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66 Oral IDN-6556, An anti-apoptotic caspase inhibitor, lowers aminotransferases in HCV patients
Parallel Session 8: Experimental Hepatology II accordance with previous kinetic studies with interferon. The rapid initial decay of HCV RNA during BILN 2061 treatment indicates the potential for protease inhibitors to exceed current interferon-based therapy of chronic HCV infection.
68 DAILY CANNABIS SMOKING AS A RISK FACTOR FOR FIBROSIS PROGRESSION IN CHRONIC HEPATITIS C
C. Hezode 1 , F. Roudot-Thoraval 2 , P. Grenard 3 , B. Julien 3 , E.S. Zafrani 4 , D. Dhumeaux 1 , S. Lotersztajn 3 , A. Mallat 1,3 . 1 Department of Hepatology, Hopital Henri Mondor, Creteil, France; 2 Department of Public Health, Hopital Henri Mondor, Creteil, France; 3 INSERM U581, Hopital Henri Mondor, Creteil, France; 4 Department of Pathology, Hopital Henri Mondor, Creteil, France We recently showed that liver fibrogenesis may be regulated by cannabinoid (CB) receptors: indeed, (i) in cirrhotic patients CB1 receptors are markedly upregulated in hepatic myofibroblasts (Grenard et al. AASLD 2002); (ii) chronic tetrachloride intoxication in invalidated CB1-/- mice results in decreased liver fibrosis, compared to controls, suggesting that CB1 receptors are profibrogenic. The present study evaluated the impact of cannabis smoking on fibrosis progression in chronic hepatitis C. 195 consecutive naïve patients with dated exposure (men/women: 140/55, aged 42±10 years) were included. Data collected were consumptions of cannabis, alcohol and tobacco during disease duration, age at contamination, gender, route of transmission, genotype, BMI, steatosis, activity and fibrosis (METAVIR), and fibrosis progression rate (median value being 0.08 U per year). By univariate analysis fibrosis progression rate >0.08 U per year was associated to daily cannabis smoking (63% versus 50% in occasional smokers versus 42% in no smokers, p=0.029), alcohol intake 30 ≥g/d (64%, p=0.03), moderate or marked steatosis (65%, p=0.004), age at contamination >25 years (63%, p=0.006), and histological activity ≥A2 (65%, p<0,001). In multivariate analysis, fibrosis progression rate >0.08 U per year was independently related to daily cannabis smoking (OR=3.8; 95% CI (1.7-8.7)), alcohol intake ≥30 g/d (OR=2.1; 95% CI (1.0-4.6)), age at contamination >25 years (OR=4.0; 95% CI (1.9-8.4)), and activity ≥A2 (OR=7.5; 95% CI (3.5-16.1)). This study discloses a link between daily cannabis consumption and fibrosis progression rate in patients with hepatitis C and supports our experimental data demonstrating the profibrogenic role of CB1 receptors.
Parallel Session 8: Experimental Hepatology II
25
metabolizing enzymes cyp2b10, cyp3a11, ugt1a1 were analyzed by real time PCR. Moreover, serum biochemistry and histology were investigated. Results: The major bile acid transporters Ntcp and Bsep remained unchanged in response to PB and PCN administration compared to controls. However, both PB and PCN induced Mrp3 (2.9- and 4.7-fold), Mrp4 (3.3and 16.6-fold) and Oatp2 (2.2- and 3.6-fold) mRNA levels while Mrp2 mRNA was moderatly induced only by PCN (1.6–fold). Cyp2b10 was stimulated by PB as well as PCN (more than 100-fold), while cyp3a11 was induced mainly by PCN (5.9-fold). Ugt1a1 showed no induction. Summary & Conclusions: Adaptive hepatic transporter expression (i.e. Mrp3 and Mrp4) and bile acid metabolizing enzymes (i.e. cyp2b10 and cyp3a11) are induced by CAR as well as PXR ligands. Co-ordinated stimulation of hepatocellular detoxification and elimination of biliary constituents should ameliorate cholestatic liver injury.
70 DIFFERENTIAL EFFECTS OF URSODEOXYCHOLIC ACID (UDCA) AND CHOLIC ACID (CA) ON HEPATIC AND RENAL EXPRESSION OF MRP4 AND MRP6, TWO NOVEL ABC TRANSPORTERS, IN MICE
G. Zollner 1 , E. Halilbasic 1 , M. Wagner 1 , P. Fickert 1 , D. Silbert 1 , J. Gumhold 1 , A. Fuchsbichler 2 , K. Zatloukal 2 , H. Denk 2 , M. Trauner 1 . 1 Department of Medicine,; 2 Department of Pathology, Background: Overexpression of hepatic Mrp3 and renal Mrp2 during cholestasis limits retention of bile acids by providing an alternative excretory route. We speculated, that induction of Mrp4 and Mrp6, which are expressed in liver and kidney, might contribute to this adaptive response and therefore assessed Mrp4 and Mrp6 expression after UDCA and CA feeding. Methods: C57/BL6 mice (FXR+/+) and FXR knockout mice (FXR-/-) were fed a UDCA and CA-enriched diet for 7 days and compared to standard diet-fed controls. Hepatic and renal Mrp4 and Mrp6 mRNA and protein expression were determined by real-time PCR and Western blotting. Results: UDCA but not CA induced hepatic and renal Mrp4 mRNA and protein (p<0.01) in FXR+/+. In FXR-/-, both bile acids up-regulated Mrp4 mRNA and protein levels in liver and kidney (p<0.01). In contrast, UDCA and CA had no effect on hepatic and renal Mrp6 expression. Summary and Conclusions: UDCA profoundly induced Mrp4 in liver and kidney in both genotypes, while CA increased Mrp4 expression only in FXR-/-. The latter may be a secondary event due to higher intrahepatic bile acid levels in these animals. Neither UDCA nor CA were able to up-regulate Mrp6, which therefore does not appear to be involved in alternative bile acid elimination. Since CA, the major bile acid retained during cholestasis, had no effect on hepatic and renal Mrp4 levels in wild type animals, administration of UDCA may be an appropriate option for stimulating Mrp4, leading to increased basolateral hepatic efflux and renal tubular excretion of bile acids.
69 NUCLEAR RECEPTOR (PXR, CAR) AGONISTS INDUCE ADAPTIVE HEPATIC TRANSPORTER AND ENZYME EXPRESSION IN MOUSE LIVER
M. Wagner 1 , E. Halilbasic 1 , P. Fickert 1 , G. Zollner 1 , A. Fuchsbichler 2 , J. Gumhold 1 , D. Silbert 1 , K. Zatloukal 2 , H. Denk 2 , M. Trauner 1 . 1 Department of Medicine, Medical University, Graz, Austria; 2 Department of Pathology, Medical University, Graz, Austria Background & Aims: Cholestasis results in hepatic accumulation of biliary constituents (e.g. bile acids and bilirubin) which may lead to induction of hepatocellular export systems and detoxification enzymes aimed at ameliorating hepatocellular damage. The aim of this study was to investigate the role of the nuclear receptors CAR and PXR in mediating this response. Material & Methods: Swiss Albino mice were treated with either the CAR agonist phenobarbital (PB; 100mg/kg body weight; i.p.) or the PXR agonist pregnenolone 16-alpha-carbonitrile (PCN; 75mg/kg body weight; i.p.) for 3 days. mRNA levels of bile acid transporters Ntcp, Oatp2, Bsep, Mrp2-4 and
71 ROLE OF P38MAPK IN TUDCA-INDUCED BILE FORMATION IN CHOLESTATIC LIVER
R. Wimmer, S. Hohenester, G.U. Denk, C. Rust, U. Beuers. Department of Medicine II - Grosshadern, University of Munich, Munich, Germany Ursodeoxycholic acid (UDCA) exerts anticholestatic effects in cholestatic patients and in experimental models of cholestasis. Stimulation of impaired hepatocellular secretion is regarded as a major mechanism of action of UDCA. Its taurine conjugate, TUDCA, stimulates hepatobiliary exocytosis and insertion of carrier proteins into canalicular membranes of cholestatic hepatocytes by putatively Ca2+ - and protein kinase Cadependent mechanisms and enhances their impaired secretory capacity (Hepatology 2001;33:1206;Gut 2002;51:113). TUDCA-induced bile acid secretion in non-cholestatic liver is mediated by activation of p38MAPK (Gastroenterology 2001;121:407). We studied the role of p38MAPK in a model of experimental cholestasis.
68 DAILY CANNABIS SMOKING AS A RISK FACTOR FOR FIBROSIS PROGRESSION IN CHRONIC HEPATITIS C
C. Hezode 1 , F. Roudot-Thoraval 2 , P. Grenard 3 , B. Julien 3 , E.S. Zafrani 4 , D. Dhumeaux 1 , S. Lotersztajn 3 , A. Mallat 1,3 . 1 Department of Hepatology, Hopital Henri Mondor, Creteil, France; 2 Department of Public Health, Hopital Henri Mondor, Creteil, France; 3 INSERM U581, Hopital Henri Mondor, Creteil, France; 4 Department of Pathology, Hopital Henri Mondor, Creteil, France We recently showed that liver fibrogenesis may be regulated by cannabinoid (CB) receptors: indeed, (i) in cirrhotic patients CB1 receptors are markedly upregulated in hepatic myofibroblasts (Grenard et al. AASLD 2002); (ii) chronic tetrachloride intoxication in invalidated CB1-/- mice results in decreased liver fibrosis, compared to controls, suggesting that CB1 receptors are profibrogenic. The present study evaluated the impact of cannabis smoking on fibrosis progression in chronic hepatitis C. 195 consecutive naïve patients with dated exposure (men/women: 140/55, aged 42±10 years) were included. Data collected were consumptions of cannabis, alcohol and tobacco during disease duration, age at contamination, gender, route of transmission, genotype, BMI, steatosis, activity and fibrosis (METAVIR), and fibrosis progression rate (median value being 0.08 U per year). By univariate analysis fibrosis progression rate >0.08 U per year was associated to daily cannabis smoking (63% versus 50% in occasional smokers versus 42% in no smokers, p=0.029), alcohol intake 30 ≥g/d (64%, p=0.03), moderate or marked steatosis (65%, p=0.004), age at contamination >25 years (63%, p=0.006), and histological activity ≥A2 (65%, p<0,001). In multivariate analysis, fibrosis progression rate >0.08 U per year was independently related to daily cannabis smoking (OR=3.8; 95% CI (1.7-8.7)), alcohol intake ≥30 g/d (OR=2.1; 95% CI (1.0-4.6)), age at contamination >25 years (OR=4.0; 95% CI (1.9-8.4)), and activity ≥A2 (OR=7.5; 95% CI (3.5-16.1)). This study discloses a link between daily cannabis consumption and fibrosis progression rate in patients with hepatitis C and supports our experimental data demonstrating the profibrogenic role of CB1 receptors.
Parallel Session 8: Experimental Hepatology II
25
metabolizing enzymes cyp2b10, cyp3a11, ugt1a1 were analyzed by real time PCR. Moreover, serum biochemistry and histology were investigated. Results: The major bile acid transporters Ntcp and Bsep remained unchanged in response to PB and PCN administration compared to controls. However, both PB and PCN induced Mrp3 (2.9- and 4.7-fold), Mrp4 (3.3and 16.6-fold) and Oatp2 (2.2- and 3.6-fold) mRNA levels while Mrp2 mRNA was moderatly induced only by PCN (1.6–fold). Cyp2b10 was stimulated by PB as well as PCN (more than 100-fold), while cyp3a11 was induced mainly by PCN (5.9-fold). Ugt1a1 showed no induction. Summary & Conclusions: Adaptive hepatic transporter expression (i.e. Mrp3 and Mrp4) and bile acid metabolizing enzymes (i.e. cyp2b10 and cyp3a11) are induced by CAR as well as PXR ligands. Co-ordinated stimulation of hepatocellular detoxification and elimination of biliary constituents should ameliorate cholestatic liver injury.
70 DIFFERENTIAL EFFECTS OF URSODEOXYCHOLIC ACID (UDCA) AND CHOLIC ACID (CA) ON HEPATIC AND RENAL EXPRESSION OF MRP4 AND MRP6, TWO NOVEL ABC TRANSPORTERS, IN MICE
G. Zollner 1 , E. Halilbasic 1 , M. Wagner 1 , P. Fickert 1 , D. Silbert 1 , J. Gumhold 1 , A. Fuchsbichler 2 , K. Zatloukal 2 , H. Denk 2 , M. Trauner 1 . 1 Department of Medicine,; 2 Department of Pathology, Background: Overexpression of hepatic Mrp3 and renal Mrp2 during cholestasis limits retention of bile acids by providing an alternative excretory route. We speculated, that induction of Mrp4 and Mrp6, which are expressed in liver and kidney, might contribute to this adaptive response and therefore assessed Mrp4 and Mrp6 expression after UDCA and CA feeding. Methods: C57/BL6 mice (FXR+/+) and FXR knockout mice (FXR-/-) were fed a UDCA and CA-enriched diet for 7 days and compared to standard diet-fed controls. Hepatic and renal Mrp4 and Mrp6 mRNA and protein expression were determined by real-time PCR and Western blotting. Results: UDCA but not CA induced hepatic and renal Mrp4 mRNA and protein (p<0.01) in FXR+/+. In FXR-/-, both bile acids up-regulated Mrp4 mRNA and protein levels in liver and kidney (p<0.01). In contrast, UDCA and CA had no effect on hepatic and renal Mrp6 expression. Summary and Conclusions: UDCA profoundly induced Mrp4 in liver and kidney in both genotypes, while CA increased Mrp4 expression only in FXR-/-. The latter may be a secondary event due to higher intrahepatic bile acid levels in these animals. Neither UDCA nor CA were able to up-regulate Mrp6, which therefore does not appear to be involved in alternative bile acid elimination. Since CA, the major bile acid retained during cholestasis, had no effect on hepatic and renal Mrp4 levels in wild type animals, administration of UDCA may be an appropriate option for stimulating Mrp4, leading to increased basolateral hepatic efflux and renal tubular excretion of bile acids.
69 NUCLEAR RECEPTOR (PXR, CAR) AGONISTS INDUCE ADAPTIVE HEPATIC TRANSPORTER AND ENZYME EXPRESSION IN MOUSE LIVER
M. Wagner 1 , E. Halilbasic 1 , P. Fickert 1 , G. Zollner 1 , A. Fuchsbichler 2 , J. Gumhold 1 , D. Silbert 1 , K. Zatloukal 2 , H. Denk 2 , M. Trauner 1 . 1 Department of Medicine, Medical University, Graz, Austria; 2 Department of Pathology, Medical University, Graz, Austria Background & Aims: Cholestasis results in hepatic accumulation of biliary constituents (e.g. bile acids and bilirubin) which may lead to induction of hepatocellular export systems and detoxification enzymes aimed at ameliorating hepatocellular damage. The aim of this study was to investigate the role of the nuclear receptors CAR and PXR in mediating this response. Material & Methods: Swiss Albino mice were treated with either the CAR agonist phenobarbital (PB; 100mg/kg body weight; i.p.) or the PXR agonist pregnenolone 16-alpha-carbonitrile (PCN; 75mg/kg body weight; i.p.) for 3 days. mRNA levels of bile acid transporters Ntcp, Oatp2, Bsep, Mrp2-4 and
71 ROLE OF P38MAPK IN TUDCA-INDUCED BILE FORMATION IN CHOLESTATIC LIVER
R. Wimmer, S. Hohenester, G.U. Denk, C. Rust, U. Beuers. Department of Medicine II - Grosshadern, University of Munich, Munich, Germany Ursodeoxycholic acid (UDCA) exerts anticholestatic effects in cholestatic patients and in experimental models of cholestasis. Stimulation of impaired hepatocellular secretion is regarded as a major mechanism of action of UDCA. Its taurine conjugate, TUDCA, stimulates hepatobiliary exocytosis and insertion of carrier proteins into canalicular membranes of cholestatic hepatocytes by putatively Ca2+ - and protein kinase Cadependent mechanisms and enhances their impaired secretory capacity (Hepatology 2001;33:1206;Gut 2002;51:113). TUDCA-induced bile acid secretion in non-cholestatic liver is mediated by activation of p38MAPK (Gastroenterology 2001;121:407). We studied the role of p38MAPK in a model of experimental cholestasis.