Involvement of Hepatic Stimulator Substance in Experimentally Induced Fibrosis and Cirrhosis in the Rat

Liver fibrosis results from sustained wound healing response to chronic liver injury. Liver cirrhosis, the end stage of the fibrotic process, is characterized by disruption of the entire liver architecture and reduced hepatocyte regenerative ability. Hepatic stimulator substance (HSS) is a liver-specific growth factor triggering hepatocyte proliferation in vitro and in vivo. Previous studies have indicated the involvement of HSS in animal models of acute liver injury. The aim of the present study was to investigate the involvement of HSS in the process of fibrosis and cirrhosis induction. Liver fibrosis and cirrhosis were induced in rats by thioacetamide (TAA) administration (300 mg/l) in the drinking water for 3 months, and animals were killed at 0, 1, 2, and 3 months of treatment. TAA administration resulted in progressively increasing liver fibrosis, leading to the onset of cirrhosis at the end of the experimental time. HSS was continuously produced during the course of fibrosis and cirrhosis induction, peaking at the 2nd month of TAA treatment, coinciding with markers of hepatic proliferative capacity, as thymidine kinase activity and DNA biosynthesis. Significantly reduced HSS activity was noted in cirrhotic liver (3rd month). In this case, the exogenous HSS administration during the 3rd month of TAA treatment suppressed the onset of liver cirrhosis, stimulating the hepatic regenerative capacity. Our data indicate the active participation of HSS in the process of fibrosis and cirrhosis induction post-TAA treatment in rats, suggesting also the beneficial effect of HSS treatment against cirrhosis induction with future possible clinical implications.     

Oxidative stress in the development of liver cirrhosis: a comparison of two different experimental models

BACKGROUND/AIMS: Oxidative stress has been implicated in liver cirrhosis. Carbon tetrachloride and thioacetamide are the most widely used models to develop cirrhosis in rats and the present study compares oxidative stress in the liver induced by these compounds at different stages of cirrhosis development. METHODS: Twice-weekly intragastric or intraperitoneal administration of carbon tetrachloride or thioacetamide, respectively, produced liver cirrhosis after 3 months. Histology, serum markers and hepatic hydroxy proline content confirmed the cirrhosis. RESULTS: An increase in oxidative stress parameters was seen in mitochondria, peroxisomes and microsomes from the liver after carbon tetrachloride or thioacetamide treatment. Oxidative stress was more severe in carbon tetrachloride treated animals than thioacetamide. Mild oxidative stress was evident at 1 and 2 months of treatment and a significant increase was seen by 3 months of treatment with either compound. By this time, frank liver cirrhosis was also observed. CONCLUSIONS: These results suggest that evidence of oxygen free radicals is also found early in the development of fibrosis and cirrhosis in both models.     

Inhibition of experimentally-induced liver cirrhosis in rats by a nonpeptidic mimetic of the extracellualr matrix-associated Arg-Gly-Asp epitope

Aims/Methods: in the present study, we examined whether a nonpeptidic mimetic of Arg-Gly-Asp (RGD), which specifically inhibits RGD-dependent adhesion of CD4⁺ T lymphocytes or fibroblasts to fibronectin, can prevent thioacetamide-induced liver cirrhosis in rats. The nonpeptidic RGD mimetic, rather than the RGD peptide was utilized, since the peptide is rapidly degraded, and therefore, relatively ineffective for in vivo use.Results: We now report that rats treated with thioacetamide and the RGD analogue SF-6,5 for 12 weeks had lower histopathologic scores than those treated with thioacetamide alone. Further improvement in liver histology was observed after another 8 weeks of treatment with the analogue SF-6,5. Quantitative microscopic analysis by computerized imaging morphometry of liver biopsies from the three groups and controls confirmed the semi-quantitative histopathologic scores (pτ0.001). After 3 months of treatment, the spleen weights in the SF-6,5-treated rats were 30% less than those of rats and that received only thioacetamide, which indicated that the analogue-treated rats were less portal hypertensive.Conclusions: The observed inhibition of the progression of cirrhosis in rats by the nonpeptidic RGD analogue suggests that RGD mimetics may be useful therapeutically in inhibiting pathological processes that involve RGD recognition.     

Retinoid metabolism in the small intestine during development of liver cirrhosis

Background and Aims:  Retinoids are important mediators of cellular differentiation and proliferation in various epithelia of the body including the small intestine. Though alterations in intestinal epithelial cell proliferation have been noted in liver cirrhosis, mechanisms involved in the process are not well understood. This study examined the levels of various retinoids and retinoid-metabolizing enzymes in the small intestine during development of liver cirrhosis.
Methods:  Four groups of animals were used (control, phenobarbitone control, thioacetamide and carbon tetrachloride treatment). Twice-weekly intragastric or i.p. administration of carbon tetrachloride or thioacetamide, respectively, produced liver cirrhosis after 3 months, which was confirmed through histology and serum markers. Retinoid levels were measured by high-performance liquid chromatography.
Results:  A decrease in the levels of retinal, retinoic acid and retinol was evident in the intestine by 3 months, when cirrhosis was evident histologically, and these remained low until 6 months. A decrease in the activities of retinaldehyde oxidase, retinaldehyde reductase and retinol dehydrogenase was also seen in intestine from cirrhotic rats.
Conclusion:  These results suggest that altered retinoid metabolism in the intestine of cirrhotic rats might have an influence on changes in intestinal epithelial cell differentiation, seen in liver cirrhosis.     

Hepatic 31P MRS in rat models of chronic liver disease: assessing the extent and progression of disease

BACKGROUND: Hepatic adenosine triphosphate (ATP) levels are an accurate reflection of functioning hepatic mass following surgical resections and acute liver injury. OBJECTIVE: To determine whether hepatic ATP levels can serve as a non-invasive means of documenting progression of chronic liver disease to cirrhosis. METHODS: In vivo phosphorus-31 magnetic resonance spectroscopy ((31)P MRS) was performed in three animal models of chronic liver disease. Sixty six adult Sprague- Dawley rats were subjected to either thioacetamide, carbon tetrachloride (CCl(4)), or common bile duct ligation (CBDL) to induce liver disease (n=35, 21, and 10, respectively). Serial MRS examinations, blood samples, and liver biopsies (when appropriate) were obtained throughout and/or on completion of the study. RESULTS: Over the course of the chronic liver disease, a progressive decrease in hepatic ATP levels was consistently observed in each model. The findings were most striking when end stage liver disease (cirrhosis) was established. The reduction in hepatic ATP levels correlated with significant changes in serum albumin concentrations (CCl(4) and CBDL models) and the extent of hepatocyte loss seen histologically (all models). CONCLUSION: The results of this study indicate that during progression of chronic liver disease to cirrhosis, there is a progressive reduction in hepatic ATP levels. In addition, changes in hepatic ATP levels correlate with changes in liver function and histology. Thus hepatic (31)P MRS provides a non-invasive means of documenting the severity and progression of parenchymal and cholestatic models of chronic liver disease in rats.     

DNA repair systems in early and persistent hepatocyte nodules in the rat

Summary The repair of three DNA lesions, namelyO ⁶-methylguanine, 7-methylguanine, and 3-methyladenine, was investigated within early and persistent hepatocyte nodules generated in Fischer 344 rats by a modified Solt-Farber procedure (diethylnitrosamine initiation followed by a 2-acetylaminofluorene/CCl₄ cycle). TheO ⁶-methylguanine-DNA methyltransferase concentration within both hepatocyte nodule types was always higher than that found in age-matched controls (normal, initiated-only and promoted-only livers). As far as 3-methyladenine and 7-methylguanine-DNA glycosylases are concerned, the early hepatocyte nodules showed far higher activities for both enzymes than were found in the controls, whereas in the persistent ones they underwent a significant decrease. In conclusion hepatocyte nodules are endowed with a high DNA repair activity, which is partly adaptive, partly constitutive; along with others, such a defence mechanism could allow transformed cells to resist many cytotoxic drugs.     

Inactivation of cholecystokinin octapeptide by normal and cirrhotic liver in rats

Summary In anesthetized rats, a marked decrease in CCK-OP activity and, to a far lesser extent, in the pancreatic secretory effect of CCK-33 were found after portal administration, compared to the femoral route. Changes in the biological activity of CCK-OP were further investigated after 30 min incubation with different subcellular liver fractions (1000×g, 12 000 ×g, microsomal fraction with or without NADPH). All the subcellular liver fractions caused an approximately 70% decrease in the CCK-effect, as calculated from dose-response relationships. The inactivation of CCK-OP after incubation with microsomal fractions of thioacetamide (TAA)-induced cirrhotic liver did not differ from that of control rats. The CCK-OP doseresponse curves were similar in cirrhotic and control rats, but the pancreatic secretion was sustained to a greater extent and the inhibitory effect of supramaximal stimulation was delayed in cirrhotic rats. It was concluded that CCK-OP can be inactivated by liver proteins present in microsomal fractions, by a NADPH-independent mechanism. This inactivation did not diminish in liver cirrhosis. There were no changes in CCK-OP elimination in cirrhotic rats in vivo, thus pancreatic hypertrophy in experimental cirrhosis must be explained by other mechanisms. The results in this paper were presented at the XVII Meeting of the European Pancreatic Club (Manchester, U.K., 22–25 September, 1985)     

Utility of a 13C‐methacetin breath test in evaluating hepatic injury in rats

Background and Aim: Methacetin is thought to be a good substrate for the evaluation of different cytochrome P450 enzymatic systems of liver microsomes because of its rapid metabolism and lack of toxicity in small doses. Recent studies indicate that a methacetin breath test may be a non‐invasive alternative for the evaluation of liver function since it correlates well with the severity of liver damage. It may also discriminate between different stages of liver cirrhosis and correlates with the Child–Pugh score. The application of this test in experimental liver damage in animal models has not yet been examined. This study aimed to evaluate the efficacy of the ¹³C‐methacetin breath test in assessing the extent of hepatic injury in models of acute liver failure, liver cirrhosis, and fatty liver in rats. Methods: Absorption of methacetin given per os or intraperitoneally in normal rats was evaluated. The association between liver mass and ¹³C‐methacetin breath test results was assessed in a 70% hepatectomy rat model. Fulminant hepatic failure was induced by three consecutive intraperitoneal injections of thioacetamide, 300 mg/kg, at 24 h intervals. For induction of liver cirrhosis, rats were given intraperitoneal injections of thioacetamide, 200 mg/kg, twice a week for 12 weeks. A methionine‐choline deficient diet was used for the induction of fatty liver. Rats were analyzed for ¹³C‐methacetin by BreathID (MBID) using molecular correlation spectrometry. BreathID continuously sampled the animal's breath for 60 min and displayed the results on the BreathID screen in real‐time. Results: Methacetin was absorbed well irrespective of the administration method in normal rats. Liver mass was associated with peak amplitude, complete percent dose recovery (CPDR) at 30 and 60 min and MBID peak time. A high degree of association was also demonstrated with MBID results in acute hepatitis (peak amplitude, 19.6 ± 3.4 vs 6.3 ± 1.63.4; CPDR30, 6.0 ± 3.3 vs 1.2 ± 0.5; CPDR60, 13.3 ± 4.5 vs 3.2 ± 1.4; and peak time, 31.0 ± 14.9 vs 46.9 ± 10.8 min) and liver cirrhosis (peak amplitude, 24.4 ± 2.3 vs 15.6 ± 6.4; CPDR30, 7.9 ± 1.2 vs 2.7 ± 1.0; CPDR60, 17.8 ± 2.6 vs 8.8 ± 2.1; and peak time, 30.2 ± 1.5 vs 59.6 ± 14.5 min), but not with grade of liver steatosis. Conclusions: Methacetin is well absorbed and exclusively metabolized in the liver. MBID is a sensitive test and may be a useful tool for the evaluation of functional liver mass in animal models of acute liver failure and cirrhosis. However, MBID could not distinguish between fatty liver and normal liver in rats.     

Influence of administration of long-chain polyunsaturated fatty acids on process of histological recovery in liver cirrhosis produced by oral intake of thioacetamide

Patients with liver cirrhosis frequently show some degree of protein-energy malnutrition and obviously require nutritional support. In this study, the treatment of rats consisted of thead libitum oral intake of a 300 mg/liter thioacetamide solution, used as drinking water for four months. Thioacetamide treatment produced a severe alteration in the plasma fatty acid profile with significant decreases of these, which mimicked changes described in human cirrhosis. This hepatotoxic agent causes nodular cirrhosis, with loss of the normal architecture of the liver and disruption of the vascular pattern. The goal of the study was to evaluate the influence of n-3 and n-6 series long-chain polyunsaturated fatty acid dietary supplementation in experimental animals and to assess the effects of those dietary components on structural recovery in the liver. Significant increases of saturated and monounsaturated fatty acids as well as n-6 polyunsaturated fatty acids were seen only in the animals given the n-6 polyunsaturated fatty acid supplemented diet. However, only rats given the standard diet exhibited some degree of histological regeneration.     

Steatosis and Collagen Content in Experimental Liver Cirrhosis Are Affected by Dietary Monounsaturated and Polyunsaturated Fatty Acids

Background and Methods: We used thioacetamide administered orally to induce cirrhosis in rats, and after these had recovered for 1 and 2 weeks we examined the effects of dietary supplementation with monounsaturated and n-3 polyunsaturated fatty acids, or with a combination of n-3 and n-6 polyunsaturated fatty acids, on the extent of steatosis and collagen content in the liver. Results: Nodular cirrhosis, increased collagen content, and lipid accumulation were established after 4 months of treatment with thioacetamide. When the animals were fed a diet rich in oleic acid for 2 weeks, the steatosis and fibrosis decreased. Supplementation with n-3 polyunsaturated fatty acids favored reductions in collagen content but did not reduce the fat accumulation. With a diet supplemented with a mixture of n-3 and n-6 fatty acids we found no reduction in either lipid accumulation or collagen content. Conclusions: Fibrosis and steatosis may be influenced by dietary fat, and monounsaturated fat appears to influence favorably the histologic recovery of the damaged liver.     

Effect of Dietary Nucleotides on Degree of Fibrosis and Steatosis Induced by Oral Intake of Thioacetamide

The administration of thioacetamide in ratsinduces nodular cirrhosis of the liver, characterized byfibrous septae, parenchymal nodules, proliferation ofthe bile ducts, and excessive deposition of connective tissue elements. Nodular cirrhosis is alsoassociated with changes in lipid metabolism, as shown bythe accumulation of lipid droplets in the hepatocytecytoplasm. Adequate nutritional support during cirrhosis is important to sustain liver function andpromote recovery after the lesions have been induced.Supplementation with nucleotides may increase cellularproliferation and thus optimize hepatic recovery. The aim of this study was to investigate theeffects of dietary nucleotide supplementation on thedegree of fibrosis and steatosis in rats with livercirrhosis induced by four months of oral intake ofthioacetamide. The use of dietary nucleotides afterthioacetamide administration was found to decrease thepercentage area of fibrous septae. In animals with livercirrhosis fed the nucleotidesupplemented diet for two weeks, the total area of fibrosis was reduced.Withdrawal of the hepatotoxic agent led to a decrease inthe degree of steatosis in cirrhotic animals, which wassignificant in rats given the nucleotide-supplemented diet during a two-week recovery period. Inconclusion, dietary nucleotides may be an importantfactor in the histological recovery of damaged liver inexperimental cirrhosis.     

Reproducible production of thioacetamide-induced macronodular cirrhosis in the rat with no mortality

BACKGROUND/AIMS: Hepatotoxin-induced rat models of liver cirrhosis are limited by the wide heterogeneity of cirrhosis produced. The present study developed a modified, reliable, and reproducible technique by which hepatic and systemic responses to thioacetamide during induction of cirrhosis were monitored by weekly weight changes. METHODS: Male Wistar rats (200-230 g) were divided into three groups. Group 1 (n=20) received continuous administration of 0.03% (w/v) thioacetamide in the drinking water for 12 weeks. Group 2 (n=20) received the same concentration of 0.03% thioacetamide as an initial concentration that was modified according to weekly weight changes in response to thioacetamide during the induction of cirrhosis. Group 3 (n=6) received normal water and served as controls. RESULTS: Mortality of Group 1 was 30% and the production of cirrhosis was only 45%. In contrast, there were no deaths in Group 2 and well-developed macronodular cirrhosis was found in 90% of the rats which was associated with significant portal hypertension, as indicated by increased portal venous pressure (13.6+/-0.4 vs. 9.1+/-0.3 mmHg, cirrhotic vs. control, respectively, P<0.01, Student's unpaired t-test). CONCLUSIONS: Variations in responses to thioacetamide can be easily monitored by weekly weight changes to reduce mortality to zero and simultaneously increase the production and quality of cirrhosis induced in rats.     

Treatment of thioacetamide-induced liver cirrhosis by the Ras antagonist, farnesylthiosalicylic acid

BACKGROUND/AIMS: Several studies have indicated increased expression of the Ras protooncogenes in liver cirrhosis. In a previous study in rats, we have shown that a synthetic Ras antagonist, S-farnesylthiosalicylic acid (FTS), could inhibit the development of liver cirrhosis. The aim of the current study was to examine whether FTS will accelerate the resolution of liver cirrhosis induced in rats by thioacetamide. METHODS: Cirrhosis was induced in male Wistar rats by intraperitoneal (i.p.) administration of thioacetamide (200 mg/kg twice weekly for 12 weeks). In the treated group, the Ras antagonist FTS (5 mg/kg, i.p./3 times/week) was administered for 8 weeks after liver cirrhosis has already been established. Control cirrhotic rats received PBS injections for 8 weeks. RESULTS: Rats treated with FTS for 8 weeks had lower histopathologic score of fibrosis (P = 0.01), lower hepatic hydroxyproline levels (P = 0.0002) and lower spleen weight (P = 0.02) than the cirrhotic rats treated with PBS. Following FTS treatment, the MMP-2 and MMP-9-induced collagenolytic activity and TIMP-2 expression, were increased in FTS-compared to PBS-treated rats. TUNEL assay of liver sections performed 8 weeks after thioacetamide withdrawal showed increased apoptotic figures in both groups (P = NS). CONCLUSIONS: These results indicate that the Ras antagonist FTS accelerates the regression of experimentally-induced hepatic cirrhosis. The mechanism may involve increased collagenolytic activity.     

Effect of granulocyte-macrophage colony-stimulating factor on hepatic regeneration after 70% hepatectomy in normal and cirrhotic rats

BackgroundPost-hepatectomy liver insufficiency is one of the most serious postoperative problems and its prevention is important after major hepatic resection, especially in the cirrhotic liver. Some growth factors and cytokines appear to play important roles in liver regeneration. In the present study we have investigated the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on hepatic regeneration after 70% partial hepatectomy (PH) in cirrhotic and non-cirrhotic rats.MethodsA rat model of liver cirrhosis was prepared using thioacetamide (TAA) (a dose of 20 mg/100 g body w, intra-peritoneally) on three days a week for 12 weeks. Adult male rats were divided into four groups:Group 1 (n=10) no cirrhosis and no GM-CSF; Group 2 (n=10) no cirrhosis and GM-CSF; Group 3 (n=10) cirrhosis and no GM-CSF; and Group 4 (n=10) cirrhosis and GM-CSF. All the rats underwent a 70% hepatectomy, and GM-CSF was administrated immediately after operation in Groups 2 and 4. On postoperative days 2 and 7, fresh samples from the remnant liver were obtained to evaluate its regenerative capacity.The liver regenerative process was estimated by DNA synthesis, using flow cytometry.ResultsProliferation index (PI) of hepatocytes at 48 h was higher in Group 4 rats than Group 3 rats (p<0.05). On postoperative day 7, PI was elevated in Group 3 rats compared with Group 4 rats, but this difference was not statistically significant. In non-cirrhotic rats given GM-CSF, PI was increased compared with Group 1 rats at day 2 (p<0.05), but not at day 7.ConclusionsThe findings suggest that the proliferative capacity of liver cells is impaired and delayed after 70% PH in cirrhotic rat liver. GM-CSF administration might enhance the liver PI in both normal and TAA-induced cirrhotic rats.     

Expression patterns of cell cycle-related proteins in a rat cirrhotic model induced by CCl4 or thioacetamide

To analyze the aberrant expression of cell cycle-related proteins and their biological significance in relation to cirrhosis, we compared the cirrhotic patterns induced by two different types of cirrhotic agents, CCl₄ and thioacetamide (TAA) in rats. CCl₄ or TAA treatment was given to rats for 8 or 30 weeks, respectively, and the livers were removed at 9, 20, and 30 weeks after the experiment began. The TAA-induced fibrotic pattern was different from the CCl₄-induced one, in terms of the formation of fibrous connective tissue and the proliferation of bile ductule cells. Cholangiofibrosis and clear cell foci were also observed in TAA-treated rats at 30 weeks. Histological examination revealed severe cirrhotic changes at 9 weeks in CCl₄-treated rats and at 30 weeks in TAA-treated rats. Immunoblotting for cyclin D1, E, A, B, and proliferating cell nuclear antigen (PCNA) and their counterpart protein kinases (CDK2, 4, and CDC2) showed significant overexpression in rats with severely cirrhotic livers. The p53 tumor suppressor protein increased dramatically in the CCl₄-treated group, while it was not detected in the livers of TAA-treated rats. Upregulation of p21^WAF1, a CDK inhibitory protein, was detected in TAA-treated rats, but not in CCl₄-treated rats. Immunohistochemical data for cyclin D1, E, and PCNA were well correlated with immunoblotting data; these proteins were increased in hepatocytes surrounding the cirrhotic lesions, suggesting that hepatocyte regeneration is correlated with cell cycle-related protein expression in cirrhotic liver. In the TAA-treated rats, the expression of these proteins was increased both in hepatocytes and in ductule cells. Our data suggest that liver cirrhosis induced by CCl₄ or TAA is associated with alterations in cell cycle-related proteins, and that the expression of these proteins is responsible for hepatocyte regeneration in the damaged liver and may be involved in liver carcinogenesis. Received: May 1, 2000 / Accepted: August 25, 2000     

Influence of dietary nucleotides on liver structural recovery and hepatocyte binuclearity in cirrhosis induced by thioacetamide.

Intake of thioacetamide in drinking water causes liver cirrhosis in rats, which exhibit many changes similar to human disease. Nucleotides play an important part in major cellular functions, and recent studies suggest that dietary nucleotides may be considered 'semi-essential' nutrients in situations when an inadequate dietary supply may affect the growth of tissues with a rapid turnover rate. The aim of this study was to assess the effect of dietary nucleotides on lesions in thioacetamide-cirrhotic rats, and to calculate the proportion of mono and binucleated hepatocytes in different experimental groups. Rats were given cirrhosis by oral intake of thioacetamide in the drinking water (300 mg/l) for four months. One group was treated with a standard nucleotide free diet, and another group was treated with the same diet supplemented with 250 mg of nucleotides per 100 g of diet for one and two weeks. A striking reduction (mean (SEM)) in the proportion of binucleated cells was seen in thioacetamide-cirrhotic rats (4.8 (1.3) v 21.4 (1.0)), showing a change in the mitotic mechanism in focal lesions. Cirrhotic rats that consumed a semipurified diet supplemented with nucleotides during two weeks showed considerable histological regeneration of the injured liver. These animals had significantly higher proportion of binucleated cells than did animals at the beginning of the recovery period (8.2 (1.2) v 4.8 (1.3)). In the second week of recovery, both types of diet (F = 5.54, p < 0.05) and the previous administration of thioacetamide (F = 142.82, p < 0.001) had significant effects on the percentage of binucleated hepatocytes.     

The sympathetic nervous system promotes carbon tetrachloride-induced liver cirrhosis in rats by suppressing apoptosis and enhancing the growth kinetics of regenerating hepatocytes

Norepinephrine is considered to possess potent anti-apoptotic action in regenerating hepatocytes. To clarify the role of the sympathetic nervous system in apoptosis that occurs in chronic liver damage and following the promotion of liver cirrhosis, we studied a carbon tetrachloride (CCl₄)-induced liver injury model, using spontaneously hypertensive rats (SHR), Wistar-Kyoto rats (WKY), and chemically sympathectomized WKY. At 24 h after CCl₄ administration, acute damage, characterized by vacuolated hepatocytes in the centrilobular zone, was greater in SHR than in WKY. This vacuolated change in WKY hepatocytes was significantly reduced by chemical sympathectomy with 6-hydroxydopamine (6-OHDA). After 48 h, the acute damage was dramatically improved in each animal, without significant differences between the three groups. In chronic damage after weekly repetition of CCl₄ treatment for 4 weeks, fibrosis was evident in SHR, while in the other groups there was only scant fibrosis in the centrilobular zone. After 8 weeks' repetition of CCl₄, liver cirrhosis was seen only in SHR. The incidence of apoptotic cells in areas of both acute and chronic damage in WKY, detected by terminal deoxynucleotidyl transferase-dUTP nick end labeling, was significantly increased in com-parison with that in SHR, and was further increased by 6-OHDA pretreatment. In contrast, there was significantly greater enhancement of the growth of hepatocytes in SHR than in WKY in both acute and chronic damage. Moreover, hepatocyte growth kinetics in WKY was significantly inhibited after sympathectomy in acute injury, as evidenced by immunohistochemistry for proliferating cell nuclear antigen (PCNA). In vitro, the amount of hepatocellular apoptosis induced by transforming growth factor-β1 was significantly decreased by incubation with norepinephrine. These findings suggest that the anti-apoptotic effect of the sympathetic nervous system increases cell growth kinetics and promotes liver cirrhosis in this animal model. Received: May 22, 2000 / Accepted: September 8, 2000     

Changes of hepatic fatty acid metabolism produced by chronic thioacetamide administration in rats.

Hepatic mitochondrial functions related to fatty acid metabolism, including the respiratory control ratio, fatty acid oxidative capacity and carnitine palmitoyltransferase I activity, were studied in vitro with mitochondria isolated from rats treated with thioacetamide for up to 12 wk. The levels of ketone bodies, carnitine, carnitine esters and malonyl-coenzyme A were also determined in liver extracts. Polarography of mitochondrial respiration from succinate or glutamate plus malate showed a lower respiratory control ratio in thioacetamide-treated rats, whereas uncoupled oxygen consumption was not altered. This suggests that the mitochondrial respiratory chain capacity remained intact in the thioacetamide-treated rats. The oxygen consumption associated with palmitoyl-coenzyme A and palmitoyl-L-carnitine oxidation by isolated liver mitochondria was increased by thioacetamide treatment on both a per-mitochondrial protein and a per-total liver basis. The carnitine palmitoyl-transferase I activity; the tissue levels of ketone bodies, carnitine and carnitine esters; and the beta-hydroxybutyrate/acetoacetate ratio were all higher in the livers of thioacetamide-treated animals than in control livers, whereas the hepatic malonyl-coenzyme A level was decreased by thioacetamide. These results indicate the increased diversion of cytosolic long-chain acyl-coenzyme As into the mitochondria for beta-oxidation rather than their esterification and use in lipogenesis. These intrahepatic metabolic changes induced by chronic thioacetamide administration may reflect the whole-body catabolic state and can be seen as adaptive for maintaining energy homeostasis under conditions of impaired glucose tolerance.     

Pancreatic trophism in experimental liver cirrhosis

Summary Pancreatic trophism and pancreatic enzyme composition, and plasma levels of cholecystokinin, insulin, glucagon, and glucose in liver cirrhosis induced by chronic thioacetamide administration (0.02% in the drinking water for 12 mo) were studied in rats. Advanced liver cirrhosis was evident in all thioacetamide-treated rats. The weight of the pancreas and its contents of DNA, protein, trypsinogen, chymotrypsinogen, proelastase, secretory trypsin inhibitor, and amylase were significantly increased as compared to the controls. The pancreatic secretory enzyme content changes showed a nonparallelism, characteristic of a cholecystokinin effect. Light and electron microscopy revealed a normal pancreatic architecture. Bioassayed plasma cholecystokinin levels in both fed and 24-h-fasted cirrhotic rats were significantly higher than in the corresponding controls. The plasma glucose, insulin, and glucagon levels demonstrated hypoglycemic tendencies with a glucagon predominance. These findings indicate that advanced liver cirrhosis in the rat is accompanied by pancreatic hypertrophy and hyperplasia, which might be attributed, at least in part, to elevated circulating cholecystokinin levels. Some parts of this work were presented at the 21 st Meeting of the European Pancreatic Club, Glasgow, UK, September 20–23, 1989.     

Differential alterations of cytochrome P450 proteins in livers from patients with severe chronic liver disease

To determine whether cytochrome P450 proteins were differentially altered in severe chronic liver diseases, we examined 50 livers removed at liver transplantation from patients with end-stage cirrhosis, including 18 with and 32 without cholestasis, and compared the results with 21 histologically normal livers. NADPH-cytochrome c reductase activities were unaltered in microsomes from cirrhotic livers. Total cytochrome P450 content was significantly reduced. The catalytic activities of four xenobiotic-metabolizing P450s and the level of the corresponding proteins were differentially altered. Thus, P450 3A-supported testosterone 6β-hydroxylase activity and 3A protein appeared to be reduced, but only in the subgroup without cholestasis was this change significant. In contrast, 2E1 and the related N,N-dimethylnitrosamine N-demethylase activity were clearly reduced in livers from patients with cholestatic forms of cirrhosis but appeared not to be changed in other cirrhotic livers. Similarly, P450 2C protein was reduced only in patients with severe chronic cholestasis. Finally, P450 1A2 and 1A2-supported ethoxyresorufin O-deethylase activity were significantly reduced in hepatic microsomes from patients with both types of advanced liver disease. In summary, these data demonstrate that cytochrome P450 proteins are selectively altered in severe chronic liver disease, some being profoundly decreased, others less so or not at all. Our results also suggest that there may be different patterns of altered hepatic P450 expression according to the presence or absence of cholestasis in patients with cirrhosis severe enough to require transplantation.