Experimental Liver Injury Research Articles

Preventive Effects of Acute Inflammation on Liver Cell Necrosis and Inhibition of Heparan Sulphate Synthesis in Hepatocytes

The hepatocytoprotective effect of acute, turpentine-induced inflammation on experimental liver injury, caused by thioacetamide and D-galactosamine, respectively, was studied in relation to the synthesis of glycosaminoglycans and of heparan sulphate in hepatocytes isolated from livers of treated rats. As judged from biochemical parameters of liver cell lesion in serum (various cytosolic and mitochondrial enzymes, bile acids) the extent of liver damage caused by either noxious agent was greatly attenuated under acute inflammatory conditions. There was an inverse statistical correlation (r = -0.63 to r = -0.84) between the functional concentration of a proteinase inhibitor protein determined with the chromogenic substrate assay for human alpha 2-macroglobulin and the catalytic concentrations of various cell leakage enzymes in serum from liver-injured rats with turpentine-generated inflammation. The strong inhibition of heparan sulphate synthesis in hepatocytes from injured livers (synthesis rate between 0.2 and 0.4 of that in control cell incubations) is abolished in hepatocytes from livers exposed to the noxious agents but with acute inflammation. The latter condition alone caused a 1.8 fold increase in heparan sulphate synthesis of hepatocytes. Heparan sulphate was the only type of glycosaminoglycan synthesized under all conditions. The results are discussed in view of the pathogenetic potential of heparan sulphate for liver cell necrosis.

Influence of thioacetamide-provoked liver injury on female rat blood and alveolar bone under stress.

Experimental liver injury with different stages was induced to adult female test rats with daily injection of thioacetamide (ThAA). The doses administered intraperitoneally were 50 mg/kg body weight. In the liver sections progressive changes of damage, regeneration and fat substitution were noticed. Kidney sections revealed enhanced glomerular atrophy, particularly in the cortical tubules, provoked in the 3-week traumatization period. The influence of ThAA on female rat blood was assayed using standard biochemical methods. The analyses done were: the percentage of blood obtainable and the serum/blood ratio; the serum alanine transferase; serum alkaline phosphatase; serum creatinine; serum hydroxyproline and serum beta-glucuronidase activity in the acute, subacute, chronic and highly chronic stage of liver injury. The biochemical findings show continuously progressing damages when traumatization proceeds. In the 3-week test period the histological findings processed showed an increase in osteoclastic resorption in the alveolar bone around the occlusally stressed tooth simultaneously with a horizontal bone loss. Some indications of recovering incidents were seen, too. Only in the histological findings was no difference seen in the deterioration between both sexes, contrarily to the biochemical results also discussed in this study.

Alizarin Red S-Stained Bone and Cartilage in Calcium Deficiency Provoked by Experimental Liver Injury in Rats

Experimental liver injury with different stages was provoked in rats with daily injected doses of thioacetamide (ThAA). The dose recommended for both male and female rats was 50 mg/kg body weight. The liver damages caused were acute, subacute, cirrhotic and necrotic, with a traumatization period of 2, 7, 14 and 21 days. The loss of body weight under traumatization, indicating osteopenia, was in the case of female rats during the first experimental week markedly accelerated, and in the two subsequent weeks apparently inhibited when compared to male rats. The loss of body weight of male rats revealed a progressive fall. Vital staining was made giving intraperitoneally 200 mg/kg body weight of alizarin red S (ARS). The staining intensity was improved in the acute stage for both calvaria and tibia and in the necrotic stage for tibia only. It was impaired in the subacute stage for calvaria and tibia and in the necrotic stage for calvaria only. Prolonged traumatization with ThAA causes pathological defects in the liver and kidneys. Furthermore, the epiphyseal cartilage of necrotic-stage rats was bright red without any ARS staining.

免疫学的機序による肝障害モデル

An experimental liver injury was produced in mice by injection of a sub-hepatotosic dose of anti-basic liver protein (BLP) antibody after immunization of rabbit IgG (RGG) . Strain DBA/2 mice indicated the highest susceptability to the disease. Typical histopathological changes in the liver were submassive hepatocellular necrosis and infiltration of granulocytes and lymphocytes into the portal tract and sinusoid in necrosis lesion. Administration of either prednisolone (10 and 20mg/kg), cyclophosphamide (5 and 10mg/kg) or cianidanol (500 and 1000mg/kg) for 10 days prior to injection of anti-BLP antibody suppressed development of liver injury. These results suggest that the experimental liver injury model in DBA/2 mice is useful for immunopharmacological studies of liver diseases.

Effects of gomisin A, a lignan component of Schizandra fruits, on experimental liver injuries and liver microsomal drug-metabolizing enzymes

Effects of oral administration of gomisin A, one of the components isolated from Schizandra fruits, on liver injuries induced by CCl4, d-galactosamine and dl-ethionine and on liver microsomal drug-metabolizing enzyme activities were investigated. Gomisin A suppressed the increase of serum transaminase activities and the appearances of histological changes such as degeneration and necrosis of hepatocyte, inflammatory cell infiltration and fatty deposition in each type of liver injury. The repeated administration of gomisin A (30 or 100 mg/kg, p.o., daily for 4 days) induced an apparent increase of liver weight in liver-injured and normal rats. Gomisin A decreased serum triglyceride and lipid contents of the liver in biochemical studies. Increases of microsomal cytochrome b5 and P-450, elevations of NADPH cytochrome C reductase, aminopyrine N-demethylase and 7-ethoxycoumarin O-deethylase activities and decrease of 3,4-benzo(a)pyrene hydroxylase activity per cytochrome P-450 were observed after the administration of gomisin A. In addition, gomisin A was found to enhance the incorporation of 14C-phenylalanine into liver protein and to shorten the hexobarbital-induced sleeping time. These changes caused by gomisin A were similar to those by phenobarbital. However, gomisin A is distinctly different from phenobarbital in the finding that phenobarbital lessened the survival ratio of CCl4-intoxicated mice, but gomisin A did not. Our observation suggest that gomisin A shows an antihepatotoxic action by oral application and also has hypolipidemic (mainly triglyceridemic) and liver protein synthesis-facilitating actions and that the enlargement of the liver seen with gomisin A is the adaptive hypertrophy which is due to the induction of drug-metabolizing enzymes.

Effects of Gomisin A on Liver Functions in Hepatotoxic Chemicals-Treated Rats

The effects of gomisin A, which is a lignan component of schizandra fruits, on liver functions in various experimental liver injuries and on bile secretion in CCl4-induced liver injury were studied. Gomisin A weakly accelerated the disappearance of plasma ICG by itself at a high dose (100 mg/kg, i.p.). All of the hepatotoxic chemicals used in this study inhibited the excretion of ICG from plasma. Gomisin A showed a tendency to prevent the delays of the disappearance of plasma ICG induced by CCl4, d-galactosamine and orotic acid, but not that by ANIT. Bile flow and biliary outputs of total bile acids and electrolytes (Na+, K+, Cl− and HCO3−) were decreased in CCl4-treated rats. Gomisin A maintained bile flow and biliary output of each electrolyte nearly to the level of the vehicle-treated group, but did not affect biliary output of total bile acids. These findings suggest that gomisin A possesses a liver function-facilitating property in normal and liver injured rats and that its preventive action on CCl4-induced cholestasis is due to maintaining the function of the bile acids-independent fraction.

Bioluminescence assays for bile acids in the detection and follow-up of experimental liver injury.

We evaluated the usefulness of recently developed bioluminescence assays for serum bile acids (BA) in the detection and follow-up of experimental liver injury. Liver damage was induced in rats by either D-galactosamine or CCl4, and BA were compared to SGPT and aminopyrine breath test (ABT). In severe liver injury, following D-galactosamine administration, all three methods revealed a significant difference from control values. The degree of abnormality was, however, far greater with SGPT and BA than with ABT. In moderate liver injury, induced by CCl4, the increase in BA was not significant. Values of SGPT and BA showed a very good correlation (3 alpha-OH: r = 0.88; 7 alpha-OH: r = 0.90; 12 alpha-OH: r = 0.83; p less than 0.001 for all correlations). Application of different assays for 3 alpha-OH, 7 alpha-OH and 12 alpha-OH BA allowed us to assess changes in individual BA. A 96-hr follow-up study in D-galactosamine-treated animals showed an increase in BA up to 48 hr and a decrease thereafter. The bioluminescence assays for BA are simple, rapid and require only 10 microliter of serum. Thus, these assays may be the method of choice in detecting and monitoring liver injury in small laboratory animals.

Influence of experimental liver injury on rat blood and alveolar bone under stress.

Experimental liver injury was induced to test rats with a daily injection of thioacetamide (ThAA). The doses used for intraperitoneal administrations were 50 mg/kg body weight. The loss of body weight in the 3-week test period was 15%. In the liver there was seen progressive changes which displayed cell necrosis and regeneration. The influence of ThAA on rat blood and serum was checked using standard biochemical assays consisting of the percentage of blood obtainable and the serum/blood ratio and analysis of serum alanine transferase, serum alkaline phosphatase, serum creatinine, and hydroxyproline in the acute, subacute, chronic, and necrotic stage of liver injury. With ThAA injections, the stimulated rate of glycosaminoglycan synthesis had its association to the serum calcium content. It decreased continuously as function of traumatization time. In the 3-week test period, histological findings show in the alveolar bone, around the teeth, when under occlusal stress and ThAA traumatization, the distinct decrease in osteoblastic activity and less osteoids indicating thus the decreased formation of new bone. Conspicuous osteoclastic resorption was also seen in the same area.

P-439) - Protective effects of Y-8845 against experimental liver injury

P-439) - Protective effects of Y-8845 against experimental liver injury

Glutathione metabolism and glucose 6-phosphate dehydrogenase activity in experimental liver injury.

Increased activities of liver glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGD, EC 1.1.1.44) in the pentose phosphate cycle were accompanied with a depletion of reduced glutathione (GSH) following an intragastric administration of carbon tetrachloride (CCl4) to rats. Oxidized glutathione (GSSG) also decreased remarkably, keeping the GSSG: GSH ratio constant. No significant alteration of glutathione reductase (EC 1.6.4.2.), glutathione peroxidase (EC 1.11.1.9) and malic enzyme (EC 1.1.1.40) activities in the supernatant and gamma-glutamyl transpeptidase (gamma-GTP, EC 2.3.2.2) activity in the homogenate of the injured liver were observed. Furthermore, no marked difference in the GSH-synthesizing activity was found between control and CCl4-intoxicated liver. An intraperitoneal injection of GSH produced a significant increase in liver GSH content in control rats but not in CCl4-treated rats; G6PD activity was not affected. Intraperitoneal injections of diethylmaleate resulted in continuously diminished levels of liver GSH without any alteration of liver G6PD activity. In vitro disappearance of GSH added to the liver homogenate from CCl4-treated rats occurred enzymatically and could not be prevented by the addition of a NADPH-generating system. The results suggest that increased G6PD activity in CCl4-injured liver does not play an important role in the maintenance of glutathione in the reduced form and that the decreased GSH content in the injured liver might be caused by enhanced GSH catabolism not due to gamma-GTP.

Beneficial effect of somatostatin on galactosamine induced liver injury

The purpose of our investigation was to study the effect of somatostatin on acute experimental liver injury induced in rats by galactosamine (1.2 g/100 g body wt.). Somatostatin (125 μg/100 g body wt.) was administered subcutaneously in a protamine sulphate/ZnCl 2 suspension either 2 h prior to the injection of galactosamine or 2 h and again 12 h following the injection. Serum transaminases (GOT, GPT) and serum concentrations of triiodothyronine and thyroxine were determined 28 h after the injection of galactosamine. Histology of the liver was performed by light microscopy. Our results showed that the administration of somatostatin significantly ( P < 0.02) reduced the elevation of GOT and GPT activity and diminished the degree of necrosis, and that although the administration of dibutyryl-cAMP (5 mg/100 g body wt.) intensified galactosamine induced liver injury, this effect of dibutyryl-cAMP could be completely prevented by somatostatin treatment. There was no difference in the serum concentrations of triiodothyronine and thyroxine in controls as compared to galactosamine and galactosamine plus somatostatin treated rats. At present the mechanism of this cytoprotection by somatostatin is unknown.

Protective effects of Y-8845 against experimental liver injury

The effects of a novel hepatoprotective compound, 8-(2-di-methylaminoethyl)-3-oxo-4-pheny1-1-thia-4,8-diazaspiro[4,5] decane dihydrochloride (Y-8845), on various kinds of experimental liver injury were investigated. CCl4 administered to rats (0.67 ml/kg i.p.) produced a marked increase in serum GOT and GPT activities, with a maximal response 24 hr after administration. Pretreatment with Y-8845 dose-dependently reduced these increases in enzyme activities and improved disorders in ICG excretion and liver weight. This protective action against CCl4 hepatotoxicity was confirmed by histopathologic evaluation: the reduction of CCl4-induced centrilobular necrosis was marked,and areas with a normal architectural pattern were more extensive. The hepatoprotective effects of Y-8845 were also found in other models of liver injury, ie., injury induced by thioacetamide, galactosamine, ct-naphtylisothiocyanate and endotoxin. Furthermore, anti-fibrotic action was observed in egg yolk-induced chronic hepatic fibrosis. These findings suggest that Y-8845 has potential as a hepatoprotective agent.

Lysyl Oxidase and Collagenase in Experimental Acute and Chronic Liver Injury

Lysyl oxidase and collagenase activities were measured in experimental acute and chronic liver injury in mice and rats, and correlated with collagen synthesis and accumulation. Acute liver injury was induced in mice and rats by a single dose of carbon tetrachloride given by gavage, and also in mice by a single injection of murine hepatitis virus. Chronic liver injury was induced in rats by repeated injections of carbon tetrachloride. Elevated plasma glutamic oxaloacetic transaminase levels, increased hepatic prolyl hydroxylase activity, and increased synthesis of collagen-bound hepatic hydroxyproline occurred in animals with acute as well as with chronic liver injury. However, only chronic liver injury appeared to be associated with fibrosis, increased collagen-bound hydroxyproline content, increased hepatic lysyl oxidase and collagenase activities, as well as with increased serum lysyl oxidase activity. These data suggest that lysyl oxidase and collagenase may play an important role in the collagen accumulation associated with hepatic fibrosis.

Endotoxin-induced hyperinsulinemia and hyperglucagonemia after experimental liver injury.

Basal portal and systemic venous hyperinsulinemia and hyperglucagonemia were present in fasted rats at 24 h after experimental liver injury by partial (67%) hepatectomy, carbon tetrachloride gavage, and intravenous D-galactosamine administration. Both enhanced pancreatic hormone secretion and depressed hepatic hormone extraction were likely responsible for the insulin and glucagon oversupply. Endogenous gut-derived endotoxin is proposed as the causative factor for the exaggerated hormonal response because intravenous exogenous endotoxin elicited an identical elevation of insulin and glucagon. Systemic endotoxemia at 24 h after liver injury was indicated by marked (78-100%) lethality in lead-sensitized rats and positive Limulus lysate gelation tests of plasma samples. Furthermore, antiendotoxin treatments, including endotoxin tolerance, polymyxin B, and gut sterilization, significantly reduced both lead-sensitized lethality and hyperinsulinemic and hyperglucagonemic responses at 24 h in most liver-injury groups. Portal versus systemic venous administration of endotoxin at a low dose implied that normal endotoxin phagocytosis by the liver suppressed the pancreatic endocrine response. A physiological negative-feedback control system involving gut-derived systemic endotoxemia after liver damage with insulin and glucagon hypersecretion by the pancreas for stimulation of hepatic regeneration is hypothesized.

The protection of coenzyme Q10 against carbon tetrachloride hepatotoxicity

It has been suggested that lipid peroxidation is an important factor in the pathogenesis of carbon tetrachloride (CCl4) hepatotoxicity. In the present study, experimental liver injury induced by CCl4 could be inhibited by Coenzyme Q10 (CoQ10) and in spite of exposure to CCl4 the liver tissue levels of thiobarbituric acid (TBA) reacting substances were not increased in rats pretreated with CoQ10. In the in vitro experiment as well, the apparent liver tissue levels of TBA were decreased after addition of CoQ10. These facts provided evidences that CoQ10 possessed a direct antioxidative effect and protected against CCl4 hepatotoxicity by this antioxidative effect.

Preservation of glucuronidation in carbon tetrachloride-induced acute liver injury in the rat

The influence of carbon tetrachloride on several routes of drug metabolism has been investigated in the rat in vivo, in isolated perfused livers, and in hepatic microsomal preparations to evaluate the hypothesis that hepatic glucuronyl transferase activity is preserved when mixed-function oxidase activity is impaired by this hepatotoxin. Impaired oxidative metabolism after acute administration of carbon tetrachloride was confirmed in vivo by a 65 per cent reduction in the rate of elimination of 14CO 2 in breath after [ 14C]aminopyrine administration and a 58 per cent reduction in the clearance of lorazepam in the whole animal. In the isolated perfused livers of control rats, glucuronide metabolism of lorazepam accounted for 26 per cent of its overall elimination; thus, by inference, oxidative metabolism accounted for the remainder. Carbon tetrachloride pretreatment resulted in a 63 per cent reduction in total lorazepam clearance. In microsomal preparations, cytochrome P-450 concentration, cytochrome P-450 reductase activity, and activities of o-demethylation of p-nitroanisole and hydroxylation of aniline were reduced by 63, 32, 85 and 95 per cent, respectively, after exposure to carbon tetrachloride. In contrast, in the carbon tetrachloride-damage isolated perfused liver the proportion of lorazepam conjugated to lorazepam glucuronide increased from 26 to 43 per cent of the dose administered. Furthermore, in vitro microsomal glucuronidation of o-aminophenol was not decreased by carbon tetrachloride pretreatment, while glucuronidation of p-nitrophenol actually increased significantly by 49 per cent. Solubilization of microsomes with Triton X-100 resulted in a 10.4-fold increase in glucuronyl transferase activity in control microsomes, but in only a 5.7-fold increase in microsomes from carbon tetrachloride-treated rats. This suggests that carbon tetrachloride enhanced basal glucuronidating activity but decreased the total amount of enzymes present. These overall results suggest that microsomal glucuronidation activity is spared in an experimental liver injury in which oxidative metabolism is impaired.

Effect of pyridoxine deficiency on serum and liver transaminases in experimental liver injury in the rat

We fed rats either a pyridoxine-deficient or normal rat chow diet for 8 wk and then determined transaminase activity in serum and liver with and without exposure to a known hepatotoxin, bromobenzene. Preincubation of serum and liver cytosol with pyridoxal phosphate in vitro resulted in a significantly greater increment of glutamic oxalacetic transaminase activity in the pyridoxine-deficient animals compared with controls. However, this effect was significantly reduced during liver necrosis. Total activities for both glutamic oxalacetic transaminase and glutamic pyruvic transaminase measured in the presence of excess pyridoxal phosphate were decreased significantly in serum and liver of the pyridoxine-deficient rats compared with controls. The magnitude of the fall in glutamic pyruvic transaminase in both serum and liver cytosol was significantly greater than that for glutamic oxalacetic transaminase. In the presence of bromobenzene-induced hepatocellular necrosis, there was a striking and nearly identical increase in serum glutamic oxalacetic transaminase in the pyridoxine-deficient rats and controls. In contrast, the concurrent increase in serum glutamic pyruvic transaminase was significantly less in the pyridoxinedeficient rats compared with controls. Therefore, the serum glutamic oxalacetic transaminase/glutamic pyruvic transaminase ratio was significantly greater in the pyridoxine-deficient vs. control rats. There was a parallel increased glutamic oxalacetic transaminase/ glutamic pyruvic transaminase ratio in liver cytosol of the pyridoxine-deficient compared with control animals, suggesting that the serum ratio in response to chemical hepatotoxicity reflects release of these enzymes from damaged hepatocytes in their existing proportions in liver.

Alcoholic liver disease. Absence of alpha-fetoprotein elevations in the recovery phase.

Serum alpha-fetoprotein (AFP) may be detected in patients with nonneoplastic liver diseases such as massive hepatic necrosis, viral hepatitis, and experimental liver injury, AFP levels have not been serially assessed in patients with alcoholic liver disease, with or without cirrhosis, during the period following cessation of alcohol. Thirty-two such patients were studied with weekly AFP determinations, an average of five such measurements being obtained per patient. The severity of alcoholic liver disease in this group varied from mild alcoholic hepatitis to advanced cirrhosis, and overall mortality was 31%. Thirty-one of the 32 patients had consistently negative AFP determinations. One patient with persistently elevated AFP levels proved to have hepatoma at autopsy. This study failed to detect AFP evelations in a group of patients with alcoholic liver disease and suggests that positive AFP determinations in such patients shoudl raise the question of underlying hepatocellular carcinoma.

Serum bile acid concentration in some experimental liver lesions of rat

The usefulness of measuring serum bile acid concentrations by RIA in a number of acute experimental liver injuries of rats was assessed by comparing the concentrations with the results of some of the routinely employed methods of examining hepatotoxic changes. Centribular liver cell injury produced by CCl 4 revealed leakage of GPT and GDH and to a lesser extent AP; along with minimal increase in serum bile acid levels. Serum bilirubin concentration remained unchanged. Surgical bile duct ligation resulted in marked rises in AP, GPT and GDH and total bilirubin levels and levels of serum bile acids. Intravenous injection of MnSO 4 induced focal necrosis of liver and bile canalicular dilation associated with elevated GDH and GPT concentrations. AP and bilirubin levels were unchanged. Bile acid levels were raised among female rats. 2,4-Xylidine induced hepatotoxicity revealed bile duct hyperplasia, liver cell enlargement, liver cell necrosis, biliary canalicular dilation and proliferation of endoplasmic reticulum. GDH and GPT levels were raised along with bile acid concentrations. This study suggested that assay of bile acid concentration is a sensitive indicator of several acute hepatic injuries.

Estrogens as disposing factors in experimental liver injury

The activity of hexobarbital metabolising enzymes of the liver is decreased as a consequence of both acute and chronical carbon tetrachloride damage. Simultaneous administration of estrogens was followed by more severe morphological and functional impairment although estrogens are not burdening the healthy liver. The beta receptor blocking propranolol prevented functional lesion, moderated fatty degeneration and liver cell necrosis in chronic carbon tetrachloride injury. A simultaneous estrogen administration decreased the therapeutic efficacy of beta blockade, however functional and morphological protective effects were not completely abolished. Estrogen medication should be rather avoided in hepatic damage.