Hepatic ischemia-reperfusion injury (HIRI) is defined as liver tissue damage and cell death caused by reperfusion during liver transplantation or hepatectomy. Oxidative stress is one of the important mechanisms of HIRI. Studies have shown that the incidence of HIRI is very high, however, the number of patients who can get timely and efficient treatment is small. The reason is not hard to explain that invasive ways of detection and lack of timely of diagnostic methods. Hence, a new detection method is urgently needed in clinic application. Reactive oxygen species (ROS), which are markers of oxidative stress in the liver, could be detected by optical imaging and offer timely and effective non-invasive diagnosis and monitoring. Optical imaging could become the most potential tool of diagnosis of HIRI in the future. In addition, optical technology can also be used in disease treatment. It found that optical therapy has the function of anti-oxidative stress. Consequently, it has possibility to treat HIRI caused by oxidative stress. In this review, we mainly summarized the application and prospect of optical techniques in oxidative stress-induced by HIRI.

Hepatic ischemia/reperfusion (IRP) injury is a significant clinical problem during tumor-resection surgery (Pringle maneuver) and liver transplantation. However, the relative contribution of necrotic and apoptotic cell death to the overall liver injury is still controversial. To address this important issue with a standard murine model of hepatic IRP injury, plasma biomarkers of necrotic cell death such as micro-RNA 122, full-length cytokeratin 18 (FK18), and high-mobility group box 1 (HMGB1) protein and plasma biomarkers of apoptosis such as plasma caspase-3 activity and caspase-cleaved fragment of cytokeratin 18 (CK18) coupled with markers of inflammation (hyperacetylated HMGB1) were compared by histological features in hematoxylin and eosin-stained and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-stained liver sections. After 45 minutes of hepatic ischemia and 1 to 24 hours of reperfusion, all necrosis markers increased dramatically in plasma by 40- to >10,000-fold over the baseline with a time course similar to that of alanine aminotransferase. These data correlated well with histological characteristics of necrosis. Within the area of necrosis, most cells were TUNEL positive; initially (≤3 hours of reperfusion), the staining was restricted to nuclei, but it later spread to the cytosol, and this is characteristic of karyorrhexis during necrotic cell death. In contrast, the lack of morphological evidence of apoptotic cell death and relevant caspase-3 activity in the postischemic liver correlated well with the absence of caspase-3 activity and CK18 (except for a minor increase at 3 hours of reperfusion) in plasma. A quantitative comparison of FK18 (necrosis) and CK18 (apoptosis) release indicated dominant cell death by necrosis during IRP and only a temporary and very minor degree of apoptosis. These data suggest that the focus of future research should be the elucidation of necrotic signaling mechanisms to identify relevant targets, which may be used to attenuate hepatic IRP injury.

Sivelestat sodium hydrate (sivelestat) is a specific neutrophil elastase inhibitor that is effective in treating acute lung injury associated with systemic inflammatory response syndrome. As such, it may be useful in treating hepatic ischemia-reperfusion injury (IRI), a condition in which neutrophils transmigrate into the interstitium, leading to release of neutrophil elastase from neutrophils and consequent damage to the affected tissue, particularly in cases of hepatic failure after liver transplantation or massive liver resection.

The purpose of this study was to examine whether treatment with sivelestat inhibits neutrophil adhesion and migration to the vessel wall and suppresses hepatic IRI.

Whether and, if so, the extent to which sivelestat suppresses the adhesion and migration of neutrophils and reduces liver damage in hepatic IRI was examined in a human umbilical vein endothelial cell (HUVEC) model and a rat hepatic IRI model.

In the HUVEC model, the extent of the adhesion and migration of neutrophils stimulated by platelet-activating factor were found to be dose-dependently inhibited by sivelestat treatment (p < 0.05). In the rat model, serum liver enzyme levels were significantly lower at 12 h after reperfusion, and the number of neutrophils that had migrated to extravascular sites was significantly less in the treatment group compared to the control group (p < 0.05).

Sivelestat inhibits the adhesion and migration of neutrophils to vascular endothelium in hepatic IRI, thereby suppressing liver injury.

Ischemia-reperfusion is a major component of injury in vascular occlusion both during liver surgery and during liver transplantation. The pathophysiology of hepatic ischemia-reperfusion includes a number of mechanisms including oxidant stress that contribute to various degrees to the overall organ damage. A large volume of recent research has focused on the use of antioxidants to ameliorate this injury, although results in experimental models have not translated well to the clinic. This review focuses on critical sources and mediators of oxidative stress during hepatic ischemia-reperfusion, the status of current antioxidant interventions, and emerging mechanisms of protection by preconditioning. While recent advances in regulation of antioxidant systems by Nrf2 provide interesting new potential therapeutic targets, an increased focus must be placed on more in-depth mechanistic investigations in hepatic ischemia-reperfusion injury and translational research in order to refine current strategies in disease management.

Not every unresectable hepatocellular carcinoma (HCC) could receive survival benefits from transcatheter arterial chemoembolization (TACE), even for intermediate HCC (Barcelona Clinic Liver Cancer stage B). The aim of this study was to investigate prognostic significance of serum γ-glutamyl transferase (GGT) in patients with intermediate HCC treated with TACE.

A total of 277 patients with intermediate HCC were consecutively treated with TACE and overall survival (OS) was evaluated with the Kaplan-Meier method. Significant difference was estimated with the Log rank method according to GGT value before treatment. Univariate and multivariate analyses were used for the study of significance of prognostic factor.

The median follow-up period was 18.7 months. The 1-year and 3-year OS rates were 71.6 and 38.5% in patients with normal GGT and 48.8 and 16.9% in patients with high GGT (P=0.002). High GGT, correlating with higher tumor size, α-fetoprotein (AFP), and alanine aminotrasferase, was an independent prognostic factor for OS (P=0.009). Others included tumor size and ascites. Furthermore, in small HCC and normal AFP subgroup, serum GGT was also correlated with OS (P=0.013 and 0.041, respectively). The combination of GGT and AFP had a better power to predict the TACE effects.

GGT level was an important prognostic factor to predict prognosis of patients with intermediate HCC treated with TACE.

Allopurinol was first introduced, in 1963, as a xanthine oxidase inhibitor when it was investigated for concomitant use with cancer chemotherapy drugs. Today it is used in gout and hyperuricemia. Due to its additive benefit in preventing oxidative damage, attention has shifted towards the use of allopurinol in organ ischemia and reperfusion.

Currently, the mechanism by which allopurinol exerts a protective benefit in ischemia reperfusion related events is not fully understood. There are various theories: it may act by inhibiting the irreversible breakdown of purine substrates, and/or by inhibiting the formation of reactive oxygen species, and/or by protecting against damage to the mitochondrial membrane.

This work focuses on liver ischemia and reperfusion injury in an effort to better understand the mechanisms associated with allopurinol and with this pathological entity.

The current research, mainly in animal models, points to allopurinol having a protective benefit, particularly if used pre-ischemically in liver ischemia reperfusion injury. Furthermore, after reviewing allopurinol dosing and administration, it was found that 50 mg/kg is statistically the most effective dose in attenuating liver ischemia reperfusion injury. Owing to the limited number of samples, the time of administration did not show statistical difference, but allopurinol was often beneficial when given around 1 h before ischemia.

In conclusion, allopurinol, through its known xanthine oxidase inhibitory effect, as only one of the potential mechanisms, has demonstrated its potential application in protecting the liver during ischemia and reperfusion.

Liver cell death induced by stresses such as ischemia-reperfusion, cholestasis and drug toxicity can trigger a sterile inflammatory response with activation of innate immune cells through release of damage-associated molecular patterns (DAMPs). A similar inflammatory response can be induced by pathogen-associated molecular patterns (PAMPs) such as endotoxin. Both DAMPs and PAMPs activate through toll-like receptors the resident macrophages (Kupffer cells) and recruit activated neutrophils and monocytes into the liver. Central to this inflammatory response is promotion of reactive oxygen species (ROS) formation by these phagocytes. ROS are the principal toxic mediators by which inflammatory cells kill their targets, e.g. bacteria during host defense but also hepatocytes and other liver cells. The mechanism of ROS-induced cell killing during inflammation involves the promotion of mitochondrial dysfunction through an intracellular oxidant stress in hepatocytes leading mainly to oncotic necrosis and less apoptosis. The additional release of cell contents amplifies the inflammatory injury. However, an inflammatory oxidant stress insufficient to directly cause cell damage can induce transcription of stress defence genes including antioxidant genes. This preconditioning effect of ROS enhances the resistance against future inflammatory oxidant stress and promotes the initiation of tissue repair processes. Despite the substantial progress in our understanding of mechanisms of inflammatory liver injury during the last decade, more research is necessary to better understand the role of ROS in acute liver inflammation and to develop clinically applicable therapeutic strategies that selectively target the detrimental effects of oxidant stress without compromising the vital function of ROS in host defense.

Ischemia-reperfusion (I/R) injury associated with liver transplantation remains a serious complication in clinical practice, in spite of several attempts to solve the problem. The present review focuses on the complexity of I/R injury, summarizing conflicting results obtained from the literature about the mechanisms responsible for it. We also review the therapeutic strategies designed in past years to reduce I/R injury, attempting to explain why most of them have not been applied clinically. These strategies include improvements in pharmacological treatments, modifications of University of Wisconsin (UW) preservation solution based on a variety of additives, and gene therapy. Finally, we will consider new potential protective strategies using trimetazidine, 5-amino-4-imidazole carboxamide riboside (AICAR), melatonin, modulators of the renin-angiotensin system (RAS) and the phosphatidylinositol-3-OH kinase (PI3K)-Akt and the p42/p44 extracellular signal-regulated kinases (Erk 1/2) pathway. These strategies have shown promising results for I/R injury but have not been tested in experimental liver transplantation to date. Moreover, we will review ischemic preconditioning, taking into account the recent clinical studies that suggest that this surgical strategy could be appropriate for liver transplantation.

The prototypical xanthine oxidase (XO) inhibitor allopurinol, has been the cornerstone of the clinical management of gout and conditions associated with hyperuricemia for several decades. More recent data indicate that XO also plays an important role in various forms of ischemic and other types of tissue and vascular injuries, inflammatory diseases, and chronic heart failure. Allopurinol and its active metabolite oxypurinol showed considerable promise in the treatment of these conditions both in experimental animals and in small-scale human clinical trials. Although some of the beneficial effects of these compounds may be unrelated to the inhibition of the XO, the encouraging findings rekindled significant interest in the development of additional, novel series of XO inhibitors for various therapeutic indications. Here we present a critical overview of the effects of XO inhibitors in various pathophysiological conditions and also review the various emerging therapeutic strategies offered by this approach.

The aim of the study was to investigate mitochondrial electron transfer during rat liver reperfusion after cold storage and hypothermic machine perfusion. Livers from male Brown Norway rats were preserved (UW) for 10h either by cold storage (CS) or by hypothermic oxygenated perfusion extracorporal (HOPE). Transhepatic photometric analysis allowed determination of the redox status of mitochondrial cytochromes during preservation, rewarming and reperfusion. Mitochondrial electron chain carriers were inhibited at different sites with rotenone and cyanide in some experiments. reversed transcriptional polymerase chain reaction (RT-PCR) was performed after reperfusion concerning transcription of TNFalpha, caspase 9, and c-jun kinase (JNK). Increased superoxide anion formation as well as transcription of TNFalpha, caspase 9, and JNK during reperfusion after cold storage (CS) were related with completely reduced cytochromes before and during reperfusion. In contrast, hypothermic oxygenated livers (HOPE) showed oxygenated cytochromes as well as decreased superoxide anion formation and no detectable transcription of TNFalpha, caspase 9, and JNK. A similar low level of superoxide anion formation was found when electron chain transfer of cold stored livers was inhibited during reperfusion with rotenone but not with cyanide. After hypothermic oxygenation (HOPE) inhibition of mitochondrial electron chain with rotenone showed no change in formation of superoxide anion formation whereas inhibition with cyanide showed increased superoxide anion formation. Thus mitochondrial cytochrome redox status is suggested to be related: (i) with the release of reactive oxygen substances as well as (ii) with the expressions of TNFalpha, caspase 9, and JNK during reperfusion and may thus be usable as predictive marker of liver grafts.

Nitric oxide (NO), synthesized from L-arginine by the enzyme nitric oxide synthase (NOS), seems to play an ambiguous role during tissue ischemia-reperfusion injury. Our objective was to evaluate the effects of L-arginine, a NO donor, and N(G)-nitro-L-arginine-methylester (L-NAME), a NOS inhibitor, on oxidative stress, renal dysfunction, histologic alterations and surgical mortality rate induced by renal ischemia-reperfusion (RIR) in uninephrectomized rats.

One-hundred and ninety-seven Wistar rats were randomized into five experimental groups. Group 1: sham operation; group 2: right uninephrectomy (UNI); group 3: UNI + RIR in the contralateral kidney; group 4: UNI + L-NAME (20 mg/kg; intraperitoneally) + RIR; and group 5: UNI + L-arginine + RIR. The effect of the drugs was evaluated by lipid peroxidation measured by the renal malondialdehyde (MD) content and chemiluminescence (CL) levels, serum creatinine (Cr) levels, urinary volume, tubular necrosis and athrophy, inflammatory infiltrate, interstitial fibrosis as histologic evaluation and surgical mortality rate after the procedures. A P value less than 0.05 was considered significant.

Right uninephrectomy did not alter the renal parameters. RIR increased Cr levels (at 24 and 96 h of reperfusion), index of lipid peroxidation (both MD and QL levels), and worsened the histologic aspects. Pretreatment with L-arginine reduced the kidney levels of QL when compared with the non-treated group (5574 +/- 909 vs. 13 660 +/- 1104 cps/mg of protein; P < 0.05) but increased the MD levels (0.97 +/- 0.24 vs. 0.79 +/- 0.06 nmol/mg of protein; P < 0.05). Moreover, L-arginine attenuated the increment of Cr levels, inflammatory infiltrate and tubular athrophy in rats subjected to RIR (P < 0.05). On the other hand, pretreatment with L-NAME increased both CL (17 482 +/- 4397 vs. 13 660 +/- 1104 cps/mg of protein; P < 0.05) and MD levels (1.16 +/- 0.11 vs. 0.79 +/- 0.06 nmol/mg of protein; P < 0.05). Furthermore, L-NAME worsened the renal dysfunction (P < 0.05) at 192 h after the RIR, and surgical mortality rates were similar (P > 0.05).

L-arginine has a tendency to exert a beneficial effect on renal damage during RIR in rats. Moreover, L-NAME seems to worsen the renal damage by increasing the kidney-levels of CL and impairment of renal function probably due to reduction of NO production.

The effect of allopurinol (an inhibitor of xanthine oxidase) on oxidative stress, renal dysfunction, and histologic alterations was evaluated during the renal ischemia--reperfusion in uninephrectomized rats. Renal malondialdehyde and serum creatinine levels significantly increased after renal ischemia--reperfusion. However, the pretreatment with allopurinol demonstrated a protector effect in these parameters. Renal ischemia--reperfusion provoked a significant renal damage in the operated group. Tubular atrophy and interstitial fibrosis were attenuated by allopurinol when given prior to the surgery. In our study, allopurinol had a strong tendency to exert a beneficial effect during renal ischemia--reperfusion in uninephrectomized rats.

The effect of geranyl-geranyl-acetone (GGA) administration before heat shock preconditioning on heat shock protein (HSP) 72 induction and on the acquisition of tolerance against ischemia-reperfusion Injury was studied in rat livers. Male Wistar rats were divided into four groups: a control group (group C); a GGA group (group G); a simple heat shock group (group VH); and a heat shock with GGA premedication group (group GH). Five-, 10-, and 15-minute periods of heat shock preconditioning at 42 degrees C were performed in groups VH and GH. Subgroups were determined according to the period of heat shock exposure. After a 48-hour recovery, rats in groups C, VH5, VH15, and GH5 received a 30-minute period of hepatic ischemia. Induction of HSP72, survival rates, and changes in biochemical and histologic parameters were compared among the groups. Five-minute heat shock preconditioning was not enough to Induce HSP72. However, livers in group GH5 expressed approximately the same amount of HSP72 as those in group VH15. The expression of HSP72 in group GH15 was stronger than that found in group VH15. The degree and location of HSP72 expression were not different between groups GH5 and VH15. Seven-day survival was significantly better in groups GH5 (16/16) and VH15 (15/16) than in group C (8/16) or VH5 (9/16). The recovery of adenosine triphosphate in liver tissue was faster, and the release of liver-related enzymes during reperfusion was lower in groups GH5 and VH15 than in group C or VH5. Administration of GGA before heat shock preconditioning augmented the induction of HSP72 by decreasing the threshold for triggering the stress response.

Discrepancies in the levels of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) following hemorrhagic shock (HS) may be due to the inconsistent rates of bleeding. The purpose of this study was to investigate the effects of rapid versus slow bleeding rates on TNF-alpha levels and if inhibition of TNF-alpha convertase by a matrix metalloproteinase inhibitor (MMPI) affects hepatic integrity in animals exposed to 35% HS. Sprague-Dawley male rats (n = 24, 300-350 g) were divided into four groups: HS 15 (produced over 15 min), HS 30 (produced over 30 min), and HS with MMPI (2.5 mg/kg British Biotech 1101: HS15 + MMPI, HS30 + MMPI). Mean arterial blood pressure (MAP), serum TNF-alpha,levels, and hepatic resting membrane potentials (E(m)) were obtained. A Student t test was performed. TNF-alpha levels for HS 15, HS15 + MMPI, HS 30, and HS 30 + MMPI were 474, 40, 32, and 50 pg/ml, respectively. The hepatic resting membrane potentials for HS 15, HS15 + MMPI, HS 30, and HS 30 + MMPI were -26, -30, -23, and -31 mV, respectively. In conclusion, circulating TNF-alpha levels are affected by the rate of bleeding in hemorrhagic shock. However, despite the differences in the magnitude of TNF-alpha in untreated animals, hepatic integrity was compromised. Interestingly, MMPI, an inhibitor of TNF-alpha convertase, stabilizes the membrane potential in both types of hemorrhagic shock.

The mechanisms underlying the initial graft dysfunction in liver transplantation are not completely understood, although much of the liver graft injury derives from the ischemia/reperfusion-induced oxidative stress. Thus, the purpose of our study was to determine the involvement of oxidative stress in the initial graft dysfunction in human liver transplantation.

Liver biopsies were taken at different times of the transplantation procedure, at the organ donor laparatomy (T1), before graft reperfusion (T2), and 5-60 min after graft reperfusion (T3), determining the levels of GSH, GSSG, as well as peroxides and malondialdehyde in liver homogenates.

Patients were graded into two groups depending on whether the peak serum alanine aminotransferases within the first 3 postoperative days were lower (group A, mild to moderate injury: 32 patients) or higher (group B, severe injury: 5 patients) than 2500 U/l. The levels of GSH at time intervals T1-T3 were similar for groups A and B, with a trend to lower GSSG levels in group B at T2 and T3 samples. This outcome was accompanied by unchanged levels of malondialdehyde and hydrogen peroxide in the same samples in both groups of patients. No patient developed primary graft nonfunction. One-year cumulative survival was 81% and 60% in groups A and B, respectively (p>0.05).

These findings indicate a lack of significant generation of reactive oxygen species and consequent oxidative stress as a major factor involved in the pathogenesis of the initial graft dysfunction in human liver transplantation.

Reperfusion injury of the liver is characterized by intravascular oxidative stress and GSH consumption. Whether mitochondria contribute to hepatocellular damage has never been elucidated. Therefore, we assessed mitochondrial function and redox state during reperfusion and the effect of glutathione monoethyl ester (GSHE) administration, which may replenish the GSH pool.

Rats were subjected to partial hepatic ischemia (90 min) followed by reperfusion. Mitochondrial function was assessed in vivo and in vitro by the KICA breath test and the ATP synthase activity. Just prior to the start of reperfusion, rats received 5 mmol/kg of GSHE or saline iv. ALT, total and oxidized (GSSG) glutathione, GSHE, and CYS were measured in plasma and liver. GSH, GSSG, malondialdehyde (MDA), and carbonyl proteins were measured in mitochondria. The extent of necrosis was also estimated. Sham-operated rats served as controls.

Reperfusion markedly increased ALT (>1500 U/L) and doubled the liver content of MDA and carbonyl proteins. Mitochondrial GSH decreased approximately 30%, without increase of GSSG. The in vivo KICA breath test was not significantly impaired by reperfusion. In contrast, both KICA decarboxylation and ATP synthase activity were both reduced by approximately 50% in mitochondria isolated from reperfused livers. GSHE administration significantly decreased ALT ( approximately 40%), protected ATP synthase activity, and reduced the extent of necrosis. Compared to controls, plasma GSHE and plasma GSH at 1 h were lower in rats subjected to ischemia. GSHE was higher in reperfused lobes than in continuously perfused ones and the concentration of GSH was significantly higher in ischemic liver than in untreated animals, indicating that the uptake of GSHE is increased in postischemic liver. GSHE prevented the reperfusion-associated increase of oxidized products in liver and mitochondria.

Reperfusion of ischemic liver is associated with oxidative modifications and functional impairment of mitochondria. GSHE protects against reperfusion injury, possibly by providing intra- and extracellular GSH.

The extracellular generation of reactive oxygen species (ROS) by Kupffer cells contributes to reperfusion injury of the liver allograft. The endogenous antioxidant glutathione (GSH) can detoxify these ROS; however, this effect might be limited by the low extracellular concentration of GSH. We therefore investigated whether an increase of extracellular GSH protects the liver against reperfusion injury after cold preservation.

Livers of male Sprague-Dawley rats subjected to 24 hours of cold ischemia in University of Wisconsin solution (4 degrees C) were reperfused for 2 hours in the absence (controls) or presence of 0.5, 1, 2, or 4 mmol/L GSH (n = 4-6 each).

Two hours after starting reperfusion of control livers, the sinusoidal release of lactate dehydrogenase and purine nucleoside phosphorylase increased to 247 +/- 96 and 27 +/- 13 mU. min(-1). g liver(-1), respectively, but only to 76 +/- 43 and 10 +/- 4 mU. min(-1). g liver(-1) in the presence of 4 mmol/L GSH. This cytoprotective effect was confirmed histologically by a marked reduction of trypan blue staining of hepatocytes. Compared with control livers, postischemic bile flow was significantly enhanced by GSH (0.15 +/- 0.02 vs. 0.41 +/- 0.11 microL. min(-1). g liver(-1)), indicating improved liver function. During reperfusion of control livers, intracellular GSH content declined from 4.5 +/- 0.3 to 2.3 +/- 0.1 micromol/g liver, but only to 3.8 +/- 0.4 micromol/g liver in the presence of 4 mmol/L GSH. Reperfusion of untreated livers was accompanied by a prolonged increase of portal pressure to maximally 12.5 +/- 1.9 cm H2O, which was significantly attenuated by 4 mmol/L GSH (7.2 +/- 1.4 cm H2O). Similar cytoprotective and hemodynamic effects were observed with 2 mmol/L GSH, but not with 0.5 and 1 mmol/L GSH.

Treatment of cold-preserved livers with GSH upon reperfusion prevents damage of hepatocytes, deterioration of the hepatic circulation, and loss of intracellular GSH. In view of these protective effects and its low toxicity in humans, GSH should be considered a candidate drug for prevention of ROS-related reperfusion injury of the liver allograft.

beta-Ethoxyacrolein (BEA), a side product that forms during the preparation of malondialdehyde (MDA) by acidic hydrolysis of tetraethoxypropane (TEP), has been found to be an inhibitor of milk xanthine oxidase (XO) several times more potent than pure MDA (NaMDA). The incubation of XO with 10 microM BEA abolished 50% of the enzyme activity within 1 min; the inhibited enzyme was totally regenerated by dialysis and filtration through Sephadex. The BEA inhibition mode of the enzyme was mixed-type with the apparent inhibition constants (Ki) of 2.4 x 10(-6) M. An HPLC method for quantitation of BEA in the crude commonly used MDA preparation was set up.

Oxidant stress has been implicated as playing a role in the pathogenesis of cholestatic liver injury. The objective of this study was to determine whether the xanthine oxidase/xanthine dehydrogenase enzyme system was involved in this oxidant stress. Adult Sprague-Dawley rats were treated with the xanthine oxidase inhibitor, oxypurinol, and randomized to bile duct ligation or sham surgery; vehicle-treated, sham-operated rats served as controls. After 5 d of bile duct ligation, serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and total and direct bilirubin concentrations were significantly elevated, and increased lipid peroxidation of hepatic mitochondria and microsomes was present. Treatment with oxypurinol reduced the aspartate aminotransferase, alanine aminotransferase, and bilirubin values by 26-47% but did not alter the increased lipid peroxidation of mitochondria and microsomes. Serum vitamin E:total lipids ratio was also reduced in both bile duct-ligated groups, consistent with oxidant injury. These data show that inhibition of xanthine oxidase reduces biochemical evidence of hepatocellular injury during bile duct ligation without affecting oxidant damage to intracellular hepatocyte organelles. Thus, in this model a component of cholestatic injury appears to have been caused by oxidant stress from a source outside of the hepatocyte.

In this investigation, we used chemiluminescence to study the ability of increasing durations of ischemia (1, 2, or 2.5 h) to induce enhanced generation of reactive oxygen species in a crystalloid perfused rat liver model. To evaluate the effect of reactive oxygen species generation upon the development of the postischemic hypoperfusion, hepatic vascular resistance was simultaneously monitored. One hour of ischemia did not produce sustained reactive oxygen species generation or development of no-reflow. Two hours of ischemia did not result in sustained reactive oxygen species generation but did produce no-reflow. Sustained reactive oxygen production was achieved after 2.5 h of ischemia and was accompanied by the development of no-reflow. We found that 2.5 h of ischemia is the threshold for sustained lipid peroxidation. Both lipid peroxidation and no-reflow could be mitigated through the administration of superoxide dismutase. Superoxide dismutase could reduce the amount of cell injury due to the enhanced lipid peroxidation induced by 2.5 h of ischemia. Limitation of reactive oxygen species generation to a critical threshold, either by restricting the duration of ischemia or by pharmacological intervention, may be an important means of preventing further cellular injury through no-reflow and lipid peroxidation.

We have investigated the involvement of MAP kinase cascades in the response of the liver to post-ischemic reperfusion. Both JNKs and ERKs are activated but the duration and magnitude of the increase in their activities appear to be different. JNK activation is more marked but shorter than that of ERKs. The increase observed in the phosphotyrosine content of the 52 kDa Shc protein, accompanied by an increased amount of co-immunoprecipitated Grb2, and the activation of Raf-1 kinase provide evidence of the involvement of a Ras-Raf-dependent pathway, with a time course that is similar to that of ERK activation. The treatment of rats with IL-1 receptor antagonist modified all of the described effects, suggesting that IL-1 plays a role in the response of the liver to reperfusion.

It is well known that ischemia causes functional and structural damage to liver cells, and that the status of energy metabolism provides an important means of assessing the functional viability of the ischemic organ. However, the specific sequence leading to ischemic liver cell injury is not yet fully understood; therefore, it is clinically and pathophysiologically important to elucidate the mechanism of cellular injury during hepatic ischemia and subsequent reperfusion. Whereas the conventional view attributes this injury process to the ischemia itself, recent studies have demonstrated that a variable but often substantial proportion of this injury is caused by reactive oxygen metabolites that are generated at the time of reperfusion. This article presents an outline of the mechanism of cellular injury caused during hepatic ischemia and subsequent reperfusion resulting from certain types of surgery, with special reference to the xanthine-xanthine oxidase system and the activation of neutrophils and macrophages.

Transplantation-related pathogenic factors such as ischemia or allograft-directed inflammation are associated with oxidative changes that might lead to cellular oxidative stress. The aim of this study was to investigate the impact of oxidative stress on: (1) CMV replication in cultured human endothelial cells and (2) the stimulation of endothelial cells by proinfiammatory cytokines. Both pathomechanisms are known to contribute to graft rejection crises in vivo. Oxidative stress was induced in endothelial cell cultures with 10-200 microM buthionine sulfoximine. Western blotting showed a significant increase in the production of CMV-specific immediate early and late proteins in buthionine sulfoximine-treated cultures. Immunocytochemical staining suggested that this effect was caused by increased numbers of CMV antigen expressing cells (66% immediate early; 78%, late). Quantitative polymerase chain reaction for CMV-specific DNA and virus titration revealed that enhanced viral replication levels correlated with increased virion production. As a measure for the endothelial cell activation status, the surface expression of HLA-ABC and HLA-DR and adhesion molecules (ICAM-1, ELAM-1, VCAM-1) was quantified by fluorometric methods. Whereas oxidative stress alone did not modulate any surface molecule expression, the IFN-gamma-mediated expression of HLA-ABC and HLA-DR and the IL-1-mediated expression of ICAM-1, but not of ELAM-1 and VCAM-1 (IL-1 + TNF-alpha), was amplified. Interestingly, the amplification of HLA molecule expression was even higher in CMV-infected endothelial cells. This study provides evidence that oxidative stress contributes to the regulation of CMV replication, virus shedding, and the activation of endothelial cells by proinflammatory cytokines as it is observed in transplant recipients.

The protective effects of a neutrophil elastase inhibitor (ONO-5046) on reperfusion injury following pancreaticoduodenal transplantation in rats were studied by measuring serum concentrations of cytokine-induced neutrophil chemoattractant (CINC). Male Wistar rats were transplanted with syngeneic pancreaticoduodenal grafts. ONO-5046 was injected intravenously 5 min before vascular clamping and immediately after reperfusion at a dose of 10 mg/kg. No significant differences were observed in the peak serum concentrations of amylase between the groups treated with and treated without ONO-5046. The serum lipase concentrations in the untreated animals increased and peaked 3 hr after reperfusion. ONO-5046 significantly decreased the peak serum lipase concentration. The serum CINC concentrations, which were determined by enzyme-linked immunosorbent assay, increased and peaked 3 hr after reperfusion, decreasing gradually thereafter. However, pretreatment with ONO-5046 significantly inhibited the rise in serum CINC concentrations after reperfusion. Expression of CICN transcripts in the pancrease grafts was evaluated by Northern blot analysis and peaked 3 hr after reperfusion in untreated animals. Pretreatment with ONO-5046 also significantly inhibited the expression of CINC mRNA transcripts in the graft. ONO-5046 significantly decreased the number of neutrophils accumulated in the pancreas graft 24 hr after transplantation. In vitro CINC production by peritoneal macrophages was increased by neutrophil elastase in dose-dependent fashion. However, ONO-5046 decreased CINC production by peritoneal macrophages in response to neutrophil elastase. These results suggest that ONO-5046 prevents early neutrophil accumulation in the pancreas following ischemia/reperfusion of pancreaticoduodenal transplantation.

Oxidant stress seems to be involved in the pathogenesis of several important gastroenterologic disorders in infants and children. The question can still be asked, in most circumstances, whether the oxidant stress precedes, and therefore is involved in, tissue or cellular injury or is a result of injury and not of clinical importance. The data favor the former situation in several inflammatory conditions of the bowel and in a variety of liver diseases. Experimental and clinical testing of this possible basic mechanism of tissue injury over the next few years will shed light on the role of antioxidants in treating gastrointestinal disorders.

To prevent oxidative tissue damage induced by strenuous exercise in the liver and kidney superoxide dismutase derivative (SM-SOD), which circulated bound to albumin with a half-life of 6 h, was injected intraperitoneally into rats. Exhausting treadmill running caused a significant increase in the activities of xanthine oxidase (XO), and glutathione peroxidase (GPX) in addition to concentrations of thiobarbituric acid-reactive substances (TBARS) in hepatic tissue immediately after running. There was a definite increase in the immunoreactive content of mitochondrial superoxide dismutase (Mn-SOD) 1 day after the running. Meanwhile, the TBARS concentration in the kidney was markedly elevated 3 days after running. The activities of GPX, and catalase in the kidney increased significantly immediately and on days 1 and 3 following the test. The immunoreactive content of Mn-SOD also increased 1 day after running. The exercise induced no significant changes in immunoreactive Cu, Zn-SOD content in either tissue. The administration of SM-SOD provided effective protection against lipid peroxidation, and significantly attenuated the alterations in XO and all the anti-oxidant enzymes, measured. In summary, the present data would suggest that exhausting exercise may induce XO-derived oxidative damage in the liver, while the increase in lipid peroxidation in the kidney might be the result of washout-dependent accumulation of peroxidised metabolites. We found that the administration of SM-SOD provided excellent protection against exercise-induced oxidative stress in both liver and kidney.

Many of the reports implicating the contribution of oxygen radicals to preservation-reperfusion injury have been based largely on indirect experiments demonstrating the effects or the consumption of various antioxidants. Investigations based on the direct measurement of the amounts of oxygen radicals that are actually formed during reoxygenation after preservation have not given satisfactory results. In this study, we attempted direct measurement of H2O2 from hepatocellular mitochondria and superoxide (O2-) from Kupffer cells, using the HRP method and cytochrome c perfusion method, respectively, for quantitative comparison of the cold preservation-induced changes in radical generation activity between these sources. H2O2 generation in mitochondria isolated after 24 h cold preservation decreased to 8% of non-preserved liver, but in the mitochondria isolated from the livers that were reperfused for 30 min after 24 h preservation H2O2 generation recovered to 60%. The respiratory control ratio also decreased significantly after 24 h preservation, and similarly recovered after an additional 30 min reperfusion. By contrast, O2- from Kupffer cells increased in time-dependent fashion until 12 h preservation and decreased after 24 h preservation. Although 12 h preservation did not cause an increase in LDH release, the lipid peroxide in the perfusate significantly increased after 12 h preservation, which indicated the occurrence of lipid peroxidation in the sinusoidal area. These results suggested that mitochondrial H2O2 was dependent upon the activity of respiratory function and so did not cause hepatocellular injury and that O2- from Kupffer cells contributed to oxidative injury to the sinusoidal lining cells. Our data support reports demonstrating the vulnerability of nonparenchymal cells.

The aim of this study was to determine the cellular source of oxygen free radicals generated by isolated hepatocytes during post-anoxic reoxygenation. Superoxide anions (O2.-) were detected by lucigenin chemiluminescence. Cell damage was assessed by LDH release. During anoxia, the chemiluminescence decreased to background levels while LDH release increased 8-fold. During reoxygenation, O2.- formation increased 15-fold within 15 min then declined towards control levels. LDH release increased from 161 to 285 mU/min in the first 30 min of reoxygenation, then declined toward the control rate. Allopurinol, an inhibitor of the xanthine-xanthine oxidase system, did not inhibit O2.- formation nor LDH release. Antimycin, a mitochondrial complex III inhibitor that does not block O2.- formation, increased both O2.- generation and LDH release 82 and 133% respectively. Diphenyleneiodonium (DPI), a mitochondrial and microsomal NADPH oxidase inhibitor, reduced O2.- and LDH release 60-70%. SOD, which catalyzes the dismutation of O2.- to H2O2, was without effect on O2.- and LDH release, but TEMPO, a stable nitroxide which mimics SOD and easily penetrates the cell membrane, decreased O2.-86% without affecting LDH. These results suggest that mitochondria or microsomes are the principal sites of O2.- production during reoxygenation of isolated hepatocytes, whereas the cytosolic xanthine/xanthine oxidase system is apparently not involved.

In the model of the perfused rat liver, we investigated the alterations of sinusoidal cells in the pathogenesis of liver injury caused by hypoxia and reperfusion. In sinusoidal endothelial cells, the activity of xanthine oxidase (XOX), a cytoplasmic marker enzyme, was located cytochemically and determined biochemically. Kupffer cells, identified by their endogenous peroxidase staining, were studied with regard to changes in their ultrastructure. In our experiments, parenchymal cells were shown to be severely damaged in contrast to sinusoidal lining cells, which showed minor signs of injury. In comparison with the control group, XOX activity increased significantly in the sinusoidal endothelial cells after low-flow hypoxia; however, after reoxygenation of only 5 minutes, that activity was lower after hypoxia but higher after control perfusion. In Kupffer cells, hypoxia resulted in a strong suppression of phagocytic and endocytotic activity and in a disappearance of the lamellopodia. Kupffer cells were flattened, resembling sinusoidal endothelial cells. After reoxygenation phagocytic vesicles, lamellopodia, and cell volume of Kupffer cells increased markedly in comparison with the control group. In the hypoxia/reperfusion injury model, our observations revealed significant alterations of sinusoidal lining cells. It appears that sinusoidal endothelial cells respond to the hypoxic phase by producing oxygen-derived free radicals and that Kupffer cells respond to the subsequent reperfusion phase by activation followed by the release of toxic mediators.

It has been proposed that xanthine oxidase-derived superoxide mediates reperfusion injury in the liver; however, there is a little direct evidence to support this hypothesis. In this paper we describe a model system to directly and noninvasively measure oxyradical formation and hepatic injury in isolated perfused rat liver. Using this sensitive chemiluminescent technique, we clearly demonstrate the theorized burst in oxygen radical production upon reperfusion of previously ischemic liver, without perturbing the system with chemical luminescence enhancers. This increase in chemiluminescence (CL) upon reperfusion was diminished by the free radical scavengers trolox and ascorbate, as well as N-2-mercaptoproprionyl-glycine (MPG), thereby confirming the oxyradical nature of this signal. Additionally, superoxide dismutase and the xanthine oxidase inhibitor allopurinol, but not catalase, attenuated the reperfusion effect, providing the most direct evidence so far that XOD derived superoxide anion is formed during liver reperfusion. Hepatic injury (AST release) did not appear to relate to increased CL, supporting the notion that the oxyradical flux may serve as a signal for other events leading to tissue injury. Further studies using this sensitive chemiluminescent technique should aid in delineating the detailed mechanism(s) of reperfusion injury.

Isolated hepatocytes incubated under hypoxic conditions were more sensitive to H2O2-mediated injury as compared to cells kept under aerobic conditions, but only for the highest H2O2 concentration tested (8 mM). At lower concentrations (2 and 4 mM) cells were still able to detoxify H2O2 even under hypoxic conditions. Reoxygenation of hypoxic hepatocytes did not result in a cytolytic effect, whereas reoxygenation in the presence of H2O2 resulted in an enhanced cytotoxicity. The duration of previous hypoxia (before H2O2 addition) did not affect the lytic effect induced by H2O2. Enzymatic activities of both catalase and glutathione peroxidase were unchanged over 2 h of incubation under hypoxic conditions. Preincubation of hepatocytes in the presence of Desferal (5 mM) resulted in the abolition of H2O2-mediated lytic effects. A role for free iron, released from intracellular stores and acting on H2O2 to yield reactive oxygen species is discussed.

Based on our current understanding, we have developed a provisional model for hepatocyte necrosis that may be applicable to cell necrosis in general (Figure 6). Damage to mitochondria appears to be a key early event in the progression to necrosis. At least two pathways may be involved. In the first, inhibition of oxidative phosphorylation in the absence of the MMPT leads to ATP depletion, ion dysregulation, and enhanced degradative hydrolase activity. If oxygen is present, toxic oxygen species may be generated and lipid peroxidation can occur. Subsequent cytoskeleton and plasma membrane damage result in plasma membrane bleb formation. These steps are reversible if the insult to the cell is removed. However, if injury continues, bleb rupture and cell lysis occur. In the second pathway, mitochondrial damage results in an MMPT. This step is irreversible and leads to cell death by as yet uncertain mechanisms. It is important to note that MMPT may occur secondary to changes in the first pathway (e.g. oxidative stress, increased Cai2+, and ATP depletion) and that all the "downstream events" occurring in the first pathway may result from MMPT (e.g., ATP depletion, ion dysregulation, or hydrolase activation). Proof of this model's applicability to cell necrosis in general awaits further validation. In this review, we have attempted to highlight the advances in our understanding of the cellular mechanisms of necrotic injury. Recent advances in this understanding have allowed scientists and clinicians a better comprehension of liver pathophysiology. This knowledge has provided new avenues of therapy and played a key role in the practice of hepatology as evidenced by advances in organ preservation. Understanding the early reversible events leading to cellular and subcellular damage will be key to prevention and treatment of liver disease. Hopefully, disease and injury specific preventive or pharmacological strategies can be developed based on this expanding data base.

Rat liver was kept at 4 degrees C or 37 degrees C in MEM, and reperfused through a closed circulation from the hepatic vein to the portal vein at 37 degrees C with the same solution. Although purine nucleoside phosphorylase and ALT activities were increased in the perfusate, depending on the duration of ischemia at both 4 degrees C and 37 degrees C, the ratio of the latter to the former was significantly higher after 37 degrees C-ischemia than after 4 degrees C-ischemia. The stimulation stage of Kupffer cells evaluated in situ by formazan deposition after liver perfusion with nitro blue tetrazolium and phorbol myristate acetate was elevated after 4 degrees C-ischemia longer than 1 h, but not after 37 degrees C-ischemia. In contrast, the degree of oxidative stress in hepatocytes assessed by formazan deposition after liver perfusion with nitro blue tetrazolium alone was greater after 37 degrees C-ischemia than after 4 degrees C-ischemia. These results suggest that oxidative stress in hepatocytes and the stimulatory state of Kupffer cells after ischemia-reperfusion may differ between 4 degrees C-ischemia and 37 degrees C-ischemia, probably leading to different development of liver damage.

The pathophysiological importance of reactive oxygen species has been extensively documented in the pathogenesis of hepatic ischemia-reperfusion injury. Kupffer cells and neutrophils were identified as the dominant sources of the postischemic oxidant stress. To test the hypothesis that a direct free radical-mediated injury mechanism (lipid peroxidation; LPO) may be involved in the pathogenesis, highly sensitive and specific parameters of LPO, i.e., hydroxy-eicosatetraenoic acids (HETES), and F2-isoprostanes, were determined by gas chromatographic-mass spectrometric analysis in liver tissue and plasma during 45 min of hepatic ischemia and up to 24 h of reperfusion. A significant 60-250% increase of F2-isoprostane levels in plasma was found at all times during reperfusion; the HETE content increased only significantly at 1 h of reperfusion and in severely necrotic liver tissue at 24 h with increases between 90-320%. On the other hand, in a model of LPO-induced liver injury (infusion of 0.8 mumol tert-butylhydroperoxide/min/g liver), the hepatic HETE content increased two to fourfold over baseline values at 45 min, i.e., before liver injury. A further increase to 12- to 30-fold of baseline was observed during moderate liver injury. Based on these quantitative comparisons of LPO and liver injury, it seems highly unlikely that LPO is the primary mechanism of parenchymal cell injury during reperfusion, although it cannot be excluded that LPO may be important as a damaging mechanism in a limited compartment of the liver, e.g., endothelial cells, close to the sources of reactive oxygen, e.g., Kupffer cells and neutrophils.

The accumulation and activation of polymorphonuclear leukocytes in the liver may play an important role in liver damage following ischemia and reperfusion. To study the effects of polymorphonuclear leukocytes on hepatic function, human polymorphonuclear leukocytes were infused into perfused rat livers. Infusion of polymorphonuclear leukocytes into continuously oxygenated livers led to an increased consumption of oxygen by the perfused liver which paralleled the production of superoxide anion radicals by activated polymorphonuclear leukocytes. The increased use of oxygen was followed by a decrease in the sinusoidal efflux of glutathione and a marked increase in the biliary excretion of glutathione disulfide, indicating that polymorphonuclear leukocytes were activated within the liver, and created a potentially deleterious oxidant stress. When polymorphonuclear leukocytes were infused into rat livers that had been subjected to 45 min of warm ischemia followed by reperfusion, the release of lactate dehydrogenase upon reperfusion of ischemic liver was significantly higher from livers exposed to polymorphonuclear leukocytes than from livers subjected to the same period of ischemia without leukocytes. This indicates that polymorphonuclear leukocytes potentiate ischemia/reperfusion injury. The present in vitro system provides a model to study pharmacological interventions designed to modulate the potentially deleterious interactions of polymorphonuclear leukocytes with the liver.

To elucidate the significance of the changes in plasma glutathione concentrations associated with carbon tetrachloride (CCl4)-induced liver damage, the changes in the concentrations of reduced (GSH) and oxidized glutathione (GSSG) in plasma as well as in the liver were investigated in rats. In the liver, the concentration of GSH decreased, and that of GSSG increased 24 hr after the intraperitoneal administration of CCl4. In the right atrial plasma, the concentration of both GSH and GSSG increased. The GSH/GSSG ratio in the plasma decreased as did that in the liver. The net sinusoidal efflux of GSH and GSSG from the liver was calculated by subtracting their concentrations in plasma of the infrahepatic inferior vena cava from those of the suprahepatic inferior vena cava. The net efflux of GSH and GSSG started to increase as early as 3-6 hr after CCl4 administration, and reached a plateau 6 and 24 hr after CCl4 administration, respectively. On the other hand, an elongation of prothrombin time and leakage of alanine aminotransferase reached a maximum 24 and 48 hr after CCl4 administration, respectively. Vacuolization in the centri-lobular region and inflammatory infiltration started 3 and 6 hr after CCl4 administration, respectively, and progressed for 48 hr. These results suggest that CCl4 induced an increase in plasma concentrations of GSH as well as GSSG by increasing their efflux from the liver, and that the changes in plasma glutathione status might be a useful and sensitive marker for CCl4-induced liver damage.

The specific activity of seven enzymes involved in protecting tissue from oxidative stress was determined in rat kidneys subjected to 0, 2, 4, or 8 h of normothermic ischemia and in isolated rat livers during control perfusion, after 2 h ischemia, and after 2 h ischemia plus 1 h of reperfusion. In general, none of the antioxidant enzymes measured showed any consistent variation throughout the ischemic period even though mitochondrial function was significantly decreased, indicating substantial cell injury. Glutathione peroxidase (Se-GSH-Px) activity remained constant during 8 h of ischemia, although a small (29%) increase above control activity was noted at 4 h of ischemia. Se-independent GSH-Px activity (non-Se-GSH-Px) and glutathione reductase (GSSG-Red) remained constant up to 8 h of ischemia, when we measured an increase of 158% above controls in non-Se-GSH-Px and a decrease of 35% relative to controls in GSSG-Red. In perfused livers, the only change in enzyme activity after 2 h of ischemia was an increased GSSG-Red activity of 21% above control. This increase persisted into the reperfusion phase (35% above control activity) and was accompanied by decreases in both forms of GSH-Px (28% Se-GSH-Px and 44% non-Se-GSH-Px).

The role of neutrophil CD11b/CD18 (Mac-1) adhesion proteins in the pathogenesis of hepatic reperfusion injury was investigated in an experimental model. Male Fischer rats were treated with a CD11b monoclonal antibody or an isotype-matched IgM control antibody and subjected to 45 min of hepatic ischemic followed by 24 hr of reperfusion. Large numbers of neutrophils were present in postischemic liver lobes (1,241 +/- 64 polymorphonuclear cells/50 high-power fields) compared with numbers in baseline measurements (14 +/- 3 polymorphonuclear cells/50 high-power fields), and severe liver injury was observed after 24 hr of reperfusion (hepatic necrosis: 88% +/- 2%). Pretreatment with the CD11b antibody (two doses of 2 mg/kg each significantly attenuated liver injury and reduced the number of polymorphonuclear cells in the post-ischemic liver by 59%. Selective treatment with the antibody only during reperfusion was similarly effective. The increased spontaneous superoxide formation of neutrophils isolated from postischemic liver (1.05 +/- 0.11 nmol O2-/hr/10(6) cells) was reduced by 56% in neutrophils from CD11b antibody-treated animals. Flow cytometric analysis of CD11b/CD18 expression on circulating neutrophils demonstrated significant upregulation at all time points during reperfusion. Clone 17 also effectively inhibited neutrophil extravasation in a glycogen peritonitis model. Our data are consistent with a dual protective effect of the CD11b antibody in hepatic reperfusion injury in vivo (i.e., reduced accumulation of neutrophils and their functional inactivation).

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

Isolated perfused livers from rats fasted overnight were subjected to 90 min of low-flow hypoxia followed by reoxygenation for 30 min. Intra-hepatic generation of superoxide anion was analysed by continuous perfusion with 40 mumol/l of oxidized cytochrome c, the reduction of which was measured spectrophotometrically in the effluate. Reduction of cytochrome c as an indicator for hepatic superoxide anion generation remained constant during pre-hypoxic perfusion and during hypoxia. Upon reperfusion, an initial peak was observed to 47.2 +/- 3.8 nmol/g/min followed by a stable plateau above pre-hypoxic values. Both peak and plateau were significantly attenuated in the presence of 80,000 U/l superoxide dismutase (SOD). Accordingly, tissue contents of lipid peroxides were significantly lower at the end of reperfusion (976 +/- 73 vs 1153 +/- 71 nmol/g*), enzyme leakage [U/g/min] from the endothelium (PNP: 8.4 +/- 4.2 vs 17.2 +/- 3.4**) and from the hepatic parenchyma (Alt: 108 +/- 35 vs 170 +/- 23*) was significantly reduced during reperfusion and oxygen consumption was elevated in the presence of SOD (3.27 +/- 0.34 vs 2.71 +/- 0.37*). It is concluded that reactive oxygen species arise in the vascular lumen or the space of Disse after prolonged hypoxia of the liver, altering the functional outcome of the organ upon reoxygenation. SOD is able to protect against these alterations. * P < 0.05; ** P < 0.01.

This study was aimed at examining the vulnerability of the liver to oxygen-free radicals upon reoxygenation after prolonged ischemia. Livers from male Wistar rats were first flushed with Ringer's and Euro-Collins solutions. After ischemic storage in Krebs-Henseleit solution at 37 degrees C for 60 min and in Euro-Collins solution at 4 degrees C for another 60 min, they were then persufflated with either gaseous O2 or N2 for 30 min at 37 degrees C, and rinsed again with Ringer's solution. Enzyme concentrations and calcium ion activities were measured in the effluent rinsing solution after passage through the liver. Treatment with superoxide dismutase (SOD) or allopurinol resulted in a significant reduction of tissue injury, determined by the enzyme loss, calcium uptake, and lipid peroxidation upon persufflation with O2. Allopurinol also improved the tissue levels of ATP and the sum of adenine nucleotides after aerobic persufflation, whereas SOD did not. Notwithstanding, neither treatment had any effect on anoxic persufflation with N2. Thus, we conclude that the postischemic liver is susceptible to oxygen-induced free radical injury and that allopurinol and SOD promote specific antioxidative protection of the liver, with the exclusion of side effects related to substrates or perfusion modalities.

A major component of the organ injury mediated by toxic oxidants, such as seen following reperfusion of the ischemic liver, is due to the peroxidation of polyunsaturated fatty acids, especially of cell membranes. We utilized the measurement of exhaled breath ethane, a metabolic product unique to oxidant-mediated lipid peroxidation, as a noninvasive indicator of this process in swine liver subjected to warm ischemia/reperfusion. Under rigorously controlled anesthesia conditions, pig livers were subjected to 2 h of warm total ischemia, followed by reperfusion in situ. Expired air was collected and its ethane content quantitated by a novel gas chromatographic technique. The time course of breath ethane generation correlated closely with the appearance of hepatocellular injury as measured by impairment of Factor VII generation and other measures of liver integrity. Moreover, the administration of the specific superoxide free radical scavenger, superoxide dismutase (SOD), significantly attenuated both the elaboration of ethane and the hepatocellular injury. These findings not only provide confirmation of the previously reported link between hepatocellular injury by free radicals generated at reperfusion, but also establish the use of expired breath ethane analysis as a sensitive, specific, and noninvasive indicator of the injury process in real time.

Isolated cells make it possible to study mechanisms of cell and tissue injury under well-defined conditions, including the interaction of different cells in coculture experiments. Isolated cells, either in suspension or in monolayer cultures, have also been used to study the mechanism of reperfusion injury--in this case better termed as reoxygenation injury in view of the experimental approach taken. In hepatocytes, Kupffer, and endothelial cells, reoxygenation injury resulted in necrosis primarily mediated by reactive oxygen species released by various sources such as mitochondria (hepatocytes) and NADPH oxidase (Kupffer cells). In contrast, contracture was a characteristic feature of reoxygenation injury occurring in cardiomyocytes without loss of cytosolic enzymes. Beside reactive oxygen species, Kupffer cells were activated to release prostanoids and a decrease in endothelial cell-mediated fibrinolysis occurred upon reoxygenation. Reoxygenation injury in endothelial cells was significantly increased when neutrophils were added at the time of reoxygenation, presumably due to additional generation of reactive oxygen species and the release of proteases. As exemplified for the liver, these experiments suggest a mechanism of reperfusion injury in which the various cell types of a given tissue differ significantly in their response to hypoxia-reoxygenation but in which they interact with each other in a complex pathobiochemical network via various mediators such as cytokines, and tissue damaging effector molecules such as reactive oxygen species. Future experiments with isolated cells will allow detailed analysis of the underlying molecular mechanisms.