Effect of oxygen free radicals on rabbit and human erythrocytes. Studies on cellular deformability

Changes in deformability of rabbit and human erythrocytes caused by exposure in vitro to the oxygen free radical generator hypoxanthine and xanthine oxidase were studied. The deformability reduction observed after 30 min of exposure to hypoxanthine-xanthine oxidase could be prevented by pretreatment with SOD, while after only 5 min of such exposure allopurinol and catalase also appeared to have a protective effect. Exposure of human erythrocytes to hypoxanthine and xanthine oxidase in Krebs solution prevented an otherwise occurring hemolysis. Exposure to both substances or to xanthine oxidase alone in Dulbeccos phosphate solution produced a reduction in deformability. The results indicate that exposure of erythrocytes to free oxygen radicals reduces deformability and that this effect may contribute to the myocardial dysfunction and the epicardial erythrostasis observed during open-heart surgery.     

Effect of oxygen free radicals on the rat pancreas in vivo]

Many reports concerning the involvement of active oxygen free radicals in the pathogenesis and progression of acute pancreatitis have been published. In this study, the direct toxic effect of active oxygen free radicals on the rat pancreas was evaluated in vivo. Superoxide anions, generated via the xanthine/xanthine oxidase (X/XO) system, and hydrogen peroxide (H2O2) were used. After continuous arterial injection of X/XO into the celiac artery hemorrhage and extensive edema developed. However, additional continuous injection of superoxide dismutase (SOD) into the external jugular vein completely suppressed the hemorrhage and relieved the edema. When hydrogen peroxide (100 microM/Kg/hour) was injected continuously through the celiac artery made hemorrhage and edema were recognized in the pancreas, both of which were suppressed by continuous injection of catalase (10 mg/Kg/hour) or gabexate mesilate (10 mg/Kg/hour) into the external jugular vein. The amylase and lipase levels in the intraperitoneal fluid rose to more than 10 times the preoperative values 5 hours after drug administration. These levels were lowered to 2 times the preoperative values by the continuous venous injection of SOD or catalase (which are specific scavengers of superoxide anions or hydrogen peroxide, respectively) or by gabexate mesilate. On the other hand, serum amylase and lipase levels remained almost constant throughout the entire experiment. Thus, the administration of active oxygen free radicals caused acute pancreatitis, which was suppressed by the systemic administration of specific scavengers for each free radical. Active oxygen free radicals were shown to have a direct, toxic effect on the pancreas.     

Xanthine:acceptor oxidoreductase activities in ischemic rat skin flaps

Xanthine:acceptor oxidoreductase activities were assayed in free skin flaps following prolonged preservation. In normal rat skin, xanthine dehydrogenase transfers electrons to NAD+ and accounts for 73% of total oxidoreductase activity, and xanthine oxidase transfers electrons to molecular oxygen and accounts for the remaining 27%. Xanthine oxidase activity increased significantly in skin flaps during ischemia: approximately 30 and 100% increases after 6 and 24 hr of ischemia, respectively. Allopurinol inhibited xanthine oxidoreductase activity: free skin flaps obtained from allopurinol-treated animals exhibited a low level of xanthine oxidoreductase activity throughout the period of preservation. Systemic allopurinol significantly improved the survival rate from 32 to 75% of free flaps transferred after 24 hr of preservation at room temperature. These observations suggest that the xanthine oxidase system is a major source of oxygen free radicals following ischemia/reperfusion in skin. The increase in xanthine oxidase is attributable to the conversion of xanthine dehydrogenase to oxidase, a conversion which involves sulfhydryl oxidation in skin flaps.     

Effect of artificial cells on hepatic function after ischemia-reperfusion injury in liver

BACKGROUND: The liver suffers from ischemia/reperfusion injury during transplantation. Reactive oxygen species generated by xanthine oxidase during reperfusion of the ischemic liver may be partially responsible for the hepatic injury. Oxygen free radicals are removed by antioxidant enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase. Using glutaraldehyde and lysine we constructed crosslinked hemoglobin, containing SOD and catalase, and assessed its ability to protect against ischemia/reperfusion injury during transplantation. METHODS: In contrast to the sham-operated control groups, blood was exchanged using crosslinked hemoglobin (polyHb) a PolyHb-SOD-catalase (PSC) group. After ischemia/reperfusion injury, several parameters of hepatic damage and oxygen free radicals were measured as well as microscopic examination. RESULTS: Alanine aminotransferase, aspartate aminotransferase, superoxide production, hydrogen peroxide, and malondialdehyde levels were higher among the PolyHb group than sham-operated controls. The PolyHb group revealed a few apoptotic bodies, some acute inflammatory infiltrates in the sinusoids, nuclear fragmentations, cell shrinkage, and chromatin clumping with formation of apoptotic bodies in the apoptotic cells under microscopic examination. Alanine aminotransferase, aspartate aminotransferase, superoxide production, and hydrogen peroxide levels were lower in the PSC than the PolyHb group. Hepatic structures were well preserved in the PSC group. CONCLUSIONS: Reactive oxygen species contribute to hepatic dysfunction with morphologic changes. PSC is effective to reduce hepatic damage by lowering oxygen free radical-mediated injury after ischemia/reperfusion in the liver.     

The role of neutrophils and oxygen free radicals in post-operative adhesions

BACKGROUND: Postoperative intra-abdominal adhesion formation remains a major surgical problem. Surgery induces an inflammatory reaction, which is responsible for adhesion formation. Neutrophils and their oxygen-free radicals are key mediators in the early post-operative inflammatory response. The present study evaluates the effect of either blocking the influx of neutrophils or its products by scavenging oxygen-free radicals on adhesion formation. MATERIALS AND METHODS: Reproducible rat models were used to induce post-surgical intra-abdominal adhesions. In the first experiment anti-neutrophil serum (ANS) was used to prevent neutrophils from entering the peritoneal cavity after surgery. In a second experiment superoxide dismutase (SOD), catalase, and mannitol were tested, to scavenge the superoxide, hydrogen peroxide, and hydroxyl radicals, respectively. RESULTS: In positive control groups 69 to 76% of the area of interest contained adhesions. In all experimental groups, except for mannitol, a significant reduction in post-surgical adhesion formation could be achieved. ANS reduced adhesion formation by 38% (P < 0.001) and SOD/catalase by 42% (P < 0.01). Mannitol could not reduce adhesion formation. CONCLUSIONS: Intra-abdominal influx of neutrophils after surgical peritoneal trauma plays an important role in post-operative adhesion formation. Preventing the intra-abdominal influx of neutrophils in the early post-operative inflammatory reaction can reduce adhesion formation, but an even more selective approach, by scavenging its products, proved as efficient.     

Calcium oxalate stone disease: role of lipid peroxidation and antioxidants

Membrane injury facilitated the fixation of calcium oxalate crystals and subsequent growth into kidney stones. Oxalate-induced membrane injury was mediated by lipid peroxidation reaction through the generation of oxygen free radicals. In urolithic rat kidney or oxalate exposed cultured cells, both superoxide anion and hydroxyl radicals were generated in excess, causing cellular injury. In hyperoxaluric rat kidney, both superoxide and H2O2-generating enzymes such as glycolic acid oxidase (GAO) and xanthine oxidase (XO) were increased, and hydroxyl radical and transition metal ions, iron, and copper were accumulated. The lipid peroxidation products, thiobarbituric acid-reactive substances (TBARS), hydroperoxides, and diene conjugates were excessively released in tissues of urolithic rats and in plasma of rats as well as stone patients. The accumulation of these products was concomitant with the decrease in the antioxidant enzymes, superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and glucose-6 phosphate dehydrogenase (G6PD) as well as radical scavengers, vitamin E, ascorbic acid, reduced glutathione (GSH), and protein thiol. All the above parameters were decreased in urolithic condition, irrespective of the agents used for the induction of urolithiasis. Oxalate binding activity and calcium oxalate crystal deposition were markedly pronounced, along with decreased adenosine triphosphatase (ATPase) activity. Lipid peroxidation positively correlated with cellular oxalate, oxalate binding, gamma-glutamyl carboxylase, and calcium level and negatively correlated with GSH, vitamin E. ascorbic acid, and total protein thiol. Antioxidant therapy to urolithic rats with vitamin E, glutathione monoester, methionine, lipoic acid, or fish oil normalised the cellular antioxidant system, enzymes and scavengers, and interrupted membrane lipid and protein peroxidation reaction, ATPase inactivation, and its associated calcium accumulation. Antioxidant therapy prevented calcium oxalate precipitation in the rat kidney and reduced oxalate excretion in stone patients. Similarly, calcium oxalate crystal deposition in vitro to urothelium was prevented by free radical scavengers such as phytic acid and mannitol by protecting the membrane from free radical-mediated damage. All these observations were suggestive of the active involvement of free radical-mediated lipid peroxidation-induced membrane damage in the pathogenesis of calcium oxalate crystal deposition and retention.     

锌-金属硫蛋白对烫伤大鼠迟延复苏红细胞膜损伤的保护作用

目的　探讨锌 金属硫蛋白 (Zn MT)对抗严重烫伤动物延迟复苏后出现的氧化应激效应。　方法　采用大鼠 30 %总体表面积Ⅲ度烫伤模型 ,将 2 7只大鼠平均分成正常对照组、延迟复苏组、及Zn MT保护组。烫伤 4h后在保护组动物所补液体中 ,加入Zn MT (1× 10 -5mol/L)。伤后 2 4h取血 ,用电子自旋共振标记物马来酰亚胺标记红细胞膜 ,检测红细胞膜蛋白构象。取血清 ,加入黄嘌呤 ,黄嘌呤氧化酶及自旋共振自旋捕集剂二甲基吡啶氮氧化物以判断血清中超氧化物歧化酶 (SOD)含量变化。　结果　各组大鼠红细胞膜蛋白强弱固定化比值分别为 0 35 2± 0 0 4 3、0 4 0 9± 0 0 2 7及0 386± 0 0 6 2 ,旋转相关时间 (s)分别为 1 30 0± 0 2 10、1 5 76± 0 190及 1 381± 0 2 10 ,与正常对照组相比 ,延迟复苏组大鼠红细胞膜蛋白构象明显改变 ,膜蛋白运动速度明显减慢 ,给予Zn MT后 ,膜蛋白构象改变及膜蛋白运动速度降低的程度明显减轻。各组大鼠血清中SOD相对含量分别为 73 2 %±1 4 %、4 8 8%± 3 8%、6 6 8%± 3 2 % ,与正常对照组相比 ,延迟复苏组动物血清SOD含量明显降低 ,而Zn MT治疗组动物血清SOD含量降低的程度明显减少 ,说明烫伤延迟复苏组动物体内生成过多的氧自由基能被Zn MT清除。　结论　Zn MT对烫伤     

Pulmonary reperfusion injury: evidence for oxygen-derived free radical mediated damage and effects of different free radical scavengers

Blood granulocyte-mediated reactions involving generation of oxygen-derived free radicals have recently been shown to be capable of causing injury to the lungs. These findings suggest a similar mechanism also to be involved in the development of pulmonary ischemia/reperfusion injury. In the present study, therefore, the effects of three oxygen-derived free radical scavengers, superoxide dismutase (SOD; 1 mg/kg), catalase (20000 IU/kg) and allopurinol (45 mg/kg), were evaluated during reperfusion in a rabbit model after 2 h normothermic ischemia of the lung. During reperfusion, ischemic lungs were found to have an elevated pulmonary vascular resistance, increased total and extravascular lung water content, and decreased arterial oxygen tension (PaO₂) compared to control animals. SOD and catalase, but not allopurinol, were able to reduce pulmonary injury by lowering the pulmonary vascular resistance, but could not prevent pulmonary damage as shown by total lung water (TLW) or PaO₂. It is concluded that oxygen-derived free radicals such as hydrogen peroxide and the superoxide anion may play an important role in precipitating pulmonary injury after ischemia. The failure of xanthine oxidase inhibition (allopurinol) to exert protective effects may suggest that oxygen-derived free radical generation following pulmonary ischemia occurs predominantly via leukocyte-mediated reactions.     

The role of antioxidant enzymes in the control of opossum gallbladder motility.

BACKGROUND: Superoxide rapidly oxidizes nitric oxide (NO) to form peroxynitrite, thus terminating the biological activity of NO. The aims of our study were to determine if superoxide alters the motor function of the gallbladder and to localize the antioxidant enzymes in the gallbladder. MATERIALS AND METHODS: Immunostaining and immunoblots were performed and enzyme activities were measured in the gallbladder. In physiologic experiments, force-displacement transducers recorded tension in gallbladder muscle strips. Superoxide was generated by the addition of xanthine with xanthine oxidase, while superoxide radicals were scavenged by the addition of superoxide dismutase (SOD) and catalase. SOD was inhibited by deithyldithiocarbamate. RESULTS: Immunostaining demonstrated superoxide dismutase and catalase immunoreactivity in ganglia situated throughout the smooth muscle. Total superoxide dismutase activity was 115 +/- 12 U/mg. Western blots detected expression of proteins of 19.4 kDa for copper/zinc SOD and 25.0 kDa for manganese SOD. Generation of superoxide increased isometric tension, while pretreatment with SOD prevented the increase in isometric tension induced by superoxide. Inhibition of SOD diminished the EFS-induced off response. CONCLUSIONS: We conclude that superoxide alters gallbladder motor function, and the presence of superoxide scavenging enzymes in enteric plexuses suggests that they may regulate gallbladder neuromuscular function by clearing endogenous superoxide.     

The primary localization of free radical generation after anoxia/reoxygenation in isolated endothelial cells

While free radical-mediated reperfusion injury is clearly important in a variety of disparate organs, the particular cellular source of these radicals is unclear. To address this question, we subjected relatively pure (92% +/- 3% by factor VIII immunoassay) cultures of rat pulmonary artery endothelial cells to 0 to 45 minutes of anoxia (95% N2, 5% CO2), followed by reoxygenation (95% air, 5% CO2), to simulate ischemia/reperfusion. Cell injury was assayed after reoxygenation by the release of previously incorporated 51chromium and/or lactate dehydrogenase, and viability was determined by means of trypan blue exclusion. These three end points correlated closely. Without anoxia, the cells remained viable, with minimal evidence of injury for the entire experimental period, while 45 minutes of hypoxia followed by 30 minutes of reoxygenation produced substantial evidence of cell injury in 71% +/- 6% of the cells. This injury was reduced to 21% +/- 2% by treatment with the highly specific free radical scavengers superoxide dismutase and catalase together, either before anoxia or after anoxia, but just before reoxygenation. Similar protection was provided by xanthine oxidase inhibition with allopurinol. The injury was mimicked (without anoxia) by the exogenous generation of superoxide radicals with xanthine and xanthine oxidase. These experiments establish the essential components of free radical generation at reperfusion to be localized within the isolated endothelial cell in the absence of neutrophils or parenchymal cells.     

Changes in xanthine oxidase in ischemic rat brain

Xanthine oxidase activity in the rat brain was measured by means of high-performance liquid chromatography with electrochemical detection of uric acid. Cerebral ischemia was produced by a four-vessel occlusion method. In the control rat, the enzyme activity was 0.87 +/- 0.13 nmol/gm wet weight/min at 25 degrees C (mean +/- standard deviation), of which 92.4% was associated with the nicotinamide adenine dinucleotide (NAD)-dependent dehydrogenase form and only 7.6% with the oxygen-dependent superoxide-producing oxidase form. However, the ratio of the latter form increased to 43.7% after 30 minutes of global ischemia, despite the total xanthine oxidase activity remaining the same. Thus, it was revealed that uric acid can be synthesized in the rat brain and that cerebral ischemia induced the conversion of xanthine oxidase from an NAD-dependent dehydrogenase to an oxygen-dependent superoxide-producing oxidase. Although the xanthine oxidase pathway has been proposed as a source of oxygen-derived free radicals in various ischemic organs other than brain, the results of the present study suggest the involvement of the oxygen free radicals generated from this pathway in the pathogenesis of the ischemic injury of the rat brain.     

Postischemic renal dysfunction: the limited role of xanthine oxidase-generated oxygen free radicals

Oxygen free radicals (OFRs) generated during reperfusion are putative mediators of postischemic renal dysfunction. To address this issue, the renal response to ischemia and reperfusion was compared to the response to OFR generation without ischemia. Isolated rat kidneys were perfused at 37 degrees C and 90-100 mm Hg with an asanguinous modified Krebs' buffer. Kidneys were subjected to 30 min of ischemia followed by reperfusion or to OFRs generated by combining 25 mumole hypoxanthine with 1 unit xanthine oxidase. Both insults caused a 50% increase in vascular resistance. This was accompanied by a 30% reduction in perfusate flow rate and an 80% reduction in glomerular filtration and urine flow rates. The OFR scavengers, superoxide dismutase (SOD, 250 units/ml) and catalase (CAT, 500 units/ml), prevented these alterations after OFR generation but not after 30 min of ischemia and reperfusion. SOD and CAT also afforded no protection against the less severe dysfunction observed after 10 or 20 min of ischemia and reperfusion. OFRs do not appear to be prominent mediators of postischemic renal dysfunction; other factors, probably associated with ischemia must be primarily responsible.     

Reactive Oxygen Species Activate the Human Elastin Promoter in a Transgenic Model of Cutaneous Photoaging

BACKGROUND: Generation of free radicals has been shown to play a role in cutaneous alterations resulting from ultraviolet radiation.OBJECTIVE: Cells from a previously described in vitro transgenic model of cutaneous photoaging were exposed to reactive oxygen species to determine if this results in elastin promoter activation.METHODS: Reactive oxygen species were generated using a hypoxanthine and xanthine oxidase system, and elastin promoter activation was measured using cells derived from transgenic mice containing the human elastin promoter.RESULTS: Free radical generation resulted in a greater than sixfold increase in elastin promoter activity, and this increase was blocked with the addition of catalase.CONCLUSION: Elastin promoter activation may play a role in the generation of solar elastosis in photoaged skin. Utilizing hypoxanthine and xanthine oxidase with the in vitro transgenic photoaging model results in a sensitive system for evaluating agents that may prevent oxidative damage.     

Minimal role of xanthine oxidase and oxygen free radicals in rat renal tubular reoxygenation injury

The role of xanthine oxidase and oxygen free radicals in postischemic reperfusion injury in the rat kidney remains controversial. Proximal tubules, the focal segment affected by ischemic renal injury, were isolated in bulk, assayed for xanthine oxidase activity, and subjected to 60 min of anoxia or hypoxia and 60 min of reoxygenation to evaluate the participation of xanthine oxidase and oxygen radicals in proximal tubule reoxygenation injury. The total xanthine oxidase in isolated rat proximal tubules was 1.1 mU/mg of protein, approximately 30% to 40% of the activity found in rat intestine and liver. Lactate dehydrogenase release, an indicator of irreversible cell damage, increased substantially during anoxia (39.8 +/- 2.3 versus 9.8 +/- 1.8% in controls) with an additional 8 to 12% release during reoxygenation. Addition of 0.2 mM allopurinol, a potent xanthine oxidase inhibitor, and dimethylthiourea, a hydroxyl radical scavenger, failed to protect against the reoxygenation lactate dehydrogenase release. Analysis of xanthine oxidase substrate levels after anoxia and flux rates during reoxygenation indicates that hypoxanthine and xanthine concentrations are in a 15-fold excess over the enzyme Km and 0.3 mU/mg of protein of xanthine oxidase activity exists during reoxygenation. Hypoxic tubule suspensions had a minimal lactate dehydrogenase release during hypoxia and failed to demonstrate accelerated injury upon reoxygenation. In conclusion, although xanthine oxidase is present and active during reoxygenation in isolated rat proximal tubules, oxygen radicals did not mediate reoxygenation injury.     

The use of hyperbaric oxygen for preservation of free flaps.

The effects of hyperbaric oxygen (HBO) during tissue preservation on flap survival have been investigated in free flaps in rats. Groin skin flaps were harvested, stored in either room air or HBO (100% oxygen at 2.9 atm absolute) at 23 degrees C for 18 hours, and transplanted to the contralateral groin. Free flaps exhibit a high incidence of complete necrosis in the room air control. The survival of free flaps stored under HBO increased from 10% to 60% (p less than 0.05) after 18 hours of preservation. Skin flaps exhibited an increase in tissue hypoxanthine by 3.6-fold normal after 18 hours of storage in room air. HBO preservation prevented the accumulation of hypoxanthine and inhibited xanthine oxidase. Inhibition of the xanthine oxidase system may be one of the mechanisms of improved success of skin flap transplantation.     

Oxygen free radical mediated renal dysfunction

Postischemic renal dysfunction (PIRD) is characterized by a reduction in glomerular filtration and tubular reabsorption of solute. The relative contribution of oxygen free radicals (OFRs) generated during reperfusion remains unclear. This study characterized the renal response to OFRs--independent of an ischemic insult. Isolated rat kidneys were perfused at 37 degrees C and 90-100 mm Hg with a modified Krebs' buffer. Hypoxanthine (25 mumole) and xanthine oxidase (1 unit) were combined and infused proximal to the kidney. There was a 50% increase in vascular resistance. This was accompanied by a 30% reduction in perfusate flow rate and a 70% reduction in glomerular filtration rate. There was also a significant reduction in urine flow rate and oxygen consumption. The percentage reabsorption of filtered water and sodium by the renal tubules was not diminished, however. This pattern was not observed when the xanthine oxidase was inactivated or when the perfusate was pretreated with superoxide dismutase (250 units/ml) and catalase (500 units/ml). The generation of OFRs, independent of an ischemic insult, causes a decrease in glomerular filtration out of proportion to the decrease in renal flow similar to that observed with PIRD. OFRs may contribute to the hemodynamic and glomerular alterations seen with PIRD. Factors other than OFRs, probably associated with ischemia, must be responsible for the tubular dysfunction.     

Effect of reactive oxygen species and the activity of antioxidant systems on human semen; association with male infertility

A range of compounds with a role in oxidative stress were measured in ejaculates from 40 normozoospermic individuals and 93 infertile males. Ejaculates were classified according to WHO criteria. Seminal plasma and the sperm cell fraction were assessed separately for superoxide dismutase (SOD), catalase, xanthine oxidase, capability for singlet oxygen trapping and content of thiobarbituric acid-reactive substances (TBARS). Pathological cases defined as oligozoospermia, asthenozoospermia, or teratozoospermia revealed different backgrounds of oxidative stress as reflected by different levels of tested substances in every type of sperm pathology. In the majority of abnormal ejaculates, a significant increase in intracellular activity of SOD, decreased intracellular levels of catalase, elevated levels of xanthine oxidase and TBARS, and severely impaired singlet oxygen trapping were observed when compared to normozoospermic ejaculates. Interrelationships between SOD and TBARS, and between xanthine oxidase and catalase, appeared to be of key importance when analysed separately in seminal plasma and in spermatozoa or in a combination of both. Elevated xanthine oxidase levels and low capacity for singlet oxygen trapping are statistically significant factors for the evaluation of male infertility which can develop as a result of persistent oxidative stress.     

Lipid peroxidation and changes in xanthine oxidase in cerebral ischemia

To verify the lipid peroxidation in the focal cerebral ischemia, the levels of alpha-tocopherol, ubiquinone and ascorbate were measured in the ischemic center in rats. The former two were endogeneous lipid soluble antioxidants and the last was a water soluble antioxidant. alpha-Tocopherol, reduced ubiquinone-9 and -10, and reduced ascorbate decreased to 79%, 73%, 66%, and 76% 0.5 hour after ischemia, respectively. alpha-Tocopherol decreased to 63% 6 hours after ischemia, and then reached a plateau, while reduced ubiquinones and reduced ascorbate declined further to 16% and 10% 12 hours after ischemia, respectively, and then reached plateau levels. These results suggest their functional and durational differences as antioxidants against lipid peroxidation in this ischemic model. Although the reciprocal increase in oxidized ubiquinones during ischemia was not observed, that in oxidized ascorbate was noted. The complementary antioxidant system between cytoplasmic and membranous components, the combination alpha-tocopherol/ascorbate, was estimated from the calculated consumption ratio of these antioxidants, assuming that the loss of these reduced antioxidants is due to neutralization of free radicals. This system was suggested to play an important role in an early ischemic period. Urate also markedly increased during ischemia. Therefore, xanthine oxidase activity was measured in rats both in normal brain and in ischemic brain induced by four-vessel occlusion method. In the control rat, the enzyme activity was 0.87 +/- 0.13 nmol/g wet brain/min at 25 degrees C (mean +/- S.D.): 92.4% was associated with the NAD-dependent dehydrogenase form and only 7.6% with the oxygen-dependent superoxide-producing oxidase form. However, the ratio of the latter form increased to 43.7% after 0.5 hour of global ischemia despite the same level in total xanthine oxidase activity. This result suggests the involvement of the oxygen free radicals generated from the xanthine oxidase pathway in the pathogenesis of the ischemic injury of the rat brain.     

Clinical and Enzymatic Investigation of Induction of Oxygen Free Radicals by Ischemia and Reperfusion in Human Hepatocellular Carcinoma and Adjacent Liver

Serum concentration of thiobarbituric acid (TBA) reactants in the hepatic vein were measured before and after transient dearterialization of the liver in five human subjects bearing unresectable hepatocellular carcinoma (HCC). During 1 hour of the occlusion of the hepatic artery, change inTBA reactants level was slight. However, the mean value of TBA reactants in 1 hour after the reflow increased to 1.50 ± 0.11 nmol/ml (mean ± S.E.) and was significantly higher (p < 0.05) than those before hepatic dearterialization (1.28 ± 0.11 nmol/ml) and just before the release of occlusion (1.32 ± 0.09 nmol/ml).Further, two endogeneous scavenger enzymes, superoxide dismutase (SOD) and catalase (CAT), and one of the major sources of oxygen free radicals, xanthine oxidase (XOD) were measured in human untreated HCC and the corresponding adjacent liver tissue. The results demonstrated an increase in SOD in 81.8% (9/11) of HCC, and a decrease in CAT in 72.7% (8/11) of HCC when compared with the corresponding adjacent liver tissue. The mean value of SOD in HCC was significantly higher (66.8 ± 6.5 vs 52.8 ± 3.8 U/mg protein; p < 0.05), and that of CAT was significantly lower (22.6 ± 2.4 vs 36.0 ± 6.1 U/mg protein; p < 0.05) than those in liver tissue. All of nine HCC samples had a significantly lower activity of XOD (6.4 ± 1.9 vs 20.3 ± 3.4 pmol/minute/mg protein; p < 0.01) than the corresponding liver tissue. There was no obvious relation between the content of SOD and CAT in HCC, or in liver tissue.These data may suggest that oxygen free radicals can be generated in human HCC by ischemia and reperfusion of the tumor- bearing liver. It is also indicated that the antioxidant system of HCC is not always impaired, and that HCC might develop several lines of defence systems against the oxidative attack. A possible strategy of the treatment for liver tumor with oxygen derived free radicals induced by ischemia and reperfusion is hypothized here.     

Modulation of endogenous nitric oxide synthase in experimental acute pancreatitis: role of anti-ICAM-1 and oxygen free radical scavengers

OBJECTIVE: To evaluate, in an experimental model of acute pancreatitis, the impact of nitric oxide on the disease process and the interaction between nitric oxide and oxygen free radicals. SUMMARY BACKGROUND DATA: Nitric oxide and oxygen free radicals are involved in the pathophysiology of acute pancreatitis. It is well established that oxygen free radicals play an important role in the development of pancreatic cell damage and remote organ failure, but the impact of nitric oxide on the disease process and the interactions between the two radical species remain controversial. METHODS: Necrotizing pancreatitis (NP) was induced in Wistar rats by intraductal sodium taurocholate infusion after pretreatment with isotonic saline (NP-S), superoxide dismutase/catalase (NP-SOD/CAT), or an anti-ICAM-1 antibody (aICAM-1). Sham-operated rats received isotonic saline (SHX). After an observation period of 5 minutes and 24 hours, the pancreas was removed for microscopy, glutathione, and myeloperoxidase (MPO) analysis. The inducible NO synthase (NOS-2) was detected by Western blotting or RT-PCR. Serum was analyzed for nitrite/nitrate (NO2-/NO3-) and S-nitrosothioles (RSNO), while plasma was used to assay for trypsinogen activation peptides (TAP). RESULTS: NP-S animals showed a significant decrease in GSH levels after NP-induction as compared with animals under therapy. Increased MPO levels in the NP-S group were significantly reduced by aICAM-1 while SOD/CAT injection showed no changes. Serum NO-derivatives peaked at 12 hours while TAP levels had a maximum at 6 hours after NP induction, and were lower after aICAM-1 application SOD/CAT treatment increased both parameters. Extended acinar cell damage and inflammatory infiltrate developed in NP-S animals and was significantly improved by SOD/CAT and aICAM-1 treatment. RT-PCR and Western-blot analysis revealed NOS-2 expression in the NP-S group, which was reduced by radical scavengers and aICAM-1. CONCLUSION: Enhanced nitric oxide synthase expression and increased nitric oxide derivatives are found during severe acute pancreatitis. Oxygen free radicals and neutrophils seem to be potent and important regulation mechanisms for nitric oxide synthase activity and nitric oxide-mediated toxicity but imply only a secondary role for nitric oxide in the local pathologic mechanism of this disease.