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Hemorrhagic shock and resuscitation cause endotoxemia and hepatocellular damage. Because lipopolysaccharide-binding protein (LBP) enhances cellular responses to endotoxin, our aim was to determine whether LBP contributes to hemorrhage/resuscitation-induced injury by comparing LBP knockout and wild-type mice. Under pentobarbital anaesthesia, wild-type and LBP-deficient mice were hemorrhaged to 30 mmHg for 3 h and then resuscitated with shed blood plus half the volume of lactated Ringer solution. Serum alanine aminotransferase (ALT) necrosis, neutrophil infltration, and 4-hydroxynonenal by histology/ cytochemistry and stress kinase activation by immunoblot analysis were then determined. ALT in wild-type mice was 2,461 [+ or -] 383 and 1,418 [+ or -] 194 IU/l (means [+ or -] SE), respectively, at 2 and 6 h after resuscitation versus sham ALT of 102 [+ or -] 6 IU/l. In LBP-deficient mice, ALT was blunted at both time points to 1,108 [+ or -] 340 and 619 [+ or -] 171 IU/l (P < 0.05). Liver necrosis after 6 h was also attenuated from 3.5 [+ or -] 0.8% in wild-type mice to 1.3 [+ or -] 0.5% in LBP-deficient mice (P < 0.05). After hemorrhage/resuscitation, neutrophil infiltration increased 71% more in wild-type than LBP knockout mice. Similarly, hepatic 4-hydroxynonenal staining, indicative of lipid peroxidation, decreased from 33.8 [+ or -] 4.5% in wild-type mice to 11.6 [+ or -] 1.9% in knockout mice (P < 0.05). After hemorrhage/resuscitation, activation of MAPKs, JNK and ERK, occurred in wild-type mice, which was largely blocked in LBP-deficient mice. However, endotoxin in portal blood after resuscitation was not significantly different between wild-type and knockout mice. In conclusion, hemorrhagic shock and resuscitation to mice cause severe, LBP-mediated hepatocellular damage. An absence of LBP blunts hepatocellular injury with decreased neutrophil infiltration, oxidative stress, and c-Jun and ERK activation. knockout mouse; hemorrhage/resuscitation; liver injury; stress kinases doi:10.1152/ajpgi.00480.2005

Intravenous infusion of glycine significantly improves survival in liver transplantation in rats. This was gleaned from the results of an experiment, which revealed that minimal dissection of the rat liver for 12 minutes plus 13 minutes without manipulation did not affect survival. Thus, pretreatment of donors with intravenous glycine minimizes reperfusion injury caused by organ manipulation during harvest and after transplantation.

The function of endotoxin in the hypermetabolic state or swift increase in alcohol metabolism (SIAM) due to acute ethanol exposure was studied. The specific Kupffer cell toxicant GdCl3 totally prevented increases in oxygen uptake in female Sprague-Dawley rats because of endotoxin. The results illustrate that endotoxin has an important function in SIAM, most probably due to the stimulation of eicosanoid release from Kupffer cells.

Alcohol ingestion results in increases in the release of endotoxin from gut bacteria or membrane permeability of the gut to endotoxin, or both. Female rats are more sensitive to these changes. Elevated levels of endotoxin activate Kupffer cells to release substances such as eicosanoids, tumor necrosis factor-[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 that lead to tissue damage in oxygen-poor pericentral regions of the liver lobule. KEY WORDS: alcohol-induced liver injury endotoxin free radicals sex differences rats

An in vivo enteral ethanol feeding model was utilized to determine the mechanisms responsible in the high susceptibility of females to alcohol-induced liver injury. Analysis of female susceptibility to alcohol-induced injury based on a novel ethanol feeding model indicated the presence of higher plasma endotoxin levels in females compared to males after exposure to ethanol. Higher endotoxin levels in female also induced a more extensive Kupffer cell activation in females compared to males.

Rats showed a marked increase in hepatic oxygen uptake and ethanol metabolism two to three hours after exposure to acute alcohol and treatment with pimonidazole. Pericentral regions of liver lobules also showed a significant accumulation of pimonidazole, a hypoxia marker, suggesting that exposure to alcohol causes hypoxia. Pimonidazole accumulation was blocked by treatment with a Kupferr cell-specific toxicant, indicating that the increase in oxygen uptake involved Kupffer cells.

Experimental studies of the inactivation of Kupffer cells by GdCl3 reveal the vital role of Kupffer cell activation in the pathogenesis of hepatic reperfusion injury. Lactate dehydrogenase (LDH) and malondialdehyde (MDA) release and oxygen uptake reach a maximum within the first 10 minutes of reperfusion. Inactivation of Kupffer cells by GdCl3 decreases the release of LDH and MDA. Electron paramagnetic resonance spectroscopic studies reveal the involvement of a carbon-centered radical during reflow and that GdCl3 inhibits the formation of the radical.

To test the hypothesis that leukocyte infiltration mediated by intercellular adhesion molecule (ICAM)-1 is involved in early alcohol-induced liver injury, male wild-type or ICAM-1 knockout mice were fed a high-fat liquid diet with either ethanol or isocaloric maltose-dextrin for 4 wk. There were no differences in mean urine alcohol concentrations between the groups fed ethanol. Alcohol administration significantly increased liver size and serum alanine aminotransferase levels in wild-type mice over high-fat controls, effects that were blunted significantly in ICAM-1 knockout mice. Dietary ethanol caused severe steatosis, mild inflammation, and focal necrosis in livers from wild-type mice. Furthermore, livers from wild-type mice fed ethanol showed significant increases in the number of infiltrating leukocytes, which were predominantly lymphocytes. These pathological changes were blunted significantly in ICAM-1 knockout mice. Tumor necrosis factor (TNF)-[Alpha] mRNA expression was increased in wild-type mice fed ethanol but not in ICAM-1 knockout mice. These data demonstrate that ICAM-1 and infiltrating leukocytes play important roles in early alcohol-induced liver injury, most likely by mechanisms involving TNF-[Alpha]. intragastric feeding; adhesion molecules

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 [0.sub.2] uptake (105 [+ or -] 5 [micro]mol [multiplied by] [g.sup.-1] [multipled by] [h.sup.-1]) measured polarigraphically; however, 2 h after organ manipulation, values increased significantly to 160 [+ or -] 8 [micro]mol [multipled by] [g.sup.-1].[h.sup.-1] and binding of pimonidazole, a hypoxia marker, increased about threefold (P [is less than] 0.05). Moreover, Kupffer cells from manipulated livers produced three- to fourfold more tumor necrosis factor-[Alpha], and [PGE.sub.2], whereas intracellular calcium concentration increased twofold after lipopolysaccharide compared with unmanipulated controls (P [is less than] 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.sub.2] from Kupffer cells. These data indicate that gentle organ manipulation during surgery activates Kupffer cells, leading to metabolic changes dependent on [PGE.sub.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. organ harvest; liver transplantation; hypoxia; primary nonfunction

Kupffer cell-derived prostaglandin [E.sub.2] is involved in alcohol-induced fat accumulation in rat liver. Am J Physiol Gastrointest Liver Physiol 279: G100-G106, 2000.--Destruction of Kupffer cells with gadolinium chloride (Gd[Cl.sub.3]) and intestinal sterilization with antibiotics diminished ethanol-induced steatosis in the enteral ethanol feeding model. However, mechanisms of ethanol-induced fatty liver remain unclear. Accordingly, the role of Kupffer cells in ethanol-induced fat accumulation was studied. Rats were given ethanol (5 g/kg body wt) intragastrically, and tissue triglycerides were measured enzymatically. Kupffer cells were isolated 0-24 h after ethanol, and [PGE.sub.2] production was measured by ELISA, whereas inducible cyclooxygenase (COX-2) mRNA was detected by RT-PCR. As expected, ethanol increased liver triglycerides about threefold. This increase was blunted by antibiotics, Gd[Cl.sub.3], the dihydropyridine-type [Ca.sup.2+] channel blocker nimodipine, and the COX inhibitor indomethacin. Ethanol also increased [PGE.sub.2] production by Kupffer cells about threefold. This increase was also blunted significantly by antibiotics, nimodipine, and indomethacin. Furthermore, tissue triglycerides were increased about threefold by [PGE.sub.2] treatment in vivo as well as by a [PGE.sub.2] [EP.sub.2]/[EP.sub.4] receptor agonist, whereas an [EP.sub.1]/[EP.sub.3] agonist had no effect. Moreover, permeable cAMP analogs also increased triglyceride content in the liver significantly. We conclude that [PGE.sub.2] derived from Kupffer cells, which are activated by ethanol, interacts with prostanoid receptors on hepatocytes to increase cAMP, which causes triglyceride accumulation in the liver. This mechanism is one of many involved in fatty liver caused by ethanol. fatty liver; triglyceride; adenosine 3',5'-cyclic monophosphate; ethanol

Kono, Hiroshi, Michael D. Wheeler, Ivan Rusyn, Min Lin, Vitor Seabra, Chantal A. Rivera, Blair U. Bradford, Donald T. Forman, and Ronald G. Thurman. Gender differences in early alcohol-induced liver injury: role of CD14, NF-[Kappa]B, and TNF-[Alpha]. Am J Physiol Gastrointest Liver Physiol 278: G652-G661, 2000.w The purpose of this study was to determine whether early alcohol-induced liver injury (ALI) in females is associated with changes in CD14 on Kupffer cells, activation of hepatic nuclear factor (NF)-[Kappa]B, and expression of tumor necrosis factor (TNF)-[Alpha] mRNA. Male and female rats were given high-fat control or ethanol-containing diets for 4 wk using the intragastric enteral protocol. Physiological parameters were similar in both genders. Ethanol was increased as tolerance developed with higher blood levels than previously observed, resulting in a fourfold increase in aspartate aminotransferase (males 389 [+ or -] 47 IU/1 vs. females 727 [+ or -] 66 IU/1). Hepatic pathology developed more rapidly and was nearly twofold greater and endotoxin levels were significant]y higher in females after ethanol. Also, expression of CD14 on Kupffer cells was 1.5-fold greater and binding of transcription factor NF-[Kappa]B in hepatic nuclear extracts and TNF-[Alpha] mRNA expression were threefold greater in females. These data are consistent with the hypothesis that elevated endotoxin after ethanol triggers more activation of Kupffer cells via enhanced CD14 expression in females. NF-[Kappa]B is activated in this process, leading to increases in TNF-[Alpha] mRNA expression in the liver and more severe liver injury in females. It is concluded that gender differences in ALI are dependent on endotoxin and a signaling cascade leading to TNF-[Alpha]. Kupffer cells; endotoxin

Kono, Hiroshi, Nobuyuki Enomoto, Henry D. Connor, Michael D. Wheeler, Blair U. Bradford, Chantal A. Rivera, Maria B. Kadiiska, Ronald P. Mason, and Ronald G. Thurman. Medium-chain triglycerides inhibit free radical formation and TNF-[Alpha] production in rats given enteral ethanol. Am. J. Physiol. Gastrointest. Liver Physiol. 278: G467-G476, 2000.--This study determined whether free radical formation by the liver, tumor necrosis factor (TNF)-[Alpha] production by isolated Kupffer cells, and plasma endotoxin are affected by dietary saturated fat. Rats were fed enteral ethanol and corn oil (E-CO) or medium-chain triglycerides (E-MCT) and control rats received corn oil (C-CO) or medium-chain triglycerides (C-MCT) for 2 wk. E-CO rats developed moderate fatty infiltration and slight inflammation; however, E-MCT prevented liver injury. Serum aspartate aminotransferase levels, gut permeability, and plasma endotoxin doubled with E-CO but were blunted ~50% with E-MCT. In Kupffer cells from E-CO rats, intracellular calcium was elevated by lipopolysaccharide (LPS) in a dose-dependent manner. In cells from E-MCT rats, increases were blunted by ~40-50% at all concentrations of LPS. The LPS-induced increase in TNF-[Alpha] production by Kupffer cells was dose dependent and was blunted by 40% by MCT. E-CO increased radical adducts and was reduced ~50% by MCT. MCT prevent early alcohol-induced liver injury, in part, by inhibition of free radical formation and TNF-[Alpha] production by inhibition of endotoxin-mediated activation of Kupffer cells. tumor necrosis factor-[Alpha]; intracellular calcium; free radicals; alcohol

Kono, Hiroshi, Blair U. Bradford, Ming Yin, Kathleen K. Sulik, Dennis R. Koop, Jeffrey M. Peters, Frank J. Gonzalez, Tasha McDonald, Anna Dikalova, Maria B. Kadiiska, Ronald P. Mason, and Ronald G. Thurman. CYP2E1 is not involved in early alcohol-induced liver injury. Am. J. Physiol. 277 (Gastrointest. Liver Physiol. 40): G1259-G1267, 1999.--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 EAinduced 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. CYP2E1 knockout mouse; intragastric feeding

Enomoto, Nobuyuki, Shunhei Yamashina, Peter Schemmer, Chantal A. Rivera, Blair U. Bradford, Ayako Enomoto, David A. Brenner, and Ronald G. Thurman. Estriol sensitizes rat Kupffer cells via gut-derived endotoxin. Am. J. Physiol. 277 (Gastrointest. Liver Physiol. 40): G671-G677, 1999.--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 [Ca.sup.2+] concentration was measured using a microspectrofiuorometer 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 ([is less than] 20 pg/ml) by antibiotics. In isolated Kupffer cells, LPS-induced increases in intracellular [Ca.sup.2+] 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. lipopolysaccharide; tumor necrosis factor-[Alpha]; CD14; intracellular calcium