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Bilirubin activates a caspase-3-dependent apoptotic signaling cascade
78
I
Poster Sessions
1589
TERLlPRESSlN VS. SOMATOSTATIN IN ACUTE VARICEAL BLEEDING: A PROSPECTIVE, RANDOMIZED TRIAL
D. Chelarescu, 0. Gabor, C. Lunca. University of Medicine and Pharmacy, Romania Aim: We compared the efficacy and safety of terlipressin and somato-
statin in treatment of cirrhotic patients with variceal bleeding. Method: We investigated a number of 59 patients with endoscopyproved oesophageal bleeding, treated with somatostatin (nl = 27) and with terlipressin-nitroglycerin (n2 = 32). Patients were randomly assign to get (double-blind) terlipressin (2 mg i.v. initially and 1 mg at every 4 h, for 24 h) - transdermal nitroglycerin (10 mg/12 h during the first 24 h) or somatostatin (0.05 mg i.v. initially and 0.05 mg/h in iv. infusion in the next 48 h). In both groups, was performed balloon tamponade for 4 hours after diagnostic setting. Patients got also transfusions, electrolyte and fluid corrections and lactulose. We assessed the Child-Pugh class and the number of rebleeding at 48 hours since beginning of the treatment, number of deaths related to bleeding. The success was considered as 24 hours bleeding free period during 48 hours and lack of further bleeding from initial control to 5 days later. Results: Success rate was 66% in terlipressin- transdermal nitroglycerin group and 64% in somatostatin group (without statistically significant difference). The efficacy was similar regarding Child-Pugh class. Mortality rate was 16% in terlipressin group and 14% in somatostatin group. Few side effects were noted in each group.
PO3 Category 3: Molecular and cell biology (gene expression, signalling, fibrosis)
07
MITOGENIC THERAPY DIRECTED AT THE LIVER
Malik Raza, Neil Mellor, Clare Selden, Humphrey Hodgson. Royal Free Hosp., Pond St., London, UK
Aim: a) Assess if thyroid hormone (T3) acts as a primary mitogen on the liver b) Characterise the effects of T3 when administered prior to a 70% partial hepatectomy (PH) Method: a) Rats (n = 7) were injected with a single dose of T3 and sacrificed at intervals of 1, 2, 4, 7, 10, 14 days. b) 70% PH was performed at various time points (1, 3, 10 days) following a single dose of T3. The rats were sacrificed 24 hrs after PH Results: a) Liver mass was increased in rats treated with T3 when compared to controls Maximum effect was seen on day 10 with a 20% increase in liver mass (p < 0.0009) and a corresponding increase in total DNA (p < 0.001) and liver protein (p < 0.001). Cell proliferation peaked when T3 was administered 24 hrs prior to sacrifice: 7% Hepatocytes BRDU labelled compared to < 1% in controls (p < 0.0001) b) In animals treated with T3 at 3 and 10 days prior to PH the liver weight at sacrifice was greater (p < 0.05) than controls with a corresponding increase in liver protein (p < 0.05) and total DNA (p < 0.05). T3 enhanced the proliferative response of PH when administered 24 hrs prior to surgery: 36% hepatocyes BRDU labelled compared to 26% with PH alone (p < 0.001) Conclusion: Thyroid hormone could potentially be used to increase liver mass and enhance the regenerative response of the liver.
053
DIFFERENTIAL ERK112 ACTIVATION IN RAT CHRONIC LIVER INJURY AND FIBROSIS
Francesco Ridolfi, Gianluca Svegliati, Lucia Perego, Luca Marucci, Antonio Di Sario, Antonio Benedetti, Franc0 Folli. Department of Gastroenterology, University of Ancona, Italy The extracellular signal-regulated kinase l/2 (ERK1/2) has been shown
to play a key role in regulating hepatic stellate cells (HSC) proliferation and collagen synthesis. Since no definitive study on ERK1/2 activation are available in vivo, we evaluated ERKlR activation in two different models of chronic liver injury leading to hepatic fibrosis: dimethylnitrosamine (DMN) administration and bile duct ligation (BDL). By immunohistochemistry using specific antibodies for activated and phosphorylated ERK112 (PERK), in normal rat liver PERK showed a granular cytoplasmic staining in zone 3 parenchymal cells, while at 6 hours after the third DMN injection a perimembranous distribution was observed. At 24 and 48 hours the reaction was evident in the nuclei of hepatocytes close to the area of necrosis and in cells with a stellate-like phenotype. In cirrhotic rats (DMN treatment for 5 weeks) PERK showed a granular cytoplasmic staining in hepatocytes and in stellate-like cells close to fibrotic septa. The subcellular modification of PERK distribution was paralleled by increased ERK activity (Western blot and MBP phosphorylation) that preceded the appearance of aSMA-positive cells, evident at 24 and 48 hours. At all times after BDL, PERK was limited to the nuclei of proliferating cholangiocytes and portal space fibroblasts and accompanied the appearance of aSMA-positive cells. This study indicates that ERK1/2 activation occurs in different cell phenotypes (i.e. hepatocytes or cholangiocytes) and involves either hepatic stellate cells or portal fibroblasts depending on the model of liver injury, providing specific targets for the selective delivery of ERK1/2 inhibitors.
066
BILIRUBIN ACTIVATES A CASPASE+DEPENDENT APOPTOTIC SIGNALING CASCADE
Cecilia M.P. Rodrigues, Susana Sol& Maria A. Brito, Jose G. Moura, Dora Brites. CPM, FFUL, Dept., University of Lisbon, Portugal Bilirubin impairs crucial aspects of cell function, often involving mitochondria. We hypothesised that the mechanism(s) by which bilirubin induces apoptosis in neuronal cells involves release of mitochondrial factors, which can trigger cytosolic pro-caspase activation. Neurones were isolated from rat foetuses and incubated with unconjugated bilirubin. Cytochrome c release and mitochondrial-dependent caspase activation in intact cells were determined by Western blot. Dynamic properties and oxidative damage of isolated mitochondrial membranes were assessed by spin-label electron paramagnetic resonance spectroscopy. In neurones, incubated with bilirubin, cytochrome c was rapidly released from mitochondria and accumulated in cytosol. Moreover, cytochrome c redistribution was followed by cleavage of pro-caspase-3 into the active 20 kDa subunit, and degradation of the full-length substrate poly(ADP)ribose polimerase. Using spectroscopic analyses, we detected major bilirubin-induced structural perturbation in isolated mitochondria, including increased polarity of the membrane lipid core and disrupted protein order. In addition, bilirubin insertion into mitochondrial membranes was accompanied by loss of spin-label paramagnetism, indicative of lipid peroxidation. Both ursodeoxycholate, a mitochondrial-membrane stabilising agent, and cyclosporine A, an inhibitor of the permeability transition, almost completely abrogated bilirubin-induced perturbation of rnitochondrial membrane structure. Thus, bilirubin directly interacts with mitochondria influencing membrane lipid polarity, protein order, and redox status, culminating with the release of caspaseactivating factors. Furthermore, these data demonstrate that bilirubin induces apoptosis by promoting cytosolic pro-caspase-3 activation, via a mitochondrial-dependent pathway, while providing novel insight into mechanisms of bilirubin cytotoxicity. Supported by EASL Fellowship and PRAXIS/C/SAU/1431 l/98.
I
Poster Sessions
1589
TERLlPRESSlN VS. SOMATOSTATIN IN ACUTE VARICEAL BLEEDING: A PROSPECTIVE, RANDOMIZED TRIAL
D. Chelarescu, 0. Gabor, C. Lunca. University of Medicine and Pharmacy, Romania Aim: We compared the efficacy and safety of terlipressin and somato-
statin in treatment of cirrhotic patients with variceal bleeding. Method: We investigated a number of 59 patients with endoscopyproved oesophageal bleeding, treated with somatostatin (nl = 27) and with terlipressin-nitroglycerin (n2 = 32). Patients were randomly assign to get (double-blind) terlipressin (2 mg i.v. initially and 1 mg at every 4 h, for 24 h) - transdermal nitroglycerin (10 mg/12 h during the first 24 h) or somatostatin (0.05 mg i.v. initially and 0.05 mg/h in iv. infusion in the next 48 h). In both groups, was performed balloon tamponade for 4 hours after diagnostic setting. Patients got also transfusions, electrolyte and fluid corrections and lactulose. We assessed the Child-Pugh class and the number of rebleeding at 48 hours since beginning of the treatment, number of deaths related to bleeding. The success was considered as 24 hours bleeding free period during 48 hours and lack of further bleeding from initial control to 5 days later. Results: Success rate was 66% in terlipressin- transdermal nitroglycerin group and 64% in somatostatin group (without statistically significant difference). The efficacy was similar regarding Child-Pugh class. Mortality rate was 16% in terlipressin group and 14% in somatostatin group. Few side effects were noted in each group.
PO3 Category 3: Molecular and cell biology (gene expression, signalling, fibrosis)
07
MITOGENIC THERAPY DIRECTED AT THE LIVER
Malik Raza, Neil Mellor, Clare Selden, Humphrey Hodgson. Royal Free Hosp., Pond St., London, UK
Aim: a) Assess if thyroid hormone (T3) acts as a primary mitogen on the liver b) Characterise the effects of T3 when administered prior to a 70% partial hepatectomy (PH) Method: a) Rats (n = 7) were injected with a single dose of T3 and sacrificed at intervals of 1, 2, 4, 7, 10, 14 days. b) 70% PH was performed at various time points (1, 3, 10 days) following a single dose of T3. The rats were sacrificed 24 hrs after PH Results: a) Liver mass was increased in rats treated with T3 when compared to controls Maximum effect was seen on day 10 with a 20% increase in liver mass (p < 0.0009) and a corresponding increase in total DNA (p < 0.001) and liver protein (p < 0.001). Cell proliferation peaked when T3 was administered 24 hrs prior to sacrifice: 7% Hepatocytes BRDU labelled compared to < 1% in controls (p < 0.0001) b) In animals treated with T3 at 3 and 10 days prior to PH the liver weight at sacrifice was greater (p < 0.05) than controls with a corresponding increase in liver protein (p < 0.05) and total DNA (p < 0.05). T3 enhanced the proliferative response of PH when administered 24 hrs prior to surgery: 36% hepatocyes BRDU labelled compared to 26% with PH alone (p < 0.001) Conclusion: Thyroid hormone could potentially be used to increase liver mass and enhance the regenerative response of the liver.
053
DIFFERENTIAL ERK112 ACTIVATION IN RAT CHRONIC LIVER INJURY AND FIBROSIS
Francesco Ridolfi, Gianluca Svegliati, Lucia Perego, Luca Marucci, Antonio Di Sario, Antonio Benedetti, Franc0 Folli. Department of Gastroenterology, University of Ancona, Italy The extracellular signal-regulated kinase l/2 (ERK1/2) has been shown
to play a key role in regulating hepatic stellate cells (HSC) proliferation and collagen synthesis. Since no definitive study on ERK1/2 activation are available in vivo, we evaluated ERKlR activation in two different models of chronic liver injury leading to hepatic fibrosis: dimethylnitrosamine (DMN) administration and bile duct ligation (BDL). By immunohistochemistry using specific antibodies for activated and phosphorylated ERK112 (PERK), in normal rat liver PERK showed a granular cytoplasmic staining in zone 3 parenchymal cells, while at 6 hours after the third DMN injection a perimembranous distribution was observed. At 24 and 48 hours the reaction was evident in the nuclei of hepatocytes close to the area of necrosis and in cells with a stellate-like phenotype. In cirrhotic rats (DMN treatment for 5 weeks) PERK showed a granular cytoplasmic staining in hepatocytes and in stellate-like cells close to fibrotic septa. The subcellular modification of PERK distribution was paralleled by increased ERK activity (Western blot and MBP phosphorylation) that preceded the appearance of aSMA-positive cells, evident at 24 and 48 hours. At all times after BDL, PERK was limited to the nuclei of proliferating cholangiocytes and portal space fibroblasts and accompanied the appearance of aSMA-positive cells. This study indicates that ERK1/2 activation occurs in different cell phenotypes (i.e. hepatocytes or cholangiocytes) and involves either hepatic stellate cells or portal fibroblasts depending on the model of liver injury, providing specific targets for the selective delivery of ERK1/2 inhibitors.
066
BILIRUBIN ACTIVATES A CASPASE+DEPENDENT APOPTOTIC SIGNALING CASCADE
Cecilia M.P. Rodrigues, Susana Sol& Maria A. Brito, Jose G. Moura, Dora Brites. CPM, FFUL, Dept., University of Lisbon, Portugal Bilirubin impairs crucial aspects of cell function, often involving mitochondria. We hypothesised that the mechanism(s) by which bilirubin induces apoptosis in neuronal cells involves release of mitochondrial factors, which can trigger cytosolic pro-caspase activation. Neurones were isolated from rat foetuses and incubated with unconjugated bilirubin. Cytochrome c release and mitochondrial-dependent caspase activation in intact cells were determined by Western blot. Dynamic properties and oxidative damage of isolated mitochondrial membranes were assessed by spin-label electron paramagnetic resonance spectroscopy. In neurones, incubated with bilirubin, cytochrome c was rapidly released from mitochondria and accumulated in cytosol. Moreover, cytochrome c redistribution was followed by cleavage of pro-caspase-3 into the active 20 kDa subunit, and degradation of the full-length substrate poly(ADP)ribose polimerase. Using spectroscopic analyses, we detected major bilirubin-induced structural perturbation in isolated mitochondria, including increased polarity of the membrane lipid core and disrupted protein order. In addition, bilirubin insertion into mitochondrial membranes was accompanied by loss of spin-label paramagnetism, indicative of lipid peroxidation. Both ursodeoxycholate, a mitochondrial-membrane stabilising agent, and cyclosporine A, an inhibitor of the permeability transition, almost completely abrogated bilirubin-induced perturbation of rnitochondrial membrane structure. Thus, bilirubin directly interacts with mitochondria influencing membrane lipid polarity, protein order, and redox status, culminating with the release of caspaseactivating factors. Furthermore, these data demonstrate that bilirubin induces apoptosis by promoting cytosolic pro-caspase-3 activation, via a mitochondrial-dependent pathway, while providing novel insight into mechanisms of bilirubin cytotoxicity. Supported by EASL Fellowship and PRAXIS/C/SAU/1431 l/98.