Oxygen-derived free radicals producing activity and survival of activated polymorphonuclear leukocytes

Summary Activation of polymorphonuclear (PMN) leukocytes is known to generate oxygen free radicals (OFR). However the fate of activated PMN leukocytes is not known. We investigated the OFR producing (chemiluminescence) activity and the survival of the activated PMN leukocytes. The study was divided into two groups. Group I, In vivo study (n = 7): zymosan (8.4 mg/kg) was administered intravenously in the anesthetized dogs and the blood samples were collected before and after 5, 15, 30, 60 and 120 min of zymosan administration. This group represents the in vivo pre-stimulated PMN leukocytes; Group II, In vitro study (n = 7): the blood were collected from dogs and further divided into two groups. Group A (n = 7): non-stimulated, without any added zymosan and group B (n = 7): zymosan was added to stimulate PMN leukocytes. Blood samples from group A and B were also collected at various time intervals similar to in vivo studies. Oxygen free radical producing activity of PMN leukocytes was monitored by measuring luminoldependent chemiluminescence (CL). Opsonized zymosan was used to activate PMN leukocytes. The studies in which the PMN leukocytes were stimulated in in vivo, both oxygen derived free radicals and superoxide dismutase (SOD) inhibitable oxygen free radical CL decreased significantly for 60 min and tended to reach thereafter to the pre-stimulated values. The resting chemiluminescence (chemiluminescence without zymosan stimulation in the assay medium) increased significantly for 15 min reaching to pre-stimulated values at 30 min and thereafter. In in vitro studies, oxygen derived free radicals CL of pre-stimulated PMN leukocytes (Group B) was depressed for the whole duration of investigation while SOD inhibitable CL was depressed for only 60 min. There was approximately a two-fold increase in the resting CL within 5 min of PMN leukocyte activation and it remained high for the whole duration of study. The chemiluminescence of non-stimulated PMN leukocytes in vitro (group A) remained practically normal throughout the period of observation. In in vivo studies, total white blood cells (WBC) and PMN leukocyte counts decreased initially and tended to approach towards pre-stimulated values at the end of the protocol. There were no changes in these counts in in vitro studies. These results indicate that the capacity to generate OFR is decreased in the in vivo and in vitro pre-stimulated PMN leukocytes. However this activity recovers with time. This study also suggests that the activated PMN leukocytes are not destroyed.     

Modification of contractile proteins by oxygen free radicals in rat heart

This study was undertaken to investigate the effects of oxygen free radicals on myofibrillar creatine kinase activity. Isolated rat heart myofibrils were incubated with xanthine+xanthine oxidase (a superoxide anion radical-generating system) or hydrogen peroxide and assayed for creatine kinase activity. To clarify the involvement of changes in sulfhydryl groups in causing alterations in myofibrillar creatine kinase activity, 1) effects of N-ethylmaleimide (sulfhydryl groups reagent) on myofibrillar creatine kinase activity, 2) effect of oxygen free radicals on myofibrillar sulfhydryl groups content, and 3) protective effects of dithiothreitol (sulfhydryl groups-reducing agent) on the changes in myofibrillar creatine kinase activity due to oxygen free radicals were also studied. Xanthine+xanthine oxidase inhibited creatine kinase activity both in a time-and a concentration-dependent manner. Superoxide dismutase (SOD) showed a protective effect on the depression in creatine kinase activity caused by xanthine+xanthine oxidase. Hydrogen peroxide inhibited creatine kinase activity in a concentration-dependent manner; this inhibition was prevented by the addition of catalase. N-ethylmaleimide reduced creatine kinase activity in a dose-dependent manner. The content of myofibrillar sulfhydryl groups was decreased by xanthine+xanthine oxidase; this reduction was protected by SOD. Furthermore, the depression in myofibrillar creatine kinase activity by xanthine+xanthine oxidase was protected by the addition of dithiothreitol. Oxygen free radicals may inhibit myofibrillar creatine kinase activity by modifying sulfhydryl groups in the enzyme protein. The reduction of myofibrillar creatine kinase activity may lead to a disturbance of energy utilization in the heart and may contribute to cardiac dysfunction due to oxygen free radicals.     

Oxygen free radicals in volume overload heart failure

It has been suggested that oxygen free radicals (OFR) depress the excitation-contraction coupling in cardiac muscle. It is possible that a decrease in the cardiac contractility in the failing heart may be due to an increased OFR producing activity of polymorphonuclear (PMN) leukocytes. We studied the OFR producing activity (chemiluminescence) of PMN leukocytes from blood in dogs with heart failure due to chronic volume overload. The animals were divided into two groups: I) normal, (n = 10): II) dogs with mitral insufficiency (MI) of 6 to 9 months duration, (n = 10). Hemodynamic studies were done to establish the presence of heart failure. Blood samples were collected to measure PMN leukocyte chemiluminescence. There was a decrease in the cardiac index and index of myocardial contractility (dp/dt/IIP) and an increase in the left ventricular end-diastolic pressure in dogs with MI indicating left ventricular failure. The peak chemiluminescent activity of the PMN leukocytes in blood of dogs with failure was about four folds greater than that in the blood from normal dogs. These results suggest that there may be an increased OFR generation in dogs with volume overload heart failure. The decrease in the myocardial contractility in the failing heart might be due to an increase in the OFR produced by the PMN leukocytes.     

Reactivity of ubiquinones and ubiquinols with free radicals

The reactivity of quinones 1–4 and of the corresponding quinols 5–8 towards carbon- and oxygen-centred radicals were studied. All quinones bearing at least one nuclear position free, readily react with alkyl and phenyl radicals to afford the alkylated quinones 12–24; however, quinones 1 and 3 reacted with 2-cyano-2-propyl radical to yield products (the mono- and di-ethers 9–11) derived from the attack on the carbonylic oxygen. The reactions carried out on quinones with the benzoyloxy radical led to no reaction products and in the case of Q₁₀, the isoprenic chain also remained unchanged. Quinols 5–8 reacted only with oxygencentred radicals (benzoyloxy and 2-cyano-2-propylperoxy radicals) to give the corresponding quinones. The isoprenic chain of Q₁₀ did not undergo attack even with peroxy radicals. Carbon-centred radicals resulted unable to abstract hydrogen from the studied quinols.     

缩短体外循环管道对新生儿和小婴儿心脏手术输血量的影响

目的回顾分析了逐步缩短体外循环管道对新生儿和小婴儿心脏手术输血量的影响。方法将2000年至2010年体重10kg以下病人4822例,分为3组：组1（n=1265）Dedico901/902/Medtronic氧合器＋Stockert II/Jostra HL20人工心肺机;组2（n=2534）Rx-05氧合器＋Stockert II/Jostra HL20人工心肺机;组3（n=1023）Rx-05氧合器＋Jostra HL30人工心肺机。比较各组预充总量,红细胞悬液的使用量和血球压积。结果组3和组2的预充量显著低于组1预充量（组1,718±15ml;组2,458±15ml;组3,334±8ml,P〈0.001）。围术期各组血球压积无显著差异;然而各组在预充液中加入的红细胞悬液有显著差异（组1,313±5 ml;组2,205±5ml;组3,167±8ml,P〈0.001）,体外循环转流中加入的红细胞悬液组间也有显著性差异（组1,117±8ml;组2,88±7ml;组3,60±8ml,P〈0.001）。结论通过改进氧合器和缩短管道长度能减少预充量和总用血量,逐步向不用血心脏外科迈进。     

Chilling response of plants: importance of galactolipase, free fatty acids and free radicals

The chilling response of plants is complex and based on the interplay of two important metabolic processes--lipolytic degradation of membrane lipids and a set of oxidative reactions leading to lipid peroxidation and membrane damage evoked in chilling-sensitive (CS) plants subjected to low temperature and light. The effects of chilling of detached leaves and intact plants differ and are often neglected during experiments. In closely-related species, the activity of several constitutive enzymes (i.e. superoxide dismutase, ascorbate peroxidase and glutathione reductase) appears to be higher in chilling-tolerant (CT) than in CS species; while in several native, closely-related CS species, lipid acyl hydrolase (galactolipase) activity is higher than in CT species. Moreover, in chilling-insensitive (CI) plants, galactolipase activity is very low and is neither activated by detachment of leaves nor under stress conditions in growing plants. Dark and low-temperature treatments of detached leaves of CS species and post-chilling recovery of growing plants in the light activate galactolipase, which is responsible for the release of free fatty acids (FFA), the main substrates of peroxidation by lipoxygenase and free radicals. In several CS species, increased galactolipase activity is an important factor contributing to chilling susceptibility. Thus, it seems likely that enhancement of chilling tolerance may be achieved by genetically suppressing galactolipase in order to reduce both the degradation of chloroplast lipids and the level of released FFA, and thereby avoiding the deleterious action of their peroxidation products on plant tissues.     

活性氧、自由基与植物的衰老

介绍近 1 0年来有关活性氧、自由基的产生 ,对植物的伤害及植物对活性氧、自由基清除的研究进展。     

Generation of free oxygen radicals in heart mitochondria: Effect of hypoxia-reoxygenation

The relationship between the rate of generation of superoxide radicals and the duration of hypoxia has been studied in isolated heart mitochondria with the use of the spin trap sodium 4,5dihydroxybenzene-1,3-disulfonate. The EPR spectra were recorded from a mitochondrial suspension placed in a gas-permeable capillary under conditions of regulated partial oxygen pressure. Earlier we have shown that the mitochondria isolated from perfused hearts after 30-min ischemia display a higher rate of superoxide generation than those from controls. However, in isolated mitochondria the EPR signal from 4,5-dihydroxybenzene-1,3-disulfonate increased already after 10-min hypoxia, but its intensity remained the same in the mitochondria subjected to 30-, 45-, and 60-min hypoxia. Thus, the isolated mitochondria in the incubation medium are less sensitive to hypoxia than the mitochondria from cardiomyocytes of an ischemic heart.     

Effects of free radicals on cytosolic creatine kinase activities and protection by antioxidant enzymes and sulfhydryl compounds

The main purpose of this study was to investigate the effect of free radicals and experimental diabetes on cytosolic creatine kinase activity in rat heart, muscle and brain. Hydrogen peroxide decreased creatine kinase activity in a dose dependent manner which was reversed by catalase. Xanthine/xanthine oxidase, which produces superoxide anion, lowered the creatine kinase activity in the same manner whose effect was protected by superoxide dismutase. N-acetylcysteine and dithiothreitol also significantly ameliorated the effect of Xanthine/xanthine oxidase and hydrogen peroxide. Experimental diabetes of twenty-one days (induced by alloxan), also caused a similar decrease in the activity of creatine kinase. This led us to the conclusion that the decrease in creatine kinase activity during diabetes could be due to the production of reactive oxygen species. The free radical effect could be on the sulfhydryl groups of the enzyme at the active sites, since addition of sulfhydryl groups like N-acetylcysteine and dithiothreitol showed a significant reversal effect.     

Changes in adrenergic receptors during the development of heart failure

Moderate and severe stages of congestive heart failure due to the loss of myocardium upon coronary occlusion in rats was associated with an increase in alpha-adrenergic receptors and a decrease in beta-adrenergic receptors in the viable left ventricle. However, at early stages of heart failure the number of beta-adrenergic receptors was decreased without any changes in the number of alpha-adrenergic receptors. The affinities of these receptors to alpha receptor antagonist (³H-prazosin) and beta receptor antagonist (³H-dihydroalprenolol) were not altered in the failing hearts. On the other hand, the pattern of changes in both alpha- and beta-adrenergic receptors in heart membranes treated with oxygen free radical generating system was different from that seen in the failing hearts. In particular, the affinities for these receptors were decreased whereas the number of beta-receptors was increased and the number of alpha-receptors was decreased or unchanged. These results indicate that alterations in the adrenergic receptors in heart failure are not due to the formation of oxygen free radicals.     

Oxidation of glycosaminoglycans by free radicals and reactive oxidative species: A review of investigative methods

Abstract

Glycosaminoglycans, in particular hyaluronan (HA), and proteoglycans are components of the extracellular matrix (ECM). The ECM plays a key role in the regulation of cellular behaviour and alterations to it can modulate both the development of human diseases as well as controlling normal biochemical processes such as cell signalling and pro-inflammatory responses. For these reasons, in vitro fragmentation studies of glycosaminoglycans by free radicals and oxidative species are seen to be relevant to the understanding of in vivo studies of damage to the ECM. A wide range of investigative techniques have therefore been applied to gain insights into the relative fragmentation effects of several reactive oxidative species with the ultimate goal of determining mechanisms of fragmentation at the molecular level. These methods are reviewed here.     

传统开腹手术与腹腔镜手术治疗子宫肌瘤的效果分析

目的探讨传统开腹手术与腹腔镜手术治疗子宫肌瘤的临床疗效。方法选择2013年2月~2015年11月在我院接受治疗的子宫肌瘤患者140例为研究对象,按患者自愿原则分为对照组和观察组各70例,对照组采用传统开腹手术治疗,观察组采用腹腔镜手术治疗,并对两组患者手术时间、术中出血量、术后感染情况和住院时间进行比较。结果观察组在手术时间、术中出血量、术后感染率及住院时间等方面均优于对照组,差异具有显著统计学意义(P0.05)。结论腹腔镜下子宫肌瘤切除术与传统开腹手术比较,具有出血量少、住院时间短、恢复快、并发症少等优点,值得临床推广应用。     

维生素E和硒水平对肉鸡不同自由基代谢的影响

选用200羽14日龄健康AA肉鸡,以电子自旋共振(ESR)捕集法和生物化学法对肉仔鸡血液和组织器官的不同自由基进行直接或间接测定,探讨VE和Se对肉鸡不同自由基代谢的作用及其动态变化.结果表明:组织一氧化氮(NO)自由基水平随日粮VE含量升高而降低,二者呈负相关关系,日粮高水平Se有诱导产生NO自由基的倾向;高VE和Se日粮显著提高血清和肝脏中SOD和GSH-Px的活性,但随处理时间的延长,组织SOD活性逐渐降低,而GSH-Px活性逐渐升高,说明日粮VE和/或Se不足均会诱导机体产生O.2-、H2O2自由基,且O2.-自由基会持续大量产生,而H2O2自由基仅在缺乏初期大量产生,而后趋于缓和;低VE和/或低Se均显著提高组织MDA含量,且低Se比低VE更为显著.VE和Se对肉鸡NO、O.2-和H2O2自由基代谢的作用存在协同效应.     

Cytotoxic effect of adriamycin and agarose-coupled adriamycin on glomerular epithelial cells: Role of free radicals

Summary It has been suggested that the generation of toxic radicals plays an important role in toxicity by Adriamycin (ADR) on cancer cell lines and in vivo. We have examined the role of free radicals in determining toxicity and resistance to ADR of rat glomerular epithelial cells in culture; this method provides a good model for analyzing the mechanisms responsible for ADR experimental nephrosis in rats. Three points were established: a) the intra- or extracellular site of ADR toxicity; b) the role of the superoxide anion and of the hydroxyl radical in determining intra- and-extracellular cytotoxicity; and c) the implication of oxido-reduction cycling as a potential route for ADR semiquinone transformation. Free ADR was found to induce the same inhibition of [³H]thymidine incorporation into DNA as ADR bound to an agarose macroporous bed which prevents the intracellular incorporation of the drug. Specific scavenging of free radical activity by the enzymes catalase and superoxide dismutase, the hydroxyl radical inhibitors dimethyl sulfoxide and dimethylthiourea (DMTU) and by chelation of intracellular free iron with deferoxamine produced only a partial restoration of [³H]thymidine incorporation into DNA, which was maximal for DMTU (30% of normal incorporation). DMTU treatment was unsuccessful in preventing the extracellular cytostatic effect of ADR. Finally, glomerular epithelial cell killing (⁵¹Cr-release method) by 5-iminodaunorubicin, an ADR analogue with a modified quinone function that prohibits oxido-reduction cycling, was higher than unmodified ADR. These results indicate that ADR may exert its cytotoxic effects on glomerular epithelial cells by interaction at the cell surface, whereas the intracellular compartment, principally DNA, does not seem to be the target of ADR effects. They also suggest that the free radicals are in part responsible for ADR intracellular cytotoxicity, but other mechanisms should also be hypothesized. Finally, the participation of the ADR semiquinone radical in oxido-reduction cycling seems not important for the induction of the cellular damage.     

Iron-citrate complexes and free radicals generation: Is citric acid an innocent additive in foods and drinks?

The generation of free radicals (Fenton chemistry) from various iron citrate complexes has been studied. Spin trapping methods have been used. The results can question concerning the innocence of added citric acid in foods and cold drinks. We concluded that in absence of pathological situation citric acid is probably not dangerous but it may become dangerous in situation of oxidative stress and/or iron overload.     

Vitamins Q and E, extracorporal circulation and hemolysis

Whole blood vitamin Q (ubiquinone), plasma vitamins Q and E(alpha-(a-)tocopherol) and free cholesterol (FC) were studied before(control or base-line value, sample I) and during open chest surgery andextracorporal circulation (samples II-IV) in 10 male IHD patients. Identityexisted between control whole blood and plasma ubiquinone. During surgery anincreased discrepancy with lower plasma vitamin Q levels were seen. Controlplasma vitamins Q, E and FC averaged 0.88 ± 0.16 (SE), 12.1 ±2.2 mg × 1-1 and 0.75 ± 0.15 g × 1-1. Corresponding molarvalues were 1.02 ± 0.17, 28.1 ± 5.1 µmol × 1-1 and1.94 ± 0.74 mmol × 1-1. Vitamin Q and E decreased continuouslyand averaged 0.64 mg × 1-1 in sample IV (0.74 µmol × 1-1,p < 0.001) and 9.4 mg × 1-1 in sample III (21.8 µmol ×1-1, p < 0.001). Hemolysis in all sample IV vials, ruined all vitamin Edeterminations. When normalized for FC (NQ and NE), decreases were found tobe 17 (IV) and 12% (III), respectively. Large interindividual variationsexisted. High control NQ and NE values allowed a larger antioxidant vitamindepletion. High NQ seemed also to be a prerequisite for NE depletion. Inaddition, signs indicated an active liver vitamin Q release for patientsrich in control antioxidant values. It was suggested that the antioxidantvitamin depletion did not prevent from radical trauma to membrane structurallipids (especially omega-3 fatty acids or vitamin F1), less membranefluidity, erythrocyte fragility and hemolysis.     

Stable free radicals in insect cuticles: electron spin resonance spectroscopy reveals differences between melanization and sclerotization

Insect cuticles (exuviae; cast skins) were examined for the first time by ESR spectroscopy for the presence of stable free radicals, as found in melanins. All cuticles, except those from a locust albino strain, irrespective of the presence of melanin, provided single-line signals of varied g-values and linewidths. The ESR signals of melanins, isolated or in cuticles, were characterized by g-values <2.004 and small linewidths in the range of 4-6G, while sclerotized cuticles, lacking melanin, showed g-values >2.004 and broad linewidths of 5-11 G. The melanin spectra were comparable to those reported for eumelanins with indol-based monomers. Minor signals ascribed to pheomelanins were found in several probes. The 'sclerotin' spectra were broader and displayed unresolved hyperfine structure in some cases. As for melanins, the location and environment of the radicals in cuticles giving rise to the two types of ESR spectra could not be assigned. Changes in the radical environment due to insecticide or solvent treatment can be detected by ESR spectroscopy.     

Involvement of oxygen free radicals in the respiratory uncoupling induced by free calcium and ADP-magnesium in isolated cardiac mitochondria: Comparing reoxygenation in cultured cardiomyocytes

Recently, we have observed that the simultaneous application of free calcium (fCa) and ADP-magnesium (Mg) reduced the ADP:O ratio in isolated cardiac mitochondria. The uncoupling was prevented by cyclosporin A, an inhibitor of the permeability transition pore. The purpose of this study was to know if the generation of oxygen free radicals (OFR) is involved in this phenomenon and if it occurs during reoxygenation (Reox) of cultured cardiomyocytes. Cardiac mitochondria were harvested from male Wistar rats. Respiration was assessed in two media with different fCa concentrations (0 or 0.6 M) with palmitoylcarnitine and ADP-Mg as respiration substrates. The production of Krebs cycle intermediates (KCI) was determined. Without fCa in the medium, the mitochondria displayed a large production of citrate + isocitrate + -ketoglutarate. fCa drastically reduced these KCI and promoted the accumulation of succinate. To know if OFR are involved in the respiratory uncoupling, the effect of 4OH-TEMPO (250 M), a hydrosoluble scavenger of OFR, was tested. 4OH-TEMPO completely abolished the fCa- and ADP-Mg-induced uncoupling. Conversely, vitamin E contributed to further decreasing the ADP:O ratio. Since no hydrosoluble electron acceptor was added in our experiment, the oxygen free radical-induced oxidized vitamin E was confined near the mitochondrial membranes, which should reduce the ADP:O ratio by opening the permeability transition pore. The generation of OFR could result from the matrix accumulation of succinate. Taken together, these results indicate that mitochondrial Ca uptake induces a slight increase in membrane permeability. Thereafter, Mg enters the matrix and, in combination with Ca, stimulates the isocitrate and/or -ketoglutarate dehydrogenases. Matrix succinate favors oxygen free radical generation that further increases membrane permeability and allows respiratory uncoupling through proton leakage. To determine whether the phenomenon takes place during Reox, cultured cardiomyocytes were subjected to hypoxia and Reox. ¹⁴C-palmitate was added during Reox to determine the KCI profile. Succinate had not increased during Reox. In conclusion, calcium- and ADP-Mg-induced respiratory uncoupling is due to oxygen free radical generation through excess matrix accumulation of succinate. The phenomenon does not occur during reoxygenation because of a total restoration of mitochondrial magnesium and/or ADP concentration.     

Application of electron spin resonance for evaluation of the level of free radicals in the myometrium in full-term pregnancy with normal labour and uterine inertia

In order to identify and quantify free radicals in the tissues of patients with normal physiological and pathological states of births, we developed a method to evaluate the amount of free radicals in myometrium of subplacental area and from body of uterus, using electron spin resonance spectroscopy. Analysis of the concentration of free radicals in the myometrium in full-term pregnancy with normal labour and during uterine inertia was studied. The activities of Ca2+-ATPase, cytochrome c oxidase and succinate dehydrogenase in samples of these tissues were tested too. Low free radical concentrations in these tissues were associated with disturbances in contractile activity of myometrium along with reduction of Ca2+-ATPase, cytochrome c oxidase and succinate dehydrogenase activity. There proved to be an association between the level of free radicals in the tissues and alteration in the physiological processes.