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Hepatic macrophages are sensitized to alcohol in 24 h due to increases in the endotoxin receptor, CD14; however, desensitization to lipopolysaccharide (LPS), which occurred earlier, could not be explained by changes in CD14. Therefore, the purpose of this work was to attempt to understand factors responsible for ethanol-induced desensitization to LPS in hepatic macrophages. Rats were given ethanol (5 g/kg body wt) intragastrically, and hepatic macrophages were isolated 2 h later. After addition of endotoxin, intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured using fura 2 and tumor necrosis factor (TNF)-alpha was measured by ELISA. Ethanol given 2 h before injection of LPS totally prevented liver injury and blunted LPS-induced increases in [Ca(2+)](i) and TNF-alpha in hepatic macrophages. Furthermore, the protein kinase C (PKC) agonist phorbol 12-myristate 13-acetate and acute ethanol treatment both activated PKC and largely prevented the influx of [Ca(2+)](i) caused by LPS. Sterilization of the gut with antibiotics completely blocked all effects of ethanol on [Ca(2+)](i) and TNF-alpha release. Thus ethanol-induced desensitization of hepatic macrophages correlates with gut-derived endotoxin after ethanol and involves the effect of PKC on voltage-dependent Ca(2+) channels.

The continuous intragastric enteral feeding protocol in the rat was a major development in alcohol-induced liver injury (ALI) research. Much of what has been learned to date involves inhibitors or nutritional manipulations that may not be specific. Knockout technology avoids these potential problems. Therefore, we used long-term intragastric cannulation in mice to study early ALI. Reactive oxygen species are involved in mechanisms of early ALI; however, their key source remains unclear. Cytochrome P-450 (CYP)2E1 is induced predominantly in hepatocytes by ethanol and could be one source of reactive oxygen species leading to liver injury. We aimed to determine if CYP2E1 was involved in ALI by adapting the enteral alcohol (EA) feeding model to CYP2E1 knockout (-/-) mice. Female CYP2E1 wild-type (+/+) or -/- mice were given a high-fat liquid diet with either ethanol or isocaloric maltose-dextrin as control continuously for 4 wk. All mice gained weight steadily over 4 wk, and there were no significant differences between groups. There were also no differences in ethanol elimination rates between CYP2E1 +/+ and -/- mice after acute ethanol administration to naive mice or mice receiving EA for 4 wk. However, EA stimulated rates 1.4-fold in both groups. EA elevated serum aspartate aminotransferase levels threefold to similar levels over control in both CYP2E1 +/+ and -/- mice. Liver histology was normal in control groups. In contrast, mice given ethanol developed mild steatosis, slight inflammation, and necrosis; however, there were no differences between the CYP2E1 +/+ and -/- groups. Chronic EA induced other CYP families (CYP3A, CYP2A12, CYP1A, and CYP2B) to the same extent in CYP2E1 +/+ and -/- mice. Furthermore, POBN radical adducts were also similar in both groups. Data presented here are consistent with the hypothesis that oxidants from CYP2E1 play only a small role in mechanisms of early ALI in mice. Moreover, this new mouse model illustrates the utility of knockout technology in ALI research.

The mechanism of the immunosuppressive effects of glycine and its pathophysiological applications are discussed in this review. Glycine has been well characterized in spinal cord as an inhibitory neurotransmitter which activates a glycine-gated chloride channel (GlyR) expressed in postsynaptic membranes. Activation of the channel allows the influx of chloride, preventing depolarization of the plasma membrane and the potentiation of excitatory signals along the axon. Glycine has recently been shown to have similar inhibitory effects on several white blood cells, including hepatic and alveolar macrophages, neutrophils, and lymphocytes. Pharmacological analysis using a GlyR antagonist strychnine, chloride-free buffer, and radiolabeled chloride has provided convincing evidence to support the hypothesis that many white blood cells contain a glycine-gated chloride channel with properties similar to the spinal cord GlyR. Molecular analysis using reverse transcription-polymerase chain reaction and Western blotting has identified the mRNA and protein for the beta subunit of the GlyR in total RNA and purified membrane protein from rat Kupffer cells. Dietary glycine is protective in rat models against endotoxemia, liver ischemia-reperfusion, and liver transplantation, most likely by inactivating the Kupffer cell via this newly identified glycine-gated chloride channel. Glycine also prevents the growth of B 16 melanomas cell in vivo. Moreover, dietary glycine is protective in the kidney against cyclosporin A toxicity and ischemia-reperfusion injury. Glycine may be useful clinically for the treatment of sepsis, adult respiratory distress syndrome, arthritis, and other diseases with an inflammatory component.

The aim of this study was to determine which PGE(2) receptors and signal transduction pathways are responsible for the stimulation of oxygen uptake in liver. Hepatic parenchymal cells isolated from female Sprague-Dawley rats were incubated either with PGE(2), 17-phenyl-omega-trinor PGE(2) (an EP(1)-specific agonist), or 11-deoxy PGE(1) (an EP(2)/EP(4)-specific agonist), and oxygen consumption was measured. Both PGE(2) and 11-deoxy PGE(1) stimulated oxygen consumption. However, an EP(1) agonist was without effect. Although PGE(2) elevated intracellular calcium, this occurred at concentrations approximately 500-fold lower than that required to stimulate oxygen uptake. PGE(2)-stimulated increases in cAMP formation correlated well with the increase in oxygen consumption. Dibutyryl cAMP also increased oxygen consumption. Furthermore, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide, a cell-permeable inhibitor of protein kinase A (PKA), reduced the stimulation of oxygen uptake by PGE(2). Incubation of isolated parenchymal cell mitochondria with the purified catalytic subunit of PKA and ATP increased both state 3 rates of oxygen uptake and the respiratory control ratio by approximately 50%. Activation of these events was prevented by incubation with the PKA inhibitory peptide, PKI. These findings are consistent with the hypothesis that PGE(2) stimulates oxygen consumption via an EP(2) and/or EP(4) subclass of receptors through the actions of cAMP on a cAMP-dependent protein kinase.

Previous studies demonstrated that dietary glycine prevents elevated rates of cell proliferation following treatment with the peroxisome proliferator and liver carcinogen WY-14,643. Since increased cell replication is associated with the development of hepatic cancer caused by peroxisome proliferators, glycine may have anti-cancer properties. Therefore, experiments were designed to test the hypothesis that dietary glycine would inhibit the hepatocarcinogenic effect of WY-14,643. Male F344 rats were fed four different NIH 07-based diets: 5% glycine; 5% valine for nitrogen balance (control); 0.1% WY-14,643 + 5% valine (WY-14,643); 0.1% WY-14,643 + 5% glycine (WY-14,643 + glycine). Food consumption did not differ among the groups, but WY-14,643-fed rats weighed 10-25% less than expected based on previous studies. Serum glycine levels were elevated 4-5-fold by glycine-containing diets; however, the 10-fold increase in peroxisomal enzyme activity caused by WY-14,643 was unaffected by the addition of 5% glycine to the diet. After 22 weeks, livers from rats fed WY-14,643 had a similar incidence and multiplicity of proliferative lesions (foci and adenomas) to those fed WY-14,643 + glycine. Moreover, cell proliferation in the surrounding 'normal' parenchyma (labeling index approximately 4%) and foci (labeling index approximately 50%) did not differ between WY-14,643 and WY-14,643 + glycine-fed rats. However, after 51 weeks of dietary exposure to WY-14,643, glycine prevented formation of small (0-5 mm diameter) tumors by 23% and inhibited the development of medium size (5-10 mm) tumors by 64%. Furthermore, glycine prevented the formation of the largest tumors (>10 mm) by nearly 80%. Thus, glycine did not inhibit early foci formation; however, it significantly decreased their ability to progress to tumors. Moreover, the inhibitory effect of glycine was greater with increasing tumor size. These studies demonstrate that dietary glycine prevents the development of hepatic tumors caused by the peroxisome proliferator WY-14,643 consistent with the idea that it may be an effective chemopreventive agent.

N-6 polyunsaturated fatty acids (N-6 PUFAs), major constituents of corn oil and natural ligands for peroxisome proliferator-activated receptors, increase the rate of growth of established tumors. It has been proposed that chemical peroxisome proliferators increase hepatocyte proliferation by mechanisms involving activation of nuclear factor-kappaB (NF-kappaB) and production of low levels of tumor necrosis factor alpha (TNFalpha) by Kupffer cells; however, how N-6 PUFAs are involved in increased cell proliferation in liver is not well understood. Here, the hypothesis that N-6 PUFAs increase production of mitogens by activation of Kupffer cell NF-kappaB was tested. A single dose of corn oil (2 ml/kg, i.g.), but not olive oil or medium-chain triglycerides (saturated fat), caused an approximately 3-fold increase in hepatocyte proliferation. Similarly, when activity of NF-kappaB in whole rat liver or isolated hepatocytes and Kupffer cells was measured at various time intervals for up to 36 h, only corn oil activated NF-kappaB. Corn oil increased NF-kappaB activity approximately 3-fold 1-2 h after treatment exclusively in the Kupffer cell fraction. In contrast, increases were small and only occurred after approximately 8 h in hepatocytes. The activation of NF-kappaB at 2 h and increases in cell proliferation at 24 h due to corn oil were prevented almost completely when rats were pretreated for 4 days with either dietary glycine (5% w/w), an agent that inactivates Kupffer cells, or the NADPH oxidase inhibitor, diphenyleneiodonium (s.c., 1 mg/kg/day). Furthermore, arachidonic acid (100 microM) activated superoxide production approximately 4-fold when added to isolated Kupffer cells in vitro. This phenomenon was not observed with oleic or linoleic acids. Interestingly, a single dose of corn oil increased TNFalpha mRNA nearly 2-fold 8 h after treatment. It is concluded that corn oil rapidly activates NF-kappaB in Kupffer cells via oxidant-dependent mechanisms. This triggers production of low levels of TNFalpha which is mitogenic in liver and promotes growth of hepatocytes.

Glycine blunts lipopolysaccharide (LPS)-induced increases in intracellular calcium concentration ([Ca(2+)](i)) and tumor necrosis factor-alpha (TNF-alpha) production by Kupffer cells through a glycine-gated chloride channel. Alveolar macrophages, which have a similar origin as Kupffer cells, play a significant role in the pathogenesis of several lung diseases including asthma, endotoxemia, and acute inflammation due to inhaled bacterial particles and dusts. Therefore, studies were designed here to test the hypothesis that alveolar macrophages could be inactivated by glycine via a glycine-gated chloride channel. The ability of glycine to prevent endotoxin [lipopolysaccharide (LPS)]-induced increases in [Ca(2+)](i) and subsequent production of superoxide and TNF-alpha in alveolar macrophages was examined. LPS caused a transient increase in intracellular calcium to nearly 200 nM, with EC(50) values slightly greater than 25 ng/ml. Glycine, in a dose-dependent manner, blunted the increase in [Ca(2+)](i), with an IC(50) less than 100 microM. Like the glycine-gated chloride channel in the central nervous system, the effects of glycine on [Ca(2+)](i) were both strychnine sensitive and chloride dependent. Glycine also caused a dose-dependent influx of radiolabeled chloride with EC(50) values near 10 microM, a phenomenon which was also inhibited by strychnine (1 microM). LPS-induced superoxide production was also blunted in a dose-dependent manner by glycine and was reduced approximately 50% with 10 microM glycine. Moreover, TNF-alpha production was also inhibited by glycine and also required nearly 10 microM glycine for half-inhibition. These data provide strong pharmacological evidence that alveolar macrophages contain glycine-gated chloride channels and that their activation is protective against the LPS-induced increase in [Ca(2+)](i) and subsequent production of toxic radicals and cytokines.

Background & Aims: Tumor necrosis factor (TNF)-alpha is associated with increased mortality in alcoholics, but its role in early alcohol-induced liver injury is not fully understood. Recently, it was shown that injury induced by the enteral alcohol delivery model of Tsukamoto and French was reduced by antibodies to TNF-alpha. To obtain clear evidence for or against the hypothesis that TNF-alpha is involved, we studied TNF receptor 1 (TNF-R1, p55) or 2 (TNF-R2, p75) knockout mice., Methods: Long-term enteral alcohol delivery was modified for male gene-targeted mice lacking TNF-R1 and TNF-R2. Animals were given a high-fat liquid diet continuously with either ethanol or isocaloric maltose-dextrin as a control for 4 weeks., Results: Ethanol elevated serum levels of alanine aminotransferase nearly 3-fold in wild-type and TNF-R2 knockout mice but not in TNF-R1 knockout mice. Likewise, ethanol caused severe liver injury in wild-type mice (pathology score, 5.5 +/- 0.6) and TNF-R2 knockout mice (pathology score, 5.0 +/- 0.4), but not in TNF-R1 knockout mice (pathology score, 0.8 +/- 0.4; P < 0.001)., Conclusions: Long-term ethanol feeding caused liver injury in wild-type and TNF-R2 knockout mice but not in TNF-R1 knockout mice, providing solid evidence in support of the hypothesis that TNF-alpha plays an important role in the development of early alcohol-induced liver injury via the TNF-R1 pathway. Moreover, the long-term enteral ethanol feeding technique we described for the first time for knockout mice provides a useful new tool for alcohol research.

Cyclosporin A (CsA) nephrotoxicity is associated with renal hypoxia and increases in free radicals in the urine. This study was designed to elucidate the mechanism of radical production caused by CsA. Pretreatment of rats with CsA (25 mg/kg, i.g.) for 5 days decreased glomerular filtration rates by 65%, an effect largely prevented by both dietary glycine (5%) or renal denervation. CsA dissolved in olive oil produced a 6-line alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (4-POBN)/free radical signal in the urine, which partitioned predominantly into the aqueous phase after chloroform extraction (i.e., it is water soluble). Dimethyl sulfoxide (DMSO) is attacked by the hydroxyl radical to produce a methyl radical; administration of CsA with [(12)C]DMSO produced two radical species in urine, one with hyperfine coupling constants similar to the 4-POBN/methyl radical adduct found in aqueous solution. CsA given with [(13)C]DMSO produced a 12-line spectrum, confirming the formation of hydroxyl radicals. The methyl radical produced by the hydroxyl radical represented 62% of radicals detected in urine but only 15% in bile. Therefore, hydroxyl radicals are produced largely in the kidney. Free radicals in urine were increased about 5-fold by CsA, an effect completely blocked by the inhibitory neurotransmitter, glycine, or by renal denervation. CsA infusion for 30 min increased efferent renal nerve activity 2-fold, and dietary glycine (5%) totally blocked this phenomenon. Taken together, these data are consistent with the hypothesis that CsA increases hydroxyl radical formation by increasing renal nerve activity resulting in vasoconstriction and hypoxia-reoxygenation. Glycine blunts the effect of CsA on the renal nerve, which explains, in part, prevention of nephrotoxicity.

The relationship between gender and alcohol-induced liver disease is complex; however, endotoxin is most likely involved. Recently, it was reported that estriol activated Kupffer cells by upregulation of the endotoxin receptor CD14. Therefore, the purpose of this work was to study how estriol sensitizes Kupffer cells. Rats were given estriol (20 mg/kg ip), and Kupffer cells were isolated 24 h later. After addition of lipopolysaccharide (LPS), intracellular Ca2+ concentration was measured using a microspectrofluorometer with the fluorescent indicator fura 2, and tumor necrosis factor-alpha was measured by ELISA. CD14 was evaluated by Western analysis. One-half of the rats given estriol intraperitoneally 24 h before an injection of a sublethal dose of LPS (5 mg/kg) died within 24 h, whereas none of the control rats died. Mortality was prevented totally by sterilization of the gut with antibiotics. A similar pattern was obtained with liver histology and serum transaminases. Translocation of horseradish peroxidase was increased about threefold in gut segments by treatment with estriol. This increase was not altered by treatment with nonabsorbable antibiotics. On the other hand, endotoxin levels were increased to 60-70 pg/ml in plasma of rats treated with estriol. As expected, this increase was prevented (<20 pg/ml) by antibiotics. In isolated Kupffer cells, LPS-induced increases in intracellular Ca2+ concentration, tumor necrosis factor-alpha production, and CD14 were increased, as previously reported. All these phenomena were blocked by antibiotics. Therefore, it is concluded that estriol treatment in vivo sensitizes Kupffer cells to LPS via mechanisms dependent on increases in CD14. This is most likely due to elevated portal blood endotoxin caused by increased gut permeability.

Background: In clinical liver transplants, grafts are frequently exposed to endotoxin (lipopolysaccharide, LPS) before harvest and may be predisposed to dysfunction. Because graft failure is linked to sinusoidal endothelial cell injury after storage/reperfusion, we investigated the effect of donor exposure to LPS on graft survival in relation to sinusoidal endothelial cell injury after storage/reperfusion in rats., Methods: Rats were injected with 0.5 mg/kg LPS. In some rats, 20 mg/kg GdCl3 or 5 mg/kg indomethacin was injected before LPS to ablate Kupffer cells and inhibit prostaglandin (PG) synthesis, respectively. Other rats were injected with 100 microg/kg dimethyl PGE2, a stable PGE2 analog. Rat livers were harvested, stored in cold UW solution and transplanted to non-treated rats for determination of survival and liver injury in recipients. Otherwise, after cold storage, the livers were reperfused briefly with physiological buffer containing trypan blue for determination of sinusoidal endothelial cell injury by counting trypan blue-positive nuclei in histological sections., Results: Donor treatment with LPS increased hepatic PGE2 production before storage and decreased recipient survival, but paradoxically decreased killing of sinusoidal endothelial cells after storage and reperfusion. Pretreatment of donors with GdCl3 or indomethacin prevented the protective preconditioning of sinusoidal endothelial cells by LPS, whereas pretreatment with dimethyl PGE2 protected sinusoidal endothelial cells to the same extent as LPS. Unlike LPS, however, PGE2 attenuated graft injury after liver transplants., Conclusion: PGE2 derived from LPS-stimulated Kupffer cells protects sinusoidal endothelial cells against storage/reperfusion injury. Unlike LPS, PGE2 improves graft function after liver transplants. Thus, donor preconditioning with PGE2 may be beneficial in liver transplants.

Disturbances in hepatic microcirculation increase graft injury and failure; therefore, this study evaluates the effects of ethanol on microcirculation after liver transplantation. Donor rats were given one dose of ethanol (5 g/kg) by gavage 20 h before explantation, and grafts were stored in University of Wisconsin solution for 24 h before implantation. Acute ethanol treatment decreased 7-day survival of grafts from about 90 to 30%, increased transaminase release nearly 4-fold, and decreased bile production by 60%. Moreover, portal pressure increased significantly and liver surface oxygen tension decreased about 50%, indicating that ethanol disturbs hepatic microcirculation. Pimonidazole, a 2-nitroimidazole hypoxia marker, was given i.v. to recipients 30 min after implantation, and grafts were harvested 1 h later. Ethanol increased hepatic pimonidazole binding about 3-fold, indicating that ethanol led to hypoxia in fatty grafts. Ethanol also significantly increased free radicals in bile. Catechin (30 mg/kg i.v. upon reperfusion), a free radical scavenger, and Carolina Rinse solution, which contains several agents that inhibit free radical formation, minimized disturbances in microcirculation and prevented pimonidazole adduct formation significantly. These treatments also blunted increases in transaminase release and improved survival of fatty grafts. Destruction of Kupffer cells with GdCl(3) (20 mg/kg i.v. 24 h before explantation) or inhibition of formation of leukotrienes with MK-886 (50 microM in University of Wisconsin or rinse solution) also minimized hypoxia and improved survival after transplantation. Taken together, these results demonstrate that ethanol disturbs hepatic microcirculation, leading to graft hypoxia after transplantation, most likely by activating Kupffer cells and increasing free radical production.

Alcohol treatment results in increases in the release of endotoxin from gut bacteria and membrane permeability of the gut to endotoxin, or both. Females are more sensitive to these changes. Elevated levels of endotoxin activate Kupffer cells to release substances such as eicosanoids, TNF-alpha and free radicals. Prostaglandins increase oxygen uptake and most likely are responsible for the hypermetabolic state in the liver. The increase in oxygen demand leads to hypoxia in the liver, and on reperfusion, alpha-hydroxyethyl free radicals are formed which lead to tissue damage in oxygen-poor pericentral regions of the liver lobule.

Reperfusion after liver transplantation results in the induction of tumor necrosis factor-alpha (TNFalpha) as well as activation of the stress-associated signaling proteins, c-Jun N-terminal kinase (JNK), activating protein-1 (AP-1), and nuclear factor-kappaB (NF-kappaB). To test the hypothesis that Kupffer cells are involved in the activation of signal transduction cascades during rat liver transplantation, Kupffer cells were depleted from donor liver using gadolinium chloride (GdCl3), and then the activation of JNK, AP-1, and NF-kappaB were assessed after transplantation. The results showed that GdCl3 treatment did not inhibit the activation of these stress signals, although transplanted livers were depleted of Kupffer cells and partially protected from reperfusion injury. Interleukin-6 (IL-6) and IL-10 messenger RNAs (mRNAs) were induced by transplantation, and the induction was suppressed by Kupffer cell depletion. The induction of TNFalpha mRNA and serum protein during liver transplantation was unaffected by GdCl3. These results show that Kupffer cells are not a major source of TNFalpha production after liver transplantation and that stress-signaling protein activation occurs independently of Kupffer cells. Transplantation strongly activates the transcription factor NF-kappaB, which blocks TNFalpha-mediated apoptosis in hepatocytes in vitro. To assess the role of NF-kappaB activation during liver transplantation, the IkappaBalpha superrepressor was expressed in donor livers using adenoviral-mediated gene transfer. Inhibition of NF-kappaB resulted in increased serum alanine aminotransferase levels after 3 hours of transplantation. In addition, the blockade of NF-kappaB resulted in increased histological tissue injury and increased hepatic terminal deoxyribonucleotide transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining, indicating apoptosis. These results show that NF-kappaB activation has a protective role in the transplanted liver.

Kupffer cell-dependent injury in livers gently manipulated during harvest develops upon transplantation; however, underlying mechanisms remain unknown. Thus, the purpose of this study was to identify factors involved in mechanisms of injury. Livers from female Sprague-Dawley rats (200-230 g) were cold stored for 24 h in University of Wisconsin solution. Subsequently, livers were perfused at 37 degrees C with oxygen-saturated Krebs-Henseleit buffer containing fluorescein-dextran to assess microcirculation. Cell death was assessed by uptake of trypan blue, a vital dye. Minimal dissection during harvest had no effects on sinusoidal lining cells; however, gentle organ manipulation dramatically increased trypan blue uptake about 5-fold (p <.05). In contrast, perfusion with N2-saturated buffer after cold storage totally prevented cell death due to manipulation. At harvest, portal venous pressure was increased significantly by 70% due to manipulation. Furthermore, vascular space and microcirculation were decreased by more than 50% (p <.05), reflecting the rate of entry and exit of fluorescein-dextran. Pimonidazole, a 2-nitroimidazole marker, was given to rats before harvest to detect hypoxia in liver. Pimonidazole adduct binding was increased significantly about 2-fold by manipulation. To detect free radical adducts by electron spin resonance (ESR) spectroscopy in bile, C-phenyl-N-tert-butylnitrone was given as spin trapping reagent to the donor before operation. Free radical formation was increased about 3-fold by organ manipulation (p <.05). Donors given gadolinium chloride, a selective Kupffer cell toxicant, or dietary glycine, which prevents activation of Kupffer cells, significantly blunted microcirculatory disturbances, hypoxia, and death of endothelial lining cells. These data indicate for the first time that gentle organ manipulation during harvest causes oxygen-dependent reperfusion injury to endothelial lining cells via mechanisms involving hepatic microcirculation, hypoxia, and Kupffer cells.

This study investigated the effect of glycine on hemorrhagic shock in the rat. Rats were bled to maintain mean arterial pressure at 30-35 mm Hg for 1 h and subsequently resuscitated with 60% shed blood and lactated Ringer's solution. Only 20% of rats receiving saline just prior to resuscitation survived 72 h after shock. Survival was increased by glycine (11.2-90.0 mg/kg, i.v.) in a dose-dependent manner (half-maximal effect = 25 mg/kg) and reached maximal values of 78% at 45 mg/kg. Eighteen hours after resuscitation, creatinine phosphokinase increased 23-fold, transaminases increased 33-fold, and creatinine was elevated 2.4-fold, indicating injury to the heart, liver, and kidney, respectively. Pulmonary edema, leukocyte infiltration, and hemorrhage were also observed. In the kidney, proximal tubular necrosis, leukocyte infiltration, and severe hemorrhage in the outer medullary area occurred in rats receiving saline. Glycine reduced these pathological alterations significantly. It has been reported that oxidative stress and tumor necrosis factor(TNF)-alpha-production are involved in the pathophysiology of multiple-organ injury after shock. In this study, free radical production was increased 4-fold during shock, an effect blocked largely by glycine. Increases in intracellular calcium and production of TNF-alpha by isolated Kupffer cells stimulated by endotoxin were elevated significantly by hemorrhagic shock, alterations which were totally prevented by glycine. Taken together, it is concluded that glycine reduces organ injury and mortality caused by hemorrhagic shock by preventing free radical production and TNF-alpha formation.

The continuous intragastric in vivo enteral feeding model in the rat developed by Tsukamoto and French has been very useful; however, it requires surgical expertise. Recently, we found that Kupffer cells isolated from rats treated only once with ethanol were sensitized to endotoxin 24 hours later. Accordingly, these experiments were designed to determine if a new, simple animal model of ethanol hepatotoxicity could be developed based on Kupffer cell sensitization. Female Wistar rats were given ethanol (5 g/kg body weight) once every 24 hours intragastrically. Livers were stained with hematoxylin-eosin to assess steatosis, inflammation, and necrosis, and tissue triglycerides, serum transaminases, and plasma endotoxin were measured. Kupffer cells were isolated 0 to 24 hours after one intragastric dose of ethanol daily, and intracellular Ca2+ ([Ca2+]i) was measured using fura-2, while tumor necrosis factor alpha (TNF-alpha) was measured by enzyme-linked immunosorbent assay. CD14 was evaluated by Western and Northern analysis. Ethanol caused steatosis, necrosis, and inflammation in only a few weeks, and after 8 weeks, serum aspartate transaminase (AST) levels were doubled. Values were similar to levels achieved in the enteral feeding model. Triglycerides were also increased significantly by ethanol as expected, and endotoxin levels were increased to 70 to 80 pg/mL. This latter increase was prevented (<20 pg/mL) by antibiotics implicating endotoxin. In isolated Kupffer cells from untreated control rats, [Ca2+]i increased to 82 +/- 7 nmol/L after addition of lipopolysaccharide (LPS) (100 ng/mL), and levels were elevated about twofold by ethanol given 24 hours earlier (174 +/- 15 nmol/L). In addition, TNF-alpha production by Kupffer cells was increased fourfold in cells isolated from rats treated with ethanol 24 hours earlier. Sterilization of the gut with antibiotics blocked all effects of ethanol on [Ca2+]i and TNF-alpha release completely. Moreover, 4 weeks after ethanol, CD14 in Kupffer cells was elevated about twofold. A new, simple chronic model of ethanol hepatotoxicity has been developed here based on sensitization of Kupffer cells to endotoxin.

The effects of estriol on oxygen uptake, glucose release, lactate and pyruvate production, beta-hydroxybutyrate and acetoacetate production in perfused rat liver as well as, carbon uptake in rat liver and intracellular calcium in isolated Kupffer cells were investigated. Basal oxygen consumption of perfused liver increased significantly in estriol or ethanol-treated rats. But these increased effects were blocked by gadolinium chloride pretreatment. In a metabolic study, pretreatment with estriol resulted in a decrease in glucose production and in glycolysis while an increase in ketogenesis. A more oxidized redox state of the mitochondria was indicated by increased ratios of perfusate [lactate]/[pyruvate] and decreased ratios of perfusate [beta-hydroxybutyrate]/[acetoacetate]. Carbon uptake of Kupffer-cell increased significantly in estriol-treated rats. But these increased uptake were not shown in rats pre-treated by gadolinium chloride blocking phagocytosis. In isolated Kupffer cells from estriol-treated rats, intracellular calcium was more significantly increased after addition of lipopolysaccharide (LPS) than in controls. These findings suggest that the metabolic effects of estriol (two mg per 100 mg body wt) can be summarized to be highly toxic in rat liver, and these findings suggest that oral administration of estrogens may induce hepatic dysfunctions and play a role in the development of liver disease.

Background: In a recent study, disturbances of hepatic microcirculation at harvesting caused by in situ organ manipulation dramatically reduced survival after a liver transplant. Because hepatic innervation is involved in the regulation of liver hemodynamics, the effect of denervation before harvesting was assessed here., Methods: The livers were harvested from female Lewis rats (200-230 g) within 25 min. Briefly, after minimal dissection during the first 12 min, the livers were either manipulated gently or left alone for 13 min. Subsequently, an orthotopic liver transplant was performed after 1 hr of storage in cold UW solution. Some donors livers underwent microsurgical denervation before harvesting or rats were given hexamethonium (10 mg/kg, i.v.), a ganglionic blocking agent., Results: In the nonmanipulated group, survival was 100% after the transplant; however, gentle manipulation decreased survival by about 50%. Furthermore, manipulation elevated serum transaminases and bilirubin 6- to 8-fold 8 hr after the transplant and caused necrosis of about 25% of hepatocytes. After organ harvesting, the rate of entry and exit of fluorescein dextran, a dye confined to the vascular space, was decreased 2- to 4-fold, and the maximal increase of surface fluorescence was blunted about 2-fold. Pimonidazole binding, which reflects tissue hypoxia, was increased 2-fold by manipulation. Denervation of the liver before organ harvesting or treatment with hexamethonium prevented the effects of organ manipulation on all parameters studied., Conclusion: These data indicate for the first time that hepatic denervation before organ harvesting prevents detrimental effects of brief, gentle manipulation of the liver during harvesting on survival after the transplant. This is consistent with the hypothesis that organ manipulation disturbs the hepatic microcirculation and causes hypoxia at harvesting using mechanisms dependent on innervation.

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.

Ethanol increases free radical formation; however, it was recently demonstrated that it also causes extensive hypoxia in rat liver in vivo. To address this issue, it was hypothesized that peroxynitrite formed in normoxic periportal regions of the liver lobule has its reactivity enhanced in hypoxic pericentral regions where the pH is lower. Via this pathway, peroxynitrite could lead to free radical formation in the absence of oxygen. Livers from fed rats were perfused at low flow rates for 75 min. Under these conditions, periportal regions were well oxygenated but pericentral areas became hypoxic. Low-flow perfusion caused a significant 6-fold increase in nitrotyrosine accumulation in pericentral regions. During the last 20 min of perfusion, the spin-trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone was infused and adducts were collected for electron-spin resonance analysis. A six-line radical adduct signal was detected in perfusate. Direct infusion of peroxynitrite produced a radical adduct with identical coupling constants, and a similar pattern of nitrotyrosine accumulation was observed. Retrograde perfusion at low rates resulted in accumulation of nitrotyrosine in periportal regions. Although the magnitude of the radical in perfusate was increased by ethanol, it was not derived directly from it. Both nitrotyrosine accumulation and radical formation were reduced by inhibition of nitric oxide synthase with N-nitro-L-arginine methyl ester, but not with the inactive D-isomer. Radical formation was decreased nearly completely by superoxide dismutase and N-nitro-L-arginine methyl ester, consistent with the hypothesis that the final prooxidant is a derivative from both NO. and superoxide (i.e., peroxynitrite). These results support the hypothesis that oxidative stress occurs in hypoxic regions of the liver lobule by mechanisms involving peroxynitrite.

Extrahepatic factors, such as increased gut permeability and bacteria from the gut, have been shown to play a role in D-galactosamine toxicity in rats. Because bacterial endotoxin activates Kupffer cells, the purpose of this study was to clarify the role of Kupffer cells in the mechanism of D-galactosamine hepatotoxicity in rats and determine whether uridine, a compound that rescues animals from D-galactosamine toxicity, affects Kupffer cells. Rats were fed control or glycine (5%) containing diets to prevent Kupffer cell activation or treated with gadolinium chloride (GdCl3, 20 mg/kg) to destroy Kupffer cells selectively before injection of D-galactosamine (500 mg/kg, intraperitoneally). D-galactosamine caused panlobular focal hepatocellular necrosis, polymorphonuclear cell infiltration, and increased serum transaminases significantly at 24 hours. Dietary glycine or pretreatment with GdCl3 prevented these effects. D-galactosamine caused a transient increase in circulating endotoxin that was maximal at 1 hour and was blunted significantly by dietary glycine. Additionally, antisera to tumor necrosis factor-alpha (TNF-alpha) prevented hepatotoxicity caused by D-galactosamine. Moreover, apoptosis in hepatocytes caused by D-galactosamine occurred before necrosis (6 hours) and was prevented by glycine, GdCl3, TNF-alpha antiserum, and uridine. Thus, it was hypothesized that TNF-alpha from Kupffer cells causes apoptosis after D-galactosamine administration in the rat. Indeed, increases in TNF-alpha messenger RNA (mRNA) were detected as early as 2.5 hours after D-galactosamine treatment. Previous work proposed that uridine blocks D-galactosamine toxicity by preventing inhibition of mRNA synthesis. In view of these results, the possibility that uridine might affect Kupffer cells was investigated. Uridine significantly blunted the increase in [Ca2+]i and release of TNF-alpha caused by endotoxin in isolated Kupffer cells and prevented apoptosis caused by D-galactosamine treatment in vivo. These data support the hypothesis that uridine prevents D-galactosamine hepatotoxicity not only by rescuing the hepatocyte in the late phases of the injury but also preventing TNF-alpha release from Kupffer cells thereby blocking apoptosis that occurs early after D-galactosamine treatment. Taken together, these data strongly support the role of Kupffer cell activation by endotoxin early after D-galactosamine treatment as an important event in the mechanism of hepatotoxicity in the rat.

CD14 is a lipopolysaccharide (LPS) receptor distributed largely in macrophages, monocytes, and neutrophils; however, the role of CD14 in activation of Kupffer cells by LPS remains controversial. The purpose of this study was to determine if different methods used to isolate Kupffer cells affect CD14. Kupffer cells were isolated by collagenase (0.025%) or collagenase-Pronase (0.02%) perfusion and differential centrifugation using Percoll gradients and cultured for 24 h before experiments. CD14 mRNA was detected by RT-PCR from Kupffer cell total RNA as well as from peritoneal macrophages. Western blotting showed that Kupffer cells prepared with collagenase possess CD14; however, it was absent in cells obtained by collagenase-Pronase perfusion. Intracellular calcium in Kupffer cells prepared with collagenase was increased transiently to levels around 300 nM by addition of LPS with 5% rat serum, which contains LPS binding protein. This increase in intracellular calcium was totally serum dependent. Moreover, LPS-induced increases in intracellular calcium in Kupffer cells were blunted significantly (40% of controls) when cells were treated with phosphatidylinositol-specific phospholipase C, which cleaves CD14 from the plasma membrane. However, intracellular calcium did not increase when LPS was added to cells prepared by collagenase-Pronase perfusion even in the presence of serum. These cells were viable, however, because ATP increased intracellular calcium to the same levels as cells prepared with collagenase perfusion. Tumor necrosis factor-alpha (TNF-alpha) mRNA was increased in Kupffer cells prepared with collagenase perfusion 1 h after addition of LPS, an effect potentiated over twofold by serum; however, serum did not increase TNF-alpha mRNA in cells isolated via collagenase-Pronase perfusion. Moreover, treatment with Pronase rapidly decreased CD14 on mouse macrophages (RAW 264.7 cells) and Kupffer cells. These findings indicate that Pronase cleaves CD14 from Kupffer cells, whereas collagenase perfusion does not, providing an explanation for why Kupffer cells do not exhibit a CD14-mediated pathway when prepared with procedures using Pronase. It is concluded that Kupffer cells indeed contain a functional CD14 LPS receptor when prepared gently.

The purpose of this study was to determine whether catalase-dependent alcohol metabolism is activated by alcohol (i.e., swift increase in alcohol metabolism). When ethanol or the selective substrate for catalase, methanol, was given (5.0 g/kg) in vivo 2 to 3 h before liver perfusion, methanol and oxygen metabolism were increased significantly. This increase was blocked when the specific Kupffer cell toxicant GdCl3 was administered 24 h before perfusion. These data support the hypothesis that catalase-dependent alcohol metabolism is activated by acute alcohol and that Kupffer cells are involved. Ethanol treatment in vivo increased ketogenesis from endogenous fatty acids nearly 3-fold and increased plasma triglycerides and hepatic acyl CoA synthetase activity; all increases were blocked by GdCl3. These findings support the hypothesis that ethanol increases H2O2 supply for catalase-dependent alcohol metabolism by increasing fatty acid supply. Infusion of oleate stimulated oxygen uptake 1.5-fold and methanol metabolism 4-fold, but these parameters were not altered by GdCl3. Moreover, the effects of ethanol treatment were blocked by the cyclooxygenase inhibitor indomethacin, and prostaglandin E2 (PGE2) was increased more than 200% in media from cultured Kupffer cells from rats treated with ethanol in vivo. Furthermore, lipoprotein lipase activity in retroperitoneal fat pads, which is known to be inhibited by PGE2, was reduced 70% by ethanol. These data are consistent with the hypothesis that Kupffer cells play a key role in activation of catalase-dependent alcohol metabolism, most likely by producing mediators (e.g., PGE2) that inhibit lipoprotein lipase, increase the supply of fatty acids to the liver, and increase generation of H2O2 via peroxisomal beta-oxidation.

Both in situ organ manipulation during harvest and steatosis reduce survival after liver transplantation via mechanisms involving Kupffer cells; thus, their effect on survival was compared here. Moderate steatosis was induced by a single dose of ethanol to Lewis rats, while long-term administration of ethanol yielded severe steatosis in donor animals. After minimal dissection during the first 12 min, livers were either manipulated gently or left alone for 13 min subsequently. Orthotopic liver transplantation was performed after 1 h of cold storage in UW solution. Ethanol increased hepatic lipid content to a level of moderate or severe steatosis that reduced survival after transplantation from 100% to approximately 70% (P < 0.05). However, gentle manipulation decreased survival to approximately 30% (P < 0.05) in livers from normal, saline-treated rats and in livers from rats fed a high-fat control diet. Moreover, after short- or long-term ethanol administration, manipulation of fatty livers decreased survival from 70% to approximately 13% (P < 0.05). Further, manipulation elevated serum transaminases, total bilirubin, and necrosis significantly about 2- to 20-fold in fatty grafts after transplantation. At the end of harvest, trypan blue distribution time and hypoxia assessed from 2-nitroimidazole binding were elevated significantly about two- to fourfold by manipulation of fatty grafts. Gadolinium chloride, a Kupffer cell toxicant, blocked the detrimental effects of manipulation. These data demonstrate for the first time that, while steatosis is detrimental for survival, organ manipulation plays a much greater role than fat in mechanisms of primary nonfunction.

There are too few reliable markers by which one can predict future function of a liver before implantation. Consequently, the purpose of this study was to test the hypothesis that amino acids in rinse-effluents could predict transplant outcome in marginal fatty livers from rats. Amino acids were measured in the rinse effluent from the livers immediately after harvest and graft preparation or cold storage. Amino acids in the effluent were twice as high in ethanol-treated animals compared to those in nonfatty controls. Ethanol-treated fatty livers survived for no longer than 7 days after transplantation while 83% of nonfatty controls survived (P < 0.05). In subsequent studies, the cold-storage time was decreased to 6 h to determine whether failing fatty livers released more amino acid than grafts that would function normally. There was a significant increase in amino acids in the effluent of fatty grafts compared to controls. Moreover, the sum of the four selected amino acids (alanine, valine, histidine, leucine) was lower than 23 nmol/g liver in functional livers, whereas failing grafts had totals significantly higher than 25 nmol/g liver. The sum of the four amino acids correlated well with 24 h post-transplant serum AST levels (r = 0.78, P < 0.0001). So we can conclude that amino acid release can serve as a useful marker of graft viability and reliably predicts survival.

Background: Ethanol increases primary graft failure after liver transplantation, yet whether it acts via mechanisms involving fat accumulation remains unclear., Methods: Rats were pair-fed a modified Lieber-DeCarli liquid diet containing 35% (high-fat) or 12% (low-fat) of calories as fat combined with 36% of calories as ethanol or isocaloric maltose-dextrin for 4-5 weeks. Reperfusion injury to the liver was studied using a low-flow, reflow perfusion model and a liver transplantation model, and free radicals were detected using electron spin resonance and the spin trapping technique., Results: As expected, basal hepatic triglycerides were similar in livers from rats fed low- and high-fat control diets. Ethanol did not alter triglyceride levels significantly in rats fed a low-fat diet, but increased values about 2.4-fold in rats fed a high-fat diet. Ethanol increased lactate dehydrogenase release during reperfusion from 10 to 26 IU/g/h in rats fed a low-fat diet and from 17 to 34 IU/g/h in rats fed a high-fat diet, respectively. Portal pressure increased from about 3 to 10.5 cm H2O upon reperfusion in livers from high-fat, ethanol-fed rats, but only reached values of 9.1 in the low-fat, ethanol-fed group. A free radical adduct signal was detected in the bile of livers from ethanol-treated rats, and the magnitude of this signal was similar in livers of ethanol-treated rats fed high- or low-fat diets. However, radical adducts could not be detected in either group in the absence of dietary ethanol. Moreover, 67-77% rats given low-fat or high-fat control diets survived after liver transplantation, but only 11% survived if treated with ethanol., Conclusions: It is concluded that ethanol plays a major role in hepatic reperfusion injury, most likely via mechanisms involving free radicals. Increased hepatic fat content alone plays only a minor role, probably by causing slight disturbances in the hepatic microcirculation.

This study investigated the role of endotoxin in the hypermetabolic state or swift increase in alcohol metabolism (SIAM) due to acute ethanol exposure. Female Sprague-Dawley rats (100-120 g) were given ethanol (5 g/kg) by gavage. Endotoxin measured in plasma from portal blood was not detectable in saline-treated controls; however, 90 min after ethanol, endotoxin was increased to 85 +/- 14 pg/ml, and endotoxin clearance was diminished by approximately 50%. Oxygen uptake in perfused livers was increased 48% by ethanol, and production of PGE2 by isolated Kupffer cells was increased similarly. These effects were blunted by elimination of gram-negative bacteria and endotoxin with antibiotics before ethanol administration. To reproduce ethanol-induced endotoxemia, endotoxin was infused via the mesenteric vein at a rate of 2 ng. kg-1. h-1. Endotoxin mimicked the effect of ethanol on oxygen uptake. The specific Kupffer cell toxicant GdCl3 completely prevented increases in oxygen uptake due to endotoxin. These findings demonstrate that endotoxin plays a pivotal role in SIAM, most likely by stimulating eicosanoid release from Kupffer cells.

The formation of protein adducts with reactive aldehydes resulting from ethanol metabolism and lipid peroxidation has been suggested to play a role in the pathogenesis of alcoholic liver injury. To gain further insight on the contribution of such aldehydes in alcoholic liver disease, we have compared the appearance of acetaldehyde, malondialdehyde, and 4-hydroxynonenal adducts with the expression of cytochrome P-450IIE1, and cytochrome P-4503A enzymes in the liver of rats fed alcohol with a high-fat diet for 2 to 4 weeks according to the Tsukamoto-French procedure and in control rats (high-fat liquid diet or no treatment). Urine alcohol and serum aminotransferase levels were recorded, and the liver pathology was scored from 0 to 10 according to the presence of steatosis, inflammation, necrosis, and fibrosis. The ethanol treatment resulted in the accumulation of fat, mild necrosis and inflammation, and a mean liver pathology score of 3 (range: 1 to 5). Liver specimens from the ethanol-fed animals with early alcohol-induced liver injury were found to contain perivenular, hepatocellular acetaldehyde adducts. Malondialdehyde and 4-hydroxynonenal adducts were also present showing a more diffuse staining pattern with occasional sinusoidal reactions. In the control animals, a faint positive reaction for the hydroxynonenal adduct occurred in some of the animals fed the high fat diet, whereas no specific staining was observed in the livers from the animals receiving no treatment. Expression of both CYP2E1 and CYP3A correlated with the amount of protein adducts in the liver of alcohol-treated rats. Distinct CYP2E1-positive immunohistochemistry was seen in 3 of 7 of the ethanol-fed animals. In 5 of 7 of the ethanol-fed animals, the staining intensities for CYP3A markedly exceeded those obtained from the controls. The present findings indicate that acetaldehyde and lipid peroxidation-derived adducts are generated in the early phase of alcohol-induced liver disease. The formation of protein adducts appears to be accompanied by induction of both CYP2E1 and CYP3A.

Activation of Kupffer cells by lipopolysaccharide (LPS) plays a pivotal role in the onset of pathophysiological events that occur during endotoxemia and intracellular calcium ([Ca2+]i) is involved in LPS-stimulated cytokine production. Recently, it was shown that Kupffer cells contain a glycine-gated chloride channel. Because taurine, a ubiquitous sulfur-containing beta-amino acid, acts similarly to glycine in neurons by causing hyperpolarization, it was hypothesized that taurine would act via a similar mechanism, blunting the LPS-induced increase in [Ca2+]i in Kupffer cells. To test this hypothesis, Kupffer cells were isolated from female Sprague-Dawley rats and cultured for 24 h. LPS-induced changes in [Ca2+]i were monitored fluorometrically in single cells, whereas levels of tumor necrosis factor alpha (TNF-alpha) released by Kupffer cells after exposure to LPS were measured by enzyme-linked immunosorbent assay. Taurine significantly blunted the LPS-induced increase in [Ca2+]i in a dose-dependent manner (IC50, 0.1 mM). This effect was reversed by strychnine (1 microM) and was prevented when chloride was removed from the extracellular media. Moreover, taurine increased 36Cl- uptake by Kupffer cells in a dose-dependent manner (EC50, 0.2 mM). Furthermore, strychnine (1 microM) reversed the effect of taurine on 36Cl- uptake. These results indicate that taurine activates a glycine-gated chloride channel in Kupffer cells causing chloride influx. In addition, LPS-induced TNF-alpha production was reduced by more than 40% by taurine, an effect that was also reversed by strychnine. In conclusion, taurine blocks the increase in [Ca2+]i due to LPS and significantly reduces TNF-alpha production by mechanisms involving chloride influx into the Kupffer cell.

This study investigated whether dietary choline can prevent endotoxin shock. Female Sprague-Dawley rats fed chow or chow plus choline chloride (0.025-0.4%) for 3 days were given lipopolysaccharide (LPS) via the tail vein. Eighty-three percent and 56% of chow-fed rats survived after 2.5 or 5.0 mg/kg LPS, respectively. Choline increased survival in a dose-dependent manner, with maximal effects observed at 0.4%; this dose of choline prevented mortality completely after 2.5 or 5 mg/kg LPS. Choline also improved the microscopic appearance of the lungs and blunted increases in serum aspartate aminotransferase levels. Intracellular Ca2+ was monitored in liver and lung macrophages during LPS exposure. Ca2+ increases in macrophages from choline-fed rats were blunted by 40-60% compared with chow-fed controls. Feeding choline also blunted tumor necrosis factor-alpha production. Feeding glycine, which prevents macrophage activation via a chloride channel, in addition to choline was even more effective than feeding choline alone, suggesting that glycine and choline act via distinct sites. These data are consistent with the hypothesis that choline diminishes endotoxin shock by preventing macrophage activation.

Juniper berry oil is rich in 5,11,14-eicosatrienoic acid, a polyunsaturated fatty acid similar to one found in fish oil, yet less prone to peroxidation. Dietary fish oil treatment has been shown to effectively reduce reperfusion injury; therefore, the effects of a diet containing juniper berry oil on hepatic reperfusion injury in a low-flow, reflow reperfusion model were investigated in the rat. Rats were fed semisynthetic diets containing either juniper berry oil, fish oil, or corn oil for 14 to 16 days. Daily food consumption averaged around 20 g/d in both the control and treatment groups; average daily weight gain was around 4 g per 100 g rat weight in all three groups studied, and there were no significant differences in these parameters. Livers were initially perfused at low-flow rates to induce pericentral hypoxia followed by a 40-minute reperfusion period. Peak lactate dehydrogenase (LDH) release during reflow averaged 44 U/g/h in the corn oil group and 32 U/g/h in the fish oil group, but was only 21 U/g/h as a result of juniper berry oil treatment. Malondialdehyde (MDA), an end-product of lipid peroxidation, reached a maximum value of 62 nmol/g/h in the corn oil group, but only reached 43 nmol/g/h and 34 nmol/g/h in the fish oil and juniper berry oil groups, respectively. Both juniper berry oil and fish oil treatment improved rates of bile flow from 25 microL/g/h (corn oil) to 36 and 38 microL/g/h, respectively. Importantly, juniper berry oil reduced cell death in pericentral regions of the liver lobule by 75%. Trypan blue distribution time, an indicator of the hepatic microcirculation, was reduced by approximately 25% with fish oil and over 50% by juniper berry oil diets compared with corn oil controls. The rates of entry of fluorescein-dextran, a dye confined to the vascular space, were increased 1.8- and 2.6-fold, and rates of outflow were increased 4.4- and 4.3-fold by fish oil and juniper berry oil, respectively, also reflecting improved microcirculation. Juniper berry oil also blunted increases in intracellular calcium and release of prostaglandin E2 (PGE2) by cultured Kupffer cells stimulated by endotoxin. These results are consistent with the hypothesis that feeding a diet containing juniper berry oil reduces reperfusion injury by inhibiting activation of Kupffer cells, thus reducing vasoactive eicosanoid release and improving the hepatic microcirculation in livers undergoing oxidant stress.

It is well known that females show a greater susceptibility to alcohol-induced liver injury than males. Additionally, females who consume alcohol regularly and have been obese for 10 years or more are at greater risk for both hepatitis and cirrhosis. Female rats on an enteral alcohol protocol exhibit injury more quickly than males, with widespread fatty changes over a larger portion of the liver lobule. Levels of plasma endotoxin, intercellular adhesion molecule-1, free radical adducts, infiltrating neutrophils, and nuclear factor-kappaB are increased about twofold more in livers from female than male rats after enteral alcohol treatment. Estrogen treatment in vivo increases the sensitivity of Kupffer cells to endotoxin. Evidence has been presented that Kupffer cells are pivotal in the development of alcohol-induced liver injury. Destruction of Kupffer cells with gadolinium chloride (GdCl3) or reduction of bacterial endotoxin by sterilization of the gut with antibiotics blocks early inflammation due to alcohol. Similar results have been obtained with anti-tumor necrosis factor-alpha antibody. These findings led to the hypothesis that alcohol-induced liver injury involves increases in circulating endotoxin, leading to activation of Kupffer cells, which causes a hypoxia-reoxygenation injury. This idea has been tested using pimonidazole, a nitroimidazole marker, to quantitate hypoxia in downstream pericentral regions of the liver lobule. After chronic enteral alcohol, pimonidazole binding increases twofold. Enteral alcohol also increases free radicals detected with electron spin resonance. Importantly, hepatic hypoxia and radical production detected in bile are decreased by destruction of Kupffer cells with GdCl3. These data are consistent with the hypothesis that Kupffer cells participate in important gender differences in liver injury caused by alcohol.

The major side effect of cyclosporin A is severe nephrotoxicity. It is likely that cyclosporin A causes vasoconstriction leading to hypoxia-reperfusion injury; therefore, these experiments were designed to attempt to obtain physical evidence for hypoxia and free radical production in kidney following cyclosporin A. Rats were treated daily with cyclosporin A (25 mg/kg ig) for 5 days, and pimonidazole, a hypoxia marker, was injected 2 h after the last dose of cyclosporin A. A dose of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) was injected 3 h after cyclosporin A to trap free radicals. Cyclosporin A doubled serum creatinine and decreased glomerular filtration rates by 65% as expected. Pimonidazole adduct binding in the kidney was increased nearly threefold by cyclosporin A, providing physical evidence for tissue hypoxia. Moreover, cyclosporin A increased 4-POBN/radical adducts nearly sixfold in the urine but did not alter levels in the serum. Glycine, which causes vasodilatation and prevents cyclosporin A toxicity, minimized hypoxia and blocked free radical production; however, it did not alter cyclosporin A blood levels. These results demonstrate for the first time that cyclosporin A causes hypoxia and increases production of a new free radical species exclusively in the kidney. Therefore, it is concluded that cyclosporin A causes renal injury by mechanisms involving hypoxia-reoxygenation, effects which can be prevented effectively by dietary glycine.

Previous research from this laboratory using a continuous enteral ethanol (EtOH) administration model demonstrated that Kupffer cells are pivotal in the development of EtOH-induced liver injury. When Kupffer cells were destroyed using gadolinium chloride (GdCl3) or the gut was sterilized with polymyxin B and neomycin, early inflammation due to EtOH was blocked. Anti-tumour necrosis factor (TNF)-alpha antibody markedly decreased EtOH-induced liver injury and increased TNF-mRNA. These findings led to the hypothesis that EtOH-induced liver injury involves increases in circulating endotoxin leading to activation of Kupffer cells. Pimonidazole, a nitro-imidazole marker, was used to detect hypoxia in downstream pericentral regions of the lobule. Following one large dose of EtOH or chronic enteral EtOH for 1 month, pimonidazole binding was increased significantly in pericentral regions of the liver lobule, which was diminished with GdCl3. Enteral EtOH increased free radical generation detected with electron spin resonance (ESR). These radical species had coupling constants matching alpha-hydroxyethyl radical and were shown conclusively to arise from EtOH based on a doubling of the ESR lines when 13C-EtOH was given. Alpha-hydroxyethyl radical production was also blocked by the destruction of Kupffer cells with GdCl3. It is known that females develop more severe EtOH-induced liver injury more rapidly and with less EtOH than males. Female rats on the enteral protocol exhibited more rapid injury and more widespread fatty changes over a larger portion of the liver lobule than males. Plasma endotoxin, ICAM-1, free radical adducts, infiltrating neutrophils and transcription factor NFkappaB were approximately two-fold greater in livers from females than males after 4 weeks of enteral EtOH treatment. Furthermore, oestrogen treatment increased the sensitivity of Kupffer cells to endotoxin. These data are consistent with the hypothesis that Kupffer cells participate in important gender differences in liver injury caused by ethanol.

The aim of this study was to determine if the effect of prostaglandin E2 (PGE2) on hepatic oxygen uptake was affected by oxygen tension. Livers from fed female Sprague-Dawley rats were perfused at normal or high flow rates (4 or 8 ml . g-1 . min-1) to vary local oxygen tension within the liver lobule. During perfusion at normal flow rates, PGE2 (5 microM) infusion increased oxygen uptake by about 50 micromol . g-1 . h-1; however, when livers were perfused at high flow rates, the increase was nearly twice as large. Simultaneously, glucose output was increased rapidly by about 50%, whereas glycolysis was decreased about 60%. When flow rate was held constant, increases in oxygen uptake due to PGE2 were proportional to oxygen delivery. Infusion of PGE2 into livers perfused at normal flow rates increased state 3 rates of oxygen uptake of subsequently isolated mitochondria by about 25%; however, rates were increased 50-75% in mitochondria isolated from livers perfused at high flow rates. Thus it is concluded that PGE2 stimulates oxygen uptake via mechanisms regulated by oxygen tension in perfused rat liver. High flow rates also increased basal rates of oxygen uptake: this increase was prevented by inactivation of Kupffer cells with GdCl3. In addition, conditioned medium from Kupffer cells incubated at high oxygen tension (75% oxygen) stimulated oxygen uptake of isolated parenchymal cells by >30% and elevated PGE2 production about twofold compared with Kupffer cells exposed to normal air-saturated buffer (21% oxygen). These effects were blocked completely by both indomethacin and nisoldipine. These data support the hypothesis that oxygen stimulates Kupffer cells to release mediators such as PGE2 which elevate oxygen consumption in parenchymal cells, possibly by mechanisms involving cyclooxygenase and calcium channels.

Background & Aims: Ethanol causes both tolerance and sensitization of Kupffer cells. This study was designed to evaluate temporal effects of ethanol in an attempt to understand this paradox., Methods: Rats were given ethanol (4 g/kg body wt) intragastrically, and Kupffer cells were isolated 0-48 hours later. After addition of lipopolysaccharide (LPS), intracellular calcium concentration ([Ca2+]i) was measured using a microspectrofluorometer with the fluorescent indicator fura-2, and tumor necrosis factor alpha (TNF-alpha) was measured by enzyme-linked immunosorbent assay. CD14 was evaluated by Western and Northern analysis., Results: Two hours after ethanol administration, the LPS-induced increase in [Ca2+]i and TNF-alpha release by Kupffer cells was diminished by 50%, and these parameters were reciprocally enhanced twofold at 24 hours. Sterilization of the gut with antibiotics blocked all effects of ethanol on [Ca2+]i and TNF-alpha release completely. Twenty-four hours after ethanol, CD14 in Kupffer cells was elevated about fivefold., Conclusions: Kupffer cells isolated from rats early after ethanol exhibited tolerance to LPS, whereas sensitization was observed later. It is likely that both of these phenomena are caused by gut-derived endotoxin and that sensitization in Kupffer cells is caused by increases in CD14.

Glycine prevents hepatic damage caused by hypoxia-reoxygenation, diminishes mortality due to endotoxin and minimizes alcoholic liver injury by decreasing blood ethanol. Our purpose was to investigate the effect of dietary glycine during recovery from early alcohol-induced injury, using a model that mimics the clinical presentation and histopathology with alcoholics. Male Wistar rats were exposed to ethanol continuously for 6 wk via intragastric feeding that resulted in typical histology of alcoholic liver injury, including steatosis, inflammation, necrosis and increased serum levels of aspartate aminotransferase and alanine aminotransferase. After cessation of ethanol, one group of rats received a control diet, the other a glycine-containing diet for 2 wk. During this period, all parameters studied tended to return to baseline values. However, serum aspartate aminotransferase and alanine aminotransferase recovered about 30% more rapidly in rats fed glycine. Further, the hepatic pathology score was also significantly lower in the glycine group than in controls (0.5 vs. 2.6). After 1 wk, steatosis was reduced significantly more in the glycine group (5. 6%) than in controls (8.9%). Glycine also diminished numbers of infiltrating leukocytes and necrotic cells significantly more than in controls. This beneficial effect of glycine may be partly explained by the fact that glycine increased influx of chloride into Kupffer cells leading to diminished tumor necrosis factor-alpha production. These results indicate that a glycine containing diet expedites the process of recovery from ethanol-induced liver injury and may lead to its clinical application in alcoholic hepatitis.

Stimulation of cell proliferation caused by peroxisome proliferators was blocked by antibodies against TNF alpha and agents that inactivate Kupffer cells, a rich source of TNF alpha, which supports the hypothesis that Kupffer cells play a pivotal role in peroxisome proliferator-induced hyperplasia. Here, the ability of the very potent peroxisome proliferator WY-14 643 to activate the transcription factor NF-kappaB in rat liver was examined since it is involved in TNF alpha production. Female Sprague-Dawley rats were treated by gavage with WY-14 643 (100 mg/kg) while control animals were given equivalent doses of vehicle (olive oil). Activation of NF-kappaB in both whole liver, non-parenchymal cells, Kupffer cells and hepatocytes was assessed for up to 36 h using an electrophoretic mobility shift assay. In whole liver, WY-14 643 transiently increased NF-kappaB binding maximally 3.5-fold in 2-8 h followed by a steady decline to near control levels at 36 h. As early as 2 h after WY-14 643 treatment, the active form of NF-kappaB was localized predominantly in Kupffer cells with values 20- to 25-times greater than in hepatocytes. In hepatocytes, a small increase in NF-kappaB binding was observed but only 8 h after WY-14 643 administration. Pre-treatment with allopurinol, a xanthine oxidase inhibitor and free radical scavenger, suppressed NF-kappaB activation by WY-14 643 almost completely. It is concluded that NF-kappaB is activated by reactive oxygen species and plays a central role in the mechanism of action of peroxisome proliferators. Moreover, these findings support the hypothesis that Kupffer cells play a pivotal role in peroxisome proliferator-induced hepatocyte proliferation through rapid NF-kappaB activation and subsequent induction of TNF alpha production. TNF alpha from Kupffer cells stimulates growth in parenchymal cells later via mechanisms that also involve NF-kappaB.

It has been proposed that the C-phenyl-N-tert-butylnitrone/trichloromethyl radical adduct (PBN/.CCl3) is metabolized to either the C-phenyl-N-tert-butylnitrone/carbon dioxide anion radical adduct (PBN/.CO2-) or the glutathione (GSH) and CCl4-dependent PBN radical adduct (PBN/[GSH-.CCl3]). Inclusion of PBN/.CCl3 in microsomal incubations containing GSH, nicotinamide adenine dinucleotide phosphate (NADPH), or GSH plus NADPH produced no electron spin resonance (ESR) spectral data indicative of the formation of either the PBN/[GSH-.CCl3] or PBN/.CO2- radical adducts. Microsomes alone or with GSH had no effect on the PBN/.CCl3 radical adduct. Addition of NADPH to a microsomal system containing PBN/.CCl3 presumably reduced the radical adduct to its ESR-silent hydroxylamine because no ESR signal was observed. The Folch extract of this system produced an ESR spectrum that was a composite of two radicals, one of which had hyperfine coupling constants identical to those of PBN/.CCl3. We conclude that PBN/.CCl3 is not metabolized into either PBN/[GSH-.CCl3] or PBN/.CO2- in microsomal systems.

2-Nitroimidazoles, such as pimonidazole, are reduced in cells with low oxygen tension and are, therefore, used as hypoxia markers. However, the effect of the pyridine nucleotide redox state on pimonidazole reduction is not known. Therefore, livers from fed or fasted rats were perfused with oxygen-saturated buffer containing pimonidazole (400 microM) in the presence and absence of an inhibitor of the mitochondrial respiratory chain, potassium cyanide; these treatments were used to modulate the mitochondrial and cytosolic pyridine nucleotide redox states. Pimonidazole-induced increases in oxygen uptake over basal values were as follows: fed, 15.1 +/- 2.4; fasted, 4.2 +/- 0.8; fed + KCN, 32.1 +/- 0.9; fasted + KCN, 0.2 +/- 0.2 micromol x g(-1) x h(-1). However, if NADPH was added in excess, microsomal oxygen uptake due to oxidative metabolism of pimonidazole was independent of treatment. These results indicate that pimonidazole-stimulated O2 uptake, due predominantly to N-oxidation and glucuronidation, is dependent on the NADPH redox state. In contrast, reduced pimonidazole adducts, detected immunochemically, accumulated in pericentral regions in liver. Increasing the NADH redox state by inhibiting the mitochondrial respiratory chain with KCN decreased protein-bound pimonidazole adducts. Concomitantly, the average O2 tension of the liver was increased at least 30%. However, KCN had no effect on total pimonidazole adducts detected by ELISA, although both cytosolic (lactate/pyruvate) and mitochondrial (3-hydroxybutyrate/acetoacetate) NADH redox states were elevated by at least a factor of eight. These results indicate that, unlike oxidative metabolism, the pyridine nucleotide redox state does not determine the rate of reductive metabolism of pimonidazole. Instead, the cellular oxygen tension regulates this process. Therefore, even in cases where the supply of reducing equivalents is increased (e.g., ethanol metabolism), accumulation of the reduced bound product of pimonidazole is oxygen dependent in liver.

Background: The etiology of primary graft nonfunction and dysfunction is unknown but most likely involves Kupffer cell-dependent reperfusion injury. However, the donor operation and surgical technique may also have an effect on the outcome after transplantation. Because liver manipulation during harvest cannot be prevented completely with standard procedures, its effect on survival was assessed here., Methods: Donor livers were harvested from female Sprague-Dawley rats (200-230 g). Briefly, after minimal dissection during the first 12 min, livers were either manipulated gently or left alone for 13 subsequent minutes. At 25 min, all livers were perfused with cold University of Wisconsin solution via the portal vein, and transplantation was performed after cold storage (1 hr). In some rats, Kupffer cells were destroyed with gadolinium chloride or inactivated with dietary glycine before harvest. Survival, proteolytic activity in the rinse effluent, serum transaminases, trypan blue distribution to index microcirculation, and histology were compared., Results: In the nonmanipulated group, survival was 100% after transplantation; however, gentle manipulation decreased survival by 70%. Further, manipulation elevated transaminases fivefold and caused about 200% necrosis. At harvest, proteolytic activity and the time for trypan blue to distribute homogeneously were elevated three- to eightfold by manipulation. Gadolinium chloride and glycine prevented the effects of manipulation on all parameters studied., Conclusion: These data indicate for the first time that brief, gentle manipulation of the donor liver has a marked detrimental effect on survival by priming or activating Kupffer cells. This may represent an important early event in pathogenesis, because Kupffer cells play an important role in primary graft nonfunction.

The relationship among gender, lipopolysaccharide (LPS), and liver disease is complex. Accordingly, the effect of estrogen on activation of Kupffer cells by endotoxin was studied. All rats given estrogen intraperitoneally 24 h before an injection of a sublethal dose of LPS (5 mg/kg) died within 24 h, whereas none of the control rats died. Mortality was prevented totally by pretreatment with gadolinium chloride, a Kupffer cell toxicant. Peak serum tumor necrosis factor-alpha (TNF-alpha) values as well as TNF-alpha mRNA in the liver after LPS were twice as high in the estrogen-treated group as in the untreated controls. Plasma nitrite levels and inducible nitric oxide synthase in the liver were also elevated significantly in estrogen-treated rats 6 h after LPS. Furthermore, Kupffer cells isolated from estrogen-treated rats produced about twice as much TNF-alpha and nitrite as controls did in response to LPS. In addition, Kupffer cells from estrogen-treated rats required 15-fold lower amounts of LPS to increase intracellular Ca2+ than controls did, and Kupffer cells from estrogen-treated animals expressed more CD14, the receptor for LPS/LPS binding protein, than controls. Moreover, estrogen treatment increased LPS binding protein mRNA dramatically in liver in 6-24 h. It is concluded that estrogen treatment in vivo sensitizes Kupffer cells to LPS, leading to increased toxic mediator production by the liver.

The etiology of primary graft non-function has been linked to storage and reperfusion injury. Recently, graft manipulation during harvest was shown to aggravate reperfusion injury in livers effecting outcome after transplantation detrimentally. Since Kupffer cells play a pivotal role in reperfusion injury, glycine (1 ml, 300 mM) was infused into donors for one hour before harvest to prevent Kupffer cell activation. Valine was used as isonitrogenous control. To assess the influence of gentle manipulation, livers from female Lewis rats were dissected for 12 minutes minimally and harvested within 25 minutes. During the last 13 minutes, the organ was gently manipulated or left alone in the controls. Minimal dissection during harvest without manipulation had no influence on survival after liver transplantation (100%); however, gentle manipulation for 13 minutes decreased survival to less than 50%. Further, transaminases were elevated seven-fold after transplantation by manipulation. Glycine infusion totally prevented the effects of organ manipulation on survival and enzyme release. These data indicate for the first time that pretreatment of donors with intravenous glycine prevents the detrimental effects of graft manipulation during harvest on outcome.

Kupffer cell-dependent reperfusion injury occurs to the liver following transplantation, most often in fatty livers which fail most frequently due to primary nonfunction. Failure was largely blocked with Carolina rinse solution, which contains glycine, and prevents the activation of Kupffer cells. Furthermore, gentle in situ organ manipulation, which cannot be prevented using standard harvesting techniques, has a detrimental effect on survival. These effects were also prevented by glycine. Since proteolytic activity is increased in both fatty and manipulated livers, amino acids were measured in rinse effluents collected at harvest. A combination of four amino acids correlated with graft function. It is concluded that glycine could be beneficial in clinical liver transplantation to prevent reperfusion injury, and that amino acids measured at harvest may predict graft function.

The analysis of amino acids in perfusion effluents in experimental liver transplantation is a valid parameter of organ viability at organ harvest. Depletion of Kupffer cells with gadolinium chloride (GdCl3) in donor animals prevents primary nonfunction of fatty livers after transplantation, diminishes amino acid release at harvest, but blocks the increased expression of the adhesion molecule ICAM-1 only after transplantation. Thus, the increased proteolysis in marginal donor livers is not induced by cytokines, but kupffer cell dependent.