Blockade of electron transport before cardiac ischemia with the reversible inhibitor amobarbital protects rat heart mitochondria

Cardiac ischemia damages the mitochondrial electron transport chain. Irreversible blockade of electron transport at complex I by rotenone decreases ischemic damage to cardiac mitochondria by decreasing the loss of cytochrome c and preserving respiration through cytochrome oxidase. Therapeutic intervention to protect myocardium during ischemia and reperfusion requires the use of a reversible inhibitor that allows resumption of oxidative metabolism during reperfusion. Amobarbital is a reversible inhibitor at the rotenone site of complex I. We asked whether amobarbital administered immediately before ischemia protected respiratory function. Isolated rat hearts were perfused for 15 min followed by 25-min global ischemia at 37 degrees C. Amobarbital-treated hearts received drug for 1 min before ischemia. Subsarcolemmal (SSM) and interfibrillar (IFM) populations of mitochondria were isolated after ischemia, and oxidative phosphorylation was measured. Amobarbital protected oxidative phosphorylation, including through cytochrome oxidase, in both SSM and IFM in a dose-dependent manner, with an optimal dose of 2 to 2.5 mM. Amobarbital also preserved cytochrome c content in both SSM and IFM. Thus, reversible blockade of the electron transport chain during ischemia protects mitochondrial respiration.     

Ischemic preconditioning protects post-ischemic oxidative damage to mitochondria in rat liver

This study examined the effect of ischemic preconditioning (IPC) in protecting against a hepatic ischemia/reperfusion (I/R) injury, with particular focus on mitochondrial damage. Rat liver was preconditioned by 10 min of ischemia and 10 min of reperfusion. Immediately after IPC, liver was subjected to 90 min of sustained ischemia followed by 5 h of reperfusion. The hepatic I/R increased serum aminotransferase activity and mitochondrial lipid peroxidation 5 h after reperfusion. IPC attenuated these increases. Whereas the mitochondrial glutathione content and glutamate dehydrogenase activities were lower in the I/R group, these decreases were attenuated by IPC. During IPC, the tissue peroxide levels increased after 10 min of ischemia and were normalized after 10 min of reperfusion. In association with the IPC-derived transient increase in the peroxide levels, the significant production of peroxides observed at 10 min of reperfusion after 90 min of ischemia was attenuated. Furthermore, whereas the mitochondria isolated from rat liver after 5 h of reperfusion were rapidly swollen, the swelling rate was attenuated in the mitochondria from rat liver subjected to IPC before the sustained ischemia. The hepatic ATP and adenosine levels were 38% and 46% lower during the reperfusion, respectively. These decreases were attenuated by IPC. Thus, these results suggest that IPC protects the mitochondria against the deleterious effects of I/R, and this protection is associated with the reduced oxidative stress.     

Time course and mechanism of oxidative stress and tissue damage in rat liver subjected to in vivo ischemia-reperfusion.

The time course of oxidative stress and tissue damage in zonal liver ischemia-reperfusion in rat liver in vivo was evaluated. After 180 min of ischemia, surface chemiluminescence decreased to zero, state 3 mitochondrial respiration decreased by 70-80%, and xanthine oxidase activity increased by 26% without change in the water content and in the activities of superoxide dismutase, catalase, and glutathione peroxidase. After reperfusion, marked increases in oxyradical production and tissue damage were detected. Mitochondrial oxygen uptake in state 3 and respiratory control as well as the activities of superoxide dismutase, catalase, and glutathione peroxidase and the level of nonenzymatic antioxidants (evaluated by the hydroperoxide-initiated chemiluminescence) were decreased. The severity of the post-reperfusion changes correlated with the time of ischemia. Morphologically, hepatocytes appeared swollen with zonal cord disarrangement which ranged from mild to severe for the tissue reperfused after 60-180 min of ischemia. Neutrophil infiltration was observed after 180 min of ischemia and 30 min of reperfusion. Mitochondria appear as the major source of hydrogen peroxide in control and in reperfused liver, as indicated by the almost complete inhibition of hydrogen peroxide production exerted by the uncoupler carbonylcyanide p-(trifluoromethoxy) phenylhydrazone. Additionally, inhibition of mitochondrial electron transfer by antimycin in liver slices reproduced the inhibition of state 3 mitochondrial respiration and the increase in hydrogen peroxide steady-state concentration found in reperfused liver. Increased rates of oxyradical production by inhibited mitochondria appear as the initial cause of oxidative stress and liver damage during early reperfusion in rat liver.     

Protective effect of allopurinol on hepatic energy metabolism in ischemic and reperfused rat liver

Reactive oxygen species generated by xanthine oxidase during reperfusion of ischemic liver might in part be responsible for ischemic organ injury. In normothermic ischemia/reperfusion rat model, we investigated whether allopurinol pretreatment improved ischemia-induced mitochondrial dysfunction. Rats were subjected to 60 min of hepatic ischemia and to 1 h and 5 h of reperfusion thereafter. At 18 h and 1 h before ischemia, the animals received 0.25 mL of either saline or allopurinol (50 mg/kg) i.p. In saline-treated ischemic rats, serum aspartate aminotransferase levels increased significantly at 5 h (4685 +/- 310 IU/L) and were significantly reduced with allopurinol pretreatment. Similarly, mitochondrial lipid peroxidation was elevated in the saline-treated ischemic group, but this elevation was prevented by allopurinol. In contrast, mitochondrial glutamate dehydrogenase activity and ketone body ratio decreased in the saline-treated group, but this decrease was also inhibited by allopurinol. Hepatic ATP levels in the saline-treated rats were 42% lower 5 h after reperfusion. However, treatment with allopurinol resulted in significantly higher ATP levels. Allopurinol treatment preserved the concentration of AMP in ischemic liver but inhibited the accumulation of xanthine in reperfused liver. Our findings suggest allopurinol protects against mitochondrial injury, which prevents a mitochondrial oxidant stress and lipid peroxidation and preserves the hepatic energy metabolism.     

Possible mechanism of hepatocyte injury induced by diphenylamine and its structurally related nonsteroidal anti-inflammatory drugs

Diphenylamine is a common structure of nonsteroidal anti-inflammatory drugs (NSAIDs) to uncouple mitochondrial oxidative phosphorylation and to cause a decrease in hepatocellular ATP content and hepatocyte injury. The mechanism for acute cell injury induced by diphenylamine and its structurally related NSAIDs was investigated with rat liver mitochondria and freshly isolated hepatocytes, focusing on the relation to the uncoupling of oxidative phosphorylation. Incubation of mitochondria with diphenylamine as well as mefenamic acid and diclofenac caused pseudoenergetic mitochondrial swelling, indicating that these compounds induce mitochondrial membrane permeability transition. Diphenylamine also caused changes in safranine-binding spectra to mitochondria that was energized by succinate oxidation. This spectral shift indicates the loss of mitochondrial membrane potentials, which is known as one of the characteristics for uncouplers of oxidative phosphorylation, and also was caused by mefenamic acid and diclofenac. Incubation of hepatocytes with mefenamic acid, diclofenac, and diphenylamine diminished cellular ATP content, followed by leakage of lactose dehydrogenase from hepatocytes. Fructose, a low K(m) substrate for glycolysis, partially protected against the ATP depletion and hepatocyte injury induced by these compounds. Further addition of oligomycin, which blocks ATPase, pronounced the protection against cell injury. These results suggested that decreases in cellular ATP content, mainly caused by uncoupling of mitochondrial oxidative phosphorylation, were responsible for acute hepatocyte injury induced by diphenylamine and structurally related NSAIDs.     

Reversible blockade of electron transport during ischemia protects mitochondria and decreases myocardial injury following reperfusion

Cardiac mitochondria sustain damage during ischemia and reperfusion, contributing to cell death. The reversible blockade of electron transport during ischemia with amobarbital, an inhibitor at the rotenone site of complex I, protects mitochondria against ischemic damage. Amobarbital treatment immediately before ischemia was used to test the hypothesis that damage to mitochondrial respiration occurs mainly during ischemia and that protection of mitochondria during ischemia leads to decreased cardiac injury with reperfusion. Langendorff-perfused Fischer-344 rat hearts were treated with amobarbital (2.5 mM) or vehicle for 1 min immediately before 25 min of global ischemia. Both groups were reperfused for 30 min without additional treatment. Subsarcolemmal (SSM) and interfibrillar (IFM) populations of mitochondria were isolated after reperfusion. Ischemia and reperfusion decreased state 3 and increased state 4 respiration rate in both SSM and IFM. Amobarbital treatment protected oxidative phosphorylation measured following reperfusion and improved the coupling of respiration. Cytochrome c content measured in SSM and IFM following reperfusion decreased in untreated, but not in amobarbital-treated, hearts. H(2)O(2) release from SSM and IFM isolated from amobarbital-treated hearts during reperfusion was markedly decreased. Amobarbital treatment before ischemia improved recovery of contractile function (percentage of preischemic developed pressure: untreated 51 +/- 4%, n = 12; amobarbital 70 +/- 4%, n = 11, p < 0.01) and substantially reduced infarct size (untreated 32 +/- 2%, n = 7; amobarbital 13 +/- 2%, n = 7, p < 0.01). Thus, mitochondrial damage occurs mainly during ischemia rather than during reperfusion. Reperfusion in the setting of preserved mitochondrial respiratory function attenuates the mitochondrial release of reactive oxygen species, enhances contractile recovery, and decreases myocardial infarct size.     

Oxidative stress induced by bloodless limb surgery on humans

Measure of oxidative stress were studied in blood samples from 10 patients undergoing bloodless lower limb surgery. Ischaemia induced a significant increase in plasma hypoxanthine concentration and xanthine oxidase activity both in the operated leg and in the systemic circulation. Five minutes after reperfusion, ratio of xanthine oxidase/total xanthine oxidase and dehydrogenase activities rose moderately, whereas at 20 min xanthine oxidase accounted for all xanthine oxidoreductase activity in the systemic circulation. A significant increase in blood glutathione redox ratio, enhanced oxidation of haemoglobin to methaemoglobin and rise in plasma haemoglobin concentration were present only in the operated limb. Thus, although the level of the potential free radical generators rose significantly both locally and in the systemic circulation, oxidative stress, as indicated by blood glutathione and erythrocyte injuries, remained limited to the reperfused leg.     

Xanthine oxidase released from reperfused hind limbs mediate kupffer cell activation, neutrophil sequestration, and hepatic oxidative stress in rats subjected to tourniquet shock

We have shown previously that rats subjected to tourniquet shock develop an acute form of remote organ injury of the liver that is both Kupffer cell (KC) and polymorphonuclear (PMN) leukocyte dependent. Circulating plasma xanthine oxidase (XO) has been shown to be responsible for the development of endothelial dysfunction and for remote organ injury of the lung and intestine after ischemia-reperfusion protocols. We now hypothesize that XO is released from rat hind limbs upon reperfusion and that it is responsible for KC and PMN leukocyte activation in this shock model. Our results show that about 30% of rat gastrocnemius muscle xanthine dehydrogenase (XD) is converted to XO during the 5-h tourniquet period and that it is released into the femoral vein within 10 min of reperfusion. Total muscle xanthine oxidoreductase activity (XO + XD) decreases within 30 min of reperfusion and is paralleled by a corresponding increase in femoral vein lactic dehydrogenase. In addition, liver tissue XO increases significantly within 30 min of reperfusion without a corresponding conversion of endogenous XD. Conversion of hepatic XD becomes evident 60 min after reperfusion is initiated, as does XO, and alanine aminotransferase (ALT) release into the hepatic vein, presumably from damaged hepatocytes as a consequence of oxidative stress. Tissue myeloperoxidase activity also increases significantly after the 60-min reperfusion period. That XO mediates KC and PMN activation is supported by the following observations: a) the close relationships between plasma XO and the time courses of tumor necrosis factor-alpha TNFalpha release into the hepatic vein and colloidal carbon clearance by KCs; b) that colloidal carbon clearance, TNFalpha and ALT release, loss of tissue free thiols, lipid peroxidation (TBARS), and liver infiltration by PMN neutrophils can also be induced by the administration of exogenous XO to normal rats; and c) pretreatment of rats with allopurinol inhibits KC activation and liver leukocyte infiltration. These results suggest that XO, released from the ischemic limb on reperfusion, is taken up by the liver were it mediates KC and PMN neutrophil activation and thus contributes to the development of multiple system organ failure after hind limb reperfusion.     

Normothermic liver ischemia in rats: xanthine oxidase is not the main source of oxygen free radicals

We studied the effect of allopurinol (ALL) on the activity of xanthine dehydrogenase (XDH), xanthine oxidase (XOX), superoxide dismutase (SOD), and catalase (CAT) in rat liver during ischemia followed by 60 min of reperfusion. We induced 60-min ischemia in the median and left lobes by clamping the hepatic artery and portal branches. The percentage XOX relative to total oxidase activity increased significantly in the control group, from 10% during the stabilization period to 18% after 60 min of reperfusion. The XDH activity decreased during reperfusion. Activity of both XDH and XOX was almost completely blocked by ALL. The activity of SOD and CAT did not differ significantly between the ALL group and controls after 60 min of reperfusion. ALL treatment did not affect liver injury parameters, as concentrations of lactate dehydrogenase (LDH) and alanine transferase (ALT) increased in plasma after ischemia, both in controls and in the ALL-treated group. We concluded that ischemia promotes conversion of XDH to XOX during reperfusion. XOX may not be the main source of free radical production, since intracellular scavengers (SOD and CAT) did not differ significantly between controls and the ALL-treated group, despite the fact that ALL blocked XOX activity completely.     

Oxygen-derived free radicals and postischemic myocardial reperfusion: therapeutic implications

Oxygen-derived free radicals have been implicated in the pathogenesis of various disease states, including myocardial ischemia and reperfusion. In this article, we review 1) the evidence linking free radical production and myocardial injury during myocardial ischemia and reperfusion and 2) results of studies of the effects of the pharmacological therapies available potentially to prevent free radical-mediated injury. Free radicals can be produced during ischemia and reperfusion by several different biochemical pathways. Of these, the xanthine oxidase reaction and the output of free radicals by neutrophils that have accumulated in damaged tissue have been studied extensively. When produced, free radicals can potentially damage myocytes or endothelial cells through peroxidation of membrane lipids or damage to proteins or nucleic acids. Using electron spin resonance spectroscopy, several studies have shown a 'burst' of oxygen free radicals immediately after reperfusion. Moreover, exogenous generation of intravascular free radicals has been shown to produce marked vascular and myocyte damage, as well as contractile dysfunction. 'Anti-free radical' interventions, such as xanthine oxidase inhibitors and free radical scavengers have been reported to prevent contractile dysfunction and reperfusion-induced arrhythmias after an episode of reversible ischemic injury. However, after more severe episodes of ischemia, such interventions have had conflicting effects on myocardial infarct size. 'Anti-free radical' interventions could be of potential use in situations where reversible ischemic injury occurs. In situations where reperfusion is achieved after irreversible ischemic injury has occurred, the potential beneficial effect of these treatments on infarct size is more doubtful.     

全脑缺血再灌注后脑线粒体功能变化及山莨菪碱的保护作用

目的：探讨全脑缺血再灌注后脑线粒体功能变化及山莨菪碱的保护作用。方法：采用家兔全脑缺血再灌注损伤模型，缺血20min、再灌注2h观察脑线粒体呼吸功能、呼吸链氧化酶活性、线粒体内Ca^2 和MDA含量的变化。结果:脑缺血再灌注后，脑线粒体呼吸控制率、磷氧比、氧化磷酸化效率及烟酰胺腺喋呤二核苷酸氧化酶、琥珀酸氧化酶、细胞色素C氧化酶活性明显降低(P＜0、01)，而线粒体Ca^2 、MDA含量明显升高(P＜0．01)；再灌注早期给予山莨菪碱治疗后，上述线粒体损伤性改变明显减轻。结论：山莨菪碱对脑缺血再灌注后线粒体损伤具有一定的保护作用，其机制可能与Ca^2 拮抗、抑制脂质过氧化及保护呼吸链酶活性有关。     

Role of purines and xanthine oxidase in reperfusion injury in perfused rat liver

The purpose of this study was to evaluate the possible involvement of xanthine and xanthine oxidase in reperfusion injury in a low-flow, reflow model of liver perfusion. Livers were perfused at flow rates around 25% of normal for 90 min and then at normal flow rates (4 ml/g/min) for 30 min. When flow was restored to normal, malondialdehyde and lactic dehydrogenase (LDH) were released into the effluent perfusate. Malondialdehyde production rapidly reached values of 300 nmol/g/hr whereas LDH increased from basal levels of 100 to 600 U/l upon reperfusion. Trypan blue was taken up exclusively in cells in pericentral regions of the liver lobule under these conditions. Xanthine and hypoxanthine in the effluent perfusate increased steadily during the low-flow period reaching values around 5 and 10 microM, respectively, and decreased rapidly after the flow was restored to normal. Perfusion with nitrogen-saturated buffer for 3 min before restoration of normal flow rates or infusion of the radical scavenger catechin (400 microM) reduced cell damage by about 50%. Infusion of allopurinol (2-6 mM), an inhibitor of xanthine oxidase, prevented reperfusion injury in a dose-dependent manner. Taken together, these data indicate that a reperfusion injury occurs in liver upon reintroduction of oxygen which is initiated by oxidation of xanthine and hypoxanthine via xanthine oxidase and ultimately leads to production of lipid peroxides. Surprisingly, infusion of xanthine (4 mM), substrate for xanthine oxidase, reduced hepatocellular injury on reperfusion. LDH release was decreased from values around 700 to less than 200 U/l and trypan blue uptake in pericentral region was prevented totally by xanthine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Free radicals and myocardial ischemia and reperfusion injury

There is a growing body of evidence for the role of free radicals in mediating myocardial tissue injury during myocardial ischemia and in particular during the phase of myocardial reoxygenation. Associated with myocardial ischemia and reperfusion is the generation of oxygen-derived free radicals from a variety of sources that include the mitochondrial electron transport chain; the biosynthesis of prostaglandins; the enzyme xanthine oxidase; and circulating elements in the blood, with the polymorphonuclear neutrophil assuming a primary focus of attention. Experimental studies have shown that free radical scavengers (e.g., N-[2-mercaptopropionyl]glycine) and enzymes that scavenge or degrade reactive species of oxygen (superoxide dismutase or catalase) can reduce the mass of myocardial tissue that undergoes irreversible injury. Additionally allopurinol, which inhibits the enzyme xanthine oxidase, reduces ultimate infarct size, putatively by reducing the xanthine oxidase generation of superoxide anion. Neutrophils that enter the ischemically injured myocardium under the influence of chemotactic attraction and activation of the complement system generate and release highly reactive and cytotoxic oxygen derivatives that are destructive to the vascular endothelium and to the cardiac myocytes. Studies have documented that neutrophil depletion or suppression of neutrophil function (ibuprofen, nafazatrom, BW 755C, or more recently with prostacyclin or iloprost) results in a significant salvage of myocardial tissue that is subjected to a period of regional ischemia followed by reperfusion. Our current understanding of the events associated with myocardial ischemia suggests that within the ischemic myocardial region or area at risk, there is a population of cells that are reversibly injured and that reperfusion within a specified period (less than 3 hours) of time is capable of restoring the majority of the jeopardized cells to a normal status, but that the act of reperfusion itself will lead to the sudden demise of a fraction of the cells because of the cytotoxic effects of reactive species of oxygen derived from one or more of the sources indicated above. The efforts to minimize the amount of tissue that undergoes cell death as a result of myocardial ischemia demand that early reperfusion be established. However, the reintroduction of molecular oxygen and the circulating elements of the blood will be associated with an "explosive" and self-limited destruction of some of the myocardial cells in the area at risk.(ABSTRACT TRUNCATED AT 400 WORDS)     

山莨菪碱对全脑缺血-再灌流后脑线粒体损伤的保护作用

目的：探讨山莨菪碱对急性全脑缺血－再灌流后脑线粒体损伤的保护作用。方法：采用家兔全脑缺血－再灌流损伤模型。缺血20min，再灌流2h,观察脑线粒体呼吸功能、呼吸缺氧化酶活性、线粒体内Ca^2 和丙二醛含量的变化。结果：脑缺血－再灌流后，脑线粒体呼吸控制率、磷氧化、氧化磷酸化效率及烟酰胺腺嘌呤二核苷酸氧化酶、琥珀酸氧化酶、细胞色素C氧化酶活性明显降低（P＜0．01），而线粒体Ca^2 、丙二醛含量明显升高（P＜0．01）；再灌流早期给予山莨菪碱治疗后，上述线粒体损伤性改变明显减轻。结论：山莨菪碱对脑缺血－再灌流后线粒体损伤具有一定的保护作用，其机制可能与Ca^2 拮抗、抑制脂质过氧化及保护呼吸链酶活性有关。     

Decreased hepatosplanchnic antioxidant uptake during hepatic ischaemia/reperfusion in patients undergoing liver resection

Oxidative stress mediates cell injury during ischaemia/reperfusion. On the other hand, experimental findings suggest that ROS (reactive oxygen species) induce processes leading to ischaemic preconditioning. The extent and source of oxidative stress and its effect on antioxidant status in the human liver during intermittent ischaemia and reperfusion remains ill-defined. Therefore the aim of the present study was to investigate the occurrence of oxidative stress in humans undergoing liver resection. Liver biopsies, and arterial and hepatic venous blood samples were taken from ten patients undergoing hepatectomy with an intermittent Pringle manoeuvre. Plasma MDA (malondialdehyde) and hepatic GSSG levels were measured as markers of oxidative stress and plasma uric acid as a marker of xanthine oxidase activity. In addition, changes in hepatosplanchnic consumption of plasma antioxidants and hepatic levels of carotenoids and glutathione (GSH) were measured. After ischaemia, hepatosplanchnic release of MDA and increased hepatic GSSG levels were found. This was accompanied by the release of uric acid, reflecting xanthine oxidase activity. During reperfusion, ongoing oxidative stress was observed by further increases in hepatic GSSG content and hepatosplanchnic MDA release. Uric acid release was minimal during reperfusion. A gradual decrease in plasma antioxidant capacity and net hepatosplanchnic antioxidant uptake was observed upon prolonged cumulative ischaemia. Oxidative stress occurs during hepatic ischaemia in man mainly due to xanthine oxidase activity. Interestingly, the gradual decline in plasma antioxidant capacity and net hepatosplanchnic antioxidant uptake during prolonged cumulative ischaemia, preserved both hydrophilic and lipophilic hepatic antioxidant levels. Decreasing plasma levels and net hepatosplanchnic uptake of plasma antioxidants may warrant antioxidant supplementation, although it should be clarified to what extent limitation of oxidative stress compromises ROS-dependent pathways of ischaemic preconditioning.     

Nitrite augments tolerance to ischemia/reperfusion injury via the modulation of mitochondrial electron transfer

Nitrite (NO(2)(-)) is an intrinsic signaling molecule that is reduced to NO during ischemia and limits apoptosis and cytotoxicity at reperfusion in the mammalian heart, liver, and brain. Although the mechanism of nitrite-mediated cytoprotection is unknown, NO is a mediator of the ischemic preconditioning cell-survival program. Analogous to the temporally distinct acute and delayed ischemic preconditioning cytoprotective phenotypes, we report that both acute and delayed (24 h before ischemia) exposure to physiological concentrations of nitrite, given both systemically or orally, potently limits cardiac and hepatic reperfusion injury. This cytoprotection is associated with increases in mitochondrial oxidative phosphorylation. Remarkably, isolated mitochondria subjected to 30 min of anoxia followed by reoxygenation were directly protected by nitrite administered both in vitro during anoxia or in vivo 24 h before mitochondrial isolation. Mechanistically, nitrite dose-dependently modifies and inhibits complex I by posttranslational S-nitrosation; this dampens electron transfer and effectively reduces reperfusion reactive oxygen species generation and ameliorates oxidative inactivation of complexes II-IV and aconitase, thus preventing mitochondrial permeability transition pore opening and cytochrome c release. These data suggest that nitrite dynamically modulates mitochondrial resilience to reperfusion injury and may represent an effector of the cell-survival program of ischemic preconditioning and the Mediterranean diet.     

Renal purine efflux and xanthine oxidase activity during experimental nephrosis in rats: difference between puromycin aminonucleoside and adriamycin nephrosis

1. The hypothesis was tested that the renal xanthine oxidase system provides a source of oxygen free radicals in puromycin aminonucleoside and adriamycin experimental nephrosis by generating uric acid from hypoxanthine and xanthine. 2. The concentrations in renal tissue of the putative intermediary products of puromycin aminonucleoside metabolism, hypoxanthine and xanthine, and of their precursors, adenosine and inosine, were lower in rats treated with puromycin aminonucleoside than in normal controls, whereas concentrations of the metabolites were normal after adriamycin intoxication. Their daily urinary excretion was lower in the 24 h after puromycin aminonucleoside administration compared with the baseline values and returned to near normal levels within 5 days. After adriamycin the 24 h urinary excretion of xanthine and uric acid was double the baseline levels (P less than 0.001). 3. When equimolar amounts of hypoxanthine were injected instead of puromycin aminonucleoside, the concentration of all bases increased slightly in renal tissue and their urinary efflux was double the baseline level: allantoin, uric acid, the unmodified nucleotide and xanthine were the most represented compounds in urine. 4. The enzymatic activities relative to xanthine oxidase (EC 1.1.3.22) and xanthine dehydrogenase (EC 1.1.1.204) in renal tissues were unchanged 1 day after puromycin aminonucleoside or hypoxanthine intoxication and only moderately increased in both groups at 13 days (the time of appearance of heavy proteinuria in the puromycin aminonucleoside-treated group). In contrast, xanthine oxidase and xanthine dehydrogenase activities were higher in adriamycin-treated rats at 1 and 15 days after the treatment (P less than 0.001). 5. Feeding rats with normoprotein diets containing tungsten induced a marked and constant decrease of renal xanthine oxidase and xanthine dehydrogenase activities to 20% of the baseline values in both puromycin aminonucleoside- and adriamycin-treated rats. Inhibition of renal xanthine oxidase and xanthine dehydrogenase activities by tungsten was associated with a marked reduction (P less than 0.001) of proteinuria in adriamycin-treated rats and the same occurred with allopurinol, a specific inhibitor of xanthine oxidase activity. In contrast, tungsten treatment did not reduce the proteinuria associated with puromycin aminonucleoside, which reached a maximum 13 days after puromycin aminonucleoside intoxication. Hypoxanthine-treated rats were normoproteinuric after 2 months of observation. 6. These data demonstrate an activation of renal xanthine oxidase and xanthine dehydrogenase after adriamycin intoxication which is relevant to the induction of proteinuria. They also argue against the involvement of the renal xanthine oxidase system as a source of free radicals in puromycin aminonucleoside nephrosis and suggest that the nucleotide cycle is not a normal route for puromycin aminonucleoside degradation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Oxygen at physiological concentrations. A potential, paradoxical mediator of reperfusion injury to mitochondria induced by phosphate

Cellular injury induced by reperfusion after myocardial ischemia is manifested by striking mitochondrial damage as well as other hallmarks such as contraction band necrosis. Calcium has been implicated as a mediator of irreversible cellular injury in several systems. To identify other potential mediators of the mitochondrial injury associated with reperfusion, interactions between inorganic phosphate, oxygen, and mitochondria harvested from rabbit hearts were evaluated in vitro. Mitochondria exhibited rapid inactivation of oxidative phosphorylation after preincubation at 25 degrees C when phosphate and oxygen were present. Inactivation was partially but not completely precluded by EDTA, EGTA, magnesium, diltiazem, or ruthenium red, results in concert with findings of others suggesting involvement of a deleterious influx of calcium into mitochondria; exogenous calcium enhanced inactivation. However, the present data indicate that inactivation is prevented by incubation of mitochondria in the absence of oxygen, and demonstrate for the first time that injury elicited by phosphate is dependent on oxygen at physiological concentrations either because calcium and/or phosphate influx is linked to aerobic metabolism or because oxygen exerts deleterious effects on mitochondria, which may render them particularly susceptible to calcium influx. Since intracellular inorganic phosphate concentration increases markedly with ischemia, reperfusion with oxygenated medium may paradoxically augment mitochondrial injury in this setting. Thus, in the presence of increased intracellular concentrations of calcium and phosphate induced by ischemia, subsequent reestablishment of physiological levels of intracellular oxygen tension may promote mitochondrial damage, which is known to increase with reperfusion.     

The beneficial effect of 2′-deoxycoformycin in renal ischemia-reperfusion is mediated both by preservation of tissue ATP and inhibition of lipid peroxidation

Renal ischemia injures the renal tubular cell by disrupting the vital cellular metabolic machinery. Further cell damage is caused when the blood flow is restored by oxygen free radicals that are generated from xanthine oxidase. Oxygen radicals cause lipid peroxidation of cell and organelle membranes, disrupting the structural integrity and capacity for cell transport and energy metabolism. In the present study, the possible therapeutic usefulness of the adenosine deaminase inhibitor, 2'-deoxycoformycin (DCF), during renal ischemia and reperfusion injury was investigated. The effects of DCF on renal malondialdehyde (MDA) and ATP levels were studied after 45 min ischemia and 15 min subsequent reperfusion in rat kidneys. MDA levels remained unchanged during ischemia, but increased after the subsequent reperfusion. DCF pretreatment (2.0 mg/kg i.m.) decreased MDA and increased ATP levels during the ischemia-reperfusion period. DCF exerts a dual protective action by facilitating purine salvage for ATP synthesis and inhibiting oxygen radical-induced lipid peroxidation. These results suggest that DCF therapy could be beneficial in the treatment of ischemia-reperfusion renal injuries.     

Diglycolic acid,the toxic metabolite of diethylene glycol,chelates calcium and produces renal mitochondrial dysfunction in vitro

Context: Diethylene glycol (DEG) has caused many cases of acute kidney injury and deaths worldwide. Diglycolic acid (DGA) is the metabolite responsible for the renal toxicity, but its toxic mechanism remains unclear. Objective: To characterize the mitochondrial dysfunction produced from DGA by examining several mitochondrial processes potentially contributing to renal cell toxicity. Materials and methods: The effect of DGA on mitochondrial membrane potential was examined in normal human proximal tubule (HPT) cells. Isolated rat kidney mitochondria were used to assess the effects of DGA on mitochondrial function, including respiratory parameters (States 3 and 4), electron transport chain complex activities and calcium-induced opening of the mitochondrial permeability transition pore. DGA was compared with ethylene glycol tetraacetic acid (EGTA) to determine calcium chelating ability. DGA cytotoxicity was assessed using lactate dehydrogenase leakage from cultured proximal tubule cells. Results: DGA decreased the mitochondrial membrane potential in HPT cells. In rat kidney mitochondria, DGA decreased State 3 respiration, but did not affect State 4 respiration or the ADP/O ratio. DGA reduced glutamate/malate respiration at lower DGA concentrations (0.5?mmol/L) than succinate respiration (100?mmol/L). DGA inhibited Complex II activity without altering Complex I, III or IV activities. DGA blocked calcium-induced mitochondrial swelling, indicating inhibition of the calcium-dependent mitochondrial permeability transition. DGA and EGTA reduced the free calcium concentration in solution in an equimolar manner. DGA toxicity and mitochondrial dysfunction occurred as similar concentrations. Discussion: DGA inhibited mitochondrial respiration, but without uncoupling oxidative phosphorylation. The more potent effect of DGA on glutamate/malate respiration and the inhibition of mitochondrial swelling was likely due to its chelation of calcium. Conclusion: These results indicate that DGA produces mitochondrial dysfunction by chelating calcium to decrease the availability of substrates and of reducing equivalents to access Complex I and by inhibiting Complex II activity at higher concentrations.