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Background and Aims: Activation of the pro-inflammatory cytokine cascade, including tumor necrosis factor alpha (TNF-alpha) is considered to play an important role in the pathophysiology and clinical outcome of severe liver injury. Kupffer cells, resident macrophages of the liver, have a transmembrane protein Toll-like receptor 4 (TLR4), which recognizes endotoxin (lipopolysaccharide; LPS) or LPS-CD14 complex and mediates macrophage activation and pro-inflammatory cytokine release. D-Galactosamine (GalN), a hepatocyte-specific inhibitor of RNA synthesis, is known to sensitize animals to the lethal effects of LPS and TNF-alpha. In the present study we seek to address TLR4-signaling in the development of GalN-induced acute hepatic failure (AHF) and explore the expression of TLR4 mRNA as compared to TNF-alpha mRNA and CD14 mRNA in the liver, spleen and lung of rats with GalN-induced hepatitis., Methods: AHF was induced in male Wistar rats by the intraperitoneal injection of 1 g/kg bodyweight GalN. Expression levels of TNF-alpha, TLR4 and CD14 mRNA in the whole liver, spleen and lung of rats were detected by reverse transcription-polymerase chain reaction analysis., Results: Expression level of TLR4 mRNA in the liver of rats with GalN-induced AHF was increased parallel with that of TNF-alpha and CD14 mRNA as compared to the control rats. However, expression levels of TNF-alpha, TLR4 and CD14 mRNA in the whole spleen and lung were not different between rats with AHF and control., Conclusions: There may be a difference of stimulatory effects of endotoxin on the innate immunity between the liver and other organs of rats with GalN-induced AHF.

The protective effect of pinitol against D-galactosamine (GalN)-induced liver damage was examined. Forty male Sprague-Dawley rats were divided into normal control, GalN control, and pinitol groups (0.5%, 1%, and 2%). After 8 weeks of feeding, a single dose of GalN (650 mg/kg) was administered 24 h before their sacrifice. The serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and tumor necrosis factor-alpha (TNF-alpha) levels were significantly increased after an injection with GalN (P<0.05), but pinitol supplementation at the level of 0.5% reversed these changes to normal levels. Significant decreases in serum triglyceride and cholesterol and increases in hepatic cholesterol were observed in GalN-intoxicated rats. However, supplementation with pinitol significantly attenuated these trends. In addition, pinitol elevated the Mn-superoxide dismutase, glutathione reductase, and catalase activities, prevented hepatic lipid peroxidation, and restored the hepatic GSH levels and cytochrome P450 2E1 function. Thus, 0.5% pinitol supplementation protected the rats from the hepatotoxicity induced by GalN, at least part of its effect being attributable to attenuation of the oxidative stress and inflammatory process promoted by GalN.

Background/aims: Pyrrolidine dithiocarbamate (PDTC) is an inhibitor of nuclear factor kappa B (NF-kappaB) activation. The present study aimed to investigate the effects of PDTC on lipopolysaccharide (LPS)-induced liver injury in two different models of fulminant hepatitis., Methods: Mice infected with Bacillus Calmette Guerin (BCG) were challenged with LPS (0.2 mg/kg) to induce the model of inflammatory liver injury. Mice were injected with D-galactosamine (GalN, 600 mg/kg) and LPS (20 microg/kg) to induce the model of apoptotic liver injury. In the treatment groups, mice were pre-treated with PDTC (100 mg/kg), initiated 24 h prior to LPS., Results: PDTC pretreatment reduced the infiltration of inflammatory cells, inhibited NF-kappaB activation and the expression of tumor necrosis factor alpha (TNF-alpha), attenuated nitric oxide production, and alleviated hepatic glutathione depletion. Correspondingly, PDTC reduced serum alanine aminotransferase, improved hepatic necrosis, and prolonged the survival in the BCG/LPS model. Conversely, PDTC accelerated death and aggravated liver apoptosis in the GalN/LPS model, although it reduced nitric oxide production, attenuated glutathione depletion, and inhibited the expression of TNF-alpha in liver., Conclusions: PDTC protects mice against BCG/LPS-induced inflammatory liver injury through the repression of NF-kappaB-mediated TNF-alpha release, while it seems to be detrimental in GalN/LPS-induced apoptotic liver damage.

Objective: To reproduce a Kunming murine endotoxin shock model suitable for the anti-endotoxin pharmaceutical research., Methods: Kunming mice were challenged with an intraperitoneal (i. p.) injection of different doses of D-galactosamine (D-Gal) and endotoxin (LPS) and divided into 10 groups: i.e, group 1 [with injection of isotonic saline solution (NS) and LPS]; group 2 (with injection of NS and 90mg/kg LPS), group 3 (with injection of NS and 500mg/kg D-Gal), group 4 (with injection of 500mg/kgD-Gal and 25 microg /kg LPS), group 5 (with injection of 500mg/kg D-Gal and 50 microg/kg LPS), group 6(with injection of 500mg/kg D-Gal and 250 microg/kg LPS), group 7( with injection of NS and 600mg/ kg D-Gal), group 8 (with injection of 600mg/kg D-Gal and 10 microg/kg LPS), group 9( with injection of 600mg/kg D-Gal and 25 microg/kg LPS), group 10 (with injection of 600mg/kg D-Gal and 50 microg/kg LPS). The death of the mice were observed and the mortality rate was recorded at 48 post-injection hour (PIH). The dose of D-Gal and LPS which caused 100% lethality was chosen for the subsequent experiment to serve as control group (with injection of NS and 600mg/kg D-Gal), LPS group (with injection of 600mg/kg D-Gal and 580mg/kg LPS for later experiment). The venous blood of the mice were collected for the detection of serum content of TNF-alpha with ELISA method at 30, 75 and 120 post-injection minutes (PIM). The tissues of lung, liver, intestine were also harvested at 5 PIH for the pathological examination., Results: The lethality of mice was 100% in the groups 2, 6 and 10 (P < 0.01). The serum content of TNF-alpha was maintained in a low level in control group, but it increased remarkably in LPS group, and it reached peak at 75 PIM (6365 +/- 2087ng/L, P < 0.01). Obvious inflammatory reaction was observed in the lung, liver and intestine in LPS group, while only mild inflammatory reaction was observed in liver in control group., Conclusion: The Kunming mice showed signs of endotoxin shock after D-galactosamine presensitizing and endotoxin challenge, and it is suitable for anti-endotoxin pharmaceutical research.

Objective: To establish a practical and reproducible animal model of human acute-on-chronic liver failure for further study of the pathophysiological mechanism of acute-on-chronic liver failure and for drug screening and evaluation in its treatment., Methods: Immunological hepatic fibrosis was induced by human serum albumin in Wistar rats. In rats with early-stage cirrhosis (fibrosis stage IV), D-galactosamine and lipopolysaccharide were administered. Mortality and survival time were recorded in 20 rats. Ten rats were sacrificed at 4, 8, and 12 hours. Liver function tests and plasma cytokine levels were measured after D-galactosamine/lipopolysaccharide administration and liver pathology was studied. Cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay., Results: Most of the rats treated with human albumin developed cirrhosis and fibrosis, and 90% of them died from acute liver failure after administration of D-galactosamine/lipopolysaccharide, with a mean survival time of (16.1+/-3.7) hours. Liver histopathology showed massive or submassive necrosis of the regenerated nodules, while fibrosis septa were intact. Liver function tests were compatible with massive necrosis of hepatocytes. Plasma level of TNFalpha increased significantly, parallel with the degree of the hepatocytes apoptosis. Plasma IL-10 levels increased similarly as seen in patients with acute-on-chronic liver failure., Conclusion: We established an animal model of acute-on-chronic liver failure by treating rats with human serum albumin and later with D-galactosamine and lipopolysaccharide. TNFalpha-mediated liver cell apoptoses plays a very important role in the pathogenesis of acute liver failure.

Previous studies in our laboratory demonstrated the repressive effect of seaweeds (Laminaria sp., Sargassum fulvellum, Eisenia bicyclis and Gelidium sp.) against D-galactosamine (D-GalN)-induced hepatopathy. However, the mechanism by which these four seaweeds attenuate the D-GalN-hepatopathy has not been completely clarified. This study was carried out to determine the constituents of these seaweeds that protect rats against the D-GalN-hepatopathy. Male Wistar rats were fed for 8 d diets containing 5% seaweeds with or without the antibiotic neomycin (NEO) in experiment 1, and typical seaweed dietary fibers (laminaran, fucoidan, alginate, agar and kappa-carrageenan) of these seaweeds in experiment 2. On the 7th day, the rats were treated with D-GalN (1,900 mg in experiment 1 and 800 mg/kg in experiment 2) and then sacrificed 24 h after the injection of D-GalN. Their serum transaminase (aspartate and alanine aminotransferases: AST and ALT) activities were then determined. In experiment 1, the serum AST and ALT levels in the rats fed the four kinds of seaweeds without NEO were significantly low in comparison to that of the control group, but those with NEO were not significantly different among the groups. In experiment 2, the serum AST and ALT levels in the rats fed fucoidan were significantly low in comparison to those of the other groups fed the dietary fibers and the control. These results suggest that the protective effect of the three kinds of brown seaweeds Laminaria sp., Sargassum fulvellum and Eisenia bicyclis against D-GalN-hepatopathy was caused at least in part by fucoidan.

Background: D-galactosamine together with lipopolysaccharide can lead to a pronounced secretion by Kupffer cells of pro-inflammatory mediators, which have been shown to be early and important mediators of liver injury. Probiotics and dietary supplementation with fruit or vegetable extracts with high content of antioxidants, such as blueberry, could be beneficial in protecting against hepatotoxicity., Aims: To investigate whether blueberry and probiotics could attenuate liver injury induced by D-galactosamine and lipopolysaccharide., Subjects: Sprague-Dawley rats were used., Methods: Six experimental groups: acute liver injury control and five groups of liver injury treated by blueberry alone or by each of the probiotics strains (Lactobacillus plantarum DSM 15313 and Bifidobacterium infantis DSM 15159) with and without blueberry. Samples were collected 24 h after induction for bacterial test, liver function test, short chain fatty acids, myeloperoxidase, cytokines, malondialdehyde and glutathione., Results: Alanine aminotransferase levels decreased significantly in all groups compared to liver injury control and DSM 15313 groups. Bilirubin, liver TNF-alpha, myeloperoxidase and acetic acid in cecum content decreased significantly in all groups, while liver glutathione values increased significantly in all groups compared to liver injury control. Liver IL-1beta and bacterial translocation to the liver and mesenteric lymph nodes decreased significantly in all groups except B. infantis DSM 15159 group compared to the liver injury control. Enterobacteriaceae count in cecum decreased significantly in the groups with blueberry plus probiotics compared to the other groups., Conclusion: Blueberry and probiotics exert protective effects on acute liver injury. They reduce the hepatocytes injury, the inflammation and the pro-inflammatory cytokines, and improve the barrier functions and antioxidant activity.

Aim: To examine the effects of adiponectin on the functions of Kupffer cells, key modulators of lipopolysaccharide (LPS) -induced liver injury., Methods: D-galactosamine (GalN) and LPS were injected intraperitoneally into adiponectin-/- mice and wild type mice. Kupffer cells, isolated from Sprague-Dawley rats, were preincubated with or without adiponectin, and then treated with LPS., Results: In knockout mice, GalN/LPS injection significantly lowered the survival rate, significantly raised the plasma levels of alanine transaminase and tumor necrosis factor-alpha (TNF-alpha) and significantly reduced IL-10 levels compared with wild type mice. TNF-alpha gene expression in the liver was which higher and those of IL-10 were lower in knockout mice than in wild type mice. In cultured adiponectin-pre-treated Kupffer cells, LPS significantly lowered TNF-alpha levels and raised IL-10 levels in the culture media and their respective gene expression levels, compared with Kupffer cells without adiponectin-pre-treatment., Conclusion: Adiponectin supresses TNF-alpha production and induces IL-10 production by Kupffer cells in response to LPS stimulation, and a lack of adiponectin enhances LPS-induced liver injury.

In acute hepatic injury tests, an adzuki bean extract decreased D-galactosamine (GalN)-induced alterations in the serum alanine aminotransferase and aspartate aminotranferase activities to about 37% and 25%, respectively, although there were no significant differences in these activities between the GalN-treated group with the adzuki bean extract and the GalN-treated group without the adzuki bean extract. Furthermore, the hepatic glutathione peroxidase, glutathione reductase, and Mn- and Cu,Zn-superoxide dismutase mRNA levels in the GalN-treated group with the adzuki bean extract were higher than those in the control group and GalN-treated group without the adzuki bean extract.

Hepatobiliary abnormalities have been described in patients with chronic inflammatory bowel diseases. Hepatic cytochrome P450 (P450)-dependent drug-metabolizing enzyme activities and susceptibility to a hepatotoxin, d-galactosamine, were determined in rats with dextran sulfate sodium (DSS)-induced colitis to assess whether liver function is affected in the model of inflammatory bowel disease. Colitis was induced by treatment of rats with 3% DSS in drinking water for 7 days. Liver microsomes for enzyme activities and serum for biological analysis were prepared from the rats with colitis, along with untreated and lipopolysaccharide (LPS)-treated rats. Other rats received intraperitoneal injection of d-galactosamine to assess their susceptibility to the toxin-induced liver injury. Treatment of rats with DSS resulted in not only colitis but also decreases in hepatic P450-dependent drug-metabolizing enzyme activities. Elevated endotoxin was found in portal blood, which was not associated with liver injury. The potency and the isoform selectivity in the suppression of the P450 enzymes by DSS treatment were similar to those of LPS-treated rats. Coadministration of antibiotics, polymyxin B or metronidazole, with DSS protected rats from decreases in some but not all P450 enzyme activities, indicating partial involvement of bacterial endotoxin in the P450 decreases. The rats with colitis were less susceptible than untreated rats to d-galactosamine-induced liver injury and TNF-alpha production, suggesting development of endotoxin tolerance in DSS-colitis. In conclusion, these results suggest that the DSS-colitis leads to endotoxin-mediated down-regulation of hepatic P450 enzymes and protection against d-galactosamine-induced liver injury, probably due to endotoxin tolerance.

Background: The extracellular matrix component hyaluronan (HA) modulates the production of various cytokines and chemokines by activated inflammatory cells. In this study, we investigated whether exogenous administration of HA influences T-cell-mediated liver injury and cytokine production., Methods: Liver injury was induced by administration of concanavalin A (Con A) or D-galactosamine/lipopolysaccharide (GalN/LPS), and 0.05%-0.35% (v/v) HA (MW 250, 470, 780, 900, and 1200 kDa) was administered intravenously 18 h before Con A or GalN/LPS injection. Plasma ALT level was determined enzymatically and plasma cytokine levels were determined by ELISA., Results: The elevated plasma levels of ALT at 8 h after Con A and at 7 h after GalN/LPS injection were significantly decreased by pretreatment with high molecular weight HAs (780, 900, and 1200 kDa) but not low molecular weight HAs (250 and 470 kDa). High molecular weight HA (900 kDa) significantly reduced plasma tumor necrosis factor-alpha, interferon gamma, macrophage inflammatory protein 2, and interleukin 4 levels after Con A injection. However, this inhibitory effect on plasma cytokines was not observed with low molecular weight HA (250 kDa) pretreatment., Conclusions: The present results suggest that high molecular weight but not low molecular weight HA prevents liver injury by reducing proinflammatory cytokines in a T-cell-mediated liver injury model. The extracellular matrix component hyaluronan (HA) modulates the production of various cytokines and chemokines by activated inflammatory cells. In this study, we investigated whether exogenous administration of HA influences T-cell-mediated liver injury and cytokine production.

Non-viral gene delivery systems utilise either amine lipids or polyamines and although non-viral gene delivery systems are said to have a superior safety profile to viruses, the polyamines such as poly(L-lysine) are toxic when used without derivatisation and usually require specific receptor mediated uptake and/or endosomolytic agents to be effective. However, the conversion of poly(L-lysine) and poly(L-ornithine) polyamino acids into amphiphilic vesicle forming polymers reduces the toxicity of the polyamino acids and enables the resulting polyamino acid vesicles to deliver genes both in vitro and in vivo in the absence of receptor specific ligands and endosomolytic agents. The incorporation of a distearoylphosphatidylethanolamine poly(ethylene glycol)-galactosamine conjugate (with the galactosamine unit at the distal end of the poly(ethylene glycol) moiety) into the polyamino acid formulations improved in vitro gene transfer in the case of the amphiphilic poly(L-ornithine) (POP) although no in vivo targeting was detected with the galactosamine formulations. We conclude that the conversion of poly(L-lysine) and poly(L-ornithine) into amphiphilic colloid forming molecules reduces their toxicity, thus allowing these systems to be used for gene transfer in vivo. It is possible that this approach may be extended to other polyamines.

Background: Tumour necrosis factor-alpha (TNF-5) has been shown to exacerbate or protect against liver injury in different experimental models. In a previous study, we observed that enhancement of TNF-alpha expression in hepatocytes by prostaglandin E1 (PGE1) pre-administration induced iNOS expression and cytoprotection against experimental liver injury in rats. Nevertheless, the reduction of TNF-alpha bioactivity by anti-TNF-alpha antibodies also reduced liver injury by D-GalN. The purpose of the present study was to evaluate whether protection by PGE1 or anti-TNF-alpha was related to a common effect on the membrane-bound TNF-alpha receptor expression., Methods: Liver injury was induced in male Wistar rats by intraperitoneal injection of D-galactosamine (D-GalN) (1 g/kg). PGE1 or anti-TNF-alpha was administered at 30 or 60 min before D-GalN, respectively. Liver injury was evaluated by alanine aminotransferase (ALT) activity in serum and histological examination in liver sections. TNF-alpha was determined by ELISA in serum. The expression of TNF-alpha receptor type 1 (TNF-R1) and TNF-alpha receptor type 2 (TNF-R2) in hepatocytes was assessed by immunohistochemistry and immunoprecipitation + Western-blot analysis., Results: PGE1 or anti-TNF-alpha reduced liver injury induced by D-GalN. Although PGE1 enhanced and anti-TNF-alpha reduced TNF-alpha concentration in serum, both protective treatments reduced the expression of TNF-R1 in hepatocytes. TNF-R2 was not detected in our experimental conditions., Conclusions: Our study showed that reduction of liver injury by PGE1 or anti-TNF-alpha antibodies was related to a reduction of TNF-R1 expression in hepatocytes.

Background: Plasma exchange may be useful for treating patients with fulminant hepatic failure but during the procedure growth factors that are important for hepatic regeneration are discarded. Addition of a selective plasma filter to the plasmapheresis circuit could eliminate protein bound toxic substances and retain growth factors for hepatic regeneration. This process is called selective plasma filtration., Aims: To determine if selective plasma filtration could be a useful treatment modality for fulminant hepatic failure., Methods: The system was tested in five groups of pigs with fulminant hepatic failure induced by galactosamine: group I, diseased control group (n=5); group II, sham control, (n=6); group III, plasma exchange (n=6); group IV, treatment with AC-1770 selective plasma filter (n=7); and group V, treatment with AC-1730 selective plasma filter which had a smaller pore size than AC-1770 (n=7). Fresh pig plasma was given to replace filtered plasma in pigs of groups III, IV, and V. Treatment was initiated 48 hours after administration of 0.75 g/kg galactosamine. The efficacy of selective plasma filtration was assessed by survival rate and improvement in haematological, biochemical, and immunohistological parameters., Results: Pigs treated with AC-1770 or AC-1730 selective plasma filters survived longer than the other groups (group I: 55 (10) hours; group II: 68 (7) hours; group III: 91 (10) hours; group IV: 269 (156) hours; group V: 950 (555) hours). One pig in group IV survived for 50 days; one pig in group V survived for 77 days and another pig in group V is still alive (>150 days). After treatment, plasma levels of aspartate aminotransferase, bilirubin, bile acid, ammonia, lactate dehydrogenase, and alpha-glutathione-S-transferase decreased. Substantial amounts of tumour necrosis factor alpha (TNF-alpha) and endotoxin were found in the filtrate. The selective plasma filtration groups retained significantly higher amounts of hepatocyte growth factor than plasma exchange alone. Similar TNF-alpha clearance was observed in the selective plasma filtration groups and the plasma exchange group. On day 4, significant improvement in liver function, as measured by the indocyanine green clearance test, was observed in groups IV and V but not in the other groups. A higher regeneration index of hepatocytes was also observed in the groups treated with AC-1770 and AC-1730 selective plasma filters., Conclusion: Selective plasma filtration improved survival time and expedited liver regeneration in pigs with fulminant hepatic failure.

The effects of 1,8-cineole on D-galactosamine/lipopolysaccharide (GalN/LPS)-induced shock model of liver injury was investigated in mice. The co-administration of GalN (700 mg kg(-1), i.p.) and LPS (5 microg kg(-1), i.p.) greatly elevated serum concentrations of tumour necrosis factor-alpha (TNF-alpha), alanine aminotransferase and aspartate aminotransferase, and induced massive hepatic necrosis and lethality in 100% of control mice. Pretreatment with 1,8-cineole (400 mg kg(-1), p.o.) and dexamethasone (1 mg kg(-1), s.c.), 60 min before GalN/LPS, offered complete protection (100%) against the lethal shock and acute elevation in serum TNF-alpha and serum transaminases. Hepatic necrosis induced by GalN/LPS was also greatly reduced by both 1,8-cineole and dexamethasone treatment. The results indicate that 1,8-cineole protects mice against GalN/LPS-induced liver injury through the inhibition of TNF-alpha production, and suggest that 1,8-cineole may be a promising agent to combat septic-shock-associated pathologies.

Extracts of various types of tea and coffee significantly suppressed lipopolysaccharide (LPS)-induced liver injury, as assessed by the plasma enzyme activities, in D-galactosamine-sensitized rats when administered orally once before injecting the drugs. There was a significant negative correlation between the caffeine levels of these extracts and liver injury. Authentic caffeine also had a hepatoprotective effect. These results suggest that caffeine-containing beverages generally suppress LPS-induced liver injury according to their caffeine content.

The antioxidant action of medicinal herbs used in Ghana for treating various ailments was evaluated in vitro and in vivo. Five plants, Desmodium adscendens, Indigofera arrecta, Trema occidentalis, Caparis erythrocarpus, and Thonningia sanguinea were tested for their free radical scavenging action by their interaction with 1,1-diphenyl-2-picrylhydrazyl (DPPH). Of these five plants, only Thonningia sanguinea was found to scavenge the DPPH radical. Lipid peroxidation in liver microsomes induced by H2O2 was also inhibited by T. sanguinea. The hepatoprotective effect of T. sanguinea was studied on acute hepatitis induced in rats by a single dose of galactosamine (GalN, 400 mg/kg, IP) and in mice by carbon tetrachloride (CCl4, 25 microl/kg, IP). GalN induced hepatotoxicity in rats as evidenced by an increase in alanine aminotransferase (ALT) and glutathione (GSH) S-transferase activities in serum was significantly inhibited when T. sanguinea extract (5 ml/kg, IP) was given to rats 12 hr and 1 hr before GalN treatment. The activity of liver microsomal GSH S-transferase, which is known to be activated by oxidative stress, was increased by the GaIN treatment and this increase was blocked by T. sanguinea pretreatment. Similarly, T. sanguinea pretreatment also inhibited CCl4-induced hepatotoxicity in mice. These data indicate that T. sanguinea is a potent antioxidant and can offer protection against GalN- or CCl4-induced hepatotoxicity.

To define a possible role for changes in the regulation of antigen presentation in fulminant hepatic failure (FHF), we studied the expression of co-stimulatory molecules CD80 (B7-1), CD86 (B7-2), and CD40 along with their ligands CD28 and CD154. We analyzed the liver tissue from patients with FHF (n = 18), chronic liver disease (n = 30), and acute hepatitis (n = 3) and from normal controls (n = 9) by immunohistochemistry and examined the temporal relationship between CD80/CD86 and CD40 expression and disease in the mouse models of galactosamine-lipopolysaccharide and galactosamine-tumor-necrosis-factor-induced FHF. In human controls, faint CD80/CD86 immunoreactivity was restricted to Kupffer cells, and CD40 expression was expressed on bile ducts, macrophages, and sinusoidal endothelial cells (SECs). In FHF, immunoreactivity for CD80 and CD86 was observed on significantly higher numbers of cells, including SECs. Increased CD80/CD86 expression corresponded to increased numbers of CD28-positive lymphocytes. The expression of CD40 was also clearly elevated on virtually all cell types in FHF. In both murine models, CD40 and CD80/CD86 expression was up-regulated before tissue damage could be detected. Our data suggest that up-regulated expression of co-stimulatory molecules might lead to an excessive antigen presentation in FHF as an early step in the pathogenesis before the onset of tissue damage.

Nicotine exerts a number of physiological effects. Nicotine is absorbed through the lungs with smoking and is rapidly metabolized in humans. Although it is mainly metabolized in the liver, the effects of liver injuries on nicotine metabolism are not clear. The purpose of this study was to clarify the effects of liver injuries on nicotine metabolism. Rats were treated with D-galactosamine (GalN) or thioacetamide (TA), to induce acute hepatitis or liver cirrhosis, respectively. Serum transaminase levels were significantly elevated in model rats with both types of liver injury. Cytochrome P450 (CYP) and cytochrome b5 contents in liver microsomes were decreased significantly in TA-treated cirrhotic rats but not in GalN-treated hepatitic rats. The major metabolic pathways of nicotine, i.e. cotinine formation catalyzed by CYP and nicotine-1'-N-oxide formation catalyzed by flavin-containing monooxygenase, were investigated in these rat liver microsomes. Formation of cotinine and nicotine-1'-N-oxide from nicotine was not changed in GalN-treated hepatitic rats, in comparison with the controls, but was significantly decreased in TA-treated cirrhotic rats. By immunoblotting, decreases in CYP1A2, CYP2B2, CYP2C, and CYP2E1 protein were recognized in liver microsomes from TA-treated cirrhotic rats. It was also shown that the maximal velocity values for nicotine-1'-N-oxide formation in TA-treated cirrhotic rats were significantly decreased, compared with the controls. These results suggested that the reduction of nicotine metabolism in cirrhosis was due to decreases in CYP and flavin-containing monooxygenase protein expression levels.

Background: During fulminant hepatic failure (FHF) metabolites normally cleared by the liver accumulate in the circulation and cause hepatic coma. It is believed that the plasma of FHF patients has an inhibitory effect on liver regeneration. Plasma exchange was used to study the effect of plasma collected from donor FHF rats on liver regeneration in two-thirds partially hepatectomized syngeneic animals., Methods: FHF and hepatic coma were induced in donors by administration of galactosamine at a dose of 1.85 g/kg. Plasma from donors in either grade-II or -IV coma was transfused by plasma exchange into partially hepatectomized animals 2h after resection., Results: The livers from donor animals showed evidence of oval cell activation 1-2 days after galactosamine, but differentiation of oval cells to hepatocytes did not occur before the development of coma. The plasma collected from animals in grade-IV coma totally abolished regeneration in the partially hepatectomized recipients., Conclusion: These results support the hypothesis that metabolites present in the plasma during FHF inhibit liver regeneration.

Imipenem and ceftazidime have different specificities for penicillin-binding proteins and cause a differential release of lipopolysaccharide (LPS) from gram-negative bacteria in vitro. In studies in mice made leukopenic by the administration of cyclophosphamide, the innate relative resistance to the lethal effects of LPS was not significantly changed, but these animals became highly sensitized to bacterial infection. When leukopenic mice were challenged with graded doses of Escherichia coli O111:B4, an LD50 was achieved at a dose of approximately 10(6) cfu. Administration of either antibiotic resulted in a shift in the LD50 of approximately 500-fold, in contrast to D-galactosamine-treated LPS-sensitized mice, in which a < 10-fold increase in the LD50 was observed with antibiotic therapy. Further, if mice were made LPS-sensitive with D-galactosamine, no differences between leukopenic and normal mice were noted with antibiotic therapy.

Background & Aims: T cell-dependent liver injury involving endogenous tumor necrosis factor (TNF) alpha can be induced by either concanavalin A in naive mice or by activating anti-CD3 antibody or staphylococcal enterotoxin B in D-galactosamine-sensitized mice. In this study, the role of interferon gamma (IFN-gamma) in these T-cell models was addressed., Methods: Mice were pretreated with a neutralizing anti-mouse IFN-gamma antiserum before injection of T cell-activating agents. Plasma cytokine and transaminase levels were determined. Apoptotic cell death was assessed by hepatic DNA fragmentation., Results: Anti-IFN-gamma antiserum significantly protected mice from concanavalin A-induced liver injury. Circulating IFN-gamma was completely suppressed, and TNF was reduced by 50%. Recombinant TNF-alpha administered to mice treated with concanavalin A and anti-IFN-gamma antiserum failed to initiate liver injury. Similar results were obtained with recombinant IFN-gamma in concanavalin A-challenged mice under the condition of TNF neutralization. Neither hepatic DNA fragmentation nor release of transaminases was inhibited by anti-IFN-gamma antiserum when liver injury was induced by staphylococcal enterotoxin B or anti-CD3 antibody in D-galactosamine-sensitized mice., Conclusions: Both TNF as well as IFN-gamma are critical mediators of liver injury in concanavalin A-treated mice, whereas hepatic DNA fragmentation and liver failure in the D-galactosamine models depend only on TNF.

In contrast to human carriers of microfilariae, filariae infected rodents generally tolerate an effective microfilaricidal treatment without obvious signs of adverse reactions. The study shows, however, that also the filariae (Litomosoides carinii, Brugia malayi) infected rodent Mastomys coucha can be rendered sensitive to side effects of the treatment by the administration of D-galactosamine (D-Gal), due to reduction of liver UTP levels. Independent of the drug (diethylcarbamazine, ivermectin, CGP 20376) and the parasite species, D-Gal-primed infected animals died within 4 days after a microfilaricidal treatment. Lethal effects did also occur in naive animals to which microfilariae had been transfused 18 h prior to the challenge with D-Gal and a microfilaricidal, provided the animals had received at least approximately 10(3) larvae/g body weight. Both infected animals and naive recipients of microfilariae could be protected from death by cyclosporin A, polyclonal antibodies to mouse TNF or suitable amounts of NG-monomethyl-L-arginine. Pentoxifylline was less protective. The results suggest that components play a role in adverse reactions after microfilaricidal treatment, which are released by dying/dead microfilariae and may interact with T lymphocytes independent of a specific state of immunity. In a sequela, TNF released by T cells seems to induce an excess synthesis of N-oxides which appear to be the final morbific agent.

Polyamine concentrations including putrescine, spermidine and spermine were documented in two brain areas of rats with mild and severe stages of hepatic encephalopathy (HE) due to fulminant hepatic failure induced by galactosamine HC1 injection (3 g kg-1 i.p.). In the mild stage of HE putrescine increased by 3-4 times whereas spermidine and spermine showed a slight increase. The scenario, however, was found to be changed going from the mild to the severe stage of HE, since in this last stage spermidine and spermine showed a further rise while putrescine was found to be significantly lower than in the mild stage of HE in both the brain areas studied. The changes in the ratio among the three polyamines with an enhanced prevalence in the severe stage of HE of spermidine and spermine are likely to be related to the exhaustion of the synthetic pathway of putrescine or to a reduction of the interconversion to this polyamine from spermidine and spermine. Considering that these last two polyamines potentiate the N-methyl-D-aspartate glutamate receptor mediated toxicity and that they might exert neurotoxic effects per se, there are clear reasons for suspecting an implication of the described changes of polyamines in the neurochemical mechanism which sustain HE and to surmise a potential therapeutic effect in this pathology of non-competitive antagonists of polyamine-site on N-methyl-D-aspartate glutamate receptors.

Rat liver regeneration was studied from 24 hours to 8 days after a single intraperitoneal injection of D-galactosamine (GalN). Morphological changes in the liver were analyzed in parallel with sequential changes in expression of histone-3 mRNA (a marker of cell proliferation), fetal alpha-fetoprotein (AFP) mRNA and gamma-glutamyl transpeptidase (GGT) (markers of fetal hepatocytes), and albumin mRNA and glucose-6-phosphatase (G6Pase) (markers of adult hepatocytes). Proliferation of nonparenchymal epithelial cells (NPC), detected in situ by [3H]thymidine labeling or histone-3 mRNA expression, began after 24 hours primarily in the portal area around the bile ducts. After 2 days, histone-3 labelling intensity increased in rows and clusters of NPC which expanded from the portal zone and invaded into the parenchyma. On days 3 and 5, NPC expressing his-3 mRNA expanded further, forming pseudo-ducts and islet-like structures (NPC structures). Proliferating NPC were positive for GGT. Some GGT positive cells were also positive for the fetal form of AFP mRNA, which lagged behind GGT by 24 hours and peaked on day 5. On day 3, some cells with the appearance of NPC expressed albumin mRNA. Double label in situ hybridization for fetal AFP and albumin mRNAs and dual histochemistry for GGT and G6Pase showed simultaneous expression of these markers in NPC on day 5. Other cells expressing fetal AFP mRNA or GGT on day 5 had a morphological appearance between NPC and hepatocytes (transitional cells). Proliferation of hepatocytes began on day 2, reached maximum on day 5 and then declined. Proliferating hepatocytes did not express fetal AFP mRNA or GGT. These findings indicate that after GalN injury, the liver responds by activation of progenitor cells that proliferate and then differentiate into mature hepatocytes. Adult hepatocytes can also proliferate after GAlN injury, but these hepatocytes do not undergo dedifferentiation/redifferentiation during regeneration of the hepatic lobule.

Arachidonate metabolism was examined in rats with experimentally induced acute and chronic liver injuries. Acute liver injury was induced by a single administration of D-galactosamine (D-Galn) and lipopolysaccharide (LPS) or carbon tetrachloride (CCl4). Chronic liver injury was produced by several administrations of CCl4 for 5 weeks. Non-parenchymal liver cells from rats with D-Galn/LPS-induced acute liver injury produced prominently leukotriene B4 and 5-hydroxy-arachidonic acid which were hardly synthesized by the normal rat liver. No apparent changes were observed in the arachidonate metabolism of the non-parenchymal cells of the acute CCl4-injured liver. In chronic liver injury, the production of 6-ketoprostaglandin F1 alpha, a stable metabolite of prostaglandin I2, by the non-parenchymal cell fraction was significantly enhanced in contrast with the fixed amount of the other arachidonate metabolites. These results suggested the arachidonate metabolism by non-parenchymal liver cells might change according to the pathogenesis of the liver disease.

We observed the effects of a chinese herb medicine Sho-saiko-to on the lethal and antitumor activities of recombinant human tumor necrosis factor (rhTNF) administered in mice. Sho-saiko-to was noted to protect the rhTNF-induced lethality in galactosamine-hypersensitized mice, and also Sho-saiko-to pretreated mice was protected against the decrease of rectal temperature after rhTNF administration. On the other hand, there was a remarkable enhancement of antitumor activity of rhTNF by Sho-saiko-to pretreatment. These results suggest that Sho-saiko-to drug may protect mice from severe shock syndrome induced by rhTNF.

Galactosamine hydrochloride induces liver disease in rats that morphologically resembles drug-induced hepatitis in man. In this study we analyzed the character of the inflammatory reaction following the toxic damage resulting from the administration of galactosamine hydrochloride using a broad panel of monoclonal antibodies to lymphocyte subsets and macrophages. Fat-storing cells were identified with a polyclonal anti-desmin antibody. Cellular proliferation was assessed by labeling S-phase cells with the thymidine analog bromodeoxyuridine. Injection of galactosamine hydrochloride was associated with conspicuous hepatocyte necrosis and parenchymal granulocyte influx in the first 24 hr. Thereafter, mononuclear inflammatory cells predominated, mainly T lymphocytes and macrophages, with maximal numbers at 48 hr. The majority of T lymphocytes were CD8-positive cells and were located in the portal tracts and parenchyma. CD4-positive T cells were scarce and confined to the portal tracts. Proliferation of fat-storing cells paralleled hepatocyte regeneration with maximal values after 48 to 72 hr. The temporal relationship between infiltrating mononuclear cells, mainly T lymphocytes of CD8 phenotype and macrophages, fat-storing cell proliferation and hepatic regeneration suggests pathophysiological interactions between these cell types in liver injury in the rat after galactosamine hydrochloride administration.

Acute liver injury was induced experimentally in rats by a single injection of a large amount of D-galactosamine. Hepatocellular damage was apparent from decrease of total serum proteins, marked release of transaminases into the circulation, precipitous decrease of total microsomal proteins, and intracellular enzymes such as cytochrome P450 and 5'-nucleotidase. In parallel with such hepatocellular damage, serum asialoglycoproteins accumulated markedly, reaching a maximum level 4 days after the injection and then decreased to the control level. In contrast to this increase, hepatic binding protein, a receptor in the liver which specifically recognizes asialoglycoproteins, decreased very much. At the same time, survival time of [125I]asialoorosomucoid intravenously administered into rats was much prolonged in inverse proportion to the decrease of the binding protein. From these results it was concluded that the decrease of the hepatic binding protein induced by galactosamine treatment is probably responsible for the marked accumulation of serum asialoglycoproteins.

The hypothesis was tested whether under the presence of a membrane defect of hepatocytes an activation of the complement system leads to massive liver cell necrosis. Low doses of galactosamine (50, 100, and 200 mg/kg) were administered to rats with and without an additional i.v. injection of sublethal doses of endotoxin (1.5 mg/kg). The latter was done in order to activate the complement system via the C3-bypath. Neither after doses of 50 mg/kg and 100 mg/kg nor after an additional administration endotoxin liver cell necrosis were observed. A dose of 200 mg/kg led to moderate liver cell necrosis, and when administered with endotoxin fulminant hepatic necrosis developed. The explanation was given that only after 200 mg/kg alterations of hepatocytes membranes were present, which prepare liver cells for complement mediated hepatocytolysis. In rats to which 1 g/kg galactosamine was given in addition to endotoxin it was demonstrated by immunohistology that the third component of complement was already fixed on hepatocyte plasma membranes at hour 3 and was accumulated within areas of necrotic liver parenchymal cells at hour 12. Thus, liver cell death is suggested as complement mediated if the membranes are altered. Clinical implications are given in concern of fulminant hepatic failure and an approach to effective treatment regims.

The morphological and biochemical studies were performed on the injured livers of female rats produced by chronic administration of D-galactosamine (GALN) (250 mg/kg, i.p.) for 7 months. Light microscopically, cirrhotic changes were observed in most of the animals characterized by the proliferation of the connective tissues from portal triads into hepatic lobules. The electron microscopic study demonstrated mitochondrial proliferation and irregularities with crenated membranes, focal hypertrophy of the smooth endoplasmic reticulum, decrease of the rough endoplasmic reticulum with partial detouchment of ribosomes, slight loss of compactness of nucleoli and no remarkable accumulation of lipid droplets in the cytoplasm. The proliferation of collagen fibers was observed around the hepatocytes and acid mucopolysaccharides were seen in the space of Disse and partly in the sinusoids histochemically using electron microscope. Biochemically chronic GALN administration, which increased hepatic weight significantly, resulted in a significant decrease in microsomal protein concentration, whereas cytochrome P450 content significantly increased. There was no change in phospholipid contents. After GALN, plasma albumin concentration was significantly decreased and the value of zinc turbidity test was increased. However, there was no change in plasma GPT level and total cholesterol concentration.

A galactosamine-induced fulminant hepatic failure (FHF) rat model was used to study the effects of homologous cross-circulation and in situ liver perfusion. Cross-circulation with homologous donors did not significantly improve the survival time or recovery rate of grade II hepatic coma rats. Homologous in situ liver perfusion significantly improved the survival time and recovery in FHF only when started in grade II coma; it has no effect in the later stage of coma.

A study was conducted to elucidate the possible role of singlet oxygen in pathogenesis of D-galactosamine-induced liver injury. Tissue and plasma levels of singlet oxygen were determined with chemiluminescence analysis. Following results were obtained: 1) Chemiluminescence as well as malondialdehyde, which is regarded as one of terminal products of lipid peroxidation, significantly increased in the liver and plasma of rats treated with D-galactosamine. 2) Elevation of plasma GPT and total bilirubin was also observed in rats with D-galactosamine-induced liver injury. Histological examination of the liver revealed submassive hepatic necrosis. 3) Administration of vitamin E, a radical scavenger of singlet oxygen, significantly inhibited the increases of chemiluminescence and MDA in the liver and plasma as well as the elevations of GPT and total bilirubin in the plasma. Histological changes of the liver were also found to improve significantly by vitamin E administration. In conclusion, singlet oxygen seems to be definitely involved, at least in part, in pathogenesis of liver damage induced by D-galactosamine. In addition, inhibition of the liver injury is possible, to some extent, by administration of vitamin E, one of the potent radical scavengers of singlet oxygen.

Present experiment was aimed to study whether 16, 16' dimethylprostaglandin E2 (dmPGE2), Hepatofalk (HF), or Orotofalk (OF) may prevent an acute liver damage induced in rats with D-galactosamine (GalN). Fifty male rats were divided into 5 groups: 1. controls, 2. rats receiving GalN 750 mg/kg b. w. intraperitoneally, 3. animals pretreated with 5 microgram/kg dmPGE2 given subcutaneously 24 hours prior to, 30 min prior to and 6 hours after GalN. Rats of group 4 received HF 0,8 ml/kg intramuscularly and group 5 OF 0,3 caps/kg intragastrically 24 hours prior to, 30 min prior to and 6 hours after GalN. Animals were sacrificed 24 hours after GalN injection. Histological, histochemical and ultrastructural studies of the liver were performed. In rats receiving GalN alone (group 2) typical severe liver damage consisting of acidophilic necrosis of hepatocytes, periportal and intralobular inflammatory infiltration, hypertrophy and hyperplasia of Browicz-Kupffer cells has been observed. Histochemical investigations showed in this group fatty degeneration and a decrease in glycogen content in hepatocytes, irregular distribution of lysosomes, numerous cytolysosomes and uneven decrease in lysosomal enzymes activity (acid phosphatase and beta-glucuronidase). Ultrastructural studies revealed depletion in glycogen, fat droplets, hypertrophy of smooth endoplasmic reticulum and numerous autophagic vacuoles. Some of these vacuoles or residual bodies were dropping out into intercellular space. Focal accumulation of lamellar cytomembranes as well as condensation of heterochromatin in nuclei were also observed. Pretreatment of animals with dmPGE2 (group 3), HF (group 4) or OF (group 5) prior to GalN prevented liver cell necrosis. Histological, histochemical and ultrastructural picture of the liver was in these groups close to normal. Only very slight hypertrophy and hyperplasia of Browicz-Kupffer cells, was seen as well as depletion of glycogen and hypertrophy of smooth endoplasmic reticulum in hepatocytes. We conclude dmPGE2, HF and OF offered impressive cytoprotection against GalN induced liver damage in rat.

Galactosamine-induced fulminant hepatic failure rats have been used as a model for statistical assessment of liver support systems. The present study reports in detail the biochemical, hematological and histological changes in these animals. They have been used to study statistically the effects of ACAC charcoal hemoperfusion, cross-circulation and liver perfusion on long-term survival in fulminant hepatic failure.

Galactosamine caused changes in the livers of rats that were similar to those seen in human viral hepatitis. In hamsters the changes were characteristic of a typical toxic hepatitis. The response by the liver to galactosamine was very variable in both the rat and the hamster and there was a degree of correlation between the raised transaminase levels and the liver cell damage.

Ethyl (Z)-(3-ethyl-4-oxo-5-piperidino-thiazolidin-2-ylidene)- acetate (piprozoline, Gö 919, Problin) is a new potent choleretic which can be classified as a true cholepoietic agent. Accordingly the substance increases bile fluid and solid content as well. A dose-dependent choleretic effect could be shown in all species investigated. The choleretic effect of piprozoline is long-lasting and superior to that of the other choleretics compared with. There was a significant inhibition of experimentally induced liver damage in animals by piprozoline given prophylactically. Our experiments did not reveal any pharmacological properties interfering with the use of piprozoline as a choleretic.

The effect of a nucleoside-nucleotide mixture on liver injury of rats induced by D-galactosamine was studied by examining changes in function and histopathology of the liver. Animals with liver damage received total parenteral nutrition with glucose and amino acids supplemented with a nucleoside-nucleotide mixture containing inosine, cytidine, GMP, uridine and thymidine, or with uridine which inhibits galactosamine injury, or with liver cell extract containing flavin adenine dinucleotide and nucleic acid derivatives. As control, animals with liver damage received total parenteral nutrition with glucose and amino acids only. The serum GOT and GPT concentrations were significantly lower in the group supplemented with nucleoside-nucleotide mixture than those in other groups. A large dose (1.2 g/kg) of uridine inhibited liver injury, but a lower dose (0.14 g/kg) did not have any effect, whereas nucleoside-nucleotide mixture containing the same amount of uridine inhibited the injury. Liver cell extract also did not improve liver function. Thus infusion of a physiological and balanced mixture of nucleosides or nucleotides may improve liver function in rats with liver injury.

The present work reports on incorporation of 14-Cglycine in the proteins of three liver fractions after the application of D-Galactosamine and D-Galactosamine + Prednisolone. 164 females albino rats were used in the experiments. The results demonstrate a higher rate of glycine incorporation in thirty months than in six weeks old animals. There is also a different age depending effect of prednisolone. The results are discussed in the light of reports from the literature.ports from the literature.

The time-course of plasma lipid alterations and of triglyceride accumulation in the liver was investigated in male and female rats 12, 24 and 48 hours after treatment with 3.48 mmole/kg (0.75 g/kg) D-galactosamine (Ga1N). In the early stages of Ga1N-induced liver injury the concentrations of triglycerides, phospholipids and total cholesterol decreased, while in the later stages these values in the plasma increased above normal, especially in male animals. In contrast, glucose concentrations continually decreased, while free fatty acid (FFA) levels rose to twice those normal in female animals. Male animals had significantly lower FFA-values throughout the experiment. Consistently, the triglyceride accumulation on liver was 75 mg/g in female animals 24 hours after Ga1N administration, while male animals in the average showed only 33 mg/g triglycerides. Similar fatty infiltrations were obtained in female animals with the rather low doses of 1.16 and 2.32 mmol/kg Ga1N. It is concluded that the increase of FFA-influx after Ga1N administration is the main cause for fatty infiltration, the sex differences in the plasma FFA concentrations explaining the net differences in liver triglyceride accumulation. Additional effects in the pathogenesis of fatty liver might stem from disturbed glycosylation reactions and/or an altered secretion and metabolism of lipoproteins after Ga1N-induced liver injury.

Control trials and statistical analysis were carried out to assess the effects of albumin-collodion microencapsulated activated charcoal (ACAC) hemoperfusion on fulminant hepatic coma. A rat model of galactosamine induced fulminant hepatic coma was used. Rats which did not recover died at 3.0+/-0.6 days after galactosamine injection. Those which survived this period recovered. Forty-eight hours after galactosamine injection, a test group of 21 rats were treated with 1 hour hemoperfusion and compared with an untreated group of 23 rats. 71.4% of the treated group survived as compared to 30.4% of the untreated rats. Statistical analysis (t test) shows a significant increase in recovery for the treated group (less than 0.01). Biochemical and histological results will be discussed.

The effect of Silymarin (Legalon) upon liver lesions was investigated using four experimental models: In acute galactosamine-hepatitis, Silymarin administration achieved protection of the liver structure (electron-microscopy included), liver cell glycogen, RNA and enzymatic activity, Galactosamine-depressed gluconeogenesis in the isolated perfused rat liver was significantly preserved by Silymarin treatment. In lead and cadmium poisoning the structural damage and histochemical and histoenzymatic changes were partly but significantly prevented. The complex noxious effects of Imuran overdoses were favourably influenced by Silymarin, without diminishing the cytostatic-immunosuppressive action of Imuran.