缺氧引起的自由基增殖及其损伤

在缺氧条件下,生物机体内自由基代谢紊乱,其浓度升高,机体抗氧化系统遭到破坏;高浓度自由基通过生物膜脂质过氧化,使生物膜流动性减小,刚性增强,通透性增大,细胞内钙超载,细胞信号传导异常;攻击蛋白质和酶,使其结构破坏,功能丧失。活性降低,导致物质代谢和能量代谢障碍;攻击DNA分子,使其发生突变甚至发生断裂,从而诱发机体病变。     

Biomarkers of free radical injury during spinal cord ischemia.

Plasma and urinary levels of 8-iso-PGF(2alpha) and 15-keto-dihydro-PGF(2alpha) were analysed at baseline and during the ischemia-reperfusion period in experimental spinal cord ischemia. A significant and immediate increase of 8-iso-PGF(2alpha) in plasma at the start and up to 60 min, and in the urine at 90-150 min following ischemia indicate an association of oxidative injury. The inflammatory response indicator 15-keto-dihydro-PGF(2alpha) in plasma increased significantly at the start and up to 60 min after ischemia. No such increase was seen in animals with no spinal cord ischemia. Thus, free radical mediated and cyclooxygenase catalysed products of arachidonic acid are increased during spinal cord ischemia as a consequence of oxidative injury and inflammation.     

Modifying pulmonary ischemia-reperfusion injury by altering ventilatory strategies during ischemia.

We used an intact in vivo canine model of pulmonary ischemia-reperfusion (IR) injury to evaluate the differential effects of alveolar hypoxia and ventilation during 2 h of unilateral warm lung ischemia. Serial measurements of regional pulmonary blood flow, extravascular density (EVD), and transcapillary protein flux were made after reperfusion with the quantitative imaging technique of positron emission tomography. Twenty-seven animals were divided into five experimental groups: VENT O2 (n = 5) in which the left lung was ventilated with 40% O2 during ischemia, STATIC O2 (n = 4) in which the left lung was statically inflated with 40% O2 during ischemia, VENT N2 (n = 5) in which the left lung was ventilated with 100% N2 during ischemia, VENT N2/CO2 (n = 5) in which the left lung was ventilated with 95% N2-5% CO2 during ischemia, and STATIC N2 (n = 8) in which the left lung was statically inflated with 100% N2 during ischemia. These groups were compared with a control group (CONT, = 3) that was studied previously. Protein flux was significantly increased in the previous ischemic lung only for the STATIC N2 group [median 175 x 10(-4) min-1 (range 53-1,217) for the STATIC N2 group vs. 50 x 10(-4) min-1 (range 40-56) for the CONT group] 0.25 h after reperfusion and did not change over 3 h. EVD also increased but not significantly. Protein flux and EVD in the other groups were not different from CONT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reactive oxygen species mediate modification of glycocalyx during ischemia-reperfusion injury

The glycocalyx (Gcx) is a complex and poorly understood structure covering the luminal surface of endothelial cells. It is known to be a determinant of vascular rheology and permeability and may be a key control site for the vascular injuries caused by ischemia-reperfusion (I/R). We used intravital-microscopy to evaluate the effects of I/R injury on two properties of Gcx in mouse cremasteric microvessels: exclusion of macromolecules (anionic-dextrans) and intracapillary distribution of red blood cells (RBC). In this model, the Gcx is rapidly modified by I/R injury with an increase in 70-kDa anionic-dextran penetration without measurable effect on the penetration of 580-kDa anionic-dextran or on RBC exclusion. The effects of I/R injury appear to be mediated by the rapid production of reactive oxygen species (ROS) because they are ameliorated by the addition of exogenous superoxide dismutase-catalase. Intravenous application of allopurinol or heparin also inhibited the effects of I/R injury, and we interpret efficacy of allopurinol as evidence for a role for xanthine-oxidoreductase (XOR) in the response to I/R injury. Heparin, which is hypothesized to displace XOR from a heparin-binding domain in the Gcx, reduced the effects of I/R. The effects of I/R injury were also partially prevented or fully reversed by the intravascular infusion of exogenous hyaluronan. These data demonstrate: 1) the liability of Gcx during I/R injury; 2) the importance of locally produced ROS in the injury to Gcx; and 3) the potential importance of heparin-binding sites in modulating the ROS production. Our findings further highlight the relations between glycosaminoglycans and the pathophysiology of Gcx in vivo.     

Protective effect of a new anti-oxidant on the rat brain exposed to ischemia-reperfusion injury: inhibition of free radical formation and lipid peroxidation.

A new oligomeric derivative was synthesized from prostaglandin B2 and ascorbic acid, and its effect on rat brain ischemia-reperfusion injury was studied. Brain ischemia was produced in the rat by the combination of bilateral common carotid artery occlusion and hemorrhagic hypotension (30 mmHg, 20 min). The cerebral cortex was homogenized in the presence of the spin trap agent, N-tert-butyl-alpha-phenyl-nitrone (PBN). Spin-adducts were detected using an electron spin resonance spectrometer (EPR). Lipid peroxidation was estimated from the amounts of both thiobarbituric acid reactive substances (TBAR) and conjugated diene. In control experiments, reperfusion induced a burst of free radical formation which peaked at 5 min reperfusion time (238 +/- 41%). Lipid peroxidation increased significantly after 20 min of reperfusion (TBAR, 161 +/- 50%; conjugated diene, 160 +/- 29%). When the oligomeric derivative was administered (9 mg/kg i.p. 30 min before ischemic insult), it significantly reduced both spin adduct formation (103 +/- 13%) and lipid peroxidation (TBAR, 109 +/- 14%; conjugated diene, 97 +/- 33%).     

Metabolic activation of sulfur mustard leads to oxygen free radical formation

We recently published electron paramagnetic resonance (EPR) spin trapping results that demonstrated the enzymatic reduction of sulfur mustard sulfonium ions to carbon-based free radicals using an in vitro system containing sulfur mustard, cytochrome P450 reductase, NADPH, and the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) in buffer (A.A. Brimfield et al., 2009, Toxicol. Appl. Pharmacol. 234:128-134). Carbon-based radicals have been shown to reduce molecular oxygen to form superoxide and, subsequently, peroxyl and hydroxyl radicals. In some cases, such as with the herbicide paraquat, a cyclic redox system results, leading to magnified oxygen free radical concentration and sustained tissue damage. Low mustard carbon radical concentrations recorded by EPR in our in vitro system, despite a robust (4.0mM) sulfur mustard starting concentration, led us to believe a similar oxygen reduction and redox cycling process might be involved with sulfur mustard. A comparison of the rate of mustard radical-POBN adduct formation in our in vitro system by EPR at atmospheric and reduced oxygen levels indicated a sixfold increase in 4-POBN adduct formation (0.5 to 3.0 μM) at the reduced oxygen concentration. That result suggested competition between oxygen and POBN for the available carbon-based mustard radicals. In parallel experiments we found that the oxygen radical-specific spin trap 5-tert-butoxycarbonyl-5-methylpyrroline-N-oxide (BMPO) detected peroxyl and hydroxyl radicals directly when it was used in place of POBN in the in vitro system. Presumably these radicals originated from O(2) reduced by carbon-based mustard radicals. We also showed that reactive oxygen species (ROS)-BMPO EPR signals were reduced or eliminated when mustard carbon radical production was impeded by systematically removing system components, indicating that carbon radicals were a necessary precursor to ROS production. ROS EPR signals were completely eliminated when superoxide dismutase and catalase were included in the complete in vitro enzymatic system, providing additional proof of oxygen radical participation. The redox cycling hypothesis was supported by density functional theory calculations and frontier molecular orbital analysis.     

Evidence for free radical formation during horseradish peroxidase-catalyzed N-demethylation of crystal violet.

Crystal violet (gentian violet) can undergo an oxidative metabolism, catalyzed by horseradish peroxidase, resulting in formaldehyde formation. The N-demethylation reaction was strongly inhibited by reduced glutathione. Evidence for the formation of a crystal violet radical during the horseradish peroxidase catalyzed reaction was the detection of thiyl and ascorbate radicals from glutathione and ascorbate, respectively. The concentration of radicals from both compounds was significantly increased in the presence of crystal violet. Oxygen uptake was stimulated when glutathione was present in the system and this oxygen uptake was dependent on the dye and enzyme concentration. Oxygen uptake did not occur when ascorbate, instead of glutathione, was present in the system. However, when glutathione was present, ascorbate totally inhibited the glutathione-stimulated oxygen uptake in the crystal violet/horseradish peroxidase/hydrogen peroxide system. Although a weak ESR spectrum from a crystal violet-derived free radical was detected when the dye reacted with H2O2 and horseradish peroxidase, using the fast flow technique, this spectrum could not be interpreted.     

Increased endogenous ascorbyl free radical formation with singlet oxygen scavengers in reperfusion injury: an EPR and functional recovery study in rat hearts.

The objective of this study was to investigate the effect of singlet oxygen ((1)O2) scavengers on functional recovery and ascorbyl free radical (AFR) formation in isolated ischemic rat hearts. Hearts were subjected to 40 min. of global ischemia followed by 30 min. of reperfusion. Hemodynamics were measured as heart rate (HR), coronary flow (CF), left ventricular developed pressure (LVDP) and contractility (dP/dt). Electron paramagnetic resonance (EPR) spectroscopy was used to measure AFR release in coronary perfusate during the first two min. of reperfusion as a function of ROS scavengers. Relative to ischemic controls the administration of the (1)O2 scavengers 2,2,6,6-tetramethyl-4-piperidone x HCl (4-oxo-TEMP), carnosine (beta-alanyl-L-histidine) or a combination of the two significantly improved functional recovery as measured by LVDP. While no AFR signal was detected in coronary perfusate collected during preischemic perfusion with and without (1)O2 scavengers, the AFR background signal due to ischemia was significantly increased with the (1)O2 and *O2- scavengers. No such increase was observed with the hydroxyl radical (*OH) scavenger mannitol. Besides the AFR increase with the (1)O2 and *O2- scavengers the functional recovery was only significantly improved with the (1)O2 scavengers. In contrast to previous AFR studies we found with endogenous AFR that an increased AFR formation is not necessarily only reflecting increased oxidative stress but can also report improved functional recovery. Combining the hemodynamic data with increased AFR formation in the presence of several different ROS scavengers gives supportive evidence for (1)O2 also being involved in reperfusion injury.     

In vivo protective effects of urocortin on ischemia-reperfusion injury in rat heart via free radical mechanisms

The aim of this study was to investigate the effects of urocortin (UCN) on oxidative stress and the mechanisms of urocortin on ischemia-reperfusion injury in vivo in the rat model. Thirty-six Sprague-Dawley rats were divided into 6 groups, including sham, control (normal saline solution), UCN1, UCN2, UCN3, and verapamil groups. The left anterior descending coronary artery of all rats except those in the sham group was treated with a 30-min occlusion followed by a 60-min reperfusion. Just before the occlusion, normal saline solution, UCN (5, 10, and 20 microg/kg body mass), or verapamil (1 mg/kg body mass) was administered. Heart rates, beating rhythm, and S-T segments were constantly monitored using an ECG. At the completion of the drug administration, blood samples were taken to measure the activity of superoxide dismutase (SOD), malonaldehyde (MDA), glutathione peroxidase (GSH-PX), and nitric oxide (NO) to evaluate the effects of UCN on oxidative stress. Finally, the size of infarction was measured. Arrhythmia rates were significantly lower, and the infarction size was significantly smaller (p < 0.01), in the UCN groups vs. the control group. Verapamil also significantly reduced arrhythmia rates and infarction size. The MDA activities were remarkably diminished, whereas the SOD, GSH-PX, and NO activities were significantly higher in the UCN and VER groups (p < 0.01). MDA, SOD, and NO activities were strongly correlated with UCN doses. These results suggest that UCN may play a protective role in ischemia-reperfusion injury in rat hearts against the oxidative stress by inhibiting free radicals' activities.     

Compounds that increase cAMP prevent ischemia-reperfusion pulmonary capillary injury.

This study evaluated the physiological effects of compounds that increase adenosine 3',5'-cyclic monophosphate (cAMP) on changes in pulmonary capillary permeability and vascular resistance induced by ischemia-reperfusion (I-R) in isolated blood-perfused rabbit lungs. cAMP was elevated by 1) beta-adrenergic stimulation with isoproterenol (ISO, 10(-5) M), 2) post-beta-receptor stimulation of adenylate cyclase with forskolin (FSK, 10(-5) M), 3) and dibutyryl cAMP (DBcAMP, 1 mM), a cAMP analogue. Vascular permeability was assessed by determining the capillary filtration coefficient (Kf,c), and capillary pressure was measured using the double occlusion technique. The total, arterial, and venous vascular resistances were calculated from measured pulmonary arterial, venous, and capillary pressures and blood flow. Reperfusion after 2 h of ischemia significantly (P less than 0.05) increased Kf,c (from 0.115 +/- 0.028 to 0.224 +/- 0.040 ml.min-1.cmH2O-1.100 g-1). These I-R-induced changes in capillary permeability were prevented when ISO, FSK, or DBcAMP was added to the perfusate at reperfusion (0.110 +/- 0.022 and 0.103 +/- 0.021, 0.123 +/- 0.029 and 0.164 +/- 0.024, and 0.153 +/- 0.030 and 0.170 +/- 0.027 ml.min-1.cmH2O-1.100 g-1, respectively). I-R significantly increased total, arterial, and venous vascular resistances. These increases in vascular resistance were also blocked by ISO, FSK, and DBcAMP. These data suggest that beta-adrenergic stimulation, post-beta-receptor activation of adenylate cyclase, and DBcAMP prevent the changes in pulmonary vascular permeability and vascular resistances caused by I-R in isolated rabbit lungs through a mechanism involving an increase in intracellular levels of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Factors influencing ascorbate free radical formation

Several constituents of serum and related substances were examined in order to find factors influencing ascorbate radical formation. Human ceruloplasmin and albumin catalyzed singly ascorbate oxidation and caused a remarkable increase in ESR intensity of the ascorbate radical. Although, however, the combination of these two factors showed synergic effects on catalysis of ascorbate oxidation, the radical intensity significantly decreased. Fibrinogen and fetuin showed inhibitory effects on catalysis of ascorbate oxidation, whereas transferrin, citric acid, or other related substances exhibited no effect. A new factor which inhibited ascorbate oxidation was found in a serum fraction. These results indicate there is a counterbalanced equilibrium in the redox process of ascorbate in serum and the intensity of ascorbate radical is influenced by the summation of the complicated effects of many factors.     

Selenium,oxygen-derived free radicals,and ischemia-reperfusion injury

Circulatory shock and its treatment have been compared to a whole-body ischemia and reperfusion with activation of oxygen-derived free radicals. A pilot study had suggested a selenium redistribution in this context. To verify this hypothesis, an experimental study was designed. Temporary occlusion of the superior mesenteric artery was performed in 18 male adult Wistar rats using clamping for 0, 10, and 20 min. Hemodynamic and biochemical data were assessed before clamping and 20 min after release of the mesenteric blood flow. After release, mean arterial pressure decreased, plasma lactate increased, and erythrocyte glutathione peroxidase decreased. Plasma and erythrocyte selenium did not change; however, a slight decrease in plasma selenium was observed when related to hematocrit (to take into account the fluid balance). Erythrocyte-reduced glutathione did not change. In contrast, liver and kidney selenium increased, whereas reduced glutathione decreased in kidney, but not in liver after 20 min of clamping as compared to the sham-operated group. These results suggest that, after temporary intestinal ischemia, the changes in selenium and reduced glutathione observed in blood and tissues, like liver or kidney, could be related to a redistribution pattern in selenium metabolism during shock injury.     

Mast cell mediation of muscle and pulmonary injury following hindlimb ischemia-reperfusion.

We have observed extensive mast cell degranulation in the reperfused hindlimb muscle of the mouse, accompanied by pathological changes within the muscle. As quantitated by the tissue:blood (125)I permeability ratio, both the hindlimbs and lungs exhibited a significant increment in permeability during hindlimb reperfusion. In lungs of the same mice, mast cell-derived chymase mMCP-1 coats alveolar macrophages, an event noted by us in acid-induced direct lung injury. Mast cells in the lung contain mMCP-1, whereas those in the muscle do not. Neither extensive muscle injury nor an increased pulmonary permeability index occurs in the mast cell-deficient W/W(v) mice. We conclude that the mast cell is a key mediator in both local ischemia-reperfusion injury (I-R) of muscle and consequent remote lung injury.     

Implication of free radical mechanisms in ethanol-induced cellular injury.

Numerous experimental data reviewed in the present article indicate that free radical mechanisms contribute to ethanol-induced liver injury. Increased generation of oxygen- and ethanol-derived free radicals has been observed at the microsomal level, especially through the intervention of the ethanol-inducible cytochrome P450 isoform (CYP2E1). Furthermore, an ethanol-linked enhancement in free radical generation can occur through the cytosolic xanthine and/or aldehyde oxidases, as well as through the mitochondrial respiratory chain. Ethanol administration also elicits hepatic disturbances in the availability of non-safely-sequestered iron derivatives and in the antioxidant defense. The resulting oxidative stress leads, in some experimental conditions, to enhanced lipid peroxidation and can also affect other important cellular components, such as proteins or DNA. The reported production of a chemoattractant for human neutrophils may be of special importance in the pathogenesis of alcoholic hepatitis. Free radical mechanisms also appear to be implicated in the toxicity of ethanol on various extrahepatic tissues. Most of the experimental data available concern the gastric mucosa, the central nervous system, the heart, and the testes. Clinical studies have not yet demonstrated the role of free radical mechanisms in the pathogenesis of ethanol-induced cellular injury in alcoholics. However, many data support the involvement of such mechanisms and suggest that dietary and/or pharmacological agents able to prevent an ethanol-induced oxidative stress may reduce the incidence of ethanol toxicity in humans.     

Allopurinol-insensitive oxygen radical formation by milk xanthine oxidase systems.

Oxygen radical generation in the xanthine- and NADH-oxygen reductase reactions by xanthine oxidase, was demonstrated using the ESR spin trap 5,5'-dimethyl-1- pyrroline-N-oxide. No xanthine-dependent oxygen radical formation was observed when allopurinol-treated xanthine oxidase was used. The significant superoxide generation in the NADH-oxygen reductase reaction by the enzyme was increased by the addition of menadione and adriamycin. The NADH-menadione and -adriamycin reductase activities of xanthine oxidase were assessed in terms of NADH oxidation. From Lineweaver-Burk plots, the Km and Vmax of xanthine oxidase were estimated to be respectively 51 microM and 5.5 s-1 for menadione and 12 microM and 0.4 s-1 for adriamycin. Allopurinol-inactivated xanthine oxidase generates superoxide and OH.radicals in the presence of NADH and menadione or adriamycin to the same extent as the native enzyme. Adriamycin radicals were observed when the reactions were carried out under an atmosphere of argon. The effects of superoxide dismutase and catalase revealed that OH.radicals were mainly generated through the direct reaction of H2O2 with semiquinoid forms of menadione and adriamycin.     

The transition metal-mediated formation of the hydroxyl free radical during the reduction of molecular oxygen by ferredoxin-ferredoxin:NADP+ oxidoreductase

The NADPH-supported enzymatic reduction of molecular oxygen by ferredoxin-ferredoxin:NADP+ oxidoreductase was investigated. The ESR spin trapping technique was employed to identify the free radical metabolites of oxygen. The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used to trap and identify the oxygen-derived free radicals. [17O]Oxygen was employed to demonstrate that the oxygen-centered radicals arose from molecular oxygen. From the data, the following scheme is proposed: (Formula:see text). The formation of the free hydroxyl radical during the reduction of oxygen was demonstrated with quantitative competition experiments. The hydroxyl radical abstracted hydrogen from ethanol or formate, and the resulting scavenger-derived free radical was trapped with known rate constants. If H2O2 was added to the enzymatic reaction, a stimulation of the production of the hydroxyl radical was obtained. This stimulation was manifested in both the concentration and the rate of formation of the DMPO/hydroxyl radical adduct. Catalase was shown to inhibit formation of the hydroxyl radical adduct, further supporting the formation of hydrogen peroxide as an intermediate during the reduction of oxygen. All three components, ferredoxin, ferredoxin:NADP+ oxidoreductase, and NADPH, were required for reduction. Ferredoxin:NADP+ oxidoreductase reduces ferredoxin, which in turn is responsible for the reduction of oxygen to hydrogen peroxide and ultimately the hydroxyl radical. The effect of transition metal chelators on the DMPO/hydroxyl radical adduct concentration suggests that the reduction of chelated iron by ferredoxin is responsible for the reduction of hydrogen peroxide to the hydroxyl radical via Fenton-type chemistry.     

Sonochemical free radical formation in aqueous solutions

The phenomena of stable and transient acoustic cavitation in liquids exposed to ultrasound are briefly explained. The role of micronuclei, resonant bubble size, and rectified diffusion in the initiation of transient cavitation is reviewed. In aqueous solutions transient cavitation initially generates hydrogen atoms and hydroxyl radicals that may recombine to form hydrogen and H2O2 or may react with solutes in the gas phase, at the gas-liquid boundary, or in the bulk of the solution. The analogies and differences between sonochemistry and ionizing radiation chemistry are explored. The use of spin trapping and electron spin resonance to conclusively identify hydrogen atoms and hydroxyl radicals and to detect cavitation produced by continuous wave and by pulsed ultrasound is described in detail.     

Effect of nickel on oxygen free radical metabolism

The effect of nickel on superoxide dismutase activity (SOD), as well as on rate of hydroxydopamine oxidation, was studied in vitro since lipid peroxidation has been implicated in cell damage by nickel, whose toxicity and carcinogenicity are well established. Nickel strongly inhibits SOD activity. The degree of inhibition is directly proportion to the nickel concentration (tested range 0.066 to 0.33 microgram/mL in the reaction mixture); to the substrate concentration (tested range 0.4 x 10 4M to 1.1 x 10 4M 6-hydroxydopamine); and to reaction mixture. Autoxidation of 6-hydroxydopamine was increased by nickel concentrations higher than 15 micrograms/mL. The combination of excessive oxygen free radical production and inhibition of their elimination by inhibition of SOD activity may contribute to the nickel toxicity that has been reported in industrial accidents, as well as to the high incidence of cancer occurring in nickel workers. It may also contribute to many complications in uremic patients, in whom increased serum nickel levels were reported to be in a similar range to those inhibiting SOD.