Endothelial Cells as A Source of Oxygen-Free Radicals An Esr Study

Endothelial cells were subjected to anoxia/reoxygenation in order to simulate some of the free radical mechanisms occurring in ischaemialreperfusion. With ESR and spin trapping using the spin traps 5.5-dimethyl-l-pyrroline-l-oxide (DMPO) and 3,3,5,5-dimethyl-l-pyrroline-l-oxide (M₄PO), the results show that upon reoxygenation of endothelial cells, following a period of anoxia, these cells generate superoxide (0₂). Cytotoxicity of the spin traps was measured by standard trypan blue exclusion methods. Cell injury or death was measured at various times during reoxygenation by lactate dehydrogenase (LDH) release. Experiments using oxypurinol, SOD, CAT and a combination of SOD and CAT show that while oxypurinol partially prevents spin adduct formation. the combination of SOD and CAT is more effective in doing so. These results suggest that the majority of the oxygen radicals produced by endothelial cells are done so exogenously. The results also suggest that endothelial cells are not only a source of oxygen radicals but also a target.     

Endothelial Cells as A Source of Oxygen-Free Radicals An Esr Study

Endothelial cells were subjected to anoxia/reoxygenation in order to simulate some of the free radical mechanisms occurring in ischaemialreperfusion. With ESR and spin trapping using the spin traps 5.5-dimethyl-l-pyrroline-l-oxide (DMPO) and 3,3,5,5-dimethyl-l-pyrroline-l-oxide (M₄PO), the results show that upon reoxygenation of endothelial cells, following a period of anoxia, these cells generate superoxide (0₂). Cytotoxicity of the spin traps was measured by standard trypan blue exclusion methods. Cell injury or death was measured at various times during reoxygenation by lactate dehydrogenase (LDH) release. Experiments using oxypurinol, SOD, CAT and a combination of SOD and CAT show that while oxypurinol partially prevents spin adduct formation. the combination of SOD and CAT is more effective in doing so. These results suggest that the majority of the oxygen radicals produced by endothelial cells are done so exogenously. The results also suggest that endothelial cells are not only a source of oxygen radicals but also a target.     

Oxygen radicals generated during anoxia followed by reoxygenation reduce the synthesis of tissue-type plasminogen activator and plasminogen activator inhibitor-1 in human endothelial cell culture

The effect of anoxia and reoxygenation on the synthesis and secretion of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) was studied in primary cultures of human umbilical vein endothelial cells. Sublethal anoxia, determined by trypan blue dye exclusion and lactate dehydrogenase release, was produced by cell culture under a 95% N2, 5% CO2 atmosphere for 2-24 h and was followed by reoxygenation with 95% air, 5% CO2 for 24 or 48 h. Anoxia did not alter the levels of mRNA for t-PA or PAI-1 in the cells or the secretion of t-PA or PAI-1 into the medium. At 24 h, t-PA secreted into conditioned medium was 7.0 +/- 1.4 ng/2 x 10(6) cells (n = 9) and PAI-1 was 300 +/- 13 IU/2 x 10(6) cells (n = 9), whereas the content of t-PA mRNA was 2.2 pg/micrograms of RNA and PAI-1 mRNA was 180 pg/micrograms of RNA. During reoxygenation, however, t-PA antigen and PAI-1 activity as well as mRNA for PAI-1 decreased proportionally to the duration of anoxia, to reach 27 +/- 1.0, 49 +/- 2.0, and 47 +/- 14% of control values, respectively, within 24 h of anoxia. t-PA mRNA also decreased significantly during reoxygenation following anoxia, but the extent could not be accurately quantitated. Addition, during anoxia, of a 200 micrograms/ml concentration of the superoxide anion radical scavenger superoxide dismutase or of a 5 mM concentration of the iron chelator deferoxamine mesylate prevented the subsequent decrease of t-PA antigen during reoxygenation; addition of these compounds during reoxygenation had no effect. Superoxide dismutase, but not deferoxamine mesylate, when added during anoxia prevented the subsequent decrease in PAI-1 activity. These studies suggest that the marked alteration of endothelial cell fibrinolysis during anoxia followed by reoxygenation is most likely mediated by a mechanism dependent on oxygen radicals. Impaired endothelial cell fibrinolysis may contribute to the pathophysiology of ischemia/reperfusion injury.     

Zinc pyrithione salvages reperfusion injury by inhibiting NADPH oxidase activation in cardiomyocytes

Zinc pyrithione (ZPT), has a strong anti-apoptotic effect when administered just before reperfusion. Because oxidative stress has been proposed to contribute to myocardial reperfusion injury, we tested whether ZPT can reduce the production of reactive oxygen species during reoxygenation in cultured neonatal rat cardiac myocytes and evaluated the role of NADPH oxidase in hypoxia/reoxygenation (H/R) injury. The cells were subjected to 8 h of simulated ischemia, followed by either 30 min or 16 h of reoxygenation. ZPT when started just before reoxygenation significantly reduced superoxide generation, LDH release and improved cell survival compared to H/R. Attenuation of the ROS production by ZPT paralleled its capacity to prevent pyknotic nuclei formation. In addition, ZPT reversed the H/R-induced expression of NOX2 and p47^phox phosphorylation indicating that ZPT directly protects cardiomyocytes from reperfusion injury by a mechanism that attenuates NADPH oxidase mediated intracellular oxidative stress.     

Electron paramagnetic resonance evidence that cellular oxygen toxicity is caused by the generation of superoxide and hydroxyl free radicals

Cells require molecular oxygen for the generation of energy through mitochondrial oxidative phosphorylation; however, high concentrations of oxygen are toxic and can cause cell death. A number of different mechanisms have been proposed to cause cellular oxygen toxicity. One hypothesis is that reactive oxygen free radicals may be generated; however free radical generation in hyperoxic cells has never been directly measured and the mechanism of this radical generation is unknown. In order to determine if cellular oxygen toxicity is free radical mediated, we applied electron paramagnetic resonance, EPR, spectroscopy using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide, DMPO, to measure free radical generation in hyperoxic pulmonary endothelial cells. Cells in air did not give rise to any detectable signal. However, cells exposed to 100% O2 for 30 min exhibited a prominent signal of trapped hydroxyl radical, DMPO-OH, while cell free buffer did not give rise to any detectable radical generation. This cellular radical generation was demonstrated to be derived from the superoxide radical since the observed signal was totally quenched by superoxide dismutase, but not by equal concentrations of the denatured enzyme. It was confirmed that the hydroxyl radical was generated since in the presence of ethanol the CH3 CH(OH) radical was formed. Loss of cell viability as measured by uptake of trypan blue dye was observed paralleling the measured free radical generation. Thus, superoxide and hydroxyl radicals are generated in hyperoxic pulmonary endothelial cells and this appears to be an important mechanism of cellular oxygen toxicity.     

Alterations of hepatocyte function with free radical generators and reparation or prevention with coffee polyphenols

Liver diseases are linked in the majority of cases to oxidative stress that antioxidants could neutralize with reducing liver injury. Chlorogenic acid, a coffee polyphenol, possesses antioxidant prosperities. The aim of this study was to evaluate in vitro preventive and corrective effects of cholorogenic acid in hepatocyte toxicity induced by free radicals. Hepatocytes were isolated from adult male Wistar rats. To determine corrective effects and reparation, cells were first exposed to two free radical generators (hydrogen peroxide/iron sulfate for hydroxyl radical formation, and phenazine methosulfate/nicotinamide adenine dinucleotide for superoxide anion formation) for 12H and thereafter treated by chlorogenic acid (1 and 10?μM final concentration) for another 12H. To show preventive effects, cells were pretreated by chlorogenic acid and thereafter exposed to free radical generators. Hepatocyte proliferation, glucose uptake, ATP contents, membrane fluidity and integrity, and intracellular redox status were investigated after 24H culture. The results showed that chlorogenic acid reversed the decrease in cell proliferation, glucose uptake and ATP levels, the increased LDH release and the reduced membrane fluidity and restored the oxidant/antioxidant status under oxidative stress. When pre-treated with chlorogenic acid, hepatocytes became very resistant to oxidative conditions and cellular homeostasis was maintained. In conclusion, chlorogenic acid displayed not only corrective but also preventive effects in hepatocytes exposed to oxidative stress and could be beneficial in patients with or at risk of liver diseases.     

Superoxide dismutase/catalase mimetics but not MAP kinase inhibitors are neuroprotective against oxygen/glucose deprivation-induced neuronal death in hippocampus

Although oxygen/glucose deprivation (OGD) has been widely used as a model of ischemic brain damage, the mechanisms underlying acute neuronal death in this model are not yet well understood. We used OGD in acute hippocampal slices to investigate the roles of reactive oxygen species and of the mitogen-activated protein kinases (MAPKs) in neuronal death. In particular, we tested the neuroprotective effects of two synthetic superoxide dismutase/catalase mimetics, EUK-189 and EUK-207. Acute hippocampal slices prepared from 2-month-old or postnatal day 10 rats were exposed to oxygen and glucose deprivation for 2 h followed by 2.5 h reoxygenation. Lactate dehydrogenase (LDH) release in the medium and propidium iodide (PI) uptake were used to evaluate cell viability. EUK-189 or EUK-207 applied during the OGD and reoxygenation periods decreased LDH release and PI uptake in slices from 2-month-old rats. EUK-189 or EUK-207 also partly blocked OGD-induced ATP depletion and extracellular signal-regulated kinases 1 and 2 (ERK1/2) dephosphorylation, and completely eliminated reactive oxygen species generation. The MEK inhibitor U0126 applied together with EUK-189 or EUK-207 completely blocked ERK1/2 activation, but had no effect on their protective effects against OGD-induced LDH release. U0126 alone had no effect on OGD-induced LDH release. EUK-207 had no effect on OGD-induced p38 or c-Jun N-terminal kinase dephosphorylation, and when the p38 inhibitor SB203580 was applied together with EUK-207, it had no effect on the protective effects of EUK-207. SB203580 alone had no effect on OGD-induced LDH release either. In slices from p10 rats, OGD also induced high-LDH release that was partly reversed by EUK-207; however, neither OGD nor EUK-207 produced significant changes in ERK1/2 and p38 phosphorylation. OGD-induced spectrin degradation was not modified by EUK-189 or EUK-207 in slices from p10 or 2-month-old rats, suggesting that their protective effects was not mediated through inhibition of calpain activation. Thus, both EUK-189 and EUK-207 provide neuroprotection in acute ischemic conditions, and this effect is related to elimination of free radical formation and partial reversal of ATP depletion, but not mediated by the activation or inhibition of the MEK/ERK or p38 pathways, or inhibition of calpain activation.     

Opioid receptor-independent protection of ischemic rat hepatocytes by morphine

We studied the role of morphine in anoxia/reoxygenation injury to hepatocytes. Overnight cultured rat hepatocytes were incubated in anoxic buffer at pH 6.2 for 4h and reoxygenated at pH 7.4 for 2h to simulate anoxia/reoxygenation. Some hepatocytes were preincubated with 50 microM morphine for 10 min prior to onset of anoxia/reoxygenation. To study the effect of morphine on nitric oxide (NO), hepatocytes were loaded with 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM). Changes in NO concentration were assessed with a multi-well fluorescence reader and confocal microscopy. Morphine substantially improved cell viability after reoxygenation and increased NO generation, which was blocked by ATP-sensitive potassium channel blockers. Confocal images revealed that the increase in NO occurred mainly at the cytosol. However, treatment with opioid receptor antagonists did not reverse cytoprotection by morphine. These results indicate that morphine prevents anoxia/reoxygenation injury to hepatocytes. Protective mechanisms are associated with the potassium channels and NO, but are independent of opioid receptor-mediated signaling.     

Antioxidants and gadolinium chloride attenuate hepatic parenchymal and endothelial cell injury induced by low flow ischemia and reperfusion in perfused rat livers

The objective of this study was to determine whether Kupffer cells contribute to parenchymal and endothelial cell damage induced by ischemia-reperfusion in perfused rat livers. Parenchymal and endothelial cell injury were determined by measuring activities of lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP), respectively, in the effluent perfusate of livers subjected to 60 min of low flow ischemia followed by 30 min of reperfusion. Upon reperfusion, LDH and PNP activities increased significantly within the first 10 min of reperfusion and remained elevated over control values throughout the duration of reperfusion. Pretreatment with gadolinium chloride, an inhibitor of Kupffer cell function, significantly decreased LDH and PNP efflux during reperfusion by approximately 60% and 50%, respectively. When Kupffer cells were stimulated by vitamin A pretreatment, PNP efflux was doubled during reperfusion. Vitamin E pretreatment attenuated LDH and PNP release by approximately 70% during reperfusion compared to enzyme release in untreated livers. Moreover, the water-soluble antioxidants superoxide dismutase and desferrioxamine reduced reperfusion injury, whereas catalase had no effect on enzyme release. These results demonstrate that superoxide anions released from Kupffer cells are involved in oxidative damage to endothelial cells as well as hepatocytes during the early stages of hepatic reperfusion.     

Antioxidants and gadolinium chloride attenuate hepatic parenchymal and endothelial cell injury induced by low flow ischemia and reperfusion in perfused rat livers

The objective of this study was to determine whether Kupffer cells contribute to parenchymal and endothelial cell damage induced by ischemia-reperfusion in perfused rat livers. Parenchymal and endothelial cell injury were determined by measuring activities of lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP), respectively, in the effluent perfusate of livers subjected to 60 min of low flow ischemia followed by 30 min of reperfusion. Upon reperfusion, LDH and PNP activities increased significantly within the first 10 min of reperfusion and remained elevated over control values throughout the duration of reperfusion. Pretreatment with gadolinium chloride, an inhibitor of Kupffer cell function, significantly decreased LDH and PNP efflux during reperfusion by approximately 60% and 50%, respectively. When Kupffer cells were stimulated by vitamin A pretreatment, PNP efflux was doubled during reperfusion. Vitamin E pretreatment attenuated LDH and PNP release by approximately 70% during reperfusion compared to enzyme release in untreated livers. Moreover, the water-soluble antioxidants superoxide dismutase and desferrioxamine reduced reperfusion injury, whereas catalase had no effect on enzyme release. These results demonstrate that superoxide anions released from Kupffer cells are involved in oxidative damage to endothelial cells as well as hepatocytes during the early stages of hepatic reperfusion.     

Superoxide Disrnutase Does Protect the Cdtured Rat Cardiac Myocytes Against Hypoxia/Reoxygenation Injury

The effect of superoxide dismutase (SOD) on membrane integrity and fluidity of the cultured neonatal rat cardiac myocytes in vitro was investigated under the condition of hypoxia and hypoxia/reoxygenation. Lactate dehydrogenase (LDH) concentration was used as the biochemical indicator for the loss of cell membrane integrity. Fluorescence polarization (FP), average microviscosity (N) and anisotropy (Ast), which are inversely proportional to the fluidity of cell membrane, were assayed. Cells were respectively exposed to hypoxia or hypoxia/reoxygenation for different periods of time in the absence or presence of SOD at various concentrations. Hypoxia alone or hypoxia/ reoxygenation brought injury to the cultured myocytes. This was demonstrated by changes in LDH and membrane fluidity. In the former LDH concentration gradually increased in a time-dependent manner and the values of FP, N and Asf were significantly increased. The changes in membrane integrity and fluidity induced by hypoxia or hypoxia/reoxygenation could be prevented by adding SOD to the culture medium. The results provide a direct evidence that SOD (740 u.ml^-1, the effective dose) was effective in protecting cultured myocytes against the injury as well as an indirect evidence of free radical generation. Based on the results obtained from this study and the establishment of concept of optimally effective dose by Bernier and Omar et al, it was suggested that some previous reports, in which no evidence was found both in protective effect of SOD and in free radical generation by using only one dose in hypoxia/reoxygenation model, should be interpreted with caution.     

Low density lipoprotein cytotoxicity induced by free radical peroxidation of lipid

Low density lipoprotein (LDL) has been reported to be injurious or toxic to cells in vitro. This injurious effect is, in some instances, due to oxidation of the lipid moiety of the lipoprotein. The objectives of this study were to determine if the oxidation rendering the lipoprotein toxic to human skin fibroblasts occurred by free radical mechanisms, and if so, which of the common free radical oxygen species were involved. The selective free radical blockers or scavengers employed included superoxide dismutase for superoxide, catalase for hydrogen peroxide, dimethylfuran for singlet molecular oxygen, and mannitol for hydroxyl radical. The presence during lipoprotein preparation of general free radical scavengers (vitamin E, butylated hydroxytoluene) or the divalent cation chelator ethylenediamine tetraacetic acid prevented the formation of cytotoxic low density lipoprotein, while the simultaneous presence of superoxide dismutase and catalase partially inhibited its formation. The results indicate that superoxide and/or hydrogen peroxide are involved in the formation of the toxic LDL lipid. The toxic action of oxidized LDL could not be prevented by inclusion of antioxidants in the culture medium, indicating that an oxidized lipid was responsible for cell injury rather than free radicals generated in culture by the action of oxidized LDL. Three separate assays for cell injury (enumeration of attached cells, cell loss of lactate dehydrogenase into the culture medium, and trypan blue uptake) indicated a sequence of events in which the fibroblasts are injured, die, and then detach.     

Suppression of reactive oxygen species production enhances neuronal survival in vitro and in vivo in the anoxia-tolerant turtle Trachemys scripta

Hypoxia-ischemia with reperfusion is known to cause reactive oxygen species-related damage in mammalian systems, yet, the anoxia tolerant freshwater turtle is able to survive repeated bouts of anoxia/reoxygenation without apparent damage. Although the physiology of anoxia tolerance has been much studied, the adaptations that permit survival of reoxygenation stress have been largely ignored. In this study, we examine ROS production in the turtle striatum and in primary neuronal cultures, and examine the effects of adenosine (AD) on cell survival and ROS. Hydroxyl radical formation was measured by the conversion of salicylate to 2,3-dihydroxybenzoic acid (2,3-DHBA) using microdialysis; reoxygenation after 1 or 4 h anoxia did not result in increased ROS production compared with basal normoxic levels, nor did H₂O₂ increase after anoxia/reoxygenation in neuronally enriched cell cultures. Blockade of AD receptors increased both ROS production and cell death in vitro , while AD agonists decreased cell death and ROS. As turtle neurons proved surprisingly susceptible to externally imposed ROS stress (H₂O₂), we propose that the suppression of ROS formation, coupled to high antioxidant levels, is necessary for reoxygenation survival. As an evolutionarily selected adaptation, the ability to suppress ROS formation could prove an interesting path to investigate new therapeutic targets in mammals.     

Beta-alanine protection against hypoxic liver injury in the rat

Liver hypoxia still represents an important cause of liver injury during shock and liver transplantation. We have investigated the protective effects of beta-alanine against hypoxic injury using isolated perfused rat livers and isolated rat hepatocyte suspensions. Perfusion with hypoxic Krebs-Henseleit buffer increased liver weight and caused a progressive release of lactate dehydrogenase (LDH) in the effluent perfusate. The addition of 5 mmol/l beta-alanine to the perfusion buffer completely prevented both weight increase and LDH leakage. These findings were confirmed by histological examinations showing that beta-alanine blocked the staining by trypan blue of either liver parenchymal and sinusoidal cells. Studies performed in isolated hepatocytes revealed that beta-alanine exerted its protective effects by interfering with Na+ accumulation induced by hypoxia. The addition of gamma-amino-butyric acid, which interfered with beta-alanine uptake by the hepatocytes or of Na+/H+ ionophore monensin, reverted beta-alanine protection in either hepatocyte suspensions or isolated perfused livers. We also observed that liver receiving beta-alanine were also protected against LDH leakage and weight increase caused by the perfusion with an hyposmotic (205 mosm) hypoxic buffer obtained by decreasing NaCl content from 118 to 60 mmol/l. This latter effect was not reverted by blocking K+ efflux from hepatocyte with BaCl(2) (1mmol/l). Altogether these results indicated that beta-alanine protected against hypoxic liver injury by preventing Na+ overload and by increasing liver resistance to osmotic stress consequent to the impairment of ion homeostasis during hypoxia.     

Cu-Zn superoxide dismutase inhibits lactate dehydrogenase release and protects against cell death in murine fibroblasts pretreated with ultraviolet radiation

The effects of adding Cu-Zn superoxide dismutase (Cu-Zn SOD) to culture medium of the murine fibroblast cell line, L-929, pretreated with UV-B (312 nm, 480 mJ/cm(2)) have been investigated. Cell injury was monitored by the release of lactate dehydrogenase (LDH) into the medium, and cell death by the trypan blue exclusion test. UV-B radiation induced cell death by apoptosis, as demonstrated by DNA fragmentation. Over the range 0.1-0.3 microm Cu-Zn SOD, a significant dose-dependent protection against cell death was obtained of the UV-B exposed cells. Cell death correlated with the amount of LDH released into the medium, and Cu-Zn SOD treatment inhibited this. Heat-denatured Cu-Zn SOD did not affect either cell viability or the release of LDH from the cells. Endogenous Cu-Zn SOD activity, monitored by chemiluminescence, decreased by 20% in UV-B-irradiated cells; the addition of 0.3 microm exogenous Cu-Zn SOD to the medium did not affect intracellular Cu-Zn SOD activity. These results establish that Cu-Zn SOD added to extracellular medium can protect cells against injury caused by UV-B exposure.     

A simplified model of hypoxic injury in primary cultured rat hepatocytes

Summary The Anaeropack system for cell culture, which was originally designed for the growth of anaerobic bacteria, was used to produce a hypoxic atmosphere for cultured hepatocytes. We measured changes in the oxygen and carbon dioxide concentrations and the atmospheric temperature in an airtight jar. We also measured changes in the pH of the medium during hypoxia to assess the accuracy of this system. Moreover, we used three durations (2, 3, and 4 h) of hypoxia and 8 h of reoxygenation in cultured rat hepatocytes, and then measured the lactate dehydrogenase (LDH), ketone body concentration (acetoacetate + β-hydroxybutyrate), and the ketone body ratio (KBR: acetoacetate/β-hydroxybutyrate) in the medium in order to assess the suitability of this system as a model for reperfusion following liver ischemia. The oxygen concentration dropped to 1% or less within 1 h. The concentration of carbon dioxide rose to about 5% at 30 min after the induction of the hypoxic conditions, and was maintained at this level for 5 h. No effect of the reaction heat produced by the oxygen absorbent in the jar was recognized. The extent of cell injury produced by changing the hypoxic parameters was satisfactorily reflected by the KBR, the ketone body concentration, and the LDH activity released into the medium. Because this model can duplicate the conditions of the hepatocytes during revascularization following ischemic liver, and the Anaeropack system for cell culture is easy to manipulate, it seems suitable for the experimental study of hypoxic injury and revascularization in vitro.     

Cell surface changes and enzyme release during hypoxia and reoxygenation in the isolated, perfused rat liver

We examined the effects of hypoxia and reoxygenation in isolated, perfused rat livers. Hypoxia induced by a low rate of perfusion led to near anoxia confined to centrilobular regions of the liver lobule. Periportal regions remained normoxic. Within 15 min, anoxic centrilobular hepatocytes developed surface blebs that projected into sinusoids through endothelial fenestrations. Periportal hepatocytes were unaffected. Both scanning and transmission electron microscopy suggested that blebs developed by transformation of preexisting microvilli. Upon reoxygenation by restoration of a high rate of perfusion, blebs disappeared. Other changes included marked shrinkage of hepatocytes, enlargement of sinusoids, and dilation of sinusoidal fenestrations. There was also an abrupt increase in the release of lactate dehydrogenase and protein after reoxygenation, and cytoplasmic fragments corresponding in size and shape to blebs were recovered by filtration of the effluent perfusate. We also studied phalloidin and cytochalasin D, agents that disrupt the cytoskeleton. Both substances at micromolar concentrations caused rapid and profound alterations of cell surface topography. We conclude that hepatic tissue is quite vulnerable to hypoxic injury. The morphological expression of hypoxic injury seems mediated by changes in the cortical cytoskeleton. Reoxygenation causes disappearance of blebs and paradoxically causes disruption of cellular volume control and release of blebs as cytoplasmic fragments. Such cytoplasmic shedding provides a mechanism for selective release of hepatic enzymes by injured liver tissue.     

Cell proliferation and gill morphology in anoxic crucian carp

Is DNA replication/cell proliferation in vertebrates possible during anoxia? The oxygen dependence of ribonucleotide reductase (RNR) could lead to a stop in DNA synthesis, thereby making anoxic DNA replication impossible. We have studied this question in an anoxia-tolerant vertebrate, the crucian carp (Carassius carassius), by examining 5'-bromo-2'-deoxyuridine incorporation and proliferating cell nuclear antigen levels in the gills, intestinal crypts, and liver. We exposed crucian carp to 1 and 7 days of anoxia followed by 7 days of reoxygenation. There was a reduced incidence of S-phase cells (from 12.2 to 5.0%) in gills during anoxia, which coincided with a concomitant increase of G(0) cells. Anoxia also decreased the number of S-phase cells in intestine (from 8.1 to 1.8%). No change in the fraction of S-phase cells ( approximately 1%) in liver was found. Thus new S-phase cells after 7 days of anoxia were present in all tissues, revealing a considerable rate of DNA synthesis. Subsequently, the oxygen-dependent subunit of crucian carp RNR (RNRR2) was cloned. We found no differences in amino acids involved in radical generation and availability of the iron center compared with mouse, which could have explained reduced oxygen dependence. Furthermore, the amount of RNRR2 mRNA in gills did not decrease throughout anoxia exposure. These results indicate that crucian carp is able to sustain some cell proliferation in anoxia, possibly because RNRR2 retains its tyrosyl radical in anoxia, and that the replication machinery is still maintained. Although hypoxia triggers a 7.5-fold increase of respiratory surface area in crucian carp, this response was not triggered in anoxia.     

Hypoxia, reactive oxygen, and cell injury

Hypoxia usually decreases the formation of reactive oxygen species by oxidases and by autoxidation of components of cellular electron transfer pathways and of quinoid compounds such as menadione. In the case of menadione reactive oxygen species are liberated to a significant extent only at non-physiologically high oxygen partial pressures (PO2). At physiological and hypoxic PO2 values electron shuttling of menadione in the mitochondrial respiratory chain predominates. In contrast, lipid peroxidation induced by halogenated alkanes, such as carbon tetrachloride, in liver leads to an increase in the formation of reactive oxygen and thus in cell injury under hypoxic conditions. Reactive oxygen species may also be generated during reoxygenation of a previously hypoxic tissue. Based on experiments with isolated hepatocytes a three-zone-model of liver injury due to hypoxia and reoxygenation is presented; 1) a zone where the cells die by hypoxia; 2) a zone where the cells are destroyed upon reoxygenation, presumably mediated by an increase in the cellular ATP content; and 3) a zone where cell injury occurs upon reoxygenation, mediated by reactive oxygen species possibly liberated by xanthine oxidase.