Mechanisms of hypoxic cell injury

The injury produced when oxygen availability is limited to aerobic tissues has been intensively studied for many years with the ultimate goal of improving tissue viability (Hochachka, 1986). Recently. the use of various new technologies (in particular fluorescent probes for various ions and intracellular processes) as well as isolated cell systems have begun to reveal in more detail some of the biochemical events which occur in response to oxygen deprivation. However, it remains unclear which (if any) of these changes are critical for cell survival and which are simply compensatory responses of a stressed system. The purpose of this symposium was to examine some of the most recent ideas regarding mechanisms by which hypoxia can induce tissue injury. Model- and/or tissue-related differences clearly are important considerations in studies on hypoxic injury. Although all systerns have the common feature of diminished aerobic respiration. they often differ in other

ways. For example, removing oxygen from a system (hypoxic hypoxia) does not mimic many of the changes associated with a lack of adequate perfusion (ischemia) (Kehrer and Starnes. 1989). “Chemical hypoxia,” which was used for some of the studies described in this overview. appears to produce biochemical changes in isolated myocytes or hepatocytes similar to those seen with hypoxic hypoxia. However, other cell types or an isolated organ system may not respond the same. and even in hepatocytes differences between hypoxia and cyanide have been reported (Aw and Jones. 1989: Gores rt cl/.. 1989a). Anoxia is strictly defined as the absence of oxygen, while hypoxia can refer to any oxygen tension below normoxia. In clinical situations, anoxia may be achieved in ischemic tissues but. perhaps more frequently, variable levels of hypoxia are obtained due to collateral circulation or intermittent interruption of blood how. The extent of hypoxia needed to induce

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tissue injury is a function of the tissue or cell type involved, the presence or absence of a glycolytic substrate, time, and temperature. The level of hypoxia producing measurable changes in a tissue is also dependent on the endpoint chosen for study, and turns out to be a continuum with normoxia since different enzyme systems have different oxygen dependencies (Jones. 1988). Unless extraordinary measures are taken, all hypoxic systems contain significant amounts of residual oxygen which may have major effects on the duration of tissue viability as well as on the various adaptive and damaging biochemical changes measured. In the studies discussed here, hypoxia ranged from less than 1 mm Hg in isolated hepatocytes incubated in a closed chamber with respiring submitochondrial particles (J.J.L.). to 5 to 35 mm Hg (depending on the length of the postoxygenator tubing) using an oxygenator with IO m of gas-permeable tubing (J.P.K. and H.J.). All tissues normally exist with a tightly controlled balance between energy production and utilization (Fig. 1). The most obvious consequence of hypoxia is to disrupt this balance and decrease cellular ATP levels. Cells and tissues attempt to compensate by increasing glycolysis, but the inefficiency of the process as compared to oxidative phosphorylation makes it incapable of generating sufficient energy for long-term tissue survival. Other compensatory mechanisms also exist in hypoxic cells (Andersson et al.. 1987; Aw rt al.. 1987a,b), and it is important to differentiate between changes which adversely affect cell function and those which are designed by the cell to prolong survival (although after extended periods of time they may also be detrimental). ATP depletion is apparently a necessary but insufficient event preceding cell death. Nevertheless, mitochondrial dysfunction appears to be the critical factor leading to hypoxic injury. This dysfunction is heterogeneous, depending on regional oxygen availability. cell type. degree of differentiation. and demand. Various cellular functions are altered and ionic homeostasis is lost during hypoxia in order to

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i FIG. I Cellular energy metabolism. A balance normally exists between energy production and utilization. This balance is disrupted by hypoxia since alternative cncrgy pathways such as glycolysis are incapable of producing suflicicnt energy for long-term survival.

preserve mitochondrial protonmotive force. The discussion of these changes presented here provides insights into the role of mitochondria in hypoxic cell injury. The extensive regulatory role of calcium has focussed a great deal of attention on this cation as a mediator of tissue injury. Although in some studies described in this symposium. increases in intracellular calcium during hypoxia were not found to occur until long after irreversible injury was evident; contrasting data from other laboratories has been published (Bellomo and Orrenius, 1985; Johnson tri., 1987; Nicotera el al., 1989). Other changes which have been observed during hypoxia include activation of a plasma membrane bound phospholipase, decreased membrane fluidity, increased permeability of the plasma membrane. formation and rupture of surface blebs, and decreased cytosolic pH. The relative importance ofthese changes in cell death has not been established but recent evidence was discussed showing that intracellular acidosis suppresses the degradative processes activated by hypoxia. This effect may delay, rather than prevent. cell injury since it was noted that restoration of normal pH accelerates cell killing by removing this inhibition-an effect which suggests that cellular pH changes may be involved in reperfusion inSjury. In addition to the factors noted above. a reductive stress caused by respiratory inhibi-

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tion may enhance the formation of reactive oxygen species (ROS) and contribute to the cell injury during hypoxia (Fig. 2). The generation of ROS has been demonstrated in hypoxic heart tissue (Maupoil and Rochette, 1988). Studies using isolated perfused liver discussed here suggest this does not occur under all. or even most, hypoxic conditions. but work in whole animals (Chang et al.. 1989), isolated perfused heart tissue (Kehrer and Park. 1990), and chemically hypoxic hepatocytes (Gores ui N/.. 1989a) has revealed changes during hypoxia consistent with oxidative stress. Whether such changes are a cause or a result of mitochondrial dysfunction and suhsequent cell injury has not been established. Howev-er, it is important to note that the oxidative changes in heart tissue occur within 5 to IO min after hypoxia is initiated and are not enhanced during more prolonged hypoxic exposures (Kehrer and Park, 1990). During the period of declining oxygen tensions, it is possible that sufficient oxygen is present to intcract with the increasingly reduced respiratory chain to enhance the generation of ROS.

MITOCHONDRIAL FUNCTION DLJRING HYPOXIA Mitochondria are essential for cellular energy production and are critical targets of chemical-induced injury. Mitochondrial function is impaired by oxygen deficiency and this results in a deficient supply of ATP. The tbcus of this presentation (D.P.J.) was on the factors that determine the intracellular O? dependence of mitochondrial function. the O? dependence of chemical detoxication, and the increased susceptibility of hypoxic cells to chemical-induced oxidative injury. Studies of the determinants of the O? dependence of mitochondrial function in freshly isolated adult cells from rat liver, heart. and kidney show that these cells are on the brink of hypoxia even under normal conditions of oxygenation. The major determinant of the O2 dependence of mitochondrial function in

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FIG. 7. Hypothesis regarding intracellular changesduring hypoxia which may be involved in the progression ofcell injury. Hypoxia leads to decreased oxidative phosphorylation. ATP depletion, and reductive stress. The reductive stress mav promote the formation of reactive oxygen species by mitochondtia under conditions where residual oxygcn is present. These reactive species may further damage mitochondria. as well as other cellular components. The loss of .ATP lcads to ionic imbalances. acidosis. and the activation of proteascs and phospholipases. Intracellular acidosis protects cells by inhibiting the activity of the activated enzymes. Eventually. intracellular pH rises and a positive feedback loop is created that accelerates the progression of cell death.

cells is the distribution and density of mitochondria (Jones and Aw, 1988). Mitochondria are not randomly distributed within cells but are moved and anchored to sites that are specific for different cell types. High density and clustering of mitochondria result in foci within cells with very high O? consumption rates, At these high consumption rates, O1 supply becomes limiting. especially under hypoxia. and results in intracellular ditlusion gradients. Recent evidence suggests that the intracellular O1 dependence of mitochondria changes in association with phenotypic changes, such as postnatal development of hepatocytes (Aw and Jones. 1987, 1988) and dedifferentiation of adult hepatocytes when placed in primary culture (Aw and Jones, 1988; Jones ct rrl., 1987). Thus, the precise conditions under which mitochondrial function becomes 0: dependent are a function of the cell type and the pathophysiological state as it defines the extent of mitochondrial clustering. Under usual conditions in adult mammalian cells, even modest decreases in cellular oxygenation

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result in measurable changes in mitochondrial across the mitochondrial inner membrane function. under anoxic and normoxic conditions Although most eukaryotic cells require a showed that electrophoretic transport systems supply of O2 for survival, all aerobic cells can and some ApH-coupled systems were inhibtolerate some period of O2 deficiency without ited by anoxia (Aw et ul., 1987a). Thus, miirreversible injury. This condition, in which tochondria contain mechanisms to suppress there is a reversible change in function due to ion movement across the inner membrane O2 deficiency. is termed neahypoxia (Jones er during anoxia, apparently to slow the rate of ~1.. 1985). Studies of the regulation of mitocollapse of ion gradients and lengthen the time chondrial function in cells under hypoxic during which cells can survive anoxia. A comconditions revealed the surprising feature that parison of cyanide and anoxic toxicity promitochondrial function does not fail during vided the important observation that cyanide short-term anoxia but is tightly regulated to does not elicit these protective responses and suppress ion flux and preserve the proton mois more toxic than anoxia (Aw and Jones, tive force. During the past several years, per1989). turbations in Cal+ homeostasis have been Further experiments showed that this anconsidered as a central mechanism of toxic oxia-induced suppression of normal functions cell killing. Because both hypoxia and chemis not without costs (Aw et al.. 1987b: Tribble ical toxicants perturb Ca’+ homeostasis, studet al.. 1988)and several factors have now been identified that can contribute to enhanced ies were initiated to define the characteristics susceptibility of hypoxic cells to oxidative inof Ca’+ homeostasis during hypoxia as a basis for exploring the combined effects of toxicants jury (Table 1). Hypoxic and postanoxic cells and hypoxia on mitochondrial function and have decreased O? consumption rates, indicell injury. cating that the functional capacity of thesecells Ca’+ uptake by mitochondria is driven by is limited. Direct measurements of detoxicamembrane potential differences (A$) and ef- tion reactions show that many are impaired flux is driven by pH differences ( ApH). Thus. by hypoxia (Tribble et al.. 1988). Further. ininitial studies were designed to measure the hibition of ion transport. such as glutamate/ effects of 3O-min anoxia on these components aspartate exchange. can impair the mecha(Andersson ef al.. 1987). Unexpectedly, A# nisms that function to transport reducing was decreased only 20%) and ApH was unafequivalents acrossthe membrane. lfthe supplq fected. Calculation of the energy available for of reducing equivalents is diminished by hypATP synthesis and the amount of energy needed for ATP synthesis at the prevailing cyTABLE 1 tosolic mitochondrial concentrations of ADP. FACTORS I-IIKI COWRIBUTF TO THE ENHANC‘YD ATP. and inorganic phosphate showed that S~JSCEPTIRILI~Y 0~ HYPOXIC CELLS TO CHFMKAI I \r sufficient energy was available for ATP synINDUCED INJURY thesis, but this apparently was not occurring (Andersson et n/.. 1987: Aw et al., 1987a). Reduced respiratory capacity Experiments were performed to assess posAltered Ca2+ homeostasis Altered pH regulation sible artifacts in measurement and the possiAltered osmotic regulation bility that the A# and ApH were being mainDecreased membrane potentials tained by utilizing ATP from glycolysis. The Decreased 4TP results indicated that A$ and ApH were mainIncreased ATP-related breakdown products tained by inhibition of ion movement across Cytoskcletal and other structural changes Decreased NADPH the membrane and not by ATP from glycolyDecreased GSH synthesis sis. Measurement of the transmembranal disDecreased glucuronidation and sulfation tribution of normal metabolites and ions

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oxia, cells could have an enhanced susceptibility to toxicity. Previous studies established two general patterns of O2 dependence of oxidative stress in which cell injury increased with increasing O7 concentration (Turrens et al.. 1982) or in which maximal damage occurred under hypoxic but not anoxic conditions (Shen et ~1.. 1982). The former is a consequence of the increased production of reactive species by enzymatic and nonenzymatic reactions as the O? concentration is increased (Jones, 1985: Jones t’( cri., 1989). The latter is a consequence of requirements for both a reductive activation and an O:-dependent step such that maximum damage occurs at a hypoxic Or, concentration. Hyperoxic injury is an example of the former while CClj and halothane toxicities are examples of the latter (DeGroot and Nell. 1986: Shen et al., 1982: Tureens et ~1.. 1982). The toxicity of a constant amount of the oxidant t-butyl hydroperoxide (tBHP) as a function of O2 concentration was examined to determine whether the susceptibility to oxidative injury varied with O1 concentration. The results revealed a new pattern of O? dependence of toxicity in which the greatest toxicity occurred under anoxia (Jones d al., 1989: Dribble el al.. 1988). Detailed examination of this process revealed two salient features (Jones (‘/ ~71.. 1989: Tribble rf (II., 1988: Tribble and Jones. 1989). First. the rate of conversion of tBHP to its corresponding alcohol and the amounts of organic radicals that can be trapped by W-f-butylphenylnitrone arc independent of O2 concentration (Tribble et ~11.. 1988). Thus. the difference in toxicity under hypoxic and normoxic conditions cannot simply bc explained by a difference in the lengths of time of exposure to tBHP or to the 0, dependence of radical production. Second. the rate of tBHP reduction. 40 nmol/ 10h cells/ min (Tribble of nl.. 1988). is about fivefold greater than the maximal rate of NADPH 5~1ppIy under aerobic conditions (about X nmol/ I@ cells/min) (Tribhle and Jones. !OX9). Ihis indicates that the major pathway

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of tBHP metabolism in hepatocytes during hypoxia and anoxia is NADPH-independent. Studies with various potential electron donors showed that lactate is converted to pyruvate at a rate sufficient to account for most of the tBHP metabolism. Of perhaps greater significance, lactate substantially protected against postanoxic oxidative injury due to tBHP. These results suggest that much of the organic peroxide metabolism by hepatocytes is NADH-dependent and that NADH-linked substrates may be very important in protecting against various toxicities involving oxidative processes. In summary, these experimental results show that hypoxia affects a large number of cell functions including ion homeostasis, drug metabolism, and glutathione (GSH) supply. In the case of GSH, the amount of NADPH available for glutathione disulfide (GSSG) reduction is decreased by hypoxia (Tribble and Jones, 1989). In addition, the synthesis of GSH from methionine, but not cysteine, is impaired under hypoxic conditions (Shan et al.. 1989). These effects may further increase the dependence of cells upon NADH-linked peroxide elimination. However, the overall effect of hypoxia is impaired mitochondrial function which renders cells more susceptible to chemical-induced injury.

DIGITIZED VIDEO MICROSCOPY OF HYPOXIC CELL INJURY: THE ROLE OF INTRACELLULAR pH Cell death from disease is rarely synchronous. and events leading to irreversible injury may be obscured when large cell populations are studied, especially if these events occur rapidly or suddenly. This presentation (J.L.L.) described measurements of single living cells during hypoxic and toxic stress using a rapidly developing technology called multiparameter digitized video microscopy (MDVM) which overcomes this drawback. Numerous cell parameters, including cytosolic free Ca” and Na’ . mitochondrial membrane potential. cy-

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toplasmic pH, plasma membrane fluidity. cell surface morphology, and cell viability, can be monitored on a quantitative basis in single living cells. By comparing the time courses of changes in individual parameters. new insights are gained into the mechanisms of anoxic and hypoxic injury. This experimental approach takes advantage of a number of different fluorescent compounds whose excitation and emission spectra are sensitive to specific environmental parameters and which preferentially accumulate into specific subcellular compartments of living cells. This enables study of multiple cellular functions in individual cells in response to external stimuli. Four or more variables can be monitored in the same living cell by selecting parameter-specific fluorophores with nonoverlapping emission and excitation spectra (Lemasters ef (II.. 1987). Fortunately, the number of parameter-sensitive probes available is large and constantly expanding indicating that future research can achieve an even greater capacity to discriminate among various time-dependent changes in a cell’s status. Several protocols were used to produce or simulate hypoxia in cultured rat hepatocytes. True anoxia to cultured hepatocytes was established by perfusing an environmental chamber mounted on the video microscope with submitochondrial particles (Herman rl (I/., 1988). In the presence of an oxidizable substrate such as succinate. the submitochondrial particles actively consumed oxygen and reduced ~0, to less than I torr within 1-3 min. Another model employed KCN and iodoacetate to inhibit ATP production by oxidativc phosphorylation and glycolysis. This treatment, called “chemical hypoxia.” mimics the ATP depletion and reductive stress that accompanies anoxia. Studies have shown that the two models are comparable in many. although not all respects (Gores rf ~1.. 1989a). The model of chemical hypoxia was used principally because it is less demanding technically and certain experiments. such as measurements ofcell volume with a Coulter counter. are simply impossible to perform under

conditions of true anoxia. The results obtained with single cells were also compared with experiments in isolated perfused livers. Cell surface bleb formation occurs in all models of hypoxic cell injury. Three stages can be recognized. Stage I is characterized by formation of numerous small blebs on cell surfaces. During Stage II, blebs grow and coalesce by fusion until one to three large terminal blebs remain. Stage III is initiated by outright rupture of one of the terminal blebs followed by uptake of an external dye such as trypan blue or propidium iodide and loss of trapped cytosolic markers such as fura- or 2’,7’-bis-(2carboxyethyl)-5carboxyfluorescein (BCECF ). In anoxia, the transition to irreversible injury (i.e., an injury from which the cell cannot rccover) also appears to occur simultaneously with bleb rupture. The time required for progression of cells through Stages I-III varies from cell to cell. For chemical hypoxia, progression to cell death takes place after as little as 30 min or as long as 90 min. Bleb growth accelerates, and cell volume (judged by cross-sectional area and Coulter counter measurements) increases prior to the lethal injury from anoxia and chemical hypoxia. However, cell swelling is not the driving force for bleb formation nor a factor leading to lethal injury (Gores rt ul., 1989a). Rather. bleb formation likely is the consequence of reorganization of the cytoskeleton. A rise of cytosolic free Ca’ ’ has been suggested to initiate a series of harmful processes including cytoskeletal disruption. activation of phospholipases. proteases. and other hydrolases. activation of free-radical cascades. and uncoupling of mitochondria which culminate in cell death (Bellomo and Orrenius, 1985: Shanne rf [I/.. 1979). Using ratio imaging of fura- fluorescence. free Cal+ was monitored during chemical hypoxia in relation to blcb formation and the onset of cell death (I.+ masters ef LI/.. 1987: Nieminen ~‘1 [I/.. 198X). Chemical hypoxia was not associated with increased cytosolic free Ca’+ up to the point of hepatocyte death despite the presence of cell surface blebbing. However. intracellular fret

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sodium was dramatically increased and thus ionic imbalances do occur in the cell which may still activate these degradative systems (Kawanishi 6’1rtl.. 1990). Acidosis is a salient feature of tissue ischemia. Extracellular pH was decreased as the metabolic inhibitors KCN and iodoacetate were added to hepatocyte suspensions to mimic the acidosis of ischemia in this model of chemical hypoxia. At pH 7.4, cell viability decreased to 10% after 120 min, whereas at pH 5.5 to 7, cell survival was nearly the same as normoxic cells (Gores ef (I/.. 1988). A similar protection by acidosis was observed in isolated perfused rat livers and cardiac myocytes during hypoxic stress (Bond cf ul.. 1990: Currin (JI al., 1989). It is noteworthy that differences of only a few tenths of a pH unit produced very substantial changes of cell viability. The hypothesis that protection against lethal cell injury by acidotic pH is mediated by cytoplasmic acidification was tested by measuring intracellular pH by ratio imaging of BCECF fluorescence using MDVM (Gores ct II/.. 1989b). After chemical hypoxia at extracellular pH of 7.4, intracellular pH decreased by more than a unit. After 40 to 50 min, intracellular pH began to rise, BCECF began to leak rapidly from the cells, and cell death ensued within I-2 min. Manipulations which extended the intracellular acidosis, such as acidic extracellular pH and inhibition of exchange of extracellular Na’ for intracellular k1’. prolonged cell survival. In contrast. experimental manipulations which prevented intracellular acidosis from developing accelcrated the onset of cell death. ATP depletion at neutral and acidic extracellular pH was identical and could not explain the prolonged cell survival observed under acidic conditions. These results indicate that intracellular acidosis protects against cell death from ATP depletion, a phenomenon which may represent a protective adaptation against ischemic injury. The mechanism of this protection is not known. although it is possible that acidic intracellular pH suppresses autolytic degradation processes (e.g., proteolysis, phospholipid hy-

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drolysis) which are initiated during ATP depletion (Fig. 3) (Gores et ul.. 1989b). Despite suppression at acidic intracellular pH, these degradative processes lead eventually to an increase of plasma membrane permeability causing H’ to leak from the cell and intracellular pH to rise. A positive feedback loop is thus created which accelerates pH-dependent degradative processes, increases membrane permeability further, and culminates rapidly in cell death. Upon reperfusion of ischemic tissues, reoxygenation and a return to physiologic pH occur simultaneously. Thus, if the hypothesis outlined in Fig. 2 is correct, then the return of pH to a physiologic level during continued hypoxia should rapidly lead to cell damage. Therefore, the effects of changing pH during chemical hypoxia were investigated. When extracellular pH was changed from 6.5 to pH 7.4, cell killing accelerated almost immediately. supporting this concept (Gores et cl/.. 1989b). Isolated rat livers were perfused with anoxic buffer at pH 6.1 and reoxygenated at pH 7.3 to simulate more closely the changes of oxygenation and pH which occur during ischemia and reperfusion (Currin rf al., 1989). These conditions simulating ischemia and reperfusion produced an abrupt release of lactate dehydrogenase into the effluent perfusate. Injury after simulated reperfusion was proportional to the time of simulated ischemia and began to occur after about 50 min of anoxia at pH 6. I. However. the increase of pH rather than the reintroduction of oxygen was the factor that precipitated cell killing. Importantly. this pH-dependent reperfusion injury (“paradox”) could be avoided if pH was slowly rather than abruptly increased after reoxygenation. Reperfusion of ischemic human tissue is commonplace after thrombolytic therapy, vascular surgery, cardioplegia, and organ transplantation. and significant reperfusion injury can occur under these conditions. The present evidence indicates that a “pH paradox” may account in large part for reperfusion injury to ischemic organs and suggests that pH

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is a critical variable which can be manipulated to diminish reperfusion injury clinically.

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Liver ischemia in the intact rat results in an accelerated degradation of membrane phospholipids. With 3 hr of ischemia, there is a loss of 30% of the total phospholipid, measured as either phosphate in a lipid extract or as fatty acyl esters after separation by thin-layer chromatography of the major lipid classes in the same extracts (Chien t’t u/.. 1978: Finkelstein ef ul.. 1985). Alterations in the structure and function of both the plasma membrane and the membranes of the endoplasmic reticulum accompany this disorder in phospholipid metabolism (Chien rf al., 1978; Chien and Farber, 1977: Farber rt al.. 1978). Accelerated phospholipid degradation and its resultant membrane dysfunction may be the critical alteration that produces irreversible liver cell injury in ischemia. The major focus of this presentation (J.L.F.) addressed two related issues; ( 1) how does the disordered phospholipid metabolism perturb the structure and thus the function of cellular membranes, and (2) what is the pathogenesis of the accelerated degradation of membrane lipids. Electron spin resonance spectroscopy was used to show that microsomal membranes isolated from 3-hr ischemic livers are significantly less fluid than control membranes (Petrovich L’I u/.. 1984). This increased molecular order of the ischemic membranes is explained by the increased cholesterol to phospholipid ratio that results from the degradation of phospholipid and the retention of cholesterol (Petrovich (‘1 N/.. 1984). Such a retention of cholesterol increases the molecular order of the remaining phospholipids. Studies were conducted to explore the hypothesis that this general decrease in fluidity is accompanied by the appearance ofdomains in the lateral plane of the membrane of differing molecular order. The interface between

FIG.

3. Hypothesis regarding intracellular changes during hypoxia which may be involved in the progression of cell injury. Hypoxia leads to a loss of mitochondrial electron transport which will decrease cellular ATP and may collapse the mitochondrtal membrane potential. Subsequent cytoskeletal alterations may activate membrane phospholipase which then degrades phospholipids. The loss of phospholipids relative to cholesterol will decrease mcmbrane fluidity which. in turn. may enhance permeability resulting in cell death. This hypothesis is consistent with acidosis the concept. as depicted in Fig. 2, that intracellular can protect cells by inhibiting the activity of the activated enzymes. The main difference between these two hypotheses is the speciftc focus on phospholipase actikit) and membrane phosphoiipids.

such domains would, in turn, represent sites of increased membrane permeability. A variety of approaches was used (electron spin resonance spectroscopy with spin-labeled phospholipids, “P nuclear magnetic resonance spectroscopy, freeze-fracture electron microscopy, and X-ray diffraction) to look for such domains. Preliminary studies with each of these modalities indicated the existence of lipid domains of differing molecular order, but clearly much more work is needed. Cultured rat hepatocytes were used to explore the mechanism of the accelerated degradation of phospholipids that characterizes the reaction of the liver to ischemia. Anoxia results in an accelerated degradation of the phospholipids of cultured hepatocytes (Farbei and Young, 198 I). This alteration in lipid metabolism. as well as the loss of viability of

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FIG. 3. Cellular defense mechanisms against reactive oxygen species. Enzymatic defenses including superoxide dismutase. glutathione peroxidase and the glutathione redox system. and catalase exist within hepatocytes.

the cells, does not seem to be the consequence of ATP depletion per se (Kane rf al.. 1985; Masaki et ~1.. 1989). Rather, there is a better correlation with collapse of the mitochondrial membrane potential than with ATP depletion alone (Masaki et al.. 1989). This conclusion is based on the fact that oligomycin depletes ATP to the same extent that anoxia or cyanide does. but does not collapse the mitochondrial membrane potential and does not kill the hepatocytes over the time course that anoxia or cyanide produce a substantial loss of viability. The killing of cultured hepatocytes by cyanide was used to model the effects of ischemia/anoxia. Cyanide intoxication of cultured hepatocytes stimulates phospholipid hydrolysis (J. L. Farber, unpublished data) supporting the importance of this effect in hypoxic cell injury. Digital imaging fluorescence microscopy was used to examine the effect of cyanide on intracellular calcium homeostasis in individual cultured hepatocytes. Cyanide intoxication was not accompanied by an increase in the cytosolic calcium ion concentration. Thus, phospholipase activation by a rise in [Ca”], cannot account for the accelerated degradation of phospholipid. Rather. these data suggest that an as yet uncharacterized alteration in the cytoskeleton is associated with phospholipase activation. The nature of this alteration in the cytoskeleton is currently undcr investigation. It is our hypothesis that changes in the association of the cytoskeleton with the plasma

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membrane of the hepatocyte that accompany the loss of mitochondrial energization and the loss of ATP activate membrane-bound phospholipases, an effect that is responsible for the accelerated hydrolysis of phospholipids (Fig. 3). This, in turn, leads to the formation of abnormal lipid domains in the plasma membrane which may allow the diffusion of molecules which are normally excluded (i.e., Ca’+ ) and ultimately cause cell death.

OXIDATIVE STRESS DURING HYPOXIA IN ISOLATED PERFUSED LIVER TISSUE The original observation by Granger et (I/. ( 198 1) that the xanthine oxidase inhibitor allopurinol. superoxide dismutase, and catalase can protect against ischemia/reperfusion (I/R) injury in feline intestine stimulated a tremendous interest in the contribution of ROS to the pathogenesis of disease states involving temporary oxygen deprivation and reoxygenation in various organs. For liver tissue, similar indirect pharmacological evidence seems to support a role for ROS in I/R injury (Adkison et ul., 1986) as well as in hypoxic liver damage (Raeder et al.. 1985; Younes and Strubelt, 1988). In order to define the exact contribution of ROS in the development of hypoxic liver injury. this presentation (H.J.) discussed the formation of GSSG during hypoxia as an index of ROS formation in isolated perfused livers of male Fischer rats. In this model. the liver is continuously perfused with Krebs-Henseleit bicarbonate buffer (37°C. pH 7.4) gassed either with 95% 02:5% CO2 (~0: = 550 mm Hg) or a 95%) N7:5’% CO2 (p02 = 35 mm Hg). The generation of ROS induces GSSG formation through the reduction of hydrogen peroxide by glutathione peroxidase (Fig. 4). GSSG formation can be followed by measurement of the hepatocellular release (about 3-5% of the total formation) (Jaeschke et al.. 1988a.b: Sies and Summer, 1975) and the tissue GSSG content. About 80-85s) of the GSSG released from hepatocytes is secreted into bile and 15-

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20’70 is exported into the perfusate (Jaeschke cf al., 1988b; Jaeschke, 1990). Impairment of the biliary export mechanism for GSSG or glutathione conjugates can be compensated by an enhanced sinusoidal release under normoxia (Jaeschke, 1990). Since hypoxia could affect GSSG release by inhibiting GSSG membrane transport or enhancing the rereduction of GSSG to GSH (Jaeschke and Mitchell, 1990), the effect of hypoxia on the physiology of GSSG formation and release was tested. Short-term hypoxia (< 15 min) that caused a decline of bile flow rates by 48% reduced the canalicular and the sinusoidal GSSG transport only by 15 and 22Y~, respectively. The individual carriers were selectively tested with the glutathione conjugates of sulfobromophthalein and 1-chloro-2,4-dinitrobenzene. Despite a reduction of hepatic ATP levels by 43%, the active transport of GSSG and glutathione Sconjugates into bile was only minimally affected (Jaeschke. 1990). Infusion of tBHP (230 nmol/min/g liver) enhanced tissue GSSG levels and stimulated cellular GSSG release. Hypoxia during peroxide-induced oxidant stress reduced GSSG secretion into bile and perfusate by 38Yo but further increased hepatic GSSG content. Quantitative calculations revealed that essentially all of the GSSG not exported during hypoxia accumulated in the tissue. Thus. no evidence was found for enhanced rereduction of GSSG during hypoxia (Jaeschke, 1990). In contrast, in situations where ROS formation was caused by the intracellular reduction of molecular oxygen, e.g.. the physiological formation of ROS in mitochondria or the chemical generation of superoxide with the redox-cycling compound diquat. the resulting basal or stimulated GSSG release declined by 80-90% during hypoxia and was accompanied by lower tissue GSSG levels. This indicates that during hypoxia the basal, as well as the chemically stimulated, formation of ROS is significantly reduced. Long-term hypoxia that caused irreversible intrahepatic cholestasis and severe hepatocellular damage (assessed as release of lactate dehydrogenase: >8 U/min/g liver wt) increased

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sinusoidal GSH and GSSG efflux rates IO-fold. but the tissue GSSG content remained 30% below control values (Jaeschke et al.. 1988a). Thus, the enhanced GSSG release was due to nonspecific leakage of cell contents rather than intracellular oxidant stress. This view is supported by the fact that reoxygenation at this time immediately reduced LDH and GSH release but further increased GSSG efflux by 300% and also enhanced tissue GSSG contents by 80%’ above normoxic control values (Jaeschke et al.. 1988a). Addition of the glycolysis substrate fructose to the perfusate prevented hypoxic injury as well as the increased GSSG formation during reoxygenation in this model (Jaeschke and Mitchell, 1989). These data indicate that there is a significant intracellular oxidant stress only during reoxygenation and only after a severe hypoxic injury. Mitochondria were identified as the predominant sustained source of intracellular ROS formation. while xanthine oxidase contributed only temporarily to the oxidant stress during the initial reperfusion period (Jaeschke and Mitchell, 1989). These results with isolated perfused liver seem to contradict reports about the enhanced formation of ROS during chemical hypoxia in isolated hepatocytes (Gores et al., 1989a). However, there are important differences bctween these models of hypoxic injury. Chemical hypoxia, as well as hypoxic hypoxia, leads to an enhanced reduction state of the mitochondrial respiratory chain by blocking cytochrome a/a3 through cyanide or lack of oxygen, respectively. The increased reduction state of the mitochondrial respiratory chain can only induce enhanced formation of ROS in the presence of molecular oxygen. The residual oxygen present during maximal hypoxic hypoxia is insufficient for this task due to the low K,, of cytochrome a/a3 for molecular oxygen. This is demonstrated by the dramatic decline in physiological GSSG formation during hypoxic hypoxia in liver (Jaeschke. 1990) as well as by the complete prevention of hydrogen peroxide formation by chemical hypoxia in the absence of oxygen (Gores CI ~1..

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1989a). Furthermore, infusion of cyanide into the perfused liver under normoxic conditions enhanced GSSG release and intracellular GSSG accumulation indicating the increased formation of ROS in combination with the inhibition of catalase (Jaeschke. unpublished data). Thus. the conflicting results obtained with both models are not due to differences between isolated cells and the intact organ, but arc model-specific differences which need to be considered when discussing the pathophysiological relevance of results obtained with these models for the pathogenesis of ischemia/reflow injury in view. In a model of no-flow ischemia in the isolated perfused liver, tissue GSSG levels remained constant throughout 120 min of ischcmia (Jaeschke et [I/.. 1988b). Similar results were reported by Siemsc’tul. ( 1983). but these investigators measured increased GSSG to GSH ratios in the solution where the ischemic liver was stored. Since the changeof the GSSG to GSH ratio could be prevented by 1 rnM allopurinol in the storagesolution. the authors interpreted their data as evidence for the enhanced formation of ROS during ischemia (Siems~‘2~11..1983). However, the stimulation of hepatic GSSG releasemust be accompanied by an increase of the tissue GSSG levels, since it is a carrier-mediated transport whoseactivity is mainly dependent on the intracellular substrate concentration. The one exception to that rule would be an enhanced nonspecific release of cell contents due to massivecell damage as mentioned above. Rather than the releaseof GSSG. it is possible that GSH was releasedinto the extracellular medium where it is subject to fairly rapid oxidation without the possibility of being rereduced. The presence of the antioxidant allopurinol in the organ bath might have partially prevented the spontaneous oxidation of GSH. Therefore, enhanced extracellular GSSG to GSH ratios in an organ bath may not necessarilyreflect an enhanced cellular relcasc of GSSG and thus an intracellular oxidant stress.This view is supported by the hepatic experiments discussedhere where an in-

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creasedwashout of extracellularly accumulated enzymes and metabolites (with elevated GSSG to GSH ratios) was observed only during the initial 20-30 set of reperfusion (Jaeschke et al.. 1988b). The releaseof metabolites accumulated inside the cells occurred with a totally different kinetics. In summary, there is no evidence for a pathophysiologically significant formation of ROS in the isolatedperfused liver during either no-flow ischemia or hypoxia. Since GSSG is a reliable parameter for the intracellular ROS formation during normoxia and also during hypoxia or ischemia (as discussedin detail: Jaeschkeand Mitchell, 1990:Jaeschke, 1990). it wasdemonstrated that even the low constant generation of ROS in a normal liver is suppressedby more than 80% during hypoxia. Thus. reactive oxygen is unlikely to be involved in the pathogenesisof ischemic or hypoxic injury in the liver.

CONCLUSIONS The research summarized in this paper servesto illustrate some of the contemporary thinking regarding mechanismsof hypoxic cell injury. Although someagreement was evident regarding various biochemical changeswhich occur as a result of hypoxia (in particular the enhancement of degradative processes.the protective role of acidosis, and the development of cytoskeletal alterations [i.e., blebbing]). it would be incorrect to assumethat conflicts do not remain (compare Table I. Figs. 3 and 3). Differences in the biochemical effects induced by cyanide versushypoxia, the occurrence of hypoxia-induced changesin the mitochondrial potential. and the role calcium plays in hypoxia-induced cell injury continue to be sourcesof contrasting results and interpretations. Most of the studies discussedin this symposium using isolated hepatocytes failed to reveal any changes in cytosolic free calcium ( [Cai]) in cellssubjectedto hypoxic or chemical hypoxia. Other work in metabolically inhib-

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ited myocytes has supported the development of injury independent of [Cai] (Cobbold et al.. 1985). In contrast, PC12 (Johnson et al., 1987) or hepatoma 1c 1c7 cells (Nicotera d al.. 1989) exhibited a rapid and sustained increase in [Cal when subjected to chemical hypoxia which appearedto be related to the subsequent injury. Studies in perfused rabbit (Lee et al., 1988), ferret (Marban et al., 1987). and rat (Steenbergen tf al.. 1990) hearts have also shown increasesin [Cq] which may contribute to cell injury. The lossof ATP which accompanies hypoxia makes the long-term maintenance of normal ionic gradients impossible. However, as suggestedby the data presented in this symposium, short-term suppressionof membrane ion transport may help cells withstand brief periods of hypoxia. Even when [Cai] is clearly increased, the importance of this change in the development of cell injury is not readily evident. A recent study where isolated hepatocytes were incubated with ATP demonstrated that high [Cai] alone does not lead to an immediate loss of cell viability (Nagelkerke et al., 1989). A similar conclusion has been reached in cultured embryonic heart cells where the Na+-K+ pump was inhibited leading to a fourfold increasein cell calcium content (Murphy rf al.. 1983). These data suggestthat the focus on [Cai] should perhaps be expanded to include other ions whose abnormal distribution during hypoxia may disrupt normal cell function. Conflicting conclusions were evident in this symposium regarding the role a lossof the mitochondrial membrane potential plays in cell death. Andersson et al. ( 1987) found that cells commit extensive resourcesto maintaining the mitochondrial potential under conditions of oxygen deprivation while Masaki et ui. ( 1989) found that a lossof the mitochondrial potential correlated with cell death. Differences were also evident regarding the production of ROS during hypoxia which was evident in heart (Kehrer and Park. 1990) but not in liver (Jaeschke. 1990) tissue. Model- and tissue-related differences (partitularly the useof cyanide versushypoxia and

ET

AL.

cell lines versus primary cultures) appear likely to explain some, if not all. of the conflicting results outlined above. It alsoseemslikely that a combination of factors are involved in damaging cells during hypoxia, and that different models reveal these to a different extent. For example, the interaction of increased [Cai] together with other ionic imbalances and energy deficiency, asseenduring hypoxia, could produce injury when each change alone is nontoxic. In general, mitochondrial dysfunction and subsequent ionic imbalances, as well as the activation of various degradative processes. would appear to be the keys to understanding hypoxic cell injury. When and where such changesoccur, which ionsare involved, and their importance in producing irreversible cell injury are not known. Separating causalfrom effector changesis a difficult processwhich must be adequately addressedbefore the mechanism of hypoxic cell injury is established. ACKNOWLEDGMENTS J.P.K. is the Gustavus Pfeiffer Centennial Fellow of Pharmacology and was supported by Grants HL4069i and HL35689. D.P.J.‘s work was supported by Grants GM36538 and HL39968. J.J.L. acknowledges the valuable contributions of Drs. B. Herman. A-L. Nieminen. G. J. Gores. T. Kawanishr, J. M. Bond, R. T. Currin, K. FlorineCasteel. T. L. Dawson. and R. G. Thurman. J.J.L.‘s work was supported by NIH Grants AGO7218 and DK3703-l and by Grant J-1433 from the Oilice of Naval Research. J.I..F.‘s work was supported by Grant HL29524. H.J.‘\ work was supported by Grants GM34130 and GM42957. H.J. thanks Dr. Jerry R. Mitchell for his contributions to this project.

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