JNK activation and translocation to mitochondria mediates mitochondrial dysfunction and cell death induced by VDAC opening and sorafenib in hepatocarcinoma cells.

The multikinase inhibitor sorafenib, and opening of voltage dependent anion channels (VDAC) by the erastin-like compound X1 promotes oxidative stress and mitochondrial dysfunction in hepatocarcinoma cells. Here, we hypothesized that X1 and sorafenib induce mitochondrial dysfunction by increasing reactive oxygen species (ROS) formation and activating c-Jun N-terminal kinases (JNKs), leading to translocation of activated JNK to mitochondria. Both X1 and sorafenib increased production of ROS and activated JNK. X1 and sorafenib caused a drop in mitochondrial membrane potential (ΔΨ), a readout of mitochondrial metabolism, after 60 min. Mitochondrial depolarization after X1 and sorafenib occurred in parallel with JNK activation, increased superoxide (O2•-) production, decreased basal and oligomycin sensitive respiration, and decreased maximal respiratory capacity. Increased production of O2•- after X1 or sorafenib was abrogated by JNK inhibition and antioxidants. S3QEL 2, a specific inhibitor of site IIIQo, at Complex III, prevented depolarization induced by X1. JNK inhibition by JNK inhibitors VIII and SP600125 also prevented mitochondrial depolarization. After X1, activated JNK translocated to mitochondria as assessed by proximity ligation assays. Tat-Sab KIM1, a peptide selectively preventing the binding of JNK to the outer mitochondrial membrane protein Sab, blocked the depolarization induced by X1 and sorafenib. X1 promoted cell death mostly by necroptosis that was partially prevented by JNK inhibition. These results indicate that JNK activation and translocation to mitochondria is a common mechanism of mitochondrial dysfunction induced by both VDAC opening and sorafenib.

Amphiregulin stimulates liver regeneration after small-for-size mouse liver transplantation.

This study investigated whether amphiregulin (AR), a ligand of the epidermal growth factor receptor (EGFR), improves liver regeneration after small-for-size liver transplantation. Livers of male C57BL/6 mice were reduced to ~50% and ~30% of original sizes and transplanted. After transplantation, AR and AR mRNA increased in 50% but not in 30% grafts. 5-Bromodeoxyuridine (BrdU) labeling, proliferating cell nuclear antigen (PCNA) expression and mitotic index increased substantially in 50% but not 30% grafts. Hyperbilirubinemia and hypoalbuminemia occurred and survival decreased after transplantation of 30% but not 50% grafts. AR neutralizing antibody blunted regeneration in 50% grafts whereas AR injection (5 µg/mouse, iv) stimulated liver regeneration, improved liver function and increased survival after transplantation of 30% grafts. Phosphorylation of EGFR and its downstream signaling molecules Akt, mTOR, p70S6K, ERK and JNK increased markedly in 50% but not 30% grafts. AR stimulated EGFR phosphorylation and its downstream signaling pathways. EGFR inhibitor PD153035 suppressed regeneration of 50% grafts and largely abrogated stimulation of regeneration of 30% grafts by AR. AR also increased cyclin D1 and cyclin E expression in 30% grafts. Together, liver regeneration is suppressed in small-for-size grafts, as least in part, due to decreased AR formation. AR supplementation could be a promising therapy to stimulate regeneration of partial liver grafts.

NIM811, a mitochondrial permeability transition inhibitor, prevents mitochondrial depolarization in small-for-size rat liver grafts.

ATP decreases markedly in small-for-size liver grafts. This study tested if the mitochondrial permeability transition (MPT) underlies dysfunction of small-for-size livers. Half-size livers were implanted into recipients of about twice the donor weight, resulting in quarter-size liver grafts. NIM811 (5 microM), a nonimmunosuppressive MPT inhibitor was added to the storage solutions. Mitochondrial polarization and cell death were assessed by confocal microscopy of rhodamine 123 (Rh123) and propidium iodide (PI), respectively. After quarter-size transplantation, alanine aminotransferase (ALT), serum bilirubin and necrosis all increased. NIM811 blocked these increases by >70%. After 38 h, BrdU labeling, a marker of cell proliferation and graft weight increase were 3% and 5%, respectively, which NIM811 increased to 30% and 42%. NIM811 also increased survival of quarter-size grafts. In sham-operated livers, hepatocytes exhibited punctate Rh123 fluorescence. By contrast, in quarter-size grafts at 18 h after implantation, mitochondria of most hepatocytes did not take up Rh123, indicating mitochondrial depolarization. Nearly all hepatocytes not taking up Rh123 continued to exclude PI at 18 h, indicating that depolarization preceded cell death. NIM811 and free radical-scavenging polyphenols strongly attenuated mitochondrial depolarization. In conclusion, mitochondria depolarized after quarter-size liver transplantation. NIM811 decreased injury and stimulated regeneration, probably by inhibiting free radical-dependent MPT onset.

Endothelial nitric oxide synthase protects transplanted mouse livers against storage/reperfusion injury: Role of vasodilatory and innate immunity pathways.

Endothelial nitric oxide synthase (eNOS) plays a role in microcirculatory and immunomodulatory responses after warm ischemia/reperfusion. We hypothesized that eNOS is essential to maintain microcirculation, attenuate macrophage infiltration and decrease graft injury after liver transplantation. Liver transplantation was performed after 18 hours of cold storage in University of Wisconsin (UW) solution from wildtype and eNOS-deficient (B6.129P2-Nos3(tm/Unc)/J) donor mice into wildtype mice. Serum ALT, necrosis by histology, apoptosis by TUNEL, and macrophage infiltration by immunostaining against F4/80 antigen were determined 2 to 8 hours after implantation. Hepatic microcirculation was investigated after 4 hours by intravital confocal microscopy following injection of fluorescein-labeled erythrocytes. After sham operation, livers of wildtype and eNOS-deficient mice were not different in ALT, necrosis, apoptosis, macrophage infiltration, and microcirculation. After transplantation, ALT increased >3 times more after transplantation of eNOS-deficient livers than wildtype livers. Necrosis was >4 times greater, and TUNEL and F4/80 immunostaining in nonnecrotic areas were 2 and 1.5 times greater in eNOS-deficient donor livers, respectively. Compared with wildtype and eNOS sham-operated mice, sinusoidal blood flow velocity increased 1.6-fold after wildtype transplantation, but sinusoidal diameter was not changed. After transplantation of eNOS-deficient livers, blood flow velocity and sinusoidal diameter decreased compared with transplanted wildtype livers. These results indicate that donor eNOS attenuates storage/reperfusion injury after mouse liver transplantation. Protection is associated with improved microcirculation and decreased macrophage infiltration. Thus, eNOS-dependent graft protection may involve both vasodilatory and innate immunity pathways.

Primary cirrhotic hepatocytes resist TGFbeta-induced apoptosis through a ROS-dependent mechanism.

BACKGROUND/AIMS: The cirrhotic liver manifests dysregulated hepatocyte growth by poor regenerative capacity, formation of regenerative nodules, and malignant transformation to hepatocellular carcinoma. The purpose of this study was to determine if dysregulated hepatocyte growth occurs through deficient apoptosis. METHODS: Hepatocytes were isolated from normal and CCl(4)-treated mice and treated with TGFbeta, TNFalpha, and UV-C, known apoptotic agents. RESULTS: Cirrhotic hepatocytes were less sensitive to TGFbeta- (45+/-5 vs. 15+/-3%; P<0.003), TNFalpha- (59+/-21 vs. 21+/-8%; P=0.02), and UV-C-induced (31+/-4 vs. 17+/-4%; P<0.03) apoptosis compared to normal hepatocytes. In normal hepatocytes, TGFbeta-induced apoptosis occurred through a ROS-, MPT-, and caspase-dependent pathway. Cirrhotic hepatocytes lacked caspase activation, had decreased procaspase-8 expression, failed to undergo the MPT, and had increased basal ROS activity compared to normal hepatocytes. After treatment with trolox, an antioxidant that reduced basal ROS activity, cirrhotic hepatocytes underwent apoptosis in response to TGFbeta treatment. CONCLUSIONS: These findings suggest that increased ROS activity in cirrhotic hepatocytes plays a critical role in mediating cirrhotic hepatocyte resistance to apoptosis. Cirrhotic hepatocyte resistance to TGFbeta-induced apoptosis is ROS-dependent and is a mechanism of dysregulated growth in the chronically inflamed liver.

Activated Kupffer cells cause a hypermetabolic state after gentle in situ manipulation of liver in rats.

Harvesting trauma to the graft dramatically decreases survival after liver transplantation. Since activated Kupffer cells play a role in primary nonfunction, the purpose of this study was to test the hypothesis that organ manipulation activates Kupffer cells. To mimic what occurs with donor hepatectomy, livers from Sprague-Dawley rats underwent dissection with or without gentle organ manipulation in a standardized manner in situ. Perfused livers exhibited normal values for O(2) uptake (105 +/- 5 micromol. g(-1). h(-1)) measured polarigraphically; however, 2 h after organ manipulation, values increased significantly to 160 +/- 8 micromol. g(-1). h(-1) and binding of pimonidazole, a hypoxia marker, increased about threefold (P < 0.05). Moreover, Kupffer cells from manipulated livers produced three- to fourfold more tumor necrosis factor-alpha and PGE(2), whereas intracellular calcium concentration increased twofold after lipopolysaccharide compared with unmanipulated controls (P < 0.05). Gadolinium chloride and glycine prevented both activation of Kupffer cells and effects of organ manipulation. Furthermore, indomethacin given 1 h before manipulation prevented the hypermetabolic state, hypoxia, depletion of glycogen, and release of PGE(2) from Kupffer cells. These data indicate that gentle organ manipulation during surgery activates Kupffer cells, leading to metabolic changes dependent on PGE(2) from Kupffer cells, which most likely impairs liver function. Thus modulation of Kupffer cell function before organ harvest could be beneficial in human liver transplantation and surgery.

Alteration in Kupffer cell function after mild hemorrhagic shock.

Functional changes in Kupffer cells occur after profound hemorrhagic shock. This study was performed to demonstrate if Kupffer cell changes also occur after mild hemorrhagic shock. Sprague-Dawley rats were bled to a systolic blood pressure of 60 to 70 mmHg and resuscitated with Lactated Ringers solution (twice the shed blood volume) after 30 min. Resuscitation produced immediate recovery of blood pressure and allowed long-term recovery of the animals. Sham animals received anesthesia and monitoring only. Thirty minutes after resuscitation, Kupffer cells were isolated by centrifugal elutriation and cultured for 48 h. In Kupffer cells isolated from shocked animals, phorbol ester-stimulated superoxide production increased 7-fold and lipopolysaccharide- (LPS) stimulated prostaglandin E2 (PGE2) production increased 4-fold. Tumor necrosis factor-alpha (TNFalpha) production, on the other hand, was decreased by 50%. A non-significant trend toward increased phagocytosis was also observed, whereas LPS-stimulated nitric oxide production was unchanged. In conclusion, mild hemorrhagic shock produced increases in superoxide and PGE2 production, and decreases in TNFalpha production by Kupffer cells, changes that may be appropriate to defend against the infectious challenges that often follows trauma and hemorrhage.

Ischemic preconditioning of rat livers against cold storage-reperfusion injury: role of nonparenchymal cells and the phenomenon of heterologous preconditioning.

Brief periods of ischemia followed by reperfusion render tissues resistant against subsequent prolonged ischemia, a phenomenon called ischemic preconditioning. The effect of ischemic preconditioning on liver transplantation was investigated in relation to sinusoidal endothelial cell injury and Kupffer-cell activation, which are prominent features of storage and reperfusion injury leading to liver graft failure. Rat livers were preconditioned by 5 or 10 minutes of ischemia and 5 minutes of reperfusion and stored in University of Wisconsin (UW) solution for 30 hours. Livers were then reperfused for 15 minutes with physiological buffer containing trypan blue. Under these conditions, injury occurs predominantly to sinusoidal endothelial cells, reflected by trypan blue staining of nonparenchymal cells in histological sections. Ischemic preconditioning decreased nonparenchymal cell killing by more than 50%. When half the liver was preconditioned, sinusoidal endothelial cells were also protected in the contralateral half. Other stored livers were reperfused with nitroblue tetrazolium, which is converted to insoluble formazan by superoxide radicals. Ischemic preconditioning decreased the intensity of formazan deposition over Kupffer cells. Finally, stored livers were transplanted into nontreated rats. Ischemic preconditioning improved recipient long-term survival after 30 hours of cold ischemic storage in UW solution from 30% to 80% and decreased serum tumor necrosis factor-alpha levels in posthepatic blood 4 hours postoperatively from 98 to 54 pg/mL. In conclusion, ischemic preconditioning protects sinusoidal endothelial cells and suppresses Kupffer-cell activation after storage and reperfusion. As a result, graft survival improves after liver transplantation. Moreover, ischemia to half the liver confers protection to the other half. Such heterologous preconditioning provides a new means to protect liver tissue against ischemia-reperfusion injury without imposing ischemia on the target tissue.

NF-kappaB stimulates inducible nitric oxide synthase to protect mouse hepatocytes from TNF-alpha- and Fas-mediated apoptosis.

BACKGROUND AND AIMS: Hepatocyte apoptosis is induced by tumor necrosis factor alpha (TNF-alpha) and Fas ligand. Although nuclear factor-kappaB (NF-kappaB) activation protects hepatocytes from TNF-alpha-mediated apoptosis, the NF-kappaB responsive genes that protect hepatocytes are unknown. Our aim was to study the role of NF-kappaB activation and inducible nitric oxide synthases (iNOSs) in TNF-alpha- and Fas-mediated apoptosis in hepatocytes. METHODS: Primary cultures of hepatocytes from wild-type and iNOS knockout mice were treated with TNF-alpha, the Fas agonistic antibody Jo2, a nitric oxide (NO) donor (S-nitroso-N-acetylpenicillamine), an NO inhibitor (N(G)-methyl-L-arginine acetate), and/or adenovirus-expressing NF-kappaB inhibitors. RESULTS: The IkappaB superrepressor and a dominant-negative form of IkappaB kinase beta (IKKbeta) inhibited NF-kappaB binding activity by TNF-alpha or Jo2 and sensitized hepatocytes to TNF-alpha- and Jo2-mediated apoptosis. TNF-alpha and Jo2 induced iNOS messenger RNA and protein levels through the induction of NF-kappaB. S-nitroso-N-acetylpenicillamine inhibited Bid cleavage, the mitochondrial permeability transition, cytochrome c release, and caspase-8 and -3 activity, and reduced TNF-alpha- and Fas-mediated death in hepatocytes expressing IkappaB superrepressor. N(G)-methyl-L-arginine acetate partially sensitized hepatocytes to TNF-alpha- and Fas-mediated cell killing. TNF-alpha alone or Jo2 alone induced moderate cell death in hepatocytes from iNOS(-)/(-) mice. CONCLUSIONS: NO protects hepatocytes from TNF-alpha- and Fas-mediated apoptosis. Endogenous iNOS, which is activated by NF-kappaB via IKKbeta, provides partial protection from apoptosis.

Proinflammatory kupffer cell alterations after femur fracture trauma and sepsis in rats.

This study examined effects of trauma and sepsis on Kupffer cell function. When CBA/J mice had femur fracture (FFx), no deaths occurred. After cecal ligation and puncture (CLP), 44% died. Following combined injuries (FFx + CLP), mortality increased to 60%, suggesting a deleterious effect between FFx + CLP. Kupffer cell ablation with GdCI3 decreased mortality to 13% after CLP and 5% after FFx + CLP. After FFx, CLP, and FFx + CLP, Kupffer cells isolated from Sprague-Dawley rats produced 720%, 1,100%, and 2,130% more O2. than sham, respectively. Phagocytosis increased 320%, 610%, and 150%. Kupffer cell PGE2 production also increased 300%, 510%, and 300% over sham. After FFx alone, TNF-alpha production decreased 40%. By contrast, CLP and FFx + CLP increased TNF-alpha release 25% and 100%, respectively. After FFx, NO. production decreased 44%, whereas NO increased 280% and 260% after CLP and FFx + CLP. These findings indicate that Kupffer cells mediate mortality after CLP and FFx + CLP. Increased mortality is associated with a more proinflammatory and less antimicrobial Kupffer cell phenotype.

Contribution of adenosine A(2) receptors and cyclic adenosine monophosphate to protective ischemic preconditioning of sinusoidal endothelial cells against Storage/Reperfusion injury in rat livers.

A brief period of liver ischemia decreases sinusoidal endothelial cell killing after cold liver storage and improves graft survival after liver transplantation, a phenomenon called ischemic preconditioning. In this study, we investigated the mechanism of sinusoidal endothelial cell protection after ischemic preconditioning. Livers were preconditioned by 5 minutes of ischemia and 5 minutes of reperfusion. Subsequently, livers were stored for 30 hours in cold University of Wisconsin (UW) solution and reperfused briefly with physiological buffer containing Trypan blue. Ischemic preconditioning decreased sinusoidal endothelial cell killing after storage/reperfusion, as assessed by Trypan blue staining of nonparenchymal cells. Adenosine A(2) receptor blockade prevented the protective effect of ischemic preconditioning. By contrast, adenosine A(1) receptor blockade did not prevent protective ischemic preconditioning. Other rat livers were treated with adenosine A(1) and A(2) receptor agonists or dibutyryl-cyclic adenosine monophosphate (DB-cAMP) before storage. The adenosine A(2) receptor agonist, CGS-21680, and DB-cAMP decreased sinusoidal endothelial cell killing to the same extent as ischemic preconditioning, but the adenosine A(1) receptor agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA), had no effect. The adenosine A(2) agonist and prostaglandin E(2), another agent that preconditions sinusoidal endothelial cells against storage/reperfusion injury, but not the adenosine A(1) agonist, increased cAMP levels in cultured sinusoidal endothelial cells. In conclusion, an adenosine A(2) receptor pathway coupled to increased cAMP mediates sinusoidal endothelial cell protection by ischemic preconditioning.

Mitochondrial calcium transients in adult rabbit cardiac myocytes: inhibition by ruthenium red and artifacts caused by lysosomal loading of Ca(2+)-indicating fluorophores.

A cold/warm loading protocol was used to ester-load Rhod 2 into mitochondria and other organelles and Fluo 3 into the cytosol of adult rabbit cardiac myocytes for confocal fluorescence imaging. Transient increases in both cytosolic Fluo 3 and mitochondrial Rhod 2 fluorescence occurred after electrical stimulation. Ruthenium red, a blocker of the mitochondrial Ca(2+) uniporter, inhibited mitochondrial Rhod 2 fluorescence transients but not cytosolic Fluo 3 transients. Thus the ruthenium red-sensitive mitochondrial Ca(2+) uniporter catalyzes Ca(2+) uptake during beat-to-beat transients of mitochondrial free Ca(2+), which in turn may help match mitochondrial ATP production to myocardial ATP demand. After ester loading, substantial amounts of Ca(2+)-indicating fluorophores localized into an acidic lysosomal/endosomal compartment. This lysosomal fluorescence did not respond to electrical stimulation. Because fluorescence arose predominantly from lysosomes after the cold loading/warm incubation procedure, total cellular fluorescence failed to track beat-to-beat changes of mitochondrial fluorescence. Only three-dimensionally resolved confocal imaging distinguished the relatively weak mitochondrial signal from the bright lysosomal fluorescence.

The mitochondrial permeability transition augments Fas-induced apoptosis in mouse hepatocytes.

Tumor necrosis factor-alpha receptor 1 and Fas recruit overlapping signaling pathways. To clarify the differences between tumor necrosis factor alpha (TNFalpha) and Fas pathways in hepatocyte apoptosis, primary mouse hepatocytes were treated with TNFalpha or an agonist anti-Fas antibody after infection with an adenovirus expressing an IkappaB superrepressor (Ad5IkappaB). Treatment with TNFalpha induced apoptosis in Ad5IkappaB-infected mouse hepatocytes, as we previously reported for rat hepatocytes. Ad5IkappaB plus anti-Fas antibody or actinomycin D plus anti-Fas antibody rapidly induced apoptosis, whereas anti-Fas antibody alone produced little cytotoxicity. The proteasome inhibitor (MG-132) and a dominant-negative mutant of nuclear factor-kappaB-inducing kinase also promoted TNFalpha- and Fas-mediated apoptosis. Expression of either crmA or a dominant-negative mutant of the Fas-associated death domain protein prevented TNFalpha- and Fas-mediated apoptosis. In addition, the caspase inhibitors, DEVD-cho and IETD-fmk, inhibited TNFalpha- and Fas-mediated apoptosis. In Ad5IkappaB-infected hepatocytes, caspases-3 and -8 were activated within 2 h after treatment with anti-Fas antibody or within 6 h after TNFalpha treatment. Confocal microscopy demonstrated onset of the mitochondrial permeability transition (MPT) and mitochondrial depolarization by 2-3 h after anti-Fas antibody treatment and 8-10 h after TNFalpha treatment, followed by cytochrome c release. The combination of the MPT inhibitors, cyclosporin A, and trifluoperazine, protected Ad5IkappaB-infected hepatocytes from TNFalpha-mediated apoptosis. After anti-Fas antibody, cyclosporin A and trifluoperazine decreased cytochrome c release but did not prevent caspase-3 activation and cell-death. In conclusion, nuclear factor-kappaB activation protects mouse hepatocytes against both TNFalpha- and Fas-mediated apoptosis. TNFalpha and Fas recruit similar but nonidentical, pathways signaling apoptosis. The MPT is obligatory for TNFalpha-induced apoptosis. In Fas-mediated apoptosis, the MPT accelerates the apoptogenic events but is not obligatory for them.

Molecular cloning and characterization of a human mitochondrial ceramidase.

We have recently purified a rat brain membrane-bound nonlysosomal ceramidase (El Bawab, S., Bielawska, A., and Y. A. Hannun (1999) J. Biol. Chem. 274, 27948-27955). Using peptide sequences obtained from the purified rat brain enzyme, we report here the cloning of the human isoform. The deduced amino acid sequence of the protein did not show any similarity with proteins of known function but was homologous to three putative proteins from Arabidospis thaliana, Mycobacterium tuberculosis, and Dictyostelium discoideum. Several blocks of amino acids were highly conserved in all of these proteins. Analysis of the protein sequence revealed the presence at the N terminus of a signal peptide followed by a putative myristoylation site and a putative mitochondrial targeting sequence. The predicted molecular mass was 84 kDa, and the isoelectric point was 6.69, in agreement with rat brain purified enzyme. Northern blot analysis of multiple human tissues showed the presence of a major band corresponding to a size of 3.5 kilobase. Analysis of this major band on the blot indicated that the enzyme is ubiquitously expressed with higher levels in kidney, skeletal muscle, and heart. The enzyme was then overexpressed in HEK 293 and MCF7 cells using the pcDNA3. 1/His-ceramidase construct, and ceramidase activity (at pH 9.5) increased by 50- and 12-fold, respectively. Next, the enzyme was characterized using lysate of overexpressing cells. The results confirmed that the enzyme catalyzes the hydrolysis of ceramide in the neutral alkaline range and is independent of cations. Finally, a green fluorescent protein-ceramidase fusion protein was constructed to investigate the localization of this enzyme. The results showed that the green fluorescent protein-ceramidase fusion protein presented a mitochondrial localization pattern and colocalized with mitochondrial specific probes. These results demonstrate that this novel ceramidase is a mitochondrial enzyme, and they suggest the existence of a topologically restricted pathways of sphingolipid metabolism.

Role of cytokines and cytokine-producing cells in reperfusion injury to the liver.

We have categorized reperfusion injury into (1) warm ischemia/reperfusion injury and (2) cold storage/reperfusion injury. Warm ischemia/reperfusion injury is relevant to shock and hepatic surgery, whereas cold storage/reperfusion injury occurs when the liver is procured and transported for transplantation. Different stages of reperfusion injury; cells that mediate each stage; and cytokines involved are described. Understanding the specific cells and cytokines involved in causing reperfusion injury and their timing will encourage the development of therapeutic strategies aimed at preventing and diminishing the consequences of reperfusion injury.

Intravenous glycine improves survival in rat liver transplantation.

In situ manipulation by touching, retracting, and moving liver lobes gently during harvest dramatically reduces survival after transplantation (P. Schemmer, R. Schoonhoven, J. A. Swenberg, H. Bunzendahl, and R. G. Thurman. Transplantation 65: 1015-1020, 1998). The development of harvest-dependent graft injury upon reperfusion can be prevented with GdCl3, a rare earth metal and Kupffer cell toxicant, but it cannot be used in clinical liver transplantation because of its potential toxicity. Thus the effect of glycine, which prevents activation of Kupffer cells, was assessed here. Minimal dissection of the liver for 12 min plus 13 min without manipulation had no effect on survival (100%). However, gentle manipulation decreased survival to 46% in the control group. Furthermore, serum transaminases and liver necrosis were elevated 4- to 12-fold 8 h after transplantation. After organ harvest, the rate of entry and exit of fluorescein dextran, a dye confined to the vascular space, was decreased about twofold, indicating disturbances in the hepatic microcirculation. Pimonidazole binding, which detects hypoxia, increased about twofold after organ manipulation, and Kupffer cells isolated from manipulated livers produced threefold more tumor necrosis factor-alpha after lipopolysaccharide than controls. Glycine given intravenously to the donor increased the serum glycine concentration about sevenfold and largely prevented the effect of gentle organ manipulation on all parameters studied. These data indicate for the first time that pretreatment of donors with intravenous glycine minimizes reperfusion injury due to organ manipulation during harvest and after liver transplantation.