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Prevention of Reoxygenation Injury by Sodium Salicylate in Isolated-Perfused Rat Liver
Free Radical Biology & Medicine, Vol. 25, No. 1, pp. 87–94, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/98 $19.00 1 .00
PII S0891-5849(98)00033-1
Original Contribution PREVENTION OF REOXYGENATION INJURY BY SODIUM SALICYLATE IN ISOLATED-PERFUSED RAT LIVER ALESSANDRA COLANTONI,*† NICOLA de MARIA,* PAOLO CARACENI,† MAURO BERNARDI,† ROBERT A. FLOYD,‡ and DAVID H. VAN THIEL* *Division of Gastroenterology, Loyola University, Maywood, IL, USA; †Dipartimento di Medicina Interna, Cardioangiologia ed Epatologia, University of Bologna, Bologna, Italy, and ‡Free Radical Biology and Aging, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA (Received 16 October 1997; Revised 30 January 1998; Accepted 30 January 1998)
Abstract—Sodium salicylate can be used as a chemical trap for hydroxyl radicals, the most damaging reactive oxygen species. Because reactive oxygen species are involved in the pathogenesis of hepatic hypoxia/reoxygenation injury, the goal of this study was to determine if trapping hydroxyl radicals with salicylate would prevent or at least ameliorate such injury. Isolated rat livers, continuously perfused with Krebs-Henseleit bicarbonate buffer in the presence or absence of salicylate (2 mM), were exposed, after 30 min of recovery, to 60 min of hypoxia, followed by 30 min of reoxygenation. During reoxygenation, control livers experienced a sharp increase in the rate of lactic dehydrogenase release, taken as index of cell injury, protein carbonyl content, and malondialdehyde, taken as index of protein oxidation and lipid peroxidation, respectively. The presence of salicylate in the solution perfusion significantly reduced the rate of lactic dehydrogenase release, protein carbonyl content, and malondialdehyde production during reoxygenation. Hepatic histology documented a significantly reduced cell injury in salicylate-perfused livers compared to control livers. These data suggest that the hydroxyl radical chemical trap sodium salicylate, acting as an antioxidant, may represents an effective agent to reduce liver injury due to hypoxia/reoxygenation in a model of isolated-perfused rat liver. © 1998 Elsevier Science Inc. Keywords—Reoxygenation, Liver, Salicylate, Hydroxyl radical, Trapping agent, Oxidative stress, Free radical
INTRODUCTION
as well as to a partial or complete loss of important membrane-bound enzymatic activities.4,5 Oxidation of proteins impairs their function and enhances their catabolism. These intracellular alterations are presumed to be important in the pathogenesis of early liver graft dysfunction and/or failure in the transplant setting.6,7 Experimental data suggest that improvement in early allograft function requires protective action not only during the preservation phase of donor organ but also during the early phase of allograft reoxygenation in the recipient. Kupffer cells within the liver have been shown to be the major source of ROS during the post-hypoxic reoxygenation period,8 although both parenchymal9 and endothelial cells are also able to produce them.6 The balance between ROS production and radical scavenging activity is critically important in determining the oxidative injury experienced by any organ.10 Thus, the identification of a substance capable of reducing ROS-induced organ injury represents one of the most important
The susceptibility of the liver to hypoxia/reoxygenation represents one of the major remaining obstacles to the continued success of clinical liver transplantation. As with other organs, also in the liver the reoxygenation phase that follows a period of oxygen deprivation is characterized by the production of reactive oxygen species (ROS) at an accelerated rate.1–3 Several ROS have been identified, such as superoxide anion, hydrogen peroxide, and hydroxyl radical, the latter being recognized as the most nocive species. ROS are responsible for peroxidation of cell membrane lipids and protein oxidation. Peroxidation of membrane unsaturated fatty acids leads to changes in membrane permeability and integrity Address correspondence to: David H. Van Thiel, M.D., Division of Gastroenterology, Loyola University Medical Center, 2106 S. 1st Avenue, Building 114, Room 54, Maywood, IL 60153; Tel: (708) 2160364; Fax: (708) 216-0423. 87
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goals of research in the field of solid organ transplantation. Theoretically, trapping of the highly toxic hydroxyl radicals may significantly reduce the allograft injury experienced during postischemic reoxygenation. Sodium salicylate is a chemical trap for hydroxyl radical. The major hydroxylation products of hydroxyl radical attack on salicylate under physiological conditions are 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA).11 Either 2,3DHBA and/or 2,5-DHBA can be measured as markers of hydroxyl radical formation by high-pressure liquid chromatography with electrochemical detection methods. Thus, aromatic hydroxylation of sodium salicylate has been used as sensitive method for the measurement of hydroxyl radical formation in vivo.12–14 In an experimental model of myocardial ischemia, salicylate has been shown to reduce the severity of organ damage and the risk of postischemic ventricular fibrillation.15 Moreover, salicylate inhibited the disruption of mitochondrial oxidative phosphorylation function in isolated rat hearts.16 In the present study the time course of hydroxyl radical production within the liver during hypoxia and reoxygenation is described using salicylate as a trapping agent, and the effect of salicylate on reoxygenation injury has been assessed in a model of isolated-perfused rat liver. MATERIALS AND METHODS
Animals Male Wistar rats weighting 260 –300 g (Harlan–Sprague–Dawley, Houston, TX) were used for this study. All animals were allowed free access to standard rat chow diet and water. Only fed animals were used in the experiments described. The present research protocol complied with the criteria for animal care and use at our institution, which conforms to National Institutes of Health guidelines. Liver perfusion The animals were anesthetized using an intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight). The abdomen was opened by a median laparotomy and the liver was exposed. After isolation, the livers were continuously perfused with protein and hemoglobin-free Krebs-Henseleit bicarbonate buffer (pH 7.4). The perfusate was pumped into the liver via a cannula placed in the portal vein and the hepatic effluent was collected from a second cannula placed in the vena cava through the right atrium. The perfusate was not recirculated. During the study period the liver temperature was monitored and maintained at 37°C using a heating lamp.
Experimental protocol The animals were divided in the following groups: (1) Aerobic controls (n 5 12). The livers were perfused continuously with oxygenated Krebs-Henseleit bicarbonate buffer solution (pH 7.4) for 1 h (group 1A) or 2 h (group 1B). For all perfusions under aerobic conditions, the perfusate was saturated with oxygen and the flow rate was 3– 4 ml/min/g of liver weight. Under these conditions, the liver is normoxic.17 Perfusate samples were collected every 10 min, and tissue samples at the end of the experiments. (2) Salicylate aerobic controls (n 5 12). The livers were perfused with oxygenated KrebsHenseleit bicarbonate buffer solution supplemented with salicylate 2 mM for 1 h (group 2A) and 2 h (group 2B). Perfusate samples were collected every 10 min during the experiments and tissue samples were obtained at the end of the experiments. (3) Hypoxia-reoxygenation (n 5 12). The livers were perfused with oxygenated KrebsHenseleit bicarbonate buffer solution for 30 min after isolation (recovery period) then subjected to 60 min of hypoxia (hypoxic phase) followed by 30 min of reoxygenation. During the recovery and reoxygenation periods, the perfusion solution was equilibrated with oxygen (95% oxygen, 5% carbon dioxide) and the flow rate was 3– 4 ml/min/g of liver. During hypoxia, the perfusate was equilibrated with nitrogen (95% nitrogen, 5% carbon dioxide). One group of livers was used for the collection of perfusate samples at the beginning and at the end of recovery and hypoxia phases and after 5, 10, 15, 20, and 30 min of reoxygenation (group 3A). Tissue samples were collected from a second group of livers, at the end of the recovery, at the end of hypoxic period and after 5, 15, and 30 min of reoxygenation (group 3B). (4) Salicylate hypoxia-reoxygenation (n 5 12). After isolation, the livers underwent 30 min of recovery followed by 60 min of hypoxia and 30 min of reoxygenation, as described above. Salicylate (2 mM) was added to the perfusion solution. Perfusate samples were collected at the beginning and at the end of recovery and hypoxia phases and after 5, 10, 15, 20, and 30 min of reoxygenation (group 4A). Tissue samples were collected from a second group of livers at the end of the recovery, at the end of hypoxic period and after 5, 15, and 30 min of reoxygenation (group 4B). Lactic dehydrogenase (LDH) release The rate of LDH release into the perfusate was measured as a marker of liver injury. LDH was assessed utilizing a spectrophotometric method based on the oxidation of lactate to pyruvate (Sigma Diagnostic, St. Louis, MO). To exclude any effect of salicylate on the assay, two standard solutions reconstituted either in
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buffer solution or in buffer solution plus salicylate were compared. The presence of salicylate in the perfusate did not affect the analytical measurements used for LDH. The rate of LDH release is expressed as mU/min. Hydroxyl radical production The attack by hydroxyl radical upon salicylate leads to the formation of 2,3-DHBA and 2,5-DHBA.11–14,18 Salicylate, 2,3-DHBA, and 2,5-DHBA concentrations in perfusate samples collected at the end of recovery time, at the beginning and end of hypoxia and after 5, 10, 15, 20, and 30 min of reoxygenation, were quantitated using high-pressure liquid chromatography with an electrochemical detector as reported by Floyd et al.19 The amount of DHBA and residual salicylate were determined by comparison of the peak heights of the samples to that of 2,3-DHBA, 2,5-DHBA, and salicylate standards. The ratio total DHBA/salicylate in the perfusate at different time points was also calculated.
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of 0.2 mM, as described by Belinsky et al.22 At the end of the hypoxia/reoxygenation experiments and in aerobic controls (after 1 h and 2 h), the livers were perfused with trypan blue for 5 min and fixed in a solution containing 1% paraformaldehyde. Tissue samples were embedded in paraffin and processed for light microscopy evaluation. Four fields were selected per liver, and the percentage of stained cells quantitated. Statistical analysis All data are expressed as the mean of six experiments 6 standard error of the mean (SEM). The level of significance between treated and untreated groups was determined using either the Student’s t-test or ANOVA as appropriate. A p-value less than .05 was considered significant. RESULTS
Effect of salicylate on liver cell injury and viability Protein carbonyl content Protein carbonyl content (PCC), a marker of protein oxidation, was measured in homogenates of liver tissue by the 2,4 dinitrophenylhydrazine procedure according to Levine and colleagues.20 The total protein content of each tissue sample was determined using the bicinchonic acid assay (Pierce Chemical Company, Rockford, IL). Malondialdehyde The extent of lipid peroxidation was assessed by measuring malondialdehyde (MDA) in liver tissue homogenates using a standard colorimetric assay (Calbiochem, La Jolla, CA) according to the method of Esterbauer et al.21 Morphological examination The histologic evaluations of liver samples collected at the end of the experiments were performed using paraffin embedded, hematoxylin/eosin stained tissue. The severity of tissue injury was assessed as number of foci of necrosis per section and was scored by a single observer not aware of the group assignment using a scale ranging from 0 (no injury) to 41 (panlobular injury). Trypan blue Trypan blue is a vital dye that stains the nuclei of non viable cells. To evaluate hepatic cell viability, the dye was added to the solution of perfusion at a concentration
To evaluate the effect of the presence of salicylate 2 mM on hepatic cells, isolated livers continuously perfused in aerobic conditions with or without salicylate being added to the solution of perfusion were studied. Cell injury was monitored by measuring the rate of LDH release in samples of perfusate collected every 10 min during the experiments. Cell viability was evaluated by trypan blue exclusion after 1 h (groups 1A and 2A) and 2 h (groups 1B and 2B) of aerobic perfusion. Under aerobic conditions, the rate of hepatic release of LDH in control and salicylate-treated livers was 237 6 48 mU/ min and 200 6 29 mU/min, respectively. This rate remained steady in both groups for the 2 h of aerobic perfusion, reaching 300 6 51 mU/min and 286 6 62 mU/min, respectively, at the end of the experiments (one-way ANOVA, NS). No differences in the rate of LDH release between groups were recorded at each time point. Trypan blue exclusion after 1 h of aerobic perfusion was 95 6 2% in the livers perfused without salicylate (group 1A) and 97 6 3% in the salicylate-treated group (group 2A). After 2 h, cell viability was 92 6 3% in group 1B and 93 6 4% in group 2B. Production of hydroxyl radicals in the liver Figure 1 shows the time course of the production of 2,3-DHBA, 2,5-DHBA, and the ratio total DHBA/salicylate in the perfusate from livers exposed to 60 min of hypoxia followed by 30 min of reoxygenation and from aerobic controls. The 2,3-DHBA concentration in the perfusate remained steady during 2 h of liver perfusion under aerobic
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0.3 nmol/ml after 15 min of reoxygenation (one-way ANOVA p 5 .002). The 2,5-DHBA concentration in the perfusate remained steady under aerobic conditions, being 3.4 6 0.3 nmol/ml at the beginning of the experiments and 3.1 6 0.2 nmol/ml after 2 h of liver perfusion where the perfusate was saturated with oxygen (group 2B). Under the imposed experimental protocol, the perfusate concentration of 2,5-DHBA was 3.2 6 0.4 nmol/ml at the end of the recovery period and 2.4 6 0.3 nmol/ml at the end of 60 min of hypoxia. However, an increase in 2,5-DHBA concentration in the perfusate to 7.7 6 0.7 nmol/ml was recorded after 15 min of reoxygenation (group 4A) (oneway ANOVA p 5 .001). The DHBA/salicylate ratio remained constant during 2 h of liver perfusion under aerobic conditions (group 2B). A sharp increase in the total DHBA/salicylate ratio was recorded during the early phase of reoxygenation, being 0.70 6 0.06 after 10 min and 1.28 6 0.05 at the end of the experiments (group 4A) (recovery period: 0.36 6 0.01, end of hypoxia 0.32 6 0.02, one-way ANOVA p , .0001). Cell injury Figure 2 shows the rate of LDH release during recovery, hypoxia, and reoxygenation in untreated livers (group 3A) and in livers perfused with salicylate being added to the solution of perfusion (group 4A). In untreated livers, LDH release increased significantly during the reoxygenation phase to a level greater than that experienced during the hypoxic period. Specifically, LDH release, being 179 6 28 mU/min at the end of recovery and 258 6 85 mU/min at the end of the hypoxic
Fig. 1. Time course of 2,3-DHBA and 2,5-DHBA production from salicylate and total DHBA/salicylate ratio in isolated perfused rat livers exposed to 60 min of hypoxia followed by 30 min of reoxygenation (dashed line). Both 2,3-DHBA and 2,5-DHBA concentrations in the perfusate are significantly higher during the reoxygenation phase of the study (90 –120 min) than during the recovery and hypoxic phases. 2,3-DHBA and 2,5-DHBA production from salicylate in the aerobic controls (solid line) remained steady for the 2 h studied. Values represent the means of six experiments 6 SEM.
condition being 1.2 6 0.3 nmol/ml at the beginning of the experiments and 1.3 6 0.3 nmol/ml after 2 h of perfusion (group 2B). A sharp increase in 2,3-DHBA concentration in the perfusate (group 4A) was recorded during the early phase of reoxygenation that followed 60 min of warm hypoxia being 1.2 6 0.3 nmol/ml at the end of the recovery period, 1.1 6 0.1 nmol/ml at the end of the period of hypoxia and 2.0 6
Fig. 2. Cell injury assessed by the rate of LDH release into the perfusate during hypoxia and reoxygenation of rat liver perfused in the presence or absence of 2 mM salicylate. In the livers perfused without salicylate (solid line), LDH release markedly increased during reoxygenation (one-way ANOVA, p 5 .0001). In the presence of salicylate (dashed line), LDH release did not increase during the reoxygenation phase of the experiment. In the salicylate group, the rate of LDH release during reoxygenation was significantly lower at each time point compared to the control experiments (*p # .001). Values represent the mean of six experiments 6 SEM.
Reoxygenation injury and sodium salicylate
Fig. 3. Protein oxidation, evaluated as protein carbonyl content, at the end of 30 min of recovery, 60 min of hypoxia, and after 5, 15, and 30 min of reoxygenation in isolated livers perfused in the presence (dark columns) or absence of salicylate 2 mM. Reoxygenation causes a significant increase in protein oxidation in the livers perfused in the absence of salicylate in the solution of perfusion (one-way ANOVA, p 5 .002). Salicylate prevents the increase in liver protein oxidation observed during reoxygenation (one-way ANOVA, p 5 .267). The mean protein carbonyl content levels were significantly lower at each time point during reoxygenation of the livers perfused in the presence of salicylate compared to control experiments (*p # .02). Values represent the mean of six experiments 6 SEM.
phase, reached a level of 2768 6 86 mU/min after 5 min of reoxygenation ( p , .0001). The presence of salicylate in the perfusion solution significantly reduced the rate of LDH release during the reoxygenation. In fact, no statistically significant changes in LDH release were recorded during hypoxia and reoxygenation phases in the salicylate-treated group 4A (one-way ANOVA, NS). For each time point during reoxygenation the rate of LDH release by the untreated livers was significantly higher than that by livers continuously perfused with a solution containing salicylate ( p # .001). Light microscopic examination of the livers at the end of each hypoxia/reoxygenation experiment showed a significantly greater number of necrotic foci in untreated livers compared to livers perfused with salicylate (score: control 3.5 6 0.5 vs salicylate 2.0 6 0.3, p 5 .04). Cell viability, as assessed by trypan blue exclusion, was 47 6 5% in the untreated group and 85 6 6% in the salicylate group ( p 5 .03) at the end of the experiments. Protein oxidation and lipid peroxidation Figure 3 shows the time course of protein oxidation, as expressed by PCC, in livers exposed to hypoxia/ reoxygenation experiments in the absence (group 3B) or presence (group 4B) of salicylate in the solution of perfusion. In the untreated group, PCC reached a value of 5.3 6 0.3 nmol/mg protein after 15 min of reoxygenation. This value was significantly greater than the values recorded at the end of the recovery and hypoxic phases
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Fig. 4. Lipid peroxidation, as evaluated by MDA production, at the end of 30 min of recovery, 60 min of hypoxia, and after 5, 15, and 30 min of reoxygenation in livers perfused in the presence (dark column) or absence of salicylate 2 mM. Reoxygenation causes a significant increase in MDA in control livers (one-way ANOVA, p 5 .0001). Salicylate prevents their increase in MDA levels observed during reoxygenation. The mean MDA levels were significantly lower at each time point during reoxygenation of the salicylate perfused livers compared to control experiments (*p # .01). Values represent the mean of six experiments 6 SEM.
(3.6 6 0.2 nmol/mg protein and 3.2 6 0.2 nmol/mg protein, respectively, one-way ANOVA p 5 .002). The livers exposed to hypoxia/reoxygenation in the presence of salicylate did not manifest such an increase in PCC during the reoxygenation phase. Rather, the level of hepatic protein oxidation remained steady throughout the experiment being 3.2 6 0.1 nmol/mg protein at end of the recovery period, 3.5 6 0.2 nmol/mg protein after 5 min, 3.8 6 0.1 nmol/mg protein after 15 min and 3.6 6 0.2 nmol/mg protein after 30 min of reoxygenation (oneway ANOVA p 5 NS). The levels of PCC of the untreated group were significantly greater than those of the salicylate group at each time point during the posthypoxic reoxygenation phase ( p # .02). Lipid peroxidation was assessed by MDA production. In livers exposed to hypoxia/reoxygenation without salicylate being added to the perfusate (group 3B), MDA formation increased during the reoxygenation phase, reaching a value of 8.9 6 0.7 nmol/mg protein after 5 min (5.7 6 0.4 nmol/mg protein at the end of recovery and 5.2 6 0.5 at the end of the hypoxic period, p , .001), and values of 10.6 6 1.1 nmol/mg protein after 15 min of reoxygenation, and further increased to 11.4 6 0.6 nmol/mg protein after 30 min of reoxygenation (one-way ANOVA, p , .0001). The livers perfused with salicylate (group 4B) maintained a steady low concentration of tissue MDA throughout the experiments, as shown in Fig. 4 (one-way ANOVA, NS). At each time point during the reoxygenation phase, the values of MDA recorded in the untreated livers were significantly greater than the values of treated livers ( p # .01).
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A. COLANTONI et al. DISCUSSION
ROS extreme reactivity makes the direct detection of their production in biological systems difficult. As a result, free radical activity is assessed commonly by indirect methods such as the measurement of various end products resulting from the interaction of the free radicals with other intracellular molecules.23,24 Several techniques have been developed to directly detect free radical production during hypoxia/reoxygenation in humans and experimental models. The aromatic hydroxylation of salicylate to DHBA has been recognized as a sensitive and reliable method to describe the time course of hydroxyl radical formation in heart, lung, brain, kidney, and plasma.11–14,25–28 The present study was aimed to describe the time course of hydroxyl radical formation during hypoxia/reoxygenation in an isolated-perfused rat liver model using salicylate as the trapping agent. A required characteristic of a reporter molecule is that it should not affect organ function at the concentrations being employed. The effect of the presence of salicylate 2 mM on the function of rat isolated liver has been assessed. In particular, livers exposed to aerobic condition, continuously perfused with or without salicylate being present in the solution of perfusion, were studied for 2 h. No differences in cell injury, as monitored by the rate of LDH release, and cell viability, as evaluated by trypan blue exclusion, were recorded between salicylate treated and untreated livers. The use of salicylate as a hydroxyl radical trapping agent in liver models may be complicated by salicylate catabolism to 2,5-DHBA by the microsomal P-450 system.18 It has been reported that 2,5-DHBA formation from salicylate is prevented by inhibiting cytochrome P-450 activity but not by the presence of hydroxyl radical scavengers or iron chelators. On the other hand, 2,3-DHBA formation from salicylate does just barely occur under normal conditions,18 and some authors suggest that only 2,3-DHBA formation should be taken as marker of hydroxyl radical production.11 In the present experience, under aerobic condition, slightly higher amounts of 2,5-DHBA than 2,3-DHBA were produced from salicylate by isolated livers throughout the experiments. Because the dissection of the portion of 2,5DHBA produced by salicylate metabolism from the one produced by the hydroxyl radical attack is extremely difficult, we took 2,3-DHBA formation as the marker of hydroxyl radical formation. In our model, the significant increase in the production of 2,3-DHBA suggests that hydroxyl radicals are produced in large amount within the liver during the early phase of the posthypoxic reoxygenation period. The time course of hydroxyl radical production in isolated perfused rat liver has been described by Togashi and colleagues29 using electron spin
resonance (ESR) spectroscopy. They reported a peak after 6 min of reoxygenation followed by a gradual decline over the following 30 min. In the present study, 2,3-DHBA and 2,5-DHBA concentrations in the perfusate reached a value two times higher than that recorded at the end of hypoxia after 5 min of reoxygenation and they increased progressively during the following 20 min. A possible explanation for this difference in patterns is the technique used. Importantly, the earlier ESR study was performed mixing hepatic effluent with the spin trapper DMPO after the collection of the perfusate sample. In our study the chemical trap (salicylate) was present in the solution of perfusion. A higher sensitivity for salicylate detection of hydroxyl radical production may also be responsible for the different pattern reported. Thus, salicylate more accurately detects and quantifies hydroxyl radical formation because it is both high specific for the radical and continuously present in the perfusate. Salicylate as a free radical trapping agent has several pragmatic advantages. Specifically, it is ready available, has a low cost, and it is easy to analyze and quantitate the various end products formed as a result of its reaction with hydroxyl radical. ROS are produced as part of normal cell metabolism.30 Under particular circumstances the high rate of ROS production exceeds the capacity of the cell to neutralize them. This is the case of post hypoxic reoxygenation, where ROS are produced in such great amounts that oxidative injury occurs.10 In particular, protein oxidation is characterized by the introduction of carbonyl groups in their amino acid structure31 and lipid peroxidation by the production of MDA.32–34 The present study reports that, in a isolated-perfused rat liver model, the early phase of reoxygenation that follows a 60-min period of warm hypoxia is characterized by the occurrence of protein oxidation and lipid peroxidation. These results are in line with what is reported consistently in literature. In particular, MDA levels have been described to reach maximal values after 10 min of reperfusion in isolated perfused rat liver.35 Both a high rate of LDH release during reoxygenation and a great amount of hepatocellular necrosis demonstrated by the light morphological examination at the end of the hypoxia/reoxygenation experiments were observed. In the present model, LDH release into the perfusate peaked after 5 min of reoxygenation and remained extremely high during the following 25 min of perfusion. The extremely high rate of LDH release during the first 5 min of reoxygenation that followed 60 min of low flow hypoxia may in part represent a washout from the liver. Other investigators have reported that the LDH released into the first 40 ml of perfusate after reperfusion following a prolonged period of no-flow ischemia is extremely high and probably represents a washout.36 In our study,
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the hypoxic phase is characterized by an extremely low flow (flow rate: 1 ml/min/g liver weight). Consequently, LDH may accumulate in the liver, and is only washed out when the flow rate increases during reoxygenation. It needs to be noted, however, that the rate of LDH release persisted elevated throughout the reoxygenation phase, suggesting a continuous release of the enzyme. Among ROS, hydroxyl radical is thought to be the most damaging species. Hydroxyl radicals are produced from both hydrogen peroxide, in the presence of iron (Fenton reaction), and superoxide anion (Haber-Weiss reaction).37 The toxicity of hydrogen peroxide and superoxide anion may be due to their conversion to hydroxyl radical. Thus, hydroxyl radical has been suggested to be the main responsible for protein oxidation and lipid peroxidation.37 The presence of salicylate, a chemical trap for hydroxyl radical, in the perfusion solution of isolated rat livers was associated with a significant reduction in both PCC and MDA production that characterizes the reoxygenation phase in the untreated organs. Moreover, the livers exposed to hypoxia/reoxygenation in the presence of the compound experienced a significantly reduced cell injury with respect to control experiments. In fact, both LDH release rate and the amount of hepatocellular necrosis reached lower levels in the livers treated with salicylate at a dose of 2 mM with respect to untreated livers. Our results show that, in the liver, salicylate effectively traps hydroxyl radicals, as manifested by the increase in its hydroxylation to 2,3DHBA during reoxygenation. Thus, such a dramatic reduction of hypoxia/reoxygenation injury of the liver by a hydroxyl radical scavenger may further underscore the putative pathogenic role of this particular reactive species in the pathogenesis of reoxygenation injury. The balance between the production and the removal of pro-oxidant agents during reoxygenation is the main determinant of their toxic action on intracellular molecules. The efficacy of salicylate in preventing liver hypoxia/reoxygenation injury may be due to its ability to trap hydroxyl radical. It can be postulated that its protective action is due to the inactivation of hydroxyl radicals, and this capacity is an antioxidant property. Local biotransformation of salicylate could play an important role in its ability to markedly attenuate liver oxidative injury due to hypoxia/reoxygenation. Iron is required for hydroxyl radical generation via Fenton reaction10 and catalyses lipid peroxidation reactions.38 It is important to remember that 2,3-DHBA produced as a result of hydroxyl radical attack on salicylate is a potent iron chelator.39 This event may contribute to salicylate protective action on reoxygenation injury: the production of hydroxyl radical via Fenton reaction could be decreased and the lipid peroxidation processes diminished either directly or indirectly by the reduced availability of
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iron. Finally, ROS are involved as signaling molecules in signal transduction processes. In particular, ROS are among the responsible for nuclear factor-kB activation (NF-kB).40 NF-kB induces the transcription of cytokines and proinflammatory agents. Salicylate has been recently shown to decrease NF-kB activation in human Jurkat T cells and mouse PD31 cells.41 This events can be involved in the explanation of the protective action of salicylate on reoxygenation injury, where inflammation mediators are thought to play an extremely important role.2,42 In conclusion, salicylate administrated to isolated rat livers exposed to hypoxia/reoxygenation protects the liver from protein oxidation and peroxidative injury acting as an antioxidant compound. It both scavenges hydroxyl radicals and reduces oxidative reactions. The mode of action of this interesting molecule with respect to the possible preventive action on liver reoxygenation injury needs to be further investigated, especially at the molecular level, to determine if salicylate can be used in vivo as a powerful tool to prevent hepatic hypoxia/ reoxygenation injury. REFERENCES 1. McCord, J. M. Oxygen derived free radicals in postischemic tissue injury. N. Engl. J. Med. 312:159 –164; 1985. 2. Land, W.; Messmer, K. The impact of ischemia/reperfusion injury on specific and nonspecific, early and late chronic events after organ transplantation. Early events. Transplant. Rev. 10:108 –121; 1996. 3. Connor, H. D.; Gao, W.; Nukina, S.; Lemasters, J. J.; Mason, R. P.; Thurman R. G. Evidence that free radicals are involved in graft failure following orthotopic liver transplantation in the rat—An electron paramagnetic resonance spin trapping study. Transplantation 54:199 –204; 1992. 4. Kalid, M. A.; Ashraf, M. Direct detection of endogenous hydroxyl radical production in cultured adult cardiomyocytes during anoxia and reoxygenation. Is the hydroxyl radical really the most damaging radical species? Circ. Res. 72:725–736; 1993. 5. Yu, B. P. Cellular defenses against damage from reactive oxygen species. Physiol. Rev. 74:139 –162; 1994. 6. Bzeizi, K. I.; Jalan, R.; Plevris, J. N.; Hayes, P. C. Primary graft dysfunction after liver transplantation: from pathogenesis to prevention. Liver Transplant. Surg. 3:137–148; 1997. 7. Clavein, P. A.; Harvey, P. R.; Strasberg, S. M. Preservation and reperfusion injuries in liver allografts. An overview and synthesis of current studies. Transplantation 53:957–978; 1992. 8. Lemasters, J. J.; Thurman, R. G. The many facets of reperfusion injury (editorial). Gastroenterology 108:1317–1320; 1995. 9. Caraceni, P.; Gasbarrini, A.; Van Thiel, D. H.; Borle, A. B. Oxygen free radical formation by hepatocytes during post-anoxic reoxygenation: scavenging effect of albumin. Am. J. Physiol. 266: G451–G458; 1994. 10. Sies, H. Oxidative stress: Oxidants and antioxidants. Exp. Physiol. 82:291–295; 1997. 11. Powell, S. R. Salicylate trapping of OH• as a tool for studying post-ischemic oxidative injury in the isolated rat heart. Free Radic. Res. 21:355–370; 1994. 12. Das, D. K.; George, A.; Liu, X. K.; Rao, P. S. Detection of hydroxyl radical in the mitochondria of ischemic–reperfused myocardium by trapping with salicylate. Biochem. Biophys. Res. Commun. 165:1004 –1009; 1989. 13. Delbarre, B.; Floyd, R. A.; Delbarre, G.; Calinon, F. Glutamate
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14. 15.
16.
17. 18. 19.
20. 21. 22.
23. 24. 25.
26. 27. 28. 29.
A. COLANTONI et al. accumulation and increased hydroxyl free radical formation in the abdominal aorta and heart of gerbil after ischemia/reperfusion injury. Free Radic. Biol. Med. 13:31–34; 1992. Kadkhodaee, M.; Endre, Z. H.; Towner, R. A.; Cross, M. Hydroxyl radical generation following ischaemia–reperfusion in cell-free perfused rat kidney. Biochem. Biophys. Acta 1243:169 –174; 1995. Liu, X.; Tosaki, A.; Engelman, R. M.; Das, D. K. Salicylate reduces ventricular dysfunction and arrythmias during reperfusion in isolated rat hearts. J. Cardiovasc. Pharmacol. 19:209 –215; 1992. Van Jaarsveld, H.; Kuyl, J. M.; Van Zyl, G. F.; Barnard, H. C. Salicylate in the perfusate during ischemia/reperfusion prevented mitochondrial injury. Res. Commun. Mol. Path. Pharmacol. 86: 287–295; 1994. Marotto, M. E.; Thurman, R. G.; Lemasters, J. J. Early midzonal cell death during low-flow hypoxia in the isolated, perfused rat liver: Protection by allopurinol. Hepatology 8:585–590; 1988. Ingelman-Sundberg, M.; Kaur, H.; Terelius, Y.; Persson, J. O.; Halliwell, B. Hydroxylation of salicylate by microsomal fractions and cytochrome P-450. Biochem. J. 276:753–757; 1991. Floyd, R. A.; Watson, J. J.; Wong, P. K. Sensitive assay of hydroxyl radical formation utilizing high pressure liquid chromatography with electrochemical detection of pheol and salicylate hydroxylation products. J. Biochem. Biophys. Methods 10:221– 235; 1984. Levine, R. L.; Williams, J. A.; Stadtman, E. R.; Shachter, E. Carbonyl assays for determination of oxydatively modified proteins. Methods Enzymol. 223:334 –339; 1994. Esterbauer, H. Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydoxynonenal. Methods Enzymol. 186:407– 421; 1990. Belinsky, S. A.; Popp, J. A.; Kauffman, F. C.; Thurman, R. G. Trypan blue uptake as a new method to investigate hepatotoxicity in periportal and pericentral regions of liver lobule. Studies with allyl alcohol in the perfused liver. J. Pharmacol. Exp. Ther. 230: 755–760; 1984. Holley, A. E.; Cheeseman, G. C. Measurement free radical reactions in vivo. Br. Med. Bull. 49:494 –505; 1993. Pryor, W. A.; Godber, S. S. Non invasive measures of oxidative stress status in humans. Free Radic. Biol. Med. 10:177–184; 1991. Coudray, C.; Talla, M.; Martin, S.; Fatome, M.; Favier, A. Highperformance liquid chromatography-electrochemical determination of salicylate hydroxylation products as an in vivo marker of oxidative stress. Anal. Biochem. 227:101–111; 1995. Grammas, P.; Liu, G. J.; Wood, K.; Floyd, R. A. Anoxia/reoxygenation induces hydroxyl free radical formation in brain microvessels. Free Radic. Biol. Med. 14:553–557; 1994. Fisher, P. W.; Hang, Y. C. T.; Kennedy, T. P.; Piantadosi, C. A. PO2-dependent hydroxyl radical production during ischemia-reperfusion lung injury. Am. J. Physiol. 265:L279 –L285; 1993. Kil, H. Y.; Zhang, J.; Piantadosi, C. A. Brain temperature alters hydroxyl radical production during cerebral ischemia/reperfusion in rats. J. Cereb. Blood Flow Metab. 16:100 –106; 1996. Togashi, H.; Shinzawa, H.; Yong, H.; Takahashi, T.; Noda, H.; Oikawa, K.; Kamada, H. Ascorbic acid radical, superoxide and hydroxyl radical are detected in reperfusion injury of rat liver using electron spin resonance spectroscopy. Arch. Biochem. Biophys. 308:1–7; 1994.
30. Kehrer, J. Free radicals as mediators of tissue injury and disease. Crit. Rev. Toxicol. 23:21– 48; 1993. 31. Stadtman, E. R. Metal ion-catalyzed oxidation of proteins: biochemical mechanism and biological consequences. Free Radic. Biol. Med. 9:315–325; 1990. 32. Tribble, D. L.; Aw, T. Y.; Jones, D. P. The pathophysiological significance of lipid peroxidation in oxidative cell injury. Hepatology 7:377–387; 1987. 33. Chazouilleres, O.; Calmus, Y.; Vaubourdolle, M.; Ballet, F. Preservation-induced liver injury. Clinical aspects, mechanisms and therapeutic approaches. J. Hepatol. 18:123–134; 1993. 34. Matthews, W. R.; Guido, D. M.; Fisher, M. A.; Jaeschke, H. Lipid peroxidation as molecular mechanism of liver cell injury during reperfusion after ischemia. Free Radic. Biol. Med. 16:763–770; 1994. 35. Zhong, Z.; Qu, W.; Connor, H. D.; Thurman, R. G. Role of Kupffer cells in the pathogenesis of hepatic reperfusion injury. Am. J. Physiol. 267:G630 –G636; 1994. 36. Fiegen, R. J.; Rauen, U.; Hartmann, M.; Decking, U. K.; deGroot, H. Decrease of ischemic injury to the isolated perfused rat liver by loop diuretics. Hepatology 25:1425–1431; 1997. 37. Kaur, H.; Halliwell, B. Detection of hydroxyl radicals by aromatic hydroxylation. Methods Enzymol. 233:67– 82; 1994. 38. Takemura, G.; Onodera, T.; Millard, R. W.; Ashraf, M. Demonstration of hydroxyl radical and its role in hydrogen peroxideinduced myocardial injury: Hydroxyl radical dependent and independent mechanisms. Free Radic. Biol. Med. 15:13–25; 1993. 39. Graziano, J. H.; Grady, R. W.; Cerami, A. The identification of 2,3-dihydroxybenzoic acid as a potentially useful iron-chelating agent. J. Pharmacol. Exp. Ther. 109:570 –575; 1974. 40. Schreck, R.; Rieber, P.; Bauerle, P. A. Reactive oxygen intermediates as apparently widely used messengers in the activation of NF-kB trascription factor and HIV-1. EMBO J. 10:2247–2258; 1991. 41. Kopp, E.; Ghosh, S. Inhibition of NF-kB by sodium salicylate and aspirin. Science 265:956 –959; 1994. 42. Colletti, L.; Kunkel, S. L.; Walz, A.; Burdick, M. D.; Kunkel, R. G.; Wilke, C. A.; Strieter, R. M. The role of cytokine networks in the local liver injury following hepatic ischemia/reperfusion in the rat. Hepatology 23:506 –514; 1996.
ABBREVIATIONS
2,3-DHBA—2,3-dihydroxybenzoic acid 2,5-DHBA—2,5-dihydroxybenzoic acid DHBA— dihydroxybenzoic acid ESR— electron spin resonance LDH—lactic dehydrogenase MDA—malondialdehyde NF-kB—nuclear factor-kB PCC—protein carbonyl content ROS—reactive oxygen species
PII S0891-5849(98)00033-1
Original Contribution PREVENTION OF REOXYGENATION INJURY BY SODIUM SALICYLATE IN ISOLATED-PERFUSED RAT LIVER ALESSANDRA COLANTONI,*† NICOLA de MARIA,* PAOLO CARACENI,† MAURO BERNARDI,† ROBERT A. FLOYD,‡ and DAVID H. VAN THIEL* *Division of Gastroenterology, Loyola University, Maywood, IL, USA; †Dipartimento di Medicina Interna, Cardioangiologia ed Epatologia, University of Bologna, Bologna, Italy, and ‡Free Radical Biology and Aging, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA (Received 16 October 1997; Revised 30 January 1998; Accepted 30 January 1998)
Abstract—Sodium salicylate can be used as a chemical trap for hydroxyl radicals, the most damaging reactive oxygen species. Because reactive oxygen species are involved in the pathogenesis of hepatic hypoxia/reoxygenation injury, the goal of this study was to determine if trapping hydroxyl radicals with salicylate would prevent or at least ameliorate such injury. Isolated rat livers, continuously perfused with Krebs-Henseleit bicarbonate buffer in the presence or absence of salicylate (2 mM), were exposed, after 30 min of recovery, to 60 min of hypoxia, followed by 30 min of reoxygenation. During reoxygenation, control livers experienced a sharp increase in the rate of lactic dehydrogenase release, taken as index of cell injury, protein carbonyl content, and malondialdehyde, taken as index of protein oxidation and lipid peroxidation, respectively. The presence of salicylate in the solution perfusion significantly reduced the rate of lactic dehydrogenase release, protein carbonyl content, and malondialdehyde production during reoxygenation. Hepatic histology documented a significantly reduced cell injury in salicylate-perfused livers compared to control livers. These data suggest that the hydroxyl radical chemical trap sodium salicylate, acting as an antioxidant, may represents an effective agent to reduce liver injury due to hypoxia/reoxygenation in a model of isolated-perfused rat liver. © 1998 Elsevier Science Inc. Keywords—Reoxygenation, Liver, Salicylate, Hydroxyl radical, Trapping agent, Oxidative stress, Free radical
INTRODUCTION
as well as to a partial or complete loss of important membrane-bound enzymatic activities.4,5 Oxidation of proteins impairs their function and enhances their catabolism. These intracellular alterations are presumed to be important in the pathogenesis of early liver graft dysfunction and/or failure in the transplant setting.6,7 Experimental data suggest that improvement in early allograft function requires protective action not only during the preservation phase of donor organ but also during the early phase of allograft reoxygenation in the recipient. Kupffer cells within the liver have been shown to be the major source of ROS during the post-hypoxic reoxygenation period,8 although both parenchymal9 and endothelial cells are also able to produce them.6 The balance between ROS production and radical scavenging activity is critically important in determining the oxidative injury experienced by any organ.10 Thus, the identification of a substance capable of reducing ROS-induced organ injury represents one of the most important
The susceptibility of the liver to hypoxia/reoxygenation represents one of the major remaining obstacles to the continued success of clinical liver transplantation. As with other organs, also in the liver the reoxygenation phase that follows a period of oxygen deprivation is characterized by the production of reactive oxygen species (ROS) at an accelerated rate.1–3 Several ROS have been identified, such as superoxide anion, hydrogen peroxide, and hydroxyl radical, the latter being recognized as the most nocive species. ROS are responsible for peroxidation of cell membrane lipids and protein oxidation. Peroxidation of membrane unsaturated fatty acids leads to changes in membrane permeability and integrity Address correspondence to: David H. Van Thiel, M.D., Division of Gastroenterology, Loyola University Medical Center, 2106 S. 1st Avenue, Building 114, Room 54, Maywood, IL 60153; Tel: (708) 2160364; Fax: (708) 216-0423. 87
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goals of research in the field of solid organ transplantation. Theoretically, trapping of the highly toxic hydroxyl radicals may significantly reduce the allograft injury experienced during postischemic reoxygenation. Sodium salicylate is a chemical trap for hydroxyl radical. The major hydroxylation products of hydroxyl radical attack on salicylate under physiological conditions are 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA).11 Either 2,3DHBA and/or 2,5-DHBA can be measured as markers of hydroxyl radical formation by high-pressure liquid chromatography with electrochemical detection methods. Thus, aromatic hydroxylation of sodium salicylate has been used as sensitive method for the measurement of hydroxyl radical formation in vivo.12–14 In an experimental model of myocardial ischemia, salicylate has been shown to reduce the severity of organ damage and the risk of postischemic ventricular fibrillation.15 Moreover, salicylate inhibited the disruption of mitochondrial oxidative phosphorylation function in isolated rat hearts.16 In the present study the time course of hydroxyl radical production within the liver during hypoxia and reoxygenation is described using salicylate as a trapping agent, and the effect of salicylate on reoxygenation injury has been assessed in a model of isolated-perfused rat liver. MATERIALS AND METHODS
Animals Male Wistar rats weighting 260 –300 g (Harlan–Sprague–Dawley, Houston, TX) were used for this study. All animals were allowed free access to standard rat chow diet and water. Only fed animals were used in the experiments described. The present research protocol complied with the criteria for animal care and use at our institution, which conforms to National Institutes of Health guidelines. Liver perfusion The animals were anesthetized using an intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight). The abdomen was opened by a median laparotomy and the liver was exposed. After isolation, the livers were continuously perfused with protein and hemoglobin-free Krebs-Henseleit bicarbonate buffer (pH 7.4). The perfusate was pumped into the liver via a cannula placed in the portal vein and the hepatic effluent was collected from a second cannula placed in the vena cava through the right atrium. The perfusate was not recirculated. During the study period the liver temperature was monitored and maintained at 37°C using a heating lamp.
Experimental protocol The animals were divided in the following groups: (1) Aerobic controls (n 5 12). The livers were perfused continuously with oxygenated Krebs-Henseleit bicarbonate buffer solution (pH 7.4) for 1 h (group 1A) or 2 h (group 1B). For all perfusions under aerobic conditions, the perfusate was saturated with oxygen and the flow rate was 3– 4 ml/min/g of liver weight. Under these conditions, the liver is normoxic.17 Perfusate samples were collected every 10 min, and tissue samples at the end of the experiments. (2) Salicylate aerobic controls (n 5 12). The livers were perfused with oxygenated KrebsHenseleit bicarbonate buffer solution supplemented with salicylate 2 mM for 1 h (group 2A) and 2 h (group 2B). Perfusate samples were collected every 10 min during the experiments and tissue samples were obtained at the end of the experiments. (3) Hypoxia-reoxygenation (n 5 12). The livers were perfused with oxygenated KrebsHenseleit bicarbonate buffer solution for 30 min after isolation (recovery period) then subjected to 60 min of hypoxia (hypoxic phase) followed by 30 min of reoxygenation. During the recovery and reoxygenation periods, the perfusion solution was equilibrated with oxygen (95% oxygen, 5% carbon dioxide) and the flow rate was 3– 4 ml/min/g of liver. During hypoxia, the perfusate was equilibrated with nitrogen (95% nitrogen, 5% carbon dioxide). One group of livers was used for the collection of perfusate samples at the beginning and at the end of recovery and hypoxia phases and after 5, 10, 15, 20, and 30 min of reoxygenation (group 3A). Tissue samples were collected from a second group of livers, at the end of the recovery, at the end of hypoxic period and after 5, 15, and 30 min of reoxygenation (group 3B). (4) Salicylate hypoxia-reoxygenation (n 5 12). After isolation, the livers underwent 30 min of recovery followed by 60 min of hypoxia and 30 min of reoxygenation, as described above. Salicylate (2 mM) was added to the perfusion solution. Perfusate samples were collected at the beginning and at the end of recovery and hypoxia phases and after 5, 10, 15, 20, and 30 min of reoxygenation (group 4A). Tissue samples were collected from a second group of livers at the end of the recovery, at the end of hypoxic period and after 5, 15, and 30 min of reoxygenation (group 4B). Lactic dehydrogenase (LDH) release The rate of LDH release into the perfusate was measured as a marker of liver injury. LDH was assessed utilizing a spectrophotometric method based on the oxidation of lactate to pyruvate (Sigma Diagnostic, St. Louis, MO). To exclude any effect of salicylate on the assay, two standard solutions reconstituted either in
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buffer solution or in buffer solution plus salicylate were compared. The presence of salicylate in the perfusate did not affect the analytical measurements used for LDH. The rate of LDH release is expressed as mU/min. Hydroxyl radical production The attack by hydroxyl radical upon salicylate leads to the formation of 2,3-DHBA and 2,5-DHBA.11–14,18 Salicylate, 2,3-DHBA, and 2,5-DHBA concentrations in perfusate samples collected at the end of recovery time, at the beginning and end of hypoxia and after 5, 10, 15, 20, and 30 min of reoxygenation, were quantitated using high-pressure liquid chromatography with an electrochemical detector as reported by Floyd et al.19 The amount of DHBA and residual salicylate were determined by comparison of the peak heights of the samples to that of 2,3-DHBA, 2,5-DHBA, and salicylate standards. The ratio total DHBA/salicylate in the perfusate at different time points was also calculated.
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of 0.2 mM, as described by Belinsky et al.22 At the end of the hypoxia/reoxygenation experiments and in aerobic controls (after 1 h and 2 h), the livers were perfused with trypan blue for 5 min and fixed in a solution containing 1% paraformaldehyde. Tissue samples were embedded in paraffin and processed for light microscopy evaluation. Four fields were selected per liver, and the percentage of stained cells quantitated. Statistical analysis All data are expressed as the mean of six experiments 6 standard error of the mean (SEM). The level of significance between treated and untreated groups was determined using either the Student’s t-test or ANOVA as appropriate. A p-value less than .05 was considered significant. RESULTS
Effect of salicylate on liver cell injury and viability Protein carbonyl content Protein carbonyl content (PCC), a marker of protein oxidation, was measured in homogenates of liver tissue by the 2,4 dinitrophenylhydrazine procedure according to Levine and colleagues.20 The total protein content of each tissue sample was determined using the bicinchonic acid assay (Pierce Chemical Company, Rockford, IL). Malondialdehyde The extent of lipid peroxidation was assessed by measuring malondialdehyde (MDA) in liver tissue homogenates using a standard colorimetric assay (Calbiochem, La Jolla, CA) according to the method of Esterbauer et al.21 Morphological examination The histologic evaluations of liver samples collected at the end of the experiments were performed using paraffin embedded, hematoxylin/eosin stained tissue. The severity of tissue injury was assessed as number of foci of necrosis per section and was scored by a single observer not aware of the group assignment using a scale ranging from 0 (no injury) to 41 (panlobular injury). Trypan blue Trypan blue is a vital dye that stains the nuclei of non viable cells. To evaluate hepatic cell viability, the dye was added to the solution of perfusion at a concentration
To evaluate the effect of the presence of salicylate 2 mM on hepatic cells, isolated livers continuously perfused in aerobic conditions with or without salicylate being added to the solution of perfusion were studied. Cell injury was monitored by measuring the rate of LDH release in samples of perfusate collected every 10 min during the experiments. Cell viability was evaluated by trypan blue exclusion after 1 h (groups 1A and 2A) and 2 h (groups 1B and 2B) of aerobic perfusion. Under aerobic conditions, the rate of hepatic release of LDH in control and salicylate-treated livers was 237 6 48 mU/ min and 200 6 29 mU/min, respectively. This rate remained steady in both groups for the 2 h of aerobic perfusion, reaching 300 6 51 mU/min and 286 6 62 mU/min, respectively, at the end of the experiments (one-way ANOVA, NS). No differences in the rate of LDH release between groups were recorded at each time point. Trypan blue exclusion after 1 h of aerobic perfusion was 95 6 2% in the livers perfused without salicylate (group 1A) and 97 6 3% in the salicylate-treated group (group 2A). After 2 h, cell viability was 92 6 3% in group 1B and 93 6 4% in group 2B. Production of hydroxyl radicals in the liver Figure 1 shows the time course of the production of 2,3-DHBA, 2,5-DHBA, and the ratio total DHBA/salicylate in the perfusate from livers exposed to 60 min of hypoxia followed by 30 min of reoxygenation and from aerobic controls. The 2,3-DHBA concentration in the perfusate remained steady during 2 h of liver perfusion under aerobic
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0.3 nmol/ml after 15 min of reoxygenation (one-way ANOVA p 5 .002). The 2,5-DHBA concentration in the perfusate remained steady under aerobic conditions, being 3.4 6 0.3 nmol/ml at the beginning of the experiments and 3.1 6 0.2 nmol/ml after 2 h of liver perfusion where the perfusate was saturated with oxygen (group 2B). Under the imposed experimental protocol, the perfusate concentration of 2,5-DHBA was 3.2 6 0.4 nmol/ml at the end of the recovery period and 2.4 6 0.3 nmol/ml at the end of 60 min of hypoxia. However, an increase in 2,5-DHBA concentration in the perfusate to 7.7 6 0.7 nmol/ml was recorded after 15 min of reoxygenation (group 4A) (oneway ANOVA p 5 .001). The DHBA/salicylate ratio remained constant during 2 h of liver perfusion under aerobic conditions (group 2B). A sharp increase in the total DHBA/salicylate ratio was recorded during the early phase of reoxygenation, being 0.70 6 0.06 after 10 min and 1.28 6 0.05 at the end of the experiments (group 4A) (recovery period: 0.36 6 0.01, end of hypoxia 0.32 6 0.02, one-way ANOVA p , .0001). Cell injury Figure 2 shows the rate of LDH release during recovery, hypoxia, and reoxygenation in untreated livers (group 3A) and in livers perfused with salicylate being added to the solution of perfusion (group 4A). In untreated livers, LDH release increased significantly during the reoxygenation phase to a level greater than that experienced during the hypoxic period. Specifically, LDH release, being 179 6 28 mU/min at the end of recovery and 258 6 85 mU/min at the end of the hypoxic
Fig. 1. Time course of 2,3-DHBA and 2,5-DHBA production from salicylate and total DHBA/salicylate ratio in isolated perfused rat livers exposed to 60 min of hypoxia followed by 30 min of reoxygenation (dashed line). Both 2,3-DHBA and 2,5-DHBA concentrations in the perfusate are significantly higher during the reoxygenation phase of the study (90 –120 min) than during the recovery and hypoxic phases. 2,3-DHBA and 2,5-DHBA production from salicylate in the aerobic controls (solid line) remained steady for the 2 h studied. Values represent the means of six experiments 6 SEM.
condition being 1.2 6 0.3 nmol/ml at the beginning of the experiments and 1.3 6 0.3 nmol/ml after 2 h of perfusion (group 2B). A sharp increase in 2,3-DHBA concentration in the perfusate (group 4A) was recorded during the early phase of reoxygenation that followed 60 min of warm hypoxia being 1.2 6 0.3 nmol/ml at the end of the recovery period, 1.1 6 0.1 nmol/ml at the end of the period of hypoxia and 2.0 6
Fig. 2. Cell injury assessed by the rate of LDH release into the perfusate during hypoxia and reoxygenation of rat liver perfused in the presence or absence of 2 mM salicylate. In the livers perfused without salicylate (solid line), LDH release markedly increased during reoxygenation (one-way ANOVA, p 5 .0001). In the presence of salicylate (dashed line), LDH release did not increase during the reoxygenation phase of the experiment. In the salicylate group, the rate of LDH release during reoxygenation was significantly lower at each time point compared to the control experiments (*p # .001). Values represent the mean of six experiments 6 SEM.
Reoxygenation injury and sodium salicylate
Fig. 3. Protein oxidation, evaluated as protein carbonyl content, at the end of 30 min of recovery, 60 min of hypoxia, and after 5, 15, and 30 min of reoxygenation in isolated livers perfused in the presence (dark columns) or absence of salicylate 2 mM. Reoxygenation causes a significant increase in protein oxidation in the livers perfused in the absence of salicylate in the solution of perfusion (one-way ANOVA, p 5 .002). Salicylate prevents the increase in liver protein oxidation observed during reoxygenation (one-way ANOVA, p 5 .267). The mean protein carbonyl content levels were significantly lower at each time point during reoxygenation of the livers perfused in the presence of salicylate compared to control experiments (*p # .02). Values represent the mean of six experiments 6 SEM.
phase, reached a level of 2768 6 86 mU/min after 5 min of reoxygenation ( p , .0001). The presence of salicylate in the perfusion solution significantly reduced the rate of LDH release during the reoxygenation. In fact, no statistically significant changes in LDH release were recorded during hypoxia and reoxygenation phases in the salicylate-treated group 4A (one-way ANOVA, NS). For each time point during reoxygenation the rate of LDH release by the untreated livers was significantly higher than that by livers continuously perfused with a solution containing salicylate ( p # .001). Light microscopic examination of the livers at the end of each hypoxia/reoxygenation experiment showed a significantly greater number of necrotic foci in untreated livers compared to livers perfused with salicylate (score: control 3.5 6 0.5 vs salicylate 2.0 6 0.3, p 5 .04). Cell viability, as assessed by trypan blue exclusion, was 47 6 5% in the untreated group and 85 6 6% in the salicylate group ( p 5 .03) at the end of the experiments. Protein oxidation and lipid peroxidation Figure 3 shows the time course of protein oxidation, as expressed by PCC, in livers exposed to hypoxia/ reoxygenation experiments in the absence (group 3B) or presence (group 4B) of salicylate in the solution of perfusion. In the untreated group, PCC reached a value of 5.3 6 0.3 nmol/mg protein after 15 min of reoxygenation. This value was significantly greater than the values recorded at the end of the recovery and hypoxic phases
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Fig. 4. Lipid peroxidation, as evaluated by MDA production, at the end of 30 min of recovery, 60 min of hypoxia, and after 5, 15, and 30 min of reoxygenation in livers perfused in the presence (dark column) or absence of salicylate 2 mM. Reoxygenation causes a significant increase in MDA in control livers (one-way ANOVA, p 5 .0001). Salicylate prevents their increase in MDA levels observed during reoxygenation. The mean MDA levels were significantly lower at each time point during reoxygenation of the salicylate perfused livers compared to control experiments (*p # .01). Values represent the mean of six experiments 6 SEM.
(3.6 6 0.2 nmol/mg protein and 3.2 6 0.2 nmol/mg protein, respectively, one-way ANOVA p 5 .002). The livers exposed to hypoxia/reoxygenation in the presence of salicylate did not manifest such an increase in PCC during the reoxygenation phase. Rather, the level of hepatic protein oxidation remained steady throughout the experiment being 3.2 6 0.1 nmol/mg protein at end of the recovery period, 3.5 6 0.2 nmol/mg protein after 5 min, 3.8 6 0.1 nmol/mg protein after 15 min and 3.6 6 0.2 nmol/mg protein after 30 min of reoxygenation (oneway ANOVA p 5 NS). The levels of PCC of the untreated group were significantly greater than those of the salicylate group at each time point during the posthypoxic reoxygenation phase ( p # .02). Lipid peroxidation was assessed by MDA production. In livers exposed to hypoxia/reoxygenation without salicylate being added to the perfusate (group 3B), MDA formation increased during the reoxygenation phase, reaching a value of 8.9 6 0.7 nmol/mg protein after 5 min (5.7 6 0.4 nmol/mg protein at the end of recovery and 5.2 6 0.5 at the end of the hypoxic period, p , .001), and values of 10.6 6 1.1 nmol/mg protein after 15 min of reoxygenation, and further increased to 11.4 6 0.6 nmol/mg protein after 30 min of reoxygenation (one-way ANOVA, p , .0001). The livers perfused with salicylate (group 4B) maintained a steady low concentration of tissue MDA throughout the experiments, as shown in Fig. 4 (one-way ANOVA, NS). At each time point during the reoxygenation phase, the values of MDA recorded in the untreated livers were significantly greater than the values of treated livers ( p # .01).
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A. COLANTONI et al. DISCUSSION
ROS extreme reactivity makes the direct detection of their production in biological systems difficult. As a result, free radical activity is assessed commonly by indirect methods such as the measurement of various end products resulting from the interaction of the free radicals with other intracellular molecules.23,24 Several techniques have been developed to directly detect free radical production during hypoxia/reoxygenation in humans and experimental models. The aromatic hydroxylation of salicylate to DHBA has been recognized as a sensitive and reliable method to describe the time course of hydroxyl radical formation in heart, lung, brain, kidney, and plasma.11–14,25–28 The present study was aimed to describe the time course of hydroxyl radical formation during hypoxia/reoxygenation in an isolated-perfused rat liver model using salicylate as the trapping agent. A required characteristic of a reporter molecule is that it should not affect organ function at the concentrations being employed. The effect of the presence of salicylate 2 mM on the function of rat isolated liver has been assessed. In particular, livers exposed to aerobic condition, continuously perfused with or without salicylate being present in the solution of perfusion, were studied for 2 h. No differences in cell injury, as monitored by the rate of LDH release, and cell viability, as evaluated by trypan blue exclusion, were recorded between salicylate treated and untreated livers. The use of salicylate as a hydroxyl radical trapping agent in liver models may be complicated by salicylate catabolism to 2,5-DHBA by the microsomal P-450 system.18 It has been reported that 2,5-DHBA formation from salicylate is prevented by inhibiting cytochrome P-450 activity but not by the presence of hydroxyl radical scavengers or iron chelators. On the other hand, 2,3-DHBA formation from salicylate does just barely occur under normal conditions,18 and some authors suggest that only 2,3-DHBA formation should be taken as marker of hydroxyl radical production.11 In the present experience, under aerobic condition, slightly higher amounts of 2,5-DHBA than 2,3-DHBA were produced from salicylate by isolated livers throughout the experiments. Because the dissection of the portion of 2,5DHBA produced by salicylate metabolism from the one produced by the hydroxyl radical attack is extremely difficult, we took 2,3-DHBA formation as the marker of hydroxyl radical formation. In our model, the significant increase in the production of 2,3-DHBA suggests that hydroxyl radicals are produced in large amount within the liver during the early phase of the posthypoxic reoxygenation period. The time course of hydroxyl radical production in isolated perfused rat liver has been described by Togashi and colleagues29 using electron spin
resonance (ESR) spectroscopy. They reported a peak after 6 min of reoxygenation followed by a gradual decline over the following 30 min. In the present study, 2,3-DHBA and 2,5-DHBA concentrations in the perfusate reached a value two times higher than that recorded at the end of hypoxia after 5 min of reoxygenation and they increased progressively during the following 20 min. A possible explanation for this difference in patterns is the technique used. Importantly, the earlier ESR study was performed mixing hepatic effluent with the spin trapper DMPO after the collection of the perfusate sample. In our study the chemical trap (salicylate) was present in the solution of perfusion. A higher sensitivity for salicylate detection of hydroxyl radical production may also be responsible for the different pattern reported. Thus, salicylate more accurately detects and quantifies hydroxyl radical formation because it is both high specific for the radical and continuously present in the perfusate. Salicylate as a free radical trapping agent has several pragmatic advantages. Specifically, it is ready available, has a low cost, and it is easy to analyze and quantitate the various end products formed as a result of its reaction with hydroxyl radical. ROS are produced as part of normal cell metabolism.30 Under particular circumstances the high rate of ROS production exceeds the capacity of the cell to neutralize them. This is the case of post hypoxic reoxygenation, where ROS are produced in such great amounts that oxidative injury occurs.10 In particular, protein oxidation is characterized by the introduction of carbonyl groups in their amino acid structure31 and lipid peroxidation by the production of MDA.32–34 The present study reports that, in a isolated-perfused rat liver model, the early phase of reoxygenation that follows a 60-min period of warm hypoxia is characterized by the occurrence of protein oxidation and lipid peroxidation. These results are in line with what is reported consistently in literature. In particular, MDA levels have been described to reach maximal values after 10 min of reperfusion in isolated perfused rat liver.35 Both a high rate of LDH release during reoxygenation and a great amount of hepatocellular necrosis demonstrated by the light morphological examination at the end of the hypoxia/reoxygenation experiments were observed. In the present model, LDH release into the perfusate peaked after 5 min of reoxygenation and remained extremely high during the following 25 min of perfusion. The extremely high rate of LDH release during the first 5 min of reoxygenation that followed 60 min of low flow hypoxia may in part represent a washout from the liver. Other investigators have reported that the LDH released into the first 40 ml of perfusate after reperfusion following a prolonged period of no-flow ischemia is extremely high and probably represents a washout.36 In our study,
Reoxygenation injury and sodium salicylate
the hypoxic phase is characterized by an extremely low flow (flow rate: 1 ml/min/g liver weight). Consequently, LDH may accumulate in the liver, and is only washed out when the flow rate increases during reoxygenation. It needs to be noted, however, that the rate of LDH release persisted elevated throughout the reoxygenation phase, suggesting a continuous release of the enzyme. Among ROS, hydroxyl radical is thought to be the most damaging species. Hydroxyl radicals are produced from both hydrogen peroxide, in the presence of iron (Fenton reaction), and superoxide anion (Haber-Weiss reaction).37 The toxicity of hydrogen peroxide and superoxide anion may be due to their conversion to hydroxyl radical. Thus, hydroxyl radical has been suggested to be the main responsible for protein oxidation and lipid peroxidation.37 The presence of salicylate, a chemical trap for hydroxyl radical, in the perfusion solution of isolated rat livers was associated with a significant reduction in both PCC and MDA production that characterizes the reoxygenation phase in the untreated organs. Moreover, the livers exposed to hypoxia/reoxygenation in the presence of the compound experienced a significantly reduced cell injury with respect to control experiments. In fact, both LDH release rate and the amount of hepatocellular necrosis reached lower levels in the livers treated with salicylate at a dose of 2 mM with respect to untreated livers. Our results show that, in the liver, salicylate effectively traps hydroxyl radicals, as manifested by the increase in its hydroxylation to 2,3DHBA during reoxygenation. Thus, such a dramatic reduction of hypoxia/reoxygenation injury of the liver by a hydroxyl radical scavenger may further underscore the putative pathogenic role of this particular reactive species in the pathogenesis of reoxygenation injury. The balance between the production and the removal of pro-oxidant agents during reoxygenation is the main determinant of their toxic action on intracellular molecules. The efficacy of salicylate in preventing liver hypoxia/reoxygenation injury may be due to its ability to trap hydroxyl radical. It can be postulated that its protective action is due to the inactivation of hydroxyl radicals, and this capacity is an antioxidant property. Local biotransformation of salicylate could play an important role in its ability to markedly attenuate liver oxidative injury due to hypoxia/reoxygenation. Iron is required for hydroxyl radical generation via Fenton reaction10 and catalyses lipid peroxidation reactions.38 It is important to remember that 2,3-DHBA produced as a result of hydroxyl radical attack on salicylate is a potent iron chelator.39 This event may contribute to salicylate protective action on reoxygenation injury: the production of hydroxyl radical via Fenton reaction could be decreased and the lipid peroxidation processes diminished either directly or indirectly by the reduced availability of
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iron. Finally, ROS are involved as signaling molecules in signal transduction processes. In particular, ROS are among the responsible for nuclear factor-kB activation (NF-kB).40 NF-kB induces the transcription of cytokines and proinflammatory agents. Salicylate has been recently shown to decrease NF-kB activation in human Jurkat T cells and mouse PD31 cells.41 This events can be involved in the explanation of the protective action of salicylate on reoxygenation injury, where inflammation mediators are thought to play an extremely important role.2,42 In conclusion, salicylate administrated to isolated rat livers exposed to hypoxia/reoxygenation protects the liver from protein oxidation and peroxidative injury acting as an antioxidant compound. It both scavenges hydroxyl radicals and reduces oxidative reactions. The mode of action of this interesting molecule with respect to the possible preventive action on liver reoxygenation injury needs to be further investigated, especially at the molecular level, to determine if salicylate can be used in vivo as a powerful tool to prevent hepatic hypoxia/ reoxygenation injury. REFERENCES 1. McCord, J. M. Oxygen derived free radicals in postischemic tissue injury. N. Engl. J. Med. 312:159 –164; 1985. 2. Land, W.; Messmer, K. The impact of ischemia/reperfusion injury on specific and nonspecific, early and late chronic events after organ transplantation. Early events. Transplant. Rev. 10:108 –121; 1996. 3. Connor, H. D.; Gao, W.; Nukina, S.; Lemasters, J. J.; Mason, R. P.; Thurman R. G. Evidence that free radicals are involved in graft failure following orthotopic liver transplantation in the rat—An electron paramagnetic resonance spin trapping study. Transplantation 54:199 –204; 1992. 4. Kalid, M. A.; Ashraf, M. Direct detection of endogenous hydroxyl radical production in cultured adult cardiomyocytes during anoxia and reoxygenation. Is the hydroxyl radical really the most damaging radical species? Circ. Res. 72:725–736; 1993. 5. Yu, B. P. Cellular defenses against damage from reactive oxygen species. Physiol. Rev. 74:139 –162; 1994. 6. Bzeizi, K. I.; Jalan, R.; Plevris, J. N.; Hayes, P. C. Primary graft dysfunction after liver transplantation: from pathogenesis to prevention. Liver Transplant. Surg. 3:137–148; 1997. 7. Clavein, P. A.; Harvey, P. R.; Strasberg, S. M. Preservation and reperfusion injuries in liver allografts. An overview and synthesis of current studies. Transplantation 53:957–978; 1992. 8. Lemasters, J. J.; Thurman, R. G. The many facets of reperfusion injury (editorial). Gastroenterology 108:1317–1320; 1995. 9. Caraceni, P.; Gasbarrini, A.; Van Thiel, D. H.; Borle, A. B. Oxygen free radical formation by hepatocytes during post-anoxic reoxygenation: scavenging effect of albumin. Am. J. Physiol. 266: G451–G458; 1994. 10. Sies, H. Oxidative stress: Oxidants and antioxidants. Exp. Physiol. 82:291–295; 1997. 11. Powell, S. R. Salicylate trapping of OH• as a tool for studying post-ischemic oxidative injury in the isolated rat heart. Free Radic. Res. 21:355–370; 1994. 12. Das, D. K.; George, A.; Liu, X. K.; Rao, P. S. Detection of hydroxyl radical in the mitochondria of ischemic–reperfused myocardium by trapping with salicylate. Biochem. Biophys. Res. Commun. 165:1004 –1009; 1989. 13. Delbarre, B.; Floyd, R. A.; Delbarre, G.; Calinon, F. Glutamate
94
14. 15.
16.
17. 18. 19.
20. 21. 22.
23. 24. 25.
26. 27. 28. 29.
A. COLANTONI et al. accumulation and increased hydroxyl free radical formation in the abdominal aorta and heart of gerbil after ischemia/reperfusion injury. Free Radic. Biol. Med. 13:31–34; 1992. Kadkhodaee, M.; Endre, Z. H.; Towner, R. A.; Cross, M. Hydroxyl radical generation following ischaemia–reperfusion in cell-free perfused rat kidney. Biochem. Biophys. Acta 1243:169 –174; 1995. Liu, X.; Tosaki, A.; Engelman, R. M.; Das, D. K. Salicylate reduces ventricular dysfunction and arrythmias during reperfusion in isolated rat hearts. J. Cardiovasc. Pharmacol. 19:209 –215; 1992. Van Jaarsveld, H.; Kuyl, J. M.; Van Zyl, G. F.; Barnard, H. C. Salicylate in the perfusate during ischemia/reperfusion prevented mitochondrial injury. Res. Commun. Mol. Path. Pharmacol. 86: 287–295; 1994. Marotto, M. E.; Thurman, R. G.; Lemasters, J. J. Early midzonal cell death during low-flow hypoxia in the isolated, perfused rat liver: Protection by allopurinol. Hepatology 8:585–590; 1988. Ingelman-Sundberg, M.; Kaur, H.; Terelius, Y.; Persson, J. O.; Halliwell, B. Hydroxylation of salicylate by microsomal fractions and cytochrome P-450. Biochem. J. 276:753–757; 1991. Floyd, R. A.; Watson, J. J.; Wong, P. K. Sensitive assay of hydroxyl radical formation utilizing high pressure liquid chromatography with electrochemical detection of pheol and salicylate hydroxylation products. J. Biochem. Biophys. Methods 10:221– 235; 1984. Levine, R. L.; Williams, J. A.; Stadtman, E. R.; Shachter, E. Carbonyl assays for determination of oxydatively modified proteins. Methods Enzymol. 223:334 –339; 1994. Esterbauer, H. Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydoxynonenal. Methods Enzymol. 186:407– 421; 1990. Belinsky, S. A.; Popp, J. A.; Kauffman, F. C.; Thurman, R. G. Trypan blue uptake as a new method to investigate hepatotoxicity in periportal and pericentral regions of liver lobule. Studies with allyl alcohol in the perfused liver. J. Pharmacol. Exp. Ther. 230: 755–760; 1984. Holley, A. E.; Cheeseman, G. C. Measurement free radical reactions in vivo. Br. Med. Bull. 49:494 –505; 1993. Pryor, W. A.; Godber, S. S. Non invasive measures of oxidative stress status in humans. Free Radic. Biol. Med. 10:177–184; 1991. Coudray, C.; Talla, M.; Martin, S.; Fatome, M.; Favier, A. Highperformance liquid chromatography-electrochemical determination of salicylate hydroxylation products as an in vivo marker of oxidative stress. Anal. Biochem. 227:101–111; 1995. Grammas, P.; Liu, G. J.; Wood, K.; Floyd, R. A. Anoxia/reoxygenation induces hydroxyl free radical formation in brain microvessels. Free Radic. Biol. Med. 14:553–557; 1994. Fisher, P. W.; Hang, Y. C. T.; Kennedy, T. P.; Piantadosi, C. A. PO2-dependent hydroxyl radical production during ischemia-reperfusion lung injury. Am. J. Physiol. 265:L279 –L285; 1993. Kil, H. Y.; Zhang, J.; Piantadosi, C. A. Brain temperature alters hydroxyl radical production during cerebral ischemia/reperfusion in rats. J. Cereb. Blood Flow Metab. 16:100 –106; 1996. Togashi, H.; Shinzawa, H.; Yong, H.; Takahashi, T.; Noda, H.; Oikawa, K.; Kamada, H. Ascorbic acid radical, superoxide and hydroxyl radical are detected in reperfusion injury of rat liver using electron spin resonance spectroscopy. Arch. Biochem. Biophys. 308:1–7; 1994.
30. Kehrer, J. Free radicals as mediators of tissue injury and disease. Crit. Rev. Toxicol. 23:21– 48; 1993. 31. Stadtman, E. R. Metal ion-catalyzed oxidation of proteins: biochemical mechanism and biological consequences. Free Radic. Biol. Med. 9:315–325; 1990. 32. Tribble, D. L.; Aw, T. Y.; Jones, D. P. The pathophysiological significance of lipid peroxidation in oxidative cell injury. Hepatology 7:377–387; 1987. 33. Chazouilleres, O.; Calmus, Y.; Vaubourdolle, M.; Ballet, F. Preservation-induced liver injury. Clinical aspects, mechanisms and therapeutic approaches. J. Hepatol. 18:123–134; 1993. 34. Matthews, W. R.; Guido, D. M.; Fisher, M. A.; Jaeschke, H. Lipid peroxidation as molecular mechanism of liver cell injury during reperfusion after ischemia. Free Radic. Biol. Med. 16:763–770; 1994. 35. Zhong, Z.; Qu, W.; Connor, H. D.; Thurman, R. G. Role of Kupffer cells in the pathogenesis of hepatic reperfusion injury. Am. J. Physiol. 267:G630 –G636; 1994. 36. Fiegen, R. J.; Rauen, U.; Hartmann, M.; Decking, U. K.; deGroot, H. Decrease of ischemic injury to the isolated perfused rat liver by loop diuretics. Hepatology 25:1425–1431; 1997. 37. Kaur, H.; Halliwell, B. Detection of hydroxyl radicals by aromatic hydroxylation. Methods Enzymol. 233:67– 82; 1994. 38. Takemura, G.; Onodera, T.; Millard, R. W.; Ashraf, M. Demonstration of hydroxyl radical and its role in hydrogen peroxideinduced myocardial injury: Hydroxyl radical dependent and independent mechanisms. Free Radic. Biol. Med. 15:13–25; 1993. 39. Graziano, J. H.; Grady, R. W.; Cerami, A. The identification of 2,3-dihydroxybenzoic acid as a potentially useful iron-chelating agent. J. Pharmacol. Exp. Ther. 109:570 –575; 1974. 40. Schreck, R.; Rieber, P.; Bauerle, P. A. Reactive oxygen intermediates as apparently widely used messengers in the activation of NF-kB trascription factor and HIV-1. EMBO J. 10:2247–2258; 1991. 41. Kopp, E.; Ghosh, S. Inhibition of NF-kB by sodium salicylate and aspirin. Science 265:956 –959; 1994. 42. Colletti, L.; Kunkel, S. L.; Walz, A.; Burdick, M. D.; Kunkel, R. G.; Wilke, C. A.; Strieter, R. M. The role of cytokine networks in the local liver injury following hepatic ischemia/reperfusion in the rat. Hepatology 23:506 –514; 1996.
ABBREVIATIONS
2,3-DHBA—2,3-dihydroxybenzoic acid 2,5-DHBA—2,5-dihydroxybenzoic acid DHBA— dihydroxybenzoic acid ESR— electron spin resonance LDH—lactic dehydrogenase MDA—malondialdehyde NF-kB—nuclear factor-kB PCC—protein carbonyl content ROS—reactive oxygen species