Effect of ozone treatment on reactive oxygen species and adenosine production during hepatic ischemia-reperfusion

This study investigates whether ozone could confer protection from hepatic ischemia reperfusion by modifying the accumulation of adenosine and xanthine during ischemia. A significant increase in both adenosine and xanthine accumulation was observed as a consequence of ATP degradation during hepatic ischemia. Adenosine exerts a protective effect on hepatic ischemia reperfusion injury since the elimination of endogenous adenosine accumulation with adenosine deaminase increased the hepatic injury associated with this process. On the other hand, the high xanthine levels observed after ischemia could exert deleterious effects during reperfusion due to reactive oxygen species generation from xanthine oxidase. The administration of allopurinol, an inhibitor of xanthine oxidase, attenuated the increase in reactive oxygen species and transaminase levels observed after hepatic reperfusion. Ozone treatment in liver maintained adenosine levels similar to those found after ischemia but led to a marked reduction in xanthine accumulation. In order to evaluate the role of both adenosine and xanthine, we tried to modify the protection confered by ozone, by modifying the concentrations of adenosine and xanthine. The metabolization of endogenous adenosine after ischemia abolished the protective effect conferred by ozone. When xanthine was administered previous to ozone treatment, the protection conferred by adenosine disappeared, showing both postischemic reactive oxygen species and transaminase levels similar to those found after hepatic ischemia reperfusion. Ozone would confer protection against the hepatic ischemia reperfusion injury by the accumulation of adenosine that in turns benefits the liver and by blocking the xanthine/xanthine oxidase pathway for reactive oxygen species generation.     

Effect of ozone treatment on reactive oxygen species and adenosine production during hepatic ischemia-reperfusion

This study investigates whether ozone could confer protection from hepatic ischemia reperfusion by modifying the accumulation of adenosine and xanthine during ischemia. A significant increase in both adenosine and xanthine accumulation was observed as a consequence of ATP degradation during hepatic ischemia. Adenosine exerts a protective effect on hepatic ischemia reperfusion injury since the elimination of endogenous adenosine accumulation with adenosine deaminase increased the hepatic injury associated with this process. On the other hand, the high xanthine levels observed after ischemia could exert deleterious effects during reperfusion due to reactive oxygen species generation from xanthine oxidase. The administration of allopurinol, an inhibitor of xanthine oxidase, attenuated the increase in reactive oxygen species and transaminase levels observed after hepatic reperfusion. Ozone treatment in liver maintained adenosine levels similar to those found after ischemia but led to a marked reduction in xanthine accumulation. In order to evaluate the role of both adenosine and xanthine, we tried to modify the protection confered by ozone, by modifying the concentrations of adenosine and xanthine. The metabolization of endogenous adenosine after ischemia abolished the protective effect conferred by ozone. When xanthine was administered previous to ozone treatment, the protection conferred by adenosine disappeared, showing both postischemic reactive oxygen species and transaminase levels similar to those found after hepatic ischemia reperfusion. Ozone would confer protection against the hepatic ischemia reperfusion injury by the accumulation of adenosine that in turns benefits the liver and by blocking the xanthine/xanthine oxidase pathway for reactive oxygen species generation.     

Purine and oxypurine production in mitochondria of ischemic and reperfused myocardium

The present study was undertaken to determine whether significant breakdown of adenine nucleotides to purine bases and oxypurines occurred in mitochondria following myocardial ischemia and ischemia followed by reperfusion, and whether allopurinol prevented this effect. The adenine nucleotides adenosine, hypoxanthine, xanthine and uric acid were measured in the mitochondria and the results suggest that breakdown did occur. Malondialdehyde concentration was determined to gauge lipid peroxidation. This substance did not increase during ischemia or reperfusion, but did so in the presence of allopurinol. Xanthine dehydrogenase was converted to xanthine oxidase during reperfusion and the activity of both enzymes were inhibited by allopurinol. The results also suggested the presence of a mitochondrial 5'-nucleotidase. We conclude that significant breakdown of adenine nucleotide took place in myocardial mitochondria during ischemia and ischemia followed by reperfusion and that allopurinol may have a protective effect.     

Free radicals and myocardial ischemia: overview and outlook

Much evidence suggests that free radicals and active oxygen species derived from molecular oxygen (superoxide, hydrogen peroxide, and hydroxyl radical) contribute to the tissue injury which accompanies myocardial ischemia and reperfusion. Three possible sources have been identified for the production of active oxygen species: the enzyme xanthine oxidase; the activated polymorphonuclear leukocyte; the disrupted mitochondrial electron transport system. These sources may be mutually interactive. Once triggered, they may lead to the loss of antioxidant enzymes and to the release of iron, both of which are exacerbatory events.     

Effects of oxygen radicals on substrate oxidation by cardiac myocytes

Freshly isolated adult rat heart cells were used to study the effects of oxygen-free radicals on the myocardial oxidation of different substrates. The calcium-tolerant quiescent cells were incubated with xanthine plus xanthine oxidase as the source of free radicals. The oxidation of exogenous glucose, lactate and octanoate was severely inhibited (approx. 70%) by products of xanthine oxidase activity. Superoxide dismutase plus catalase effectively prevented the inhibition of oxidation. Cellular high energy phosphate levels were decreased in the presence of the oxygen free radical generating system although cell viability determined by Trypan blue exclusion and light microscopic assessment of normal morphology was not affected. These data suggest that oxygen free radicals decrease myocardial substrate oxidation which may contribute to the functional and ultrastructural changes in the myocardium under conditions such as reoxygenation after hypoxia and reperfusion after ischemia.     

Evaluation of the role of xanthine oxidase in myocardial reperfusion injury

The free radical-generating enzyme xanthine oxidase has been hypothesized to be a central mechanism of the injury which occurs in postischemic tissues; however, its importance remains controversial. Much attention has focused on the role of this enzyme in myocardial reperfusion injury. While xanthine oxidase has been observed in ischemic tissue homogenates, the presence and importance of radical generation by the enzyme in intact tissues are unknown. Therefore, we performed electron paramagnetic resonance, nuclear magnetic resonance and hemodynamic studies to measure the presence and significance of xanthine oxidase-mediated free radical generation in the isolated rat heart. When isolated perfused rat hearts were reperfused after 30 min of global ischemia, myocardial function and coronary flow were significantly improved in the presence of the definitive xanthine oxidase blocker oxypurinol. Free radical concentrations measured by spin-trapping with 5,5'-dimethyl-1-pyrroline-N-oxide were significantly decreased by oxypurinol and the energetic state of the heart was improved as reflected by an increased recovery of phosphocreatine and a higher phosphocreatine/Pi ratio. ATP recovery, however, was not altered, indicating that the improved functional and metabolic state of the heart was not due to ATP salvage. Spectrophotometric assays for the enzyme showed an increase in the amount of xanthine oxidase relative to dehydrogenase following ischemia, and a total available xanthine oxidase pool in the rat heart of approximately 150 milliunits/g of protein. Thus, xanthine oxidase is a significant source of the oxidative injury which occurs upon reperfusion of the ischemic rat heart.     

Free radical scavengers in myocardial ischemia

Reperfusion of ischemic myocardium is recognized as potentially beneficial because mortality is directly related to infarct size, and the latter is related to the severity and duration of ischemia. However, reperfusion is associated with extension of the injury that is additive to that produced by ischemia alone. The phenomenon of reperfusion injury is caused in large part by oxygen-derived free radicals from both extracellular and intracellular sources. The loci of oxygen-free radical formation include: myocardial sources (mitochondria), vascular endothelial sources (xanthine oxidase and other oxidases), or the inflammatory cellular infiltrate (neutrophils). Experimental studies have shown that free radical scavengers and agents that prevent free radical production can reduce myocardial infarct size in dogs subjected to temporary regional ischemia followed by reperfusion. Superoxide dismutase and catalase, which catalyze the breakdown of superoxide anion and hydrogen peroxide, respectively, limit experimental myocardial infarct size. The free radical scavenging agent N-(2-mercaptopropionyl)glycine (MPG) is reported to be effective in limiting infarct size. The ischemic-reperfused myocardium derives significant protection when experimental animals are pretreated with the xanthine oxidase inhibitor allopurinol. Neutrophils also serve as a significant source of oxygen-derived free radicals at the site of tissue injury. A number of agents have been shown to directly inhibit neutrophil-derived oxygen free radical formation and neutrophil accumulation within the reperfused myocardium. These agents include ibuprofen, nafazatrom, BW755C, prostacyclin, and iloprost. Thus, free radical scavengers and agents that prevent free radical formation can provide significant protection to the ischemic-reperfused myocardium.     

Effects of allopurinol pretreatment on myocardial ultrastructure and arrhythmias following coronary artery occlusion and reperfusion

The effect of the xanthine oxidase inhibitor, allopurinol, on myocardial ultrastructure after left circumflex coronary artery occlusion (40 min) with or without reperfusion (60 min) was examined in rabbits. Pretreatment of rabbits for 7 days with allopurinol (0.1% in the drinking water) resulted in a lower incidence of ventricular fibrillation in both ischemic and reperfusion phases. However, the number of Q waves, ST-segment elevation and premature ventricular contractions were similar in both groups of animals. Examination of hearts from allopurinol-treated animals revealed a distinct decrease in ultrastructural alterations following ischemia and reperfusion. Among the subcellular organelles studied, allopurinol had a preferential protective effect on the mitochondria both during the ischemic and reperfusion phases. In the allopurinol-treated animals, most mitochondria were intact and the cristae network preserved. Our study suggests that the preservation of mitochondrial structural and functional integrity by allopurinol may be an important determinant of its protective actions in myocardial ischemic/reperfusion injury.     

Modulation of electron transport protects cardiac mitochondria and decreases myocardial injury during ischemia and reperfusion

Mitochondria are increasingly recognized as lynchpins in the evolution of cardiac injury during ischemia and reperfusion. This review addresses the emerging concept that modulation of mitochondrial respiration during and immediately following an episode of ischemia can attenuate the extent of myocardial injury. The blockade of electron transport and the partial uncoupling of respiration are two mechanisms whereby manipulation of mitochondrial metabolism during ischemia decreases cardiac injury. Although protection by inhibition of electron transport or uncoupling of respiration initially appears to be counterintuitive, the continuation of mitochondrial oxidative phosphorylation in the pathological milieu of ischemia generates reactive oxygen species, mitochondrial calcium overload, and the release of cytochrome c. The initial target of these deleterious mitochondrial-driven processes is the mitochondria themselves. Consequences to the cardiomyocyte, in turn, include oxidative damage, the onset of mitochondrial permeability transition, and activation of apoptotic cascades, all favoring cardiomyocyte death. Ischemia-induced mitochondrial damage carried forward into reperfusion further amplifies these mechanisms of mitochondrial-driven myocyte injury. Interruption of mitochondrial respiration during early reperfusion by pharmacologic blockade of electron transport or even recurrent hypoxia or brief ischemia paradoxically decreases cardiac injury. It increasingly appears that the cardioprotective paradigms of ischemic preconditioning and postconditioning utilize modulation of mitochondrial oxidative metabolism as a key effector mechanism. The initially counterintuitive approach to inhibit mitochondrial respiration provides a new cardioprotective paradigm to decrease cellular injury during both ischemia and reperfusion. cardiolipin; cytochrome c; complex I; cytochrome oxidase     

Role of TNF-alpha-induced reactive oxygen species in endothelial dysfunction during reperfusion injury

We hypothesized that neutralization of TNF-alpha at the time of reperfusion exerts a salubrious role on endothelial function and reduces the production of reactive oxygen species. We employed a mouse model of myocardial ischemia-reperfusion (I/R, 30 min/90 min) and administered TNF-alpha neutralizing antibodies at the time of reperfusion. I/R elevated TNF-alpha expression (mRNA and protein), whereas administration of anti-TNF-alpha before reperfusion attenuated TNF-alpha expression. We detected TNF-alpha expression in vascular smooth muscle cells, mast cells, and macrophages, but not in the endothelial cells. I/R induced endothelial dysfunction and superoxide production. Administration of anti-TNF-alpha at the onset of reperfusion partially restored nitric oxide-mediated coronary arteriolar dilation and reduced superoxide production. I/R increased the activity of NAD(P)H oxidase and of xanthine oxidase and enhanced the formation of nitrotyrosine residues in untreated mice compared with shams. Administration of anti-TNF-alpha before reperfusion blocked the increase in activity of these enzymes. Inhibition of xanthine oxidase (allopurinol) or NAD(P)H oxidase (apocynin) improved endothelium-dependent dilation and reduced superoxide production in isolated coronary arterioles following I/R. Interestingly, I/R enhanced superoxide generation and reduced endothelial function in neutropenic animals and in mice treated with a neutrophil NAD(P)H oxidase inhibitor, indicating that the effects of TNF-alpha are not through neutrophil activation. We conclude that myocardial ischemia initiates TNF-alpha expression, which induces vascular oxidative stress, independent of neutrophil activation, and leads to coronary endothelial dysfunction.     

Pathophysiology of ischemic skin flaps: differences in xanthine oxidase levels among rats, pigs, and humans

Oxygen-derived free radicals have been implicated in a variety of diseases and pathologic processes, including ischemia reperfusion injury (IRI). Based on experimental work with rat skin-flap models, the enzyme xanthine oxidase (XO) has been proposed as a major source of free radicals responsible for tissue injury and flap necrosis. The presence of this enzyme is variable within different tissues of a specific species and between species. Xanthine oxidase levels in pig and human skin have not previously been reported. The activity of xanthine oxidase in the skin of rats (N = 16), pigs (N = 7), and humans (N = 8) was measured after varying intervals of ischemia and in the rat also following reperfusion. Control pig and human skin were found to contain minimal enzyme activity, almost 40 times less than that of the rat. In the rat, xanthine oxidase activity was stable throughout a prolonged period of ischemia, and a significant decrease in activity was found after 12 hours of reperfusion (p less than 0.05). In humans, xanthine oxidase activity was unaffected by ischemia time, and in the pig, it did not increase until 24 hours of ischemia (p less than 0.05). The potential sources of free radicals and the mechanism of action of xanthine oxidase and its inhibitor allopurinol in improving flap survival in different species are reviewed.     

Tourniquet shock in rats: effects of allopurinol on biochemical changes of the gastrocnemius muscle subjected to ischemia followed by reperfusion

Recent data suggest that oxygen free radicals are implicated in the pathogenesis of ischemic injury. This study evaluates the effects of allopurinol, a xanthine oxidase (XO) inhibitor, on malonaldehyde generation, free sulfhydryl levels, oxygen consumption, and water contents of rat gastrocnemius muscles of female Sprague-Dawley rats subjected to tourniquet shock and after hind-limb reperfusion. Serum lactic dehydrogenase isozyme patterns after ligature release were also examined. Our results show that the four muscle parameters were not altered during 5 hr of ischemia, but that on hind-limb reperfusion, malonaldehyde production, SH levels, O2 consumption, and water contents were significantly altered in the control animals, but not in those pretreated with allopurinol. LDH serum patterns of the untreated animals showed the presence of all five isoforms; these were much less evident in the drug-protected rats. Our data suggest that following ischemia, the affected muscles are unable to recover their normal function when reperfusion is resumed. The subsequent damage is probably due to the generation of cytotoxic superoxide radicals formed during the XO-catalyzed transformation of hypoxanthine to uric acid on tissue reoxygenation. The severity of tissue damage is related to the duration of the ischemic episode possibly due to hypoxanthine accumulation during ischemia.     

Endothelial dysfunction and reactive oxygen species production in ischemia/reperfusion and nitrate tolerance

Reactive oxygen species (ROS), as superoxide and its metabolites, have important roles in vascular homeostasis as they are involved in various signaling processes. In many cardiovascular disease states, however, the release of ROS is increased. Uncontrolled ROS production leads to impaired endothelial function and consequently to vascular dysfunction. This review focuses on two clinical conditions associated with elevated ROS levels: ischemia/reperfusion and nitrate tolerance. Injury caused by ischemia/reperfusion is an important limitation of transplantations, and complicates the management of stroke and myocardial infarction. Nitrates, which are used to treat transient myocardial ischemia (angina pectoris), decrease in efficacy in long-term continuous administration. There are several enzyme systems, such as xanthine oxidase, cyclooxygenase, uncoupled endothelial nitric oxide synthase, NAD(P)H oxidase, cytochrome P450 and the mitochondrial electron transport chain, which are responsible for the increased vascular production of superoxide. The contribution of particular ROS producing enzymes and the effect of antioxidant treatment are discussed in both pathological conditions.     

Modification of the xanthine-converting enzyme of perfused rat heart during ischemia and oxidative stress

The reversible and irreversible conversion of xanthine dehydrogenase to xanthine oxidase during ischemia/reperfusion and oxidative stress induced by hydrogen peroxide or diamide and its relationship with glutathione and protein SH groups were studied. The direct spectrophotometric measurement of the various forms of the xanthine-converting enzyme indicates that, in the fresh rat heart or after normoxic perfusion, there always is a basal level of 80% xanthine dehydrogenase and 20% of xanthine oxidase (15% irreversible and 5% reversible) that could contribute to the background production of free radicals. There is no significant increase of irreversible xanthine oxidase during ischemia nor during reperfusion. After global ischemia the reversible oxidase shows almost no increase while, when ischemia is followed by reperfusion, there is a limited increase (less then 9%) of the reversible xanthine oxidase. In the latter conditions there is a decrease of glutathione and of SH groups of about 70% and 25%, respectively. Perfusion for 1 h with oxidizing agents like hydrogen peroxide (60 microM) or diamide (100 microM) determines a marked conversion of xanthine dehydrogenase to reversible xanthine oxidase of about 40% and 60%, respectively; this oxidase activity partially reconverts to the dehydrogenase after withdrawing the oxidizing agents from the perfusion medium. The level of irreversible xanthine oxidase remains unchanged in all the conditions tested. Both hydrogen peroxide and diamide induce a strong decrease in SH groups and depletion of glutathione. The xanthine dehydrogenase----xanthine oxidase conversion thus appears to be sensitive to the redox state of thiol groups.     

Blockade of electron transport during ischemia protects cardiac mitochondria

Subsarcolemmal mitochondria sustain progressive damage during myocardial ischemia. Ischemia decreases the content of the mitochondrial phospholipid cardiolipin accompanied by a decrease in cytochrome c content and a diminished rate of oxidation through cytochrome oxidase. We propose that during ischemia mitochondria produce reactive oxygen species at sites in the electron transport chain proximal to cytochrome oxidase that contribute to the ischemic damage. Isolated, perfused rabbit hearts were treated with rotenone, an irreversible inhibitor of complex I in the proximal electron transport chain, immediately before ischemia. Rotenone pretreatment preserved the contents of cardiolipin and cytochrome c measured after 45 min of ischemia. The rate of oxidation through cytochrome oxidase also was improved in rotenone-treated hearts. Inhibition of the electron transport chain during ischemia lessens damage to mitochondria. Rotenone treatment of isolated subsarcolemmal mitochondria decreased the production of reactive oxygen species during the oxidation of complex I substrates. Thus, the limitation of electron flow during ischemia preserves cardiolipin content, cytochrome c content, and the rate of oxidation through cytochrome oxidase. The mitochondrial electron transport chain contributes to ischemic mitochondrial damage that in turn augments myocyte injury during subsequent reperfusion.     

Identification of Hypoxanthine Transport and Xanthine Oxidase Activity in Brain Capillaries

Microvessel segments were isolated from rat brain and used for studies of hypoxanthine transport and metabolism. Compared to an homogenate of cerebral cortex, the isolated microvessels were 3.7-fold enriched in xanthine oxidase. Incubation of the isolated microvessels with labeled hypoxanthine resulted in its rapid uptake followed by the slower accumulation of hypoxanthine metabolites including xanthine and uric acid. The intracellular accumulation of these metabolites was inhibited by the xanthine oxidase inhibitor allopurinol. Hypoxanthine transport into isolated capillaries was inhibited by adenine but not by representative pyrimidines or nucleosides. Similar results were obtained when blood to brain transport of hypoxanthine in vivo was measured using the intracarotid bolus injection technique. Thus, hypoxanthine is transported into brain capillaries by a transport system shared with adenine. Once inside the cell, hypoxanthine can be metabolized to xanthine and uric acid by xanthine oxidase. Since this reaction leads to the release of oxygen radicals, it is suggested that brain capillaries may be susceptible to free radical mediated damage. This would be most likely to occur in conditions where the brain hypoxanthine concentration is increased as following ischemia.     

Ischemic defects in the electron transport chain increase the production of reactive oxygen species from isolated rat heart mitochondria

Cardiac ischemia decreases complex III activity, cytochrome c content, and respiration through cytochrome oxidase in subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). The reversible blockade of electron transport with amobarbital during ischemia protects mitochondrial respiration and decreases myocardial injury during reperfusion. These findings support that mitochondrial damage occurs during ischemia and contributes to myocardial injury during reperfusion. The current study addressed whether ischemic damage to the electron transport chain (ETC) increased the net production of reactive oxygen species (ROS) from mitochondria. SSM and IFM were isolated from 6-mo-old Fisher 344 rat hearts following 25 min global ischemia or following 40 min of perfusion alone as controls. H(2)O(2) release from SSM and IFM was measured using the amplex red assay. With glutamate as a complex I substrate, the net production of H(2)O(2) was increased by 178 +/- 14% and 179 +/- 17% in SSM and IFM (n = 9), respectively, following ischemia compared with controls (n = 8). With succinate as substrate in the presence of rotenone, H(2)O(2) increased by 272 +/- 22% and 171 +/- 21% in SSM and IFM, respectively, after ischemia. Inhibitors of electron transport were used to assess maximal ROS production. Inhibition of complex I with rotenone increased H(2)O(2) production by 179 +/- 24% and 155 +/- 14% in SSM and IFM, respectively, following ischemia. Ischemia also increased the antimycin A-stimulated production of H(2)O(2) from complex III. Thus ischemic damage to the ETC increased both the capacity and the net production of H(2)O(2) from complex I and complex III and sets the stage for an increase in ROS production during reperfusion as a mechanism of cardiac injury.     

肾缺血再灌流损伤过程中肾小管上皮细胞色素氧化酶活性变化及TAD防治作用的研究

本实验选用大鼠一侧肾切除,对侧肾缺血６０ｍｉｎ动物模型,用组织化学方法观察再灌流１５ｍｉｎ,２４ｈ髓质外带肾小管上皮细胞色素氧化酶活性的变化及还原型谷胱甘肽（ＴＡＤ）对它们的影响。结果发现６０ｍｉｎ肾缺血后再灌流可致细胞色素氧化酶活性呈进行性降低。给ＴＡＤ能一定程度地保护细胞色素氧化酶活性。提示氧自由基可能损害细胞色素氧化酶活性,细胞色素氧化酶活性降低在肾缺血再灌流损伤中可能起重要作用。     

Oxygen free radical induced damage during intestinal ischemia/reperfusion in normal and xanthine oxidase deficient rats

This study looks at the role of xanthine oxidase (XO) in ischemia/reperfusion (I/R) induced intestinal mucosal damage using normal and xanthine oxidase deficient rats. Tungstate feeding for 3 days depleted the intestinal mucosal XO by 80%. A ligated loop of the rat small intestine (both normal and XO-deficient) was subjected to 1 h of total ischemia followed by 5 min revascularisation. The ensuing mucosal damage was assessed by biochemical and histological studies. Ischemia or I/R increased the XO levels in normal rats without any change in XO-deficient rats. Myeloperoxidase (a neutrophil marker) level was increased in both group of rats but it was comparatively higher in the XO-deficient rats. Accumulation of peroxidation products such as malondialdehyde, conjugated diene and increased production of hydroxyl radicals by microsomes were seen after ischemia and I/R and were similar in normal and XO-deficient rats. Studies on other parameters of peroxidation showed a decrease in polyunsaturated fatty acids and alpha-tocopherol, an increase in cysteine and cystine levels after I/R and were similar in both normal and XO-deficient rats. Histological results indicated gross morphological changes in the intestinal mucosa due to ischemia and I/R, and the damage was more severe in XO-deficient rats. These observations suggest that oxygen-derived free radicals are involved in the intestinal mucosal damage during I/R and infiltrated neutrophils rather than XO may be the primary source of free radicals under these conditions.     

Enterocyte viability and mitochondrial function after graded intestinal ischemia and reperfusion in rats

Ischemia/reperfusion of the small intestine can lead to metabolic and structural alterations in the mucosa. Cellular dysfunction occurs when mitochondrial metabolism is compromised, which may ultimately lead to impaired organ function. The aims of this study were to assess the suppression of cellular and mitochondrial oxidative metabolism and involvement of mitochondria in the ischemia/reperfusion injury. The mitochondria were prepared from isolated enterocytes obtained from the small intestine of anesthetized adult rats following different time periods of ischemia and ischemia followed by 5 min reperfusion. Cellular and mitochondrial function were assessed using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reduction assay. Ischemia of increasing time periods caused a progressive decrease in cellular and mitochondrial MTT reduction in enterocytes and reperfusion showed further decrease of MTT formazan formation. Inclusion of 1 mM succinate, as respiratory subs trate, showed reversal of suppression of mitochondrial function in 30-60 min ischemia whereas 90 min ischemia or short time period ischemia followed by 5 min reperfusion indicated an irreversible damage to mitochondria. This study indicated that mitochondria are a sensitive target of damage due to oxygen deficiency and possibly due to sudden burst of oxygen free radicals. Mitochondria can withstand short periods of ischemia whereas long duration ischemia or reperfusion results in irreversible damage to mitochondrial function. (Mol Cell Biochem 167: 81-87, 1997)