signaling components that interact with cellular compartments. As an unfortunate consequence of aerobic life for higher plants, reactive oxygen species (ROS) are formed by partial reduction of molecular oxygen. The above enzymatic and non-enzymatic antioxidants in higher plant cells can protect their cells from oxidative damage by scavenging ROS. In addition to crucial roles in defense system and as enzyme cofactors, antioxidants influence higher plant growth and development by modifying processes from miotosis and cell elongation to senescence and death. Most importantly, they provide essential information on cellular redox state, and regulate gene expression associated with biotic and abiotic stress responses to optimize defense and survival. An overview of the literature is presented in terms of primary antioxidant free radical scavenging and redox signaling in plant cells. Special attention is given to ROS and ROS-anioxidant interaction as a metabolic interface for different types of signals derived from metabolisms and from the changing environment. This interaction regulates the appropriate induction of acclimation processes or execution of cell death programs, which are the two essential directions for higher plant cells.     

Zinc protects HeLa-tat cells against free radical cytotoxicity induced by glucose

In the present study, we investigated the protective effect of zinc on the glucose-induced cytotoxicity in HeLa wild and HeLa-tat cells (30 and 20 mmol/l glucose, respectively). HeLa cells transfected with the protein Tat exhibit a lower antioxidant defense system. Incubation of HeLa wild and HeLa-tat cells with high glucose levels led to a rapid increase in generation of reactive oxygen species (ROS). As expected in the presence of high glucose concentrations, the viability was reduced for both cell lines. The redox status essentially regulated by thiol groups may play an important role in the apoptotic process. Thus, we developed a new method using the p-nitrophenyl disulfide to measure cytosolic thiol groups in intact cells. Cellular zinc was measured using inductively coupled plasma mass spectrometry. Intracellular thiol groups and intracellular zinc concentrations were significantly lower in HeLa cells cultured in hyperglycemic conditions, and their concentrations were significantly lower in HeLa-tat cells than in HeLa wild cells. However, the generation of ROS and the induction of apoptosis by a glucose specific mechanism were prevented by zinc (50 micromol/l) and the intracellular thiol groups and zinc concentrations significantly increased in both cell lines to become similar to the initial values. These results suggest that the glucose oxidation and its subsequent effects on the cells can be prevented by a biological antioxidant such as zinc.     

活性氧簇在脑缺血-再灌注损伤中的损伤与保护作用

活性氧簇是细胞有氧代谢过程中产生的一类化学基团。线粒体是活性氧簇的主要生成位点。一般观点认为,在脑缺血-再灌注损伤过程中,活性氧簇发挥神经细胞损伤作用。活性氧簇不仅直接参与神经细胞氧化损伤过程,也可通过外源性途径和内源性途径,引起神经细胞凋亡。然而,除神经细胞损伤作用外,活性氧簇也可发挥神经细胞保护作用。活性氧簇可激活低氧诱导因子、核转录因子κB、PI3K/Akt通路和MAPK通路等,参与神经细胞存活机制,减轻神经细胞损伤。本文对活性氧簇在脑缺血-再灌注损伤中的双重作用进行综述。     

Developmental regulation of beta-1,3-galactosyltransferase-1 gene expression in mouse brain

Preadipocytes are present and can proliferate to increase fat mass throughout adult life. The importance of mitochondria in these cells has never been investigated, although we recently reported that mitochondrial oxidative metabolism is non-negligible in white preadipocytes. Mitochondrial reactive oxygen species generation is intimately associated with respiratory chain function. An increasing number of reports support their role as signalling molecules. The aim of this work was to study the effects of mitochondrial reactive oxygen species on proliferation of white preadipocytes. Rotenone and oligomycin, inhibitors of complex I and of ATP synthase respectively, increased H₂O₂ and inhibited cell growth of preadipocytes (without inducing necrosis or apoptosis). These effects were partly prevented by addition of radical scavengers. A chemical uncoupler had opposite effects on reactive oxygen species generation and cell growth. Propofol, which inhibits complex I but also scavenges free radicals, had effects similar to those of the uncoupler on both parameters. Thus, mitochondrial reactive oxygen species can influence development of adipose tissue by affecting the size of the white preadipocyte pool.     

Variation of 1-pyrenebutyric acid fluorescence lifetime in single living cells treated with molecules increasing or decreasing reactive oxygen species levels

1-Pyrenebutyric acid (PBA) is a fluorescent probe whose fluorescence lifetime depends on local oxygen and free radical concentrations. We propose to use PBA fluorescence lifetime to quantify reactive oxygen species (ROS) in biological samples. Time-resolved microfluorimetry was used to record the fluorescence decay of single living cells loaded with this probe. We measured intracellular PBA fluorescence lifetimes and reduced nicotinamide adenine dinucleotide phosphate intensities under various oxygen concentrations. To confirm the feasibility of the new method, CCRF-CEM cells were treated with drugs that are known to increase or decrease ROS production. After treatment with adriamycin, we observed a decrease of PBA fluorescence lifetime. This corresponded to an increase of ROS concentration (80%). After treatment with cysteamine, we observed a reduction of the ROS concentration by 67%. Moreover, addition of exogenous H(2)O(2) solution resulted in a decrease of PBA fluorescence lifetime due to a raising of the intracellular ROS concentration. These results support our hypothesis linking a part of PBA fluorescence lifetime variations to intracellular fluctuation of ROS.     

Identification of Catechol-Protein Conjugates in Neostriatal Slices Incubated with [3H]Dopamine: Impact of Ascorbic Acid and Glutathione

Abstract: There is evidence to suggest that degeneration of dopaminergic neurons in Parkinson's disease and certain other conditions results from the action of reactive species generated during the oxidation of dopamine. We, therefore, have begun to explore the conditions under which such reactive species are formed. Tissue slices prepared from rat neostriatum were incubated in a standard Krebs bicarbonate buffer for up to 120 min. In the presence of [³H]dopamine (0.01–100 µ M ), binding of tritium to the acid-insoluble protein fraction was detected. Binding was attenuated by the addition of ascorbate (0.085–0.85 m M ) or glutathione (0.01–1.0 m M ) to the buffer. Acid hydrolysis of the protein revealed the presence of cysteinyl-dopamine and cysteinyl-dihydroxyphenylacetic acid residues. These results suggest that dopamine oxidizes to form reactive metabolites, presumably quinones, that then bind to nucleophilic sulfhydryl groups on protein cysteinyl residues. The findings further suggest that the extent to which reactive metabolites are formed is determined in part by the balance between the availability of dopamine and the antioxidant environment.     

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.     

Dietary oxidative stress and antioxidant defense with an emphasis on plant extract administration

Eukaryotic cells generally function in a reduced state, but an amount of reactive species is essential for several biochemical processes. The antioxidant network is the defensive mechanism that occurs when the concentration of reactive species exceeds a threshold. Polyphenolic compounds present in plant extracts are potent antioxidants in vitro, but they may promote oxidative stress when administered in animals and humans, especially when given as supplements in exercise, a modality usually adopted as an oxidant stimulus. This is mainly observed when antioxidant molecules are administered separately and not as part of a diet. Exercise is usually adopted as a physiological model for examining the effects of reactive species in human or animal physiology. The use of exercise as a model demonstrates that reactive species do not always have adverse effects, but are necessary in physiological processes that are beneficial for human health. This review summarizes what is known about antioxidant supplementation and demonstrates the need for a meticulous examination of the in vitro findings before applying them to in vivo models. The term “antioxidant” seems elusive, and it is more appropriate to characterize a compound as “antioxidant” if we know in which concentration it is used, when it is used, and under which conditions.     

Tert-butyl-2(4,5-dihydrogen-4,4,5,5-tetramethyl-3-O-1H-imidazole-3-cationic-1-oxyl-2-pyrrolidine-1-carboxylic ester displays novel cytotoxicity through reactive oxygen species-mediated oxidative damage in MCF-7 and MDA-MB-231 cells

The cytotoxicity of a new nitroxyl nitroxide radical, tert-butyl-2 (4,5-dihydrogen-4,4,5,5-tetramethyl-3-O-1H-imidazole-3-cationic-1-oxyl-2-pyrrolidine-1-carboxylic ester (L-NNP) was examined in MCF-7 and MDA-MB-231 cells. L-NNP treatment resulted in a significant growth inhibition in MCF-7 and MDA-MB-231 cells. Compared with control, 10, 30, and 50 μg/ml L-NNP treatments for 48 h induced significant cell and nuclei swelling, and organelle distension. The marked cell death was seen in a concentration- and time-dependant manner in L-NNP treated groups. The L-NNP treated group displayed a concentration-dependant increase in DNA double strand damage compared to the control and the 1 Gy γ-rays exposure groups. These results suggest that L-NNP could result in more lethal genotoxicity than 1 Gy γ-radiation. Based on mitochondrial alteration (membrane potential loss and SDH activity descend), DNA damage, an increase in MDA production, and GSH-PX inactivation, we predicate that L-NNP induces lipid oxidation and oxidative damage in MCF-7 and MDA-MB-231 cells. Since L-NNP initiated a significant increase in reactive oxygen species, which could largely be inhibited by NAC pretreatment, the overall data strongly suggest that the mechanism of cytotoxicity of L-NNP was its ability to act as a strong free radical, and significantly increase intracellular reactive oxygen species production. This led to intracellular oxidative damage, and antioxidant enzyme inactivation, resulting in cell death. We hypothesize that the greater cytotoxicity of L-NNP in MDA-MB-231 cells than in MCF-7 cells might be due to more ROS production in MDA-MB-231 cells, leading to more oxidative damage.     

Gamma radiolysis as a tool to study lipoprotein oxidation mechanisms

Well-defined quantities of *OH, O2*-,HO2* or RO2*)radicals (reactive oxygen species) can be specifically produced by radiolysis of water or ethanol. Such radical species can initiate one-electron oxidation or one-electron reduction reactions on numerous biological systems. The oxidative hypothesis of atherosclerosis classically admits the involvement of the oxidation of low density lipoproteins (LDLs) but also of high density lipoproteins (HDLs) in the development of the atherosclerotic process. The initiation mechanisms of this oxidation are still incompletely defined, although free radicals are likely involved. Therefore, gamma-radiolysis appears as a method of choice for the in vitro study of the mechanisms of oxidation of LDLs and HDLs by oxygen-centred free radicals (*OH, O2*-,HO2* and RO2*). Radiolytically oxidized lipoproteins exhibited a very well defined oxidation status (radiation dose-dependent quantification of vitamin E, beta-carotene, lipid peroxidation, protein carbonylation ...). gamma-Radiolysis is a less drastic method than other oxidation procedures such as for example copper ions. Moreover, gamma-radiolysis is also especially suitable for studying the reducing properties of antioxidant compounds with regard to their scavenging capacity.     

Cobalt-mediated generation of reactive oxygen species and its possible mechanism

Electron spin resonance spin trapping was utilized to investigate free radical generation from cobalt (Co) mediated reactions using 5,5-dimethyl-l-pyrroline (DMPO) as a spin trap. A mixture of Co with water in the presence of DMPO generated 5,5-dimethylpyrroline-(2)-oxy(1) DMPOX, indicating the production of strong oxidants. Addition of superoxide dismutase (SOD) to the mixture produced hydroxyl radical (OH). Catalase eliminated the generation of this radical and metal chelators, such as desferoxamine, diethylenetriaminepentaacetic acid or 1,10-phenanthroline, decreased it. Addition of Fe(II) resulted in a several fold increase in the OH generation. UV and O₂ consumption measurements showed that the reaction of Co with water consumed molecular oxygen and generated Co(II). Since reaction of Co(II) with H₂O₂ did not generate any significant amount of OH radicals, a Co(I) mediated Fenton-like reaction [Co(I) + H₂O₂ → Co(II) + OH + OH⁻] seems responsible for OH generation. H₂O₂ is produced from O₂⁻ via dismutation. O₂⁻ is produced by one-electron reduction of molecular oxygen catalyzed by Co. Chelation of Co(II) by biological chelators, such as glutathione or β-ananyl-3-methyl- -histidine alters, its oxidation–reduction potential and makes Co(II) capable of generating OH via a Co(II)-mediated Fenton-like reaction [Co(II) + H₂O₂ → Co(III) + OH + OH⁻]. Thus, the reaction of Co with water, especially in the presence of biological chelators, glutathione, glycylglycylhistidine and β-ananyl-3-methyl- -histidine, is capable of generating a whole spectrum of reactive oxygen species, which may be responsible for Co-induced cell injury.     

Oxygen free radicals and redox biology of organelles

The presence and supposed roles of reactive oxygen species (ROS) were reported in literature in a myriad of instances. However, the breadth and depth of their involvement in cellular physiology and pathology, as well as their relationship to the redox environment can only be guessed from specialized reports. Whatever their circumstances of formation or consequences, ROS seem to be conspicuous components of intracellular milieu. We sought to verify this assertion, by collecting the available evidence derived from the most recent publications in the biomedical field. Unlike other reviews with similar objectives, we centered our analysis on the subcellular compartments, namely on organelles, grouped according to their major functions. Thus, plasma membrane is a major source of ROS through NAD(P)H oxidases located on either side. Enzymes of the same class displaying low activity, as well as their components, are also present free in cytoplasm, regulating the actin cytoskeleton and cell motility. Mitochondria can be a major source of ROS, mainly in processes leading to apoptosis. The protein synthetic pathway (endoplasmic reticulum and Golgi apparatus), including the nucleus, as well as protein turnover, are all exquisitely sensitive to ROS-related redox conditions. The same applies to the degradation pathways represented by lysosomes and peroxisomes. Therefore, ROS cannot be perceived anymore as a mere harmful consequence of external factors, or byproducts of altered cellular metabolism. This may explain why the indiscriminate use of anti-oxidants did not produce the expected beneficial results in many medical applications attempted so far, underlying the need for a deeper apprehension of the biological roles of ROS, particularly in the context of the higher cellular order of organelles.