Association of Antioxidant Systems in the Protection of Human Fibroblasts Against Oxygen Derived Free Radicals

The protection of human diploid fibroblasts against high oxygen tension was investigated using various combinations of the three major antioxidant enzymes: superoxide dismutase, catalase and gluthathione peroxidase. α-Tocopherol, a well-known hydrophobic antioxidant, was also tested in combination with the different enzymes. Microinjection of solutions containing different combinations of the three enzymes was compared with the injection of each single enzyme. We observed that the protections given by catalase or superoxide dismutase on the one hand, and by glutathione peroxidase on the other hand, were additive. Surprisingly, the combinations of catalase and superoxide dismutase were less effective than catalase alone and was even toxic at low SOD concentrations. Addition of α-tocopherol following the injection of any of the three enzymes was highly beneficial, but the strongest synergistic effect was obtained with glutathione peroxidase. These results stress the importance of membrane protection by α-tocopherol and indirectly by glutathione peroxidase. They also showed that any injection leading to the decrease in the O₂^?. or H₂ O ₂ concentration combined with one of these two protectors is very beneficial for the cells probably by decreasing the OH concentration. This is also proven by the very good protective effect obtained with desferrioxamine.     

Nutritional Effect and Expression of SODs: Induction and Gene Expression; Diagnostics; Prospective Protection Against Oxygen Toxicity

The effect of micronutrient stress (either deficiency or toxicity) on the expression or different superoxide dismutase isoenzymes in plants is reviewed. The induction of Fe-SOD and Mn-SOD by different metals and the potential use of the metalloentyme system SOD lor the appraisal of the micronutrient status of plants, is examined. At subcellular level, evidence for the participation of peroxisomal SOD in the molecular mechanism of plant tolerance to Cu is presented, and the activated oxygen-dependent toxicity of a xenobiotic (clofibrate) in plant peroxisomes is examined.     

Chloroplast protection in greening leaves

Changes in photosynthetic activity, leaf pigments and the activities of enzymes that scavenge damaging oxygen species in chloroplasts were followed during the greening of 8-day-old etiolated pea (Pisum sativum L. cv. Meteor) seedlings. Accumulation of chlorophyll and carotenoids was accompanied by development of photosynthetic activity. Carotenoids present in etiolated leaves, and the high ratio of carotenoid to chlorophyll detected during the early hours of greening are suggested to provide important protection against singlet oxygen. Superoxide dismutase, ascor-bate peroxidase and glutathione reductase, which scavenge superoxide and hydrogen peroxide in chloroplasts, are present at high activities in etiolated leaves and throughout greening. The mechanisms by which developing chloroplasts may generate damaging oxygen species, and the role of these scavengers during greening is discussed.     

极谱氧电极法测定超氧化物歧化酶活性的改进

对SOD的极谱氧电极测定法做了如下修改:a.室温测定,b.酶活性用标准SOD标定,c.反应在磷酸缓冲液中进行,d.增大邻苯三酚的用量.改进后克服了易在电极薄膜表面产生气泡等问题,测定灵敏度及线性范围增大.     

Alcohol-Induced Hepatotoxicity: A Role for Oxygen Free Radicals

Perfusion of isolated rat livers with ethanol at a concentration of 2g/l (%o) resulted in a release of glutamate-pyruvate-transaminase (GPT) and sorbitol dehydrogenase (SDH) into the perfusate as markers of toxicity. Inhibition of alcohol dehydrogenase by 4-methylpyrazole or of aldehyde dehydrogenase by cyanamide totally abolished ethanol hepatotoxicity despite of a severalfold increase in acetaldehyde concentration in the perfusate. Addition of superoxide dismutase or catalase clearly suppressed the ethanol-induced release of GPT and SDH, suggesting that 0₂∼ and H₂0, are involved in this process. Also. chelation of iron ions by means of desferrioxamine displayed a clear inhibitory action, suggesting the involvement of an iron-catalyzed Haber-WeiB-reaction leading to the formation of OH radicals in the hepatotoxic response to ethanol. Our data suggest that during the metabolism of acetaldehyde primary reactive oxygen species ('0₂∼, H₂0₂) are produced which may interact to yield hydroxyl or OH-like radicals, which possibly represent the hepatotoxic principle of ethanol.     

Alcohol-Induced Hepatotoxicity: A Role for Oxygen Free Radicals

Perfusion of isolated rat livers with ethanol at a concentration of 2g/l (%o) resulted in a release of glutamate-pyruvate-transaminase (GPT) and sorbitol dehydrogenase (SDH) into the perfusate as markers of toxicity. Inhibition of alcohol dehydrogenase by 4-methylpyrazole or of aldehyde dehydrogenase by cyanamide totally abolished ethanol hepatotoxicity despite of a severalfold increase in acetaldehyde concentration in the perfusate. Addition of superoxide dismutase or catalase clearly suppressed the ethanol-induced release of GPT and SDH, suggesting that 0₂～ and H₂0, are involved in this process. Also. chelation of iron ions by means of desferrioxamine displayed a clear inhibitory action, suggesting the involvement of an iron-catalyzed Haber-WeiB-reaction leading to the formation of OH radicals in the hepatotoxic response to ethanol. Our data suggest that during the metabolism of acetaldehyde primary reactive oxygen species ('0₂～, H₂0₂) are produced which may interact to yield hydroxyl or OH-like radicals, which possibly represent the hepatotoxic principle of ethanol.     

Commentary: Oxygen Radicals, A Failure Or A Success of Evolution?

Oxygen radicals are no doubt involved in the development of many pathological states. Nevertheless, the possibility that oxygen radical production was selected for during biological evolution in order to perform useful roles in relation to cellular metabolism is contemplated; previous data on this subject are briefly reviewed. The concept of an “oxygen radical cycle” is proposed as a useful theoretical model.     

Commentary: Oxygen Radicals,A Failure Or A Success of Evolution?

Oxygen radicals are no doubt involved in the development of many pathological states. Nevertheless, the possibility that oxygen radical production was selected for during biological evolution in order to perform useful roles in relation to cellular metabolism is contemplated; previous data on this subject are briefly reviewed. The concept of an “oxygen radical cycle” is proposed as a useful theoretical model.     

活性氧所致超氧化物歧化酶肽链断裂的观察

探究活性氧所致铜锌超氧化物歧化酶（ＳＯＤ）肽链断裂的情况。将过氧化氢或抗坏血酸－Ｆｅ（Ⅲ）分别作用于马来酰亚胺标记的ＳＯＤ,然后用高效液相反相色谱（ＲＰＨＰＬＣ）分析,经１ｍｍｏｌ／ＬＨ２Ｏ２处理后ＳＯＤ用ＲＰ－ＨＰＬＣ分离出二个肽段,用顺磁共振检测显示只有一个肽段具有马来酰亚胺信号,经５ｍｍｏｌ／ＬＨ２Ｏ２处理后ＳＯＤ有四个肽段生成,其中有一个肽段具有马来酰亚胺信号,用５ｍｍｏｌ／Ｌ抗坏血酸和０．０１ｍｍｏｌ／ＬＦｅＣｌ３处理后ＳＯＤ有三个肽段生成,用５０ｍｍｏｌ／Ｌ抗坏血酸及１．０ｍｍｏｌ／ＬＦｅＣｌ３处理后ＳＯＤ也产生相同的三个肽段,只是肽段的量多．结果提示Ｈ２Ｏ２所致ＳＯＤ肽链断裂无“定点”现象,而抗坏血酸－Ｆｅ（Ⅲ）所致ＳＯＤ肽链断裂呈“定点”断裂。     

Chloroplast manganese and superoxide.

Particles prepared from spinach chloroplast membranes with Triton X-100 inhibited the superoxide-mediated reduction of nitro-blue tetrazolium by riboflavin. This superoxide dismutase-like activity was of two kinds, one inactivated by heating and inhibited by H₂O₂ and the other insensitive to both of these treatments; both activities were destroyed by washing with concentrated Tris buffer or with EDTA. Attempts at reconstitution with transition metal ions suggested that two different forms of bound manganese may be responsible and it is proposed that the inhibition by H₂O₂ is indicative of three different oxidation states of particle-bound manganese. The possibility that the photosynthetic water-splitting system and superoxide dismutase have evolved from a single precursor is discussed.     

Superoxide Production by the Mitochondrial Respiratory Chain

This mini-review describes the role of different mitochondrial components in the formation of reactive oxygen species under normal and pathological conditions and the effect of inhibitors and uncouplers on superoxide formation.     

活性氧所致超氧化物歧化酶疏水性的变化

用抗坏血酸-Fe(Ⅲ)和过氧化氢分别作用于铜锌超氧化物歧化酶(SOD),经疏水层析分离得到亲水型和疏水型SOD.用胰蛋白酶和胃蛋白酶分别作用于天然SOD,亲水型SOD及疏水型SOD,结果表明疏水型SOD较亲水型SOD及天然SOD易被降解,提示活性氧氧化修饰后的SOD对蛋白水解酶敏感性提高与其疏水性增高有关.     

The Role of Oxygen in Thermo-Photodynamic Processes Leading to Sunscald-Like Damages in Green Tissues

Sunscald is a physiological disorder causing, in warm climates. severe damages to green tissues. Earlier studies established the essential role of heat, light and chlorophyll in the development of the disorder. In the present work, the role of oxygen in the development of the injury was investigated. Green cucumber fruit were exposed to heat and light under controlled atmosphere, and the development of the injury was followed. No damage occurred in the absence of either heat or light. Tissues exposed to heat, light and either nitrogen or air, suffered marked electrolyte leakage, indicative of membrane damage. Chlorophyll fluorescence values of cucumbers treated with high temperatures, oxygen and light, were considerably lower than those measured for the respective dark treated tissues. Irradiated cucumbers flushed with either 100% N₂ or < 2.5% O₂, lost about 50% of their chlorophyll. These tissues turned olive-green but did not bleach. At higher conc., oxygen treated tissues lost their plastid pigments and bleached within 7 hr of exposure to heat and light. A significant decrease in chlorophyll was evident also in atmosphere containing 1 % CO₂ in nitrogen. Malondialdehyde conc was significantly higher in the presence of O₂ as compared with N₂ atmosphere. The results clearly show, that sunscald damage is a result of thermo-photooxidative processes.     

Toxic oxygen species and protective systems of the chloroplast

Salin, M. L. 1988. Toxic oxygen species and protective systems of the chloroplast. -Physiol. Plant. 72: 681–689.
As a consequence of living in an environment enriched in oxygen, which they themselves at least partially generate, photosynthetic organisms are exposed to large fluxes of oxyradicals and reactive oxygen species. Among these are superoxide, hydrogen peroxide, hydroxyl radical and singlet oxygen. These highly reactive intermediates pose the threat of toxicity unless neutralized by scavenger substrates or enzymes. The production of oxyradicals and intermediates by chloroplasts as well as the means of protection are discussed in this review.     

Evidence for Superoxide Dismutase and Catalase in Mollicutes and Release of Reactive Oxygen Species

The presence of superoxide dismutase was demonstrated in 21 strains of mollicutes, including achuloplas-mas, mycoplasmas and ureaplasmas. Additionally, catalase activities were demonstrated in nearly 50% of the cell lysates. whereas no peroxide activities were detectable. The production of O₂-and H₂O₂ with glucose as substrate was demonstrated for 8 strains of 10 strains tested. Anaerobic mycoplasmas showed the highest amount of radical production, whereas superoxide dismutase and catalase activities were in the range of activities estimated for aerobic mollicutes. Some pathogenic strains additionally released compounds into the culture medium, which stimulated O₂-production by PMNs.     

Oxygen effects on maize leaf superoxide dismutase and glutathione reductase

Exposure of maize seedlings to an atmosphere containing 75% O₂ and 350 ppm CO₂ resulted in a two- to three-fold increase in glutathione reductase activity in leaf tissue within 48 hr after initiation of the O₂ treatment. This elevated level of glutathione reductase was still evident in plants maintained in the hyperopic environment for 7 days. Superoxide dismutase activity was not altered from its level in control tissue during the 7-day experimental period. These results suggest a key role for glutathione reductase in the protection of photosynthetic tissue against detrimental effects of intermediate reduction products of O₂.     

Activities of Antioxidant Enzymes during Senescence of Prunus Armeniaca Leaves

During the period of senescence of apricot leaves changes in photosynthetic pigment contents and in the activities of some antioxidant enzymes (superoxide dismutase, catalase, peroxidase and ascorbate peroxidase) were analysed. Significant changes in pigment contents were, in most cases, correlated with changes in activities of the antioxidant enzymes. Modifications in superoxide dismutase and catalase isoform patterns were also observed during the progression of senescence. Both enzyme activities and isoenzyme patterns proved to be genotype-dependent.     

Increased Hepatic Lipid Peroxidation with Methionine Toxicity in the Rat

Consumption of excess methionine by rats is known to cause membrane damage, liver enlargement and accumulation of iron in the spleen. In this study two groups (n = 5) of male, Wistar rats were pair-fed either a methionine supplemented (20.0 g/kg) or control (2.0 g/kg) diet for 7 weeks. Hepatic and erythrocyte copper-zinc superoxide dismutase activities were significantly reduced (P < 0.05 and P < 0.001 respectively) by methionine supplementation while the activities of catalase (P < 0.01 and 0.05) and glutathione peroxidase (P < 0.05) were significantly increased. Methionine supplementation also increased hepatic lipid peroxidation (P < 0.01), as measured by the level of thiobarbituric acid reactive substances, and iron (P < 0.001) concentrations. These changes are indicative of increased oxidative stress resulting from methionine toxicity.     

Percentage of phagocytosis,production of O2·−, H2O2 and NO,and antioxidant enzyme activities of rat neutrophils in culture

Changes in the integrity, ultrastructure, phagocytosis capacity, and production of H₂O₂, O₂^{· −}and NO₂⁻ were evaluated in cultured neutrophils. The activities of the antioxidant enzymes (catalase—CAT, superoxide dismutase—SOD and glutathione-dependent peroxidase—GSH-Px) were measured under similar conditions. The integrity of the cells remained unchanged up to 18 h. After 24 h, the number of viable cells in culture dropped by 16 per cent. The percentage of viable cells in culture was of 72 per cent even after 72 h. An ultrastructural analysis of the cells was carried out after 3, 6, 12, 24, 48, and 72 h in culture. Neutrophils started developing morphologic changes after 24 h: decreased cell volume, abundant vacuoles (mainly around the nucleus), and also the presence of autophagic vacuoles. This period was then chosen for the study of neutrophil function and antioxidant enzyme activities. Neutrophils cultured for 24 h presented reduced phagocytosis capacity. The rates of production of H₂O₂ and O₂^{· −} remained unchanged after 24 h in culture. Concomitantly, these cells were also able to produce NO in significant amounts. The production of O₂^·− in response to PMA stimulus was lowered in 24-h cultured cells. Possibly, the production of oxygen and nitrogen reactive species accomplished with a decrease in the activities of CAT and GSH-Px play a key role for the process of apoptosis which takes place in neutrophils under these conditions. © 1998 John Wiley & Sons, Ltd.