Oxygen Free Radicals in Cardiac Transplantation

Abstract After prolonged ischemia, reperfusion of the myocardium with oxygenated blood results in high levels of superoxide anions. Several mechanisms for superoxide anion generation have been proposed, including increased xanthine oxidase activity, neutrophil activation, and arachidonate cascade activation. Superoxide anion accumulation may cause enzyme inactivation and lipid peroxidation in the sarcolemma with resultant intracellular calcium accumulation and excitation-contraction uncoupling. A review of a number of animal studies has shown that free radical scavengers such as superoxide dismutase and catalase can preserve myocardial function and metabolism during transplantation. In addition, other data indicate a role for inhibitors of free radical generation (i.e., allopurinol or oxypurinol), iron chelators (i.e., deferoxamine), or metabolic substrates such as L-glutamate in the inhibition of free radical myocardial injury. In addition, glutathione has been demonstrated to produce faster recovery of ventricular function in hypothermia preserved and reperfused rat hearts, presumably by inhibiting free radical production. Confirmatory data for human cardiac transplantation is not yet available.     

Role of neutrophil and hydroxyl radical in shock-induced gut origin infection]

The relative roles of hydroxyl radical and neutrophils in the pathogenesis of shock-induced mucosal injury and gut origin infection (GOI) were determined. The incidence of GOI was higher in the shocked rats (30 mmHg for 30 min) than the sham-shock controls (87% vs 12.5%; P less than 0.01). Administration of the hydroxyl radical scavenger, dimethyl sulfoxide (DMSO) or iron chelator and deferoxamine reduced the incidence of GOI from 87% to 20% and 40% respectively (P less than 0.05). DMSO and deferoxamine appeared to prevent shock-induced GOI by blunting the magnitude of shock-induced mucosal injury. In contrast, neutrophil depletion did not prevent GOI or protect the intestinal mucosal in the shocked rats. Instead, the incidence of systemic spread of bacteria past the mesenteric lymph nodes to the livers and spleens of the shocked rats was higher in the neutrophil depleted rats (56%) than any other group (7%) (P less than 0.01). Thus, shock-induced GOI and intestinal injury appears to be mediated by xanthine oxidase generated oxidants such as hydroxyl radical rather than neutrophil-generated factors. In addition, neutrophil depletion may be clinically deleterious, since it promotes systemic sepsis rather than preventing shock-induced GOI.     

Neutrophil-dependent, oxygen-radical mediated lung injury associated with acute pancreatitis.

Cerulein-induced acute pancreatitis in rats is associated with a reversible lung injury that is characterized by alveolar capillary endothelial-cell injury, increased microvascular permeability, interstitial edema formation, and intraalveolar hemorrhage and fibrin deposition. The role of mediators in this injury was analyzed using gravimetric data, microvascular permeability indices, electron microscopy, and a quantitative morphometric analysis. Neutrophil depletion induced by a specific antibody was highly protective against lung injury. Interruption of the complement pathway (using low dose Naja naja cobra venom factor) also protected against lung injury. Catalase and superoxide dismutase were also protective. The iron chelator deferoxamine and the hydroxyl radical scavenger, dimethylsulfoxide, were not protective against acute lung injury. These data suggest that complement, neutrophils, and neutrophil-derived (H2O2-dependent) oxygen products mediate lung injury that occurs secondary to cerulein-induced pancreatitis. In contrast to other models of neutrophil-dependent, oxygen-radical-mediated lung injury, this lung injury does not appear to be an iron-dependent and hydroxyl-radical mediated injury. We postulate that the process of acute pancreatitis leads to complement activation followed by neutrophil recruitment, sequestration, and adherence to alveolar capillary endothelial cells. Ultimately lung injury appears to result from local endothelial-cell injury secondary to neutrophil-generated oxygen products that may be myeloperoxidase dependent.     

Reactive oxygen molecule-mediated injury in endothelial and renal tubular epithelial cells in vitro

To investigate renal tubular epithelial cell injury mediated by reactive oxygen molecules and to explore the relative susceptibility of epithelial cells and endothelial cells to oxidant injury, we determined cell injury in human umbilical vein endothelial cells and in four renal tubular epithelial cell lines including LLC-PK1, MDCK, OK and normal human kidney cortical epithelial cells (NHK-C). Cells were exposed to reactive oxygen molecules including superoxide anion, hydrogen peroxide and hydroxyl radical generated by xanthine oxidase and hypoxanthine. We determined early sublethal injury with efflux of 3H-adenine metabolites and a decline in ATP levels, while late lytic injury and cell detachment were determined by release of 51chromium. When the cells were exposed to 25, 50, and 100 mU/ml xanthine oxidase with 5.0 mM hypoxanthine, ATP levels were significantly lower (P less than 0.001) in LLC-PK1, NHK-C and OK cells compared to MDCK cells while ATP levels were significantly lower (P less than 0.01) in endothelial cells compared to all tubular cell lines. A similar pattern of injury was seen with efflux of 3H-adenine metabolites. When the cells were exposed to 50 mU/ml xanthine oxidase with 5.0 mM hypoxanthine for five hours, total 51chromium release was significantly (P less than 0.001) greater in LLC-PK1, NHK-C and OK cells compared to MDCK cells, while total 51chromium release was significantly (P less than 0.001) greater in endothelial cells compared to all tubular cells. However, lytic injury was the greatest in LLC-PK1 cells and NHK-C cells while cell detachment was the greatest in endothelial cells. MDCK cells were remarkably resistant to oxidant-mediated cell detachment and cell lysis. In addition, we determined ATP levels, 3H-adenine release and 51chromium release in LLC-PK1, NHK-C and endothelial cells in the presence of superoxide dismutase to dismute superoxide anion, catalase to metabolize hydrogen peroxide, DMPO to trap hydroxyl radical and DMTU to scavenge hydrogen peroxide and hydroxyl radical. We found that catalase and DMTU (scavengers of hydrogen peroxide) provided significant protection from ATP depletion, prevented efflux of 3H-adenine metabolites and cell detachment while DMPO (scavenger of hydroxyl radical) prevented lytic injury. In addition, we found that the membrane-permeable iron chelator, phenanthroline, and preincubation with deferoxamine prevented cell detachment and cell lysis, confirming the role of hydroxyl radical in cell injury.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hemorrhagic shock-induced bacterial translocation: the role of neutrophils and hydroxyl radicals

We previously documented a relationship between xanthine oxidase activation, intestinal injury, and bacterial translocation (BT) in rats subjected to hemorrhagic shock. The current experiments were performed to determine the relative roles of hydroxyl radicals and neutrophils in the pathogenesis of shock-induced mucosal injury and BT. The incidence of BT was higher in the shocked rats (30 mm Hg for 30 min) than the sham-shock controls (87% vs 12.5%; p less than 0.01). Administration of the hydroxyl radical scavenger, dimethyl sulfoxide (DMSO), or the iron chelator, deferoxamine, reduced the incidence of BT from 87% to 20% and 40%, respectively (p less than 0.05). DMSO and deferoxamine appear to prevent shock-induced BT by blunting the magnitude of shock-induced mucosal injury. In contrast, neutrophil depletion did not prevent BT or protect the intestinal mucosa in shocked rats. Instead, the incidence of systemic spread of translocating bacteria past the mesenteric lymph nodes to the livers and spleens of the shocked rats was higher in the neutrophil-depleted rats (56%) than in any other group (p less than 0.01). Thus, shock-induced BT and intestinal injury appear to be mediated by oxidants (.OH) derived from xanthine oxidase, rather than granulocytes.     

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.     

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.     

Hydroxyl radical mediation of immune renal injury by desferrioxamine

The acute phase of glomerular injury in a model of antiglomerular basement membrane, antibody-induced glomerulonephritis (antiGBM-GN) in rabbits was shown to be neutrophil-dependent using nitrogen mustard depletion studies. Administration of desferrioxamine (DFX) prevented the development of proteinuria in this model of renal injury [24 hr protein excretion (mean +/- SEM): antiGBM-GN/DFX = 16.2 +/- 2.9 mg compared with antiGBM-GN control = 271.5 +/- 92.2 mg, P less than 0.01]. Antibody binding levels, glomerular filtration rates, circulating complement and neutrophil counts, glomerular C3 deposition, and neutrophil infiltration did not differ between DFX treated and antiGBM-GN groups. In vitro assay systems to assess oxygen radical production [superoxide anion (O2-) and hydroxyl radical (OH.)] by neutrophils activated via the interaction of antiGBM antibody, GBM and complement were established. In these assays, DFX inhibited OH. production by immunologically-stimulated neutrophils (ISN) [nM diphenol/hr/10(6) cells, mean +/- SEM, ISN/DFX = 8 +/- 2 compared with ISN = 191 +/- 22, P less than 0.01] while production of O2- was not affected [nM O2-/hr/10(6) cells, mean +/- SEM, ISN/DFX = 29.1 +/- 4.3 compared with ISN = 32.6 +/- 2.5, P greater than 0.05]. These studies demonstrate that the iron chelator desferrioxamine can prevent neutrophil-dependent immune renal injury by interfering with neutrophil function. Treatment with the hydroxyl radical scavenger dimethylthiourea also significantly attenuated renal injury in antiGBM-GN. Together, the in vivo and in vitro data strongly suggest that neutrophil-dependent immunological renal injury is mediated via hydroxyl radical production by activated neutrophils within glomeruli.     

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.     

Prevention of free radical-induced myocardial reperfusion injury with allopurinol

Growing evidence supports the concept that oxygen free radicals are an important cause of myocardial ischemic and reperfusion injury. This study was designed to determine if toxic oxygen metabolites may exacerbate ischemic injury upon reoxygenation. Left ventricular function was studied in a group of seven dogs receiving intermittent, 4 degrees C, hyperosmolar, hyperkalemic (KCI 25 mEq/L) saline cardioplegic solution. This group was compared to a group (n = 7) receiving a hyperkalemic (KCI 25 mEq/L) cardioplegic solution designed to scavenge superoxide anion and hydroxyl radical: superoxide dismutase (3,000 U/ml) and mannitol (325 mOsm/L). A third group of five animals received allopurinol pretreatment (50 mg/kg/day) for 72 hours and hyperkalemic saline cardioplegic solution. After 60 minutes of ischemia (10 degrees to 15 degrees C) and 45 minutes of reperfusion, left ventricular mechanical function was better in the groups receiving free radical scavengers and allopurinol pretreatment than in the group receiving only hyperkalemic saline cardioplegic solution. Free radical scavengers preserved myocardial function in this model of hypothermic global ischemia and reperfusion. Our data support the concept that injury occurs primarily during reperfusion with the generation of oxygen free radicals via the hypoxanthine-xanthine oxidase reaction. Allopurinol has potential clinical application in the prevention of reperfusion injury.     

Modification of oxidative stress in response to intestinal preconditioning

Previous studies have demonstrated that intestinal preconditioning protects the organ from ischemia reperfusion damage. Xanthine oxidase mediating free radical generation contributes to the development of injury associated to ischemia reperfusion. Thus, any process able to modulate the oxygen free radical generation system could attenuate the injury. Also, it is known that nitric oxide is implicated in the preconditioning response. The aim of this work is to determine: (1) the effect of intestinal preconditioning on the xanthine oxidase system, (2) the relevance of this system in the development of injury, and (3) its relationship with nitric oxide. For this purpose, we have determined the activity of the xanthine dehydrogenase/xanthine oxidase system, the levels of its substrate (xanthine), and end-product (uric acid) and oxidant stress status in rat small intestine subjected to ischemic pre-conditioning. The effects of nitric oxide inhibition have also been evaluated. Results show that the percentage of xanthine dehydrogenase to xanthine oxidase conversion, xanthine, uric acid concentration, lipoperoxides, and reduced glutathione were significantly reduced in preconditioned rats irrespectively of nitric oxide inhibition. In summary, this work shows that oxidative stress in intestinal preconditioning is reduced as consequence of the diminished conversion of xanthine dehydrogenase to xanthine oxidase, and also as a consequence of the reduced availability of xanthine.     

Allopurinol and lodoxamide in complement-induced hepatic ischemia

Intravascular complement activation with either zymosan or cobra venom factor (CVF) impairs hepatic blood flow. Oxygen radical scavengers given at the time of complement activation attenuate the resulting hepatic ischemia. It is not clear whether complement-stimulated phagocytes or transiently ischemic then reperfused endothelial and parenchymal cells generated the toxic oxygen radicals. In this study, a group of rats were given allopurinol (50 mg/kg/day postoperatively X 3 days plus 100 mg/kg iv at t = 0), a specific inhibitor of xanthine oxidase, prior to complement activation with CVF (20 units/kg iv at t = 30 and 60 min) to determine whether xanthine oxidase-derived oxygen radicals contributed significantly to the hepatic perfusion abnormalities. Additional rats received lodoxamide tromethamine (10 mg/kg iv bolus at t = 0 followed by 20 mg/kg/hr iv infusion), a novel and potent inhibitor of mast cell release and inhibitor of xanthine oxidase, prior to the same CVF challenge to determine whether mast cell mediators were involved in the flow disturbance. Thermodilution cardiac output, mean arterial pressure, heart rate, hematocrit, and effective hepatic blood flow (EHBF) by galactose clearance were determined at t = 2 hr. The percentage change in total hemolytic complement activity (% delta CH50) was determined between serum obtained prior to sacrifice and at t = 0. Systemic hemodynamics and HCT were for the most part unaffected regardless of pretreatment group or challenge with CVF or saline. CVF challenge produced a 25% reduction (P less than 0.05) in EHBF in vehicle-pretreated rats compared to saline challenge. Neither allopurinol nor lodoxamide tromethamine significantly improved EHBF when given prior to CVF challenge.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Leukocyte depletion ameliorates free radical-mediated lung injury after cardiopulmonary bypass

Activated leukocytes and oxygen free radicals have been implicated in the pathogenesis of lung injury associated with cardiopulmonary bypass. To determine whether leukocyte depletion could prevent this injury, we used a dog model simulating routine cardiac operations. Mongrel dogs (11 to 17 kg) were subjected to cardiopulmonary bypass with a bubble oxygenator and cooled to 27 degrees C. After aortic crossclamping and cardioplegic arrest for 90 minutes, control animals (n = 5) were rewarmed and weaned from bypass, and their condition was then stabilized for 90 minutes. Leukocyte-depleted animals (n = 5) had a leukocyte filter incorporated in the bypass circuit. During bypass, circulating leukocyte counts decreased by 60% in control dogs, and by 97% in leukocyte-depleted animals. Free radical generation (estimated by spectrophotometric assay of plasma conjugated dienes) was significantly reduced by leukocyte depletion during and after bypass. Total hemolytic complement activity and the titer of C5 decreased markedly immediately after the onset of bypass in both the control and leukocyte-depleted animals. Pulmonary function after bypass was better preserved in leukocyte-depleted animals. These data suggest that depletion of circulating leukocytes contributes to lung injury during cardiopulmonary bypass and is associated with increased oxygen radical activity, pulmonary edema, and vasoconstriction. Leukocyte depletion substantially reduced the pulmonary injury seen after cardiopulmonary bypass.     

Regulation of neutrophil migratory function in burn injury by complement activation products.

Polymorphonuclear neutrophils were isolated from patients with burn injury and random mobility, chemotaxis in response to C5adesArg (as agarose-activated control serum) and to N-formyl-methionyl-leucyl-phenylalanine (F-Met-Leu-Phe) were assessed. For a group of eight patients identified as not experiencing systemic infection, all three neutrophil migratory functions were observed to fall below control levels, beginning 4 to 6 days following burn injury, and to return to control levels after 21 to 30 days of hospitalization. Over this time the chemotactic differential (distance chemotactic migration-distance random migration) for F-Met-Leu-Phe remained positive, while the chemotactic differential for activated serum became nil after postburn day 4. This temporal, specific loss of a chemotactic response to activated serum was associated with rises in immunoreactive plasma C3a and C5a. This pattern of loss of chemotactic function was associated with a selective loss of C5a but not F-Met-Leu-Phe binding activity. These results demonstrate that burn injury can alter neutrophil migratory functions generally, and specifically depress chemotactic responsiveness to activated serum. The mechanism of the latter phenomenon appears to be related to desensitization of circulating neutrophils to C5a due to complement activation.     

Burn wound sepsis may be promoted by a failure of local antibacterial host defenses.

Little attention has been focused on the local burn wound environment, even though burn wound sepsis is a common cause of death in the burn victim. To characterize the effect of the local burn wound environment on neutrophil function and metabolism, the opsonic activity of blister fluid specimens against Pseudomonas aeruginosa was measured as was the effect of blister fluid on control neutrophil oxygen consumption using preopsonized zymosan and f-met-leu-phe (FMLP) as stimuli. Blister fluid did not support the killing of P. aeruginosa by normal neutrophils as well as normal serum. Additionally, blister fluid inhibited zymosan-stimulated, but not FMLP-stimulated, neutrophil oxygen consumption. The inhibitory effect of blister fluid on zymosan-stimulated oxygen consumption correlated with the extent of complement activation, measured as C3d or C3AI (p less than 0.01). That blister fluid did not inhibit the FMLP-mediated respiratory burst supports the concept that the blister fluid inhibitory effect on the zymosan-mediated respiratory burst was mediated through the complement receptor. These findings that blister fluid can affect the bactericidal and metabolic activity of normal neutrophils support the concept that cellular function can be altered by the microenvironment in which the cells are bathed. This potential impairment of host defenses within the burn wound could predispose the burn victim to burn wound sepsis.     

Role of oxygen free radicals in shock, ischemia, and organ preservation

Oxygen radicals appear to be involved in the microvascular and parenchymal cell injury associated with various pathologic disorders. Studies indicate that oxygen radicals increase microvascular permeability by creating large leakage sites predominantly in the small venules. The highly reactive hydroxyl radical appears to be responsible for the microvascular alterations associated with oxygen radical production. There is considerable indirect evidence implicating oxygen radicals in the pathogenesis of circulatory shock. The oxygen radicals are probably formed by the enzyme xanthine oxidase when intravascular volume is restored. Similar biochemical processes appear to be involved in reperfusion injury to the kidney and skin. Evidence is also presented that implicates oxygen radicals in the reperfusion injury associated with organ preservation and transplantation.     

Oxidant mechanisms in gentamicin nephrotoxicity.

Acute renal failure is a major complication of aminoglycoside antibiotics, which are widely used in the treatment of gram-negative infections. Sequential reduction of oxygen along the univalent pathway leads to the generation of superoxide anion, hydrogen peroxide, hydroxyl radical, and water. A large body of in vitro and in vivo evidence indicates that these partially reduced oxygen metabolites are important mediators of gentamicin nephrotoxicity. Gentamicin has been shown to enhance the generation of superoxide anion and hydrogen peroxide by renal cortical mitochondria. The interaction between superoxide anion and hydrogen peroxide in the presence of metal catalyst can lead to the generation of hydroxyl radical. Gentamicin has been shown to lead to release of iron from renal cortical mitochondria and to enhance generation of hydroxyl radical. These in vitro observations have been supported by in vivo studies in which scavengers of reactive oxygen metabolites and iron chelators have shown to be protective in gentamicin induced acute renal failure. There is evidence to suggest that studies may have broader implication in being relevant to other aminoglycosides including streptomycin and being applicable to other major toxicity of aminoglycoside such as ototoxicity.     

Neutrophil activation after burn injury: contributions of the classic complement pathway and of endotoxin

We attempt to elucidate the mechanisms of neutrophil (PMN) activation after burn injury. We previously reported prolonged elevations of PMN cell surface complement (C) opsonin receptor levels after burn trauma with a corresponding period of depressed PMN chemotaxis to C5a, which suggests that the C product, C5a, was responsible for PMN activation. However, a lack of direct correlation of C activation with C receptor levels soon after injury raised the possibility of a second PMN-activating substance. We therefore investigated the effect of endotoxin (LPS) on the expression of the C receptors (CR1 and CR3) by normal human PMNs. Concentrations from 0 to 50 ng/ml of LPS 026:B6 caused a dose response increase in the PMN surface expression of CR1 and CR3 as assessed by monoclonal antibody binding and indirect immunofluorescence. The relative CR1-dependent fluorescence rose from a mean of 50 to 385 and CR3 from 50 to 300. Chelation by ethylenediaminetetra acetic acid (EDTA) did not influence this dose response, thus ruling out the possibility of C activation by LPS--an inference supported by the lack of complement activation observed with these concentrations of LPS in normal serum. A similar dose response was obtained in the absence of other cell types or serum, which implies a direct effect that mimicked that of C5a. To determine the mechanism of the later, prolonged C activation after burn injury, we next examined C activation products in 22 patients with burn injuries. Elevations of plasma C3a desArg were present and persisted for 50 days. Elevations were at maximum levels on days 9 through 13 postburn (mean +/- standard error of mean [SEM], 496 +/- 47 ng/ml versus normal 113 +/- 32; p less than 0.01). These were accompanied by elevations of C4a desArg (917 +/- 154 ng/ml versus normal 424 +/- 50; p less than 0.01), which are indicative of classic pathway activation. Finally, we examined PMN function, phagocytosis and percentage killing of Staphylococcus aureus, and found PMN function to be unaltered in the 22 patients. Thus PMN activation after burn injury appears to be caused by LPS soon after injury and by C5a later after injury and affects only selected PMN functions.     

Ischemic preconditioning: a defense mechanism against the reactive oxygen species generated after hepatic ischemia reperfusion

BACKGROUND: Preconditioning protects against both liver and lung damage after hepatic ischemia-reperfusion (I/R). Xanthine and xanthine oxidase (XOD) may contribute to the development of hepatic I/R. OBJECTIVE: To evaluate whether preconditioning could modulate the injurious effects of xanthine/XOD on the liver and lung after hepatic I/R. METHODS: Hepatic I/R or preconditioning previous to I/R was induced in rats. Xanthine and xanthine dehydrogenase/xanthine oxidase (XDH/XOD) in liver and plasma were measured. Hepatic injury and inflammatory response in the lung was evaluated. RESULTS: Preconditioning reduced xanthine accumulation and conversion of XDH to XOD in liver during sustained ischemia. This could reduce the generation of reactive oxygen species (ROS) from XOD, and therefore, attenuate hepatic I/R injury. Inhibition of XOD prevented postischemic ROS generation and hepatic injury. Administration of xanthine and XOD to preconditioned rats led to hepatic MDA and transaminase levels similar to those found after hepatic I/R. Preconditioning, resulting in low circulating levels of xanthine and XOD activity, reduced neutrophil accumulation, oxidative stress, and microvascular disorders seen in lung after hepatic I/R. Inhibition of XOD attenuated the inflammatory damage in lung after hepatic I/R. Administration of xanthine and XOD abolished the benefits of preconditioning on lung damage. CONCLUSIONS: Preconditioning, by blocking the xanthine/XOD pathway for ROS generation, would confer protection against the liver and lung injuries induced by hepatic I/R.