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Molecular structure of the bile salt export pump gene (Abcb11) reveals a polymorphism between galistone-susceptible and resistant inbred mice
feeding started after CBDL resulted in a 100% mortality (6/6 animals) after 5 days of UDCA feeding. UOCAfeeding in sham operated animals had no effect on liver histology and liver enzymes. SUMMARY & CONCLUSIONS:UDCAfeeding unexpectedlyaggravatesliver injury and increases mortality in obstructive cholestasis in mice. Further clinical studies will have to determine whether UDCA could also have potential side effects in patients with obstructive cholestasis.
1 Overexpression of Sterol 12oz-Hydroxylase(CYP8bl): Effect on Rates of Cholic Acid (CA) Synthesis and Alteration in Bile Acid Pool Composition. William M. PandakJr., McGuire VAMC, Richmond, VA; Phillip B. Hylemon, Patricia 8ohdan, Virginia Commonwealth Univ, Richmond, VA; Ingemar Bjorkhem, Gosta Eggertsen, Karolinska Inst, Stockholm Sweden;Zdravko R. Vlahcevic, Virginia Commonwealth Univ, Richmond, VA
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Background: 12~-Hydmxylation of bile acid precumors by CYP8bf is necessary for the formation of cholic acid (CA), and, therefore,the ratio of CA to chenodeoxycholicacid (CDCA) synthesis. Objective:To study the effects of increasedexpressionof CYPSbf and cholesterol 7~hydmxylase (CYP7al) on rates of CA synthesis and total bile acid synthesis, and on bile acid pool composition. Methods: Overexpression of CYP8bl and CYP7al was mediated through infection of primary rat hepatocytes(PRH) or intact male Sprague-Dawleyrats with replication defective recombinant adenovirusesencoding CMV promoter driven CYP8bl or CYP7al. PRHcultures containeddexamethasone(0.1/~M) alone, or with L-thyroxineT4 (1/zM) and were harvested48 h following infection. Whole animals were sacrificed 3 days following infection, in vitro,ratesof bile acid synthesiswere determinedas conversionof [14C]cholesterol to [~4C]bile acids; beingfurther identified by TLC. In vivo,bile acid composition was determined by HPLC, Results: Data presented as % of paired controls (mean+SE). Overexpressionof either CYP8bl and CYP7al in PRH in the presence or absence of T4, markedly increased respective mRNA levels and specific activities without effecting the other. Overexpressionof CYP7al in PRH markedly increasedbile acid synthesis (457%+_18%), but did not alter the ratio of CA/CDCAplus its muricholic acid derivatives.Overexpressionof CYP8bl only slightly increased total bile acid synthesis (114%+-6.9%), but significantly increased (306% to 723%)CA synthesis and altered the CNCOCA plus its muricholic acid derivatives ratio from 1:3.4 to 2.9:1. Overexpressionof CYP8bl in rats increased CYP8bl mRNA levels (67-fold), specific activity (20-fold), and % of CA in the bile acid pool (41 -+5.6% to 64+-5%), while decreasing CDCA and its muricholic acid derivatives (59+-5.6% to 36+-4.9%). Conclusion: Overexpressionof CYP8bl markedlyincreasedCAsynthesis both in PRHand in whole animals. Overexprassionof CYP7al did not alter bile acid composition, but increased total bile acid synthesis. In PRH cultures in the absenceof T4, CYP7al specific activity is undetectablewith no significant bile acid synthesis occurring via the "classic pathway". The ability to generate CA in the absence of this pathway with CYP8bl overexpmssiondemonstratesthat CYP8bl is capable of 12a-hydroxylating bile acid precursom from the alternative pathway(s).
Bile Acid Stimulation of Small Bile Duct Proliferation Requires De Novo Expression of the Apical Rile Acid Transporter (ABAT) Gianfranco Alpioi, The Texas A&M Univ System Health Science Ctr, Temple, IX; Shannon Glaser, Jo Lynne Phinizy, Scott & White Memorial Hosp, Temple, IX; Noriatsu Kanno, The Texas A&M Uetv System Health Science Ctr, Temple, IX; Heather Francis, Scott & White Memorial Hosp, Temple, TX; Mikel Ludvik, The Texas A&M Univ System Health Science Ctr, Temple, lX; Gone Lesage,Scott & White Memorial Hosp, Temple, TX Cholangiopathies(e.g. primary biliary cirrhosis) are associatedwith ductal damageand compensatory cholangiocyte proliferation limited to small ducts. The accumulation of bile acids (BA) in cholestatic liver diseasesmay induce cholengiocyteproliferation since we havefound in vitro bile acids may stimulate proliferation in cholangiocytes isolated from rats (Am J Physiol. 1997 273:G518-29). BA stimulation of proliferation in large cholangiocytas (which line large > 15 ~ diameter ducts) requires bile acid uptake by cholangiocyte apical bile acid transporter (ABAT). In contrast, bile acids do not acutely stimulate proliferation in small cholangiocytas (which line small < 15 p.m ducts) possibly due to the absence of ABAT in small cholangiocytes.We propose the hypothesis that accumulation of bile acids in chronic cholestati¢ liver diseasescauses bile duct proliferation only in bile ducts that express ABAT. OBJECTIVES:To determinethe chronic effects of increasedbile acid concentrationon cholangiocyte proliferation and ABAT expression in large and small bite ducts. METHODS:Normal rats were fed 1% taumcholate (TC) for one week. Small and large cholangiocytes were isolated and ABAT protein and gene expression and indices of cholangiocyte proliferation (PCNA and H3 histone gone expression) were determined. RESULTS:TC feeding induced a 3-fold increase in biliary TC concentration. Comparedto controls, large cholangiocytesfrom TC fed rats showed increased proliferation, increasedABAT protein and gene expression. In small cholangiocytes,TC feeding induced de novo ABAT protein and gone expression and increasedproliferation. In vitro, exposureto TC (20 p,M) for 60 minutes increasedcholangiocyte proliferation in both small and large cholangiocyteisolated from TC fed rats but only in large cholangiocytes isolated from normal rats. CONCLUSIONS:Chronic increasedbiliary bile acid concentrationsdue to feeding TC increasescholangiocyteproliferation in both large and small cholangiocytesand induces de novo expressionof ABAT in small bile ducts. We propose that the de novo expression of ABAT in small ducts provides a route for bile acid entry into cholangiocytesto trigger the proliferation of small ducts (ductular proliferation) that is commonly observed in chronic cholestatic liver disease.These data show that the expression of ABAT in cholangiocytesdetermines their sensitivity to bile acid -stimulated proliferation.
2 Rat Ileal Apical Sodium-DependentRile Acid Transporter (ASBT): Carboxyl Terminal 14 Amino Acids ACt as an Apical Sorting Motif. An-Qiang Sun, I'Kyori Swaby, FrederickJ. Suchy, Mount Sinai Sch of Medicine, New York, NY The rat ileum ASBTis a polytopic membraneglycoprotein. Previousstudiesfrom our laboratory demonstrated that ASBT is localized to the apical domain of the stably transfected MDCK cells, mirroring its localization in rat ileum. Truncation of the entire cytoplasmic tail of 40 amino acids of ASBT results in loss of sorting to the plasma membrane. To identify further the apical sorting signal and mechanismof the membranelocalization,truncated and chimeric ASBT were generated.Taurocholateinflux assays in a transwell culture system and confocal microscopy were used to detect polarized expression of wild type and mutant ASBT in transfected cells. The results demonstrate that removal of the N-glycans present on the Nterminus of ASBT by tunicamycin treatment did not alter the polarized apical expression of ASBTin transfected MDCKcell, as is the casefor some other apically sorted proteins. Deletion of the last 14 amino acids (aa 334-348) from the C-terminus of ASBT abolished the apical expressionof the mutant ASBT. Moreover, replacementof the cytoplasmic tail of Ntcp, a liver basolateral protein, with the last 14 aa peptide of ASBT redirectedthe chimera predominantly to the apical compartment.The resultsfrom the treatments with brefeldinA (BFA)or monensin, which inhibit vesicular transport, demonstrated that BFA disrupts the polarized localization of ASBT, while monensin had no effect. In addition, low temperature shift (20°C), which blocks membrane sorting of some polytopic proteins at Golgi complex, did not affect the polarized apical surface sorting of ASBT. In summary, the above results suggest that (1) a novel 14 aa peptide apical sorting signal is present within ASBT cytoplasmic tail. (2) ASBT follows an apical sorting pathway that is delivered via BFA-ssnsitive mechanism and is independent of protein glycosylation and low temperature shift.
5 Molecular Structure of the Bile Salt Export Pump Gene (Abcb11) Reveals a Polymorphism Between Gallstone-Susceptible and Resistant Inbred Mice Anne Figge, B.~rbelTaenzler, Dept of Medicine III, Univ of Technology (RWTH), Aachen Germany; BeverlyJ. Paigen,The Jackson Lab, Bar Harbor, ME; Siegfried Matern, Frank Lammert, Oept of Medicine III, Univ of Technology (RWTH), Aachen Germany Background:The mouse geneencoding the hepatic canalicularbile salt export pump (Abcb11, formerly Spgp) is a candidate for the major gallstone gene Lithl. Abcb11 is differentially expressed in gallstone-susceptible(C57UJ) and resistant (AKR/J) inbred mice, The aim of this study was to investigatethe identity of Lithl and Abcb11 by dissecting the structure of the gene and conducting a molecular comparison of the mouse strains. Methods: Adaptor ligated PCRwas usedto generateunknown 5' flanking sequenceof the Abcb11gene. Bacterial Artificial Chromosomes (BAC) containing the Abcbll gene were identified by screening a genomic BAC library from the gallstone-susceptibleinbred mouse strain C57BU6J (RPCI23) and characterizedby pulse-field gel electmphoresis. A single BAC clone was submitted to the Trans-NIH Mouse Initiative for complete sequencing. Genomic DNA from strains C57L andAKR was comparedby SingleStrand ConformationPolymorphism(SSCP)analysis,which employsgel electrophoresisto detect nucleotidesequencepolymorphismsthat alter secondary structures of single-strandedDNA,and direct sequencingof PCRfragments. Results:Screening of the BAC library resulted in 8 Abcb11positive clones.A clone with a 180 kb insert containing both the 5' flanking region and the 3' end of the gene was chosen for sequencing.Alignment of the BAC sequence and the Abcbll cDNA revealed that the gene spans 66 kb and is comprised of 28 exons and introos ranging from 91 to 15285 bp. Sequencecomparison of the gallstone-susceptiblestrains C57L and C57BU6 and the resistant strain AKR showed no difference in the coding sequenceor the minimal promoter. However,a single polymorphism (C-T transition) was detected in the 5' flanking region of the gene. Conclusions: (1) The complete genestructure of the murine Abcb11 gene provides a basic framework for the study of molecular regulation of the bile salt export pump in geneticallydefined mouse models. (2) The detected polymorphism between the gallstone-susceptibleand resistant inbred strains bears a putative regulatory element for gene transcription of Abcb11, which could result in both differential Abcb11 expression and gallstone susceptibility. Supportedby Trans-NIH Mouse Inib'ative
3 UrsodeoxycholicAcid Feeding Aggravates Liver Injury in Bile Duct-ligated Mice Peter Fickert, Gernot Zollner, Andrea Fuchsbichler, Cenny Stumptner, KarI-FranzensUniv, Graz Austria; Frank Lammert, Univ of Aachen, Aachen Germany; Kurt Zatloukal, Helmut Denk, Michael Trauner, KarI-FranzensUniv, Graz Austria BACKGROUND& OBJECTIVES:Ursodeoxycholicacid (UDCA) has beneficialeffects in various cholestatic liver diseases(e.g., pimary biliary cirrhosis, primary sclerosing cholangitis, intrahepatic cholastasis of pregnancy, cystic fibrosis, etc.). However, it is unclear whether UDCA also improves liver injury caused by biliary obstruction. Therefore, it was the aim of this study to determine the effects of UDCA in obstructive cholestasis in mice. METHODS:Mice were fed a UDCA (0.5% w/w) supplementeddiet or control diet for 7 days. Thereafter mice were subjected to common bile duct ligation (CBDL; n=24)or selective bile duct ligation (SBDL; n = f 2)and continuedon UDCAor control diet for further 3 days. In anotherexperimental series, mice were fed UDCA only after CBDL (n=6), Mortality rates, serum liver enzymes, total serum bile acid levels, bile composition, and liver histology were investigated.RESULTS: CBDLand SBDL(ligated lobes) in control diet-fed mice resulted in disseminatedhepatocellular necrosis, bile infarct, and periductular fibrosis, whereas non-ligated lobes (SBDL) remained unchanged. CBDL in UDCA-preted mice caused more severe hepatocellular necrosis and significantly increasedmortality rates comparedwith CBDL in control mice receivingstandard diet (7/12 vs. 1/12 in controls; p
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1 Overexpression of Sterol 12oz-Hydroxylase(CYP8bl): Effect on Rates of Cholic Acid (CA) Synthesis and Alteration in Bile Acid Pool Composition. William M. PandakJr., McGuire VAMC, Richmond, VA; Phillip B. Hylemon, Patricia 8ohdan, Virginia Commonwealth Univ, Richmond, VA; Ingemar Bjorkhem, Gosta Eggertsen, Karolinska Inst, Stockholm Sweden;Zdravko R. Vlahcevic, Virginia Commonwealth Univ, Richmond, VA
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Background: 12~-Hydmxylation of bile acid precumors by CYP8bf is necessary for the formation of cholic acid (CA), and, therefore,the ratio of CA to chenodeoxycholicacid (CDCA) synthesis. Objective:To study the effects of increasedexpressionof CYPSbf and cholesterol 7~hydmxylase (CYP7al) on rates of CA synthesis and total bile acid synthesis, and on bile acid pool composition. Methods: Overexpression of CYP8bl and CYP7al was mediated through infection of primary rat hepatocytes(PRH) or intact male Sprague-Dawleyrats with replication defective recombinant adenovirusesencoding CMV promoter driven CYP8bl or CYP7al. PRHcultures containeddexamethasone(0.1/~M) alone, or with L-thyroxineT4 (1/zM) and were harvested48 h following infection. Whole animals were sacrificed 3 days following infection, in vitro,ratesof bile acid synthesiswere determinedas conversionof [14C]cholesterol to [~4C]bile acids; beingfurther identified by TLC. In vivo,bile acid composition was determined by HPLC, Results: Data presented as % of paired controls (mean+SE). Overexpressionof either CYP8bl and CYP7al in PRH in the presence or absence of T4, markedly increased respective mRNA levels and specific activities without effecting the other. Overexpressionof CYP7al in PRH markedly increasedbile acid synthesis (457%+_18%), but did not alter the ratio of CA/CDCAplus its muricholic acid derivatives.Overexpressionof CYP8bl only slightly increased total bile acid synthesis (114%+-6.9%), but significantly increased (306% to 723%)CA synthesis and altered the CNCOCA plus its muricholic acid derivatives ratio from 1:3.4 to 2.9:1. Overexpressionof CYP8bl in rats increased CYP8bl mRNA levels (67-fold), specific activity (20-fold), and % of CA in the bile acid pool (41 -+5.6% to 64+-5%), while decreasing CDCA and its muricholic acid derivatives (59+-5.6% to 36+-4.9%). Conclusion: Overexpressionof CYP8bl markedlyincreasedCAsynthesis both in PRHand in whole animals. Overexprassionof CYP7al did not alter bile acid composition, but increased total bile acid synthesis. In PRH cultures in the absenceof T4, CYP7al specific activity is undetectablewith no significant bile acid synthesis occurring via the "classic pathway". The ability to generate CA in the absence of this pathway with CYP8bl overexpmssiondemonstratesthat CYP8bl is capable of 12a-hydroxylating bile acid precursom from the alternative pathway(s).
Bile Acid Stimulation of Small Bile Duct Proliferation Requires De Novo Expression of the Apical Rile Acid Transporter (ABAT) Gianfranco Alpioi, The Texas A&M Univ System Health Science Ctr, Temple, IX; Shannon Glaser, Jo Lynne Phinizy, Scott & White Memorial Hosp, Temple, IX; Noriatsu Kanno, The Texas A&M Uetv System Health Science Ctr, Temple, IX; Heather Francis, Scott & White Memorial Hosp, Temple, TX; Mikel Ludvik, The Texas A&M Univ System Health Science Ctr, Temple, lX; Gone Lesage,Scott & White Memorial Hosp, Temple, TX Cholangiopathies(e.g. primary biliary cirrhosis) are associatedwith ductal damageand compensatory cholangiocyte proliferation limited to small ducts. The accumulation of bile acids (BA) in cholestatic liver diseasesmay induce cholengiocyteproliferation since we havefound in vitro bile acids may stimulate proliferation in cholangiocytes isolated from rats (Am J Physiol. 1997 273:G518-29). BA stimulation of proliferation in large cholangiocytas (which line large > 15 ~ diameter ducts) requires bile acid uptake by cholangiocyte apical bile acid transporter (ABAT). In contrast, bile acids do not acutely stimulate proliferation in small cholangiocytas (which line small < 15 p.m ducts) possibly due to the absence of ABAT in small cholangiocytes.We propose the hypothesis that accumulation of bile acids in chronic cholestati¢ liver diseasescauses bile duct proliferation only in bile ducts that express ABAT. OBJECTIVES:To determinethe chronic effects of increasedbile acid concentrationon cholangiocyte proliferation and ABAT expression in large and small bite ducts. METHODS:Normal rats were fed 1% taumcholate (TC) for one week. Small and large cholangiocytes were isolated and ABAT protein and gene expression and indices of cholangiocyte proliferation (PCNA and H3 histone gone expression) were determined. RESULTS:TC feeding induced a 3-fold increase in biliary TC concentration. Comparedto controls, large cholangiocytesfrom TC fed rats showed increased proliferation, increasedABAT protein and gene expression. In small cholangiocytes,TC feeding induced de novo ABAT protein and gone expression and increasedproliferation. In vitro, exposureto TC (20 p,M) for 60 minutes increasedcholangiocyte proliferation in both small and large cholangiocyteisolated from TC fed rats but only in large cholangiocytes isolated from normal rats. CONCLUSIONS:Chronic increasedbiliary bile acid concentrationsdue to feeding TC increasescholangiocyteproliferation in both large and small cholangiocytesand induces de novo expressionof ABAT in small bile ducts. We propose that the de novo expression of ABAT in small ducts provides a route for bile acid entry into cholangiocytesto trigger the proliferation of small ducts (ductular proliferation) that is commonly observed in chronic cholestatic liver disease.These data show that the expression of ABAT in cholangiocytesdetermines their sensitivity to bile acid -stimulated proliferation.
2 Rat Ileal Apical Sodium-DependentRile Acid Transporter (ASBT): Carboxyl Terminal 14 Amino Acids ACt as an Apical Sorting Motif. An-Qiang Sun, I'Kyori Swaby, FrederickJ. Suchy, Mount Sinai Sch of Medicine, New York, NY The rat ileum ASBTis a polytopic membraneglycoprotein. Previousstudiesfrom our laboratory demonstrated that ASBT is localized to the apical domain of the stably transfected MDCK cells, mirroring its localization in rat ileum. Truncation of the entire cytoplasmic tail of 40 amino acids of ASBT results in loss of sorting to the plasma membrane. To identify further the apical sorting signal and mechanismof the membranelocalization,truncated and chimeric ASBT were generated.Taurocholateinflux assays in a transwell culture system and confocal microscopy were used to detect polarized expression of wild type and mutant ASBT in transfected cells. The results demonstrate that removal of the N-glycans present on the Nterminus of ASBT by tunicamycin treatment did not alter the polarized apical expression of ASBTin transfected MDCKcell, as is the casefor some other apically sorted proteins. Deletion of the last 14 amino acids (aa 334-348) from the C-terminus of ASBT abolished the apical expressionof the mutant ASBT. Moreover, replacementof the cytoplasmic tail of Ntcp, a liver basolateral protein, with the last 14 aa peptide of ASBT redirectedthe chimera predominantly to the apical compartment.The resultsfrom the treatments with brefeldinA (BFA)or monensin, which inhibit vesicular transport, demonstrated that BFA disrupts the polarized localization of ASBT, while monensin had no effect. In addition, low temperature shift (20°C), which blocks membrane sorting of some polytopic proteins at Golgi complex, did not affect the polarized apical surface sorting of ASBT. In summary, the above results suggest that (1) a novel 14 aa peptide apical sorting signal is present within ASBT cytoplasmic tail. (2) ASBT follows an apical sorting pathway that is delivered via BFA-ssnsitive mechanism and is independent of protein glycosylation and low temperature shift.
5 Molecular Structure of the Bile Salt Export Pump Gene (Abcb11) Reveals a Polymorphism Between Gallstone-Susceptible and Resistant Inbred Mice Anne Figge, B.~rbelTaenzler, Dept of Medicine III, Univ of Technology (RWTH), Aachen Germany; BeverlyJ. Paigen,The Jackson Lab, Bar Harbor, ME; Siegfried Matern, Frank Lammert, Oept of Medicine III, Univ of Technology (RWTH), Aachen Germany Background:The mouse geneencoding the hepatic canalicularbile salt export pump (Abcb11, formerly Spgp) is a candidate for the major gallstone gene Lithl. Abcb11 is differentially expressed in gallstone-susceptible(C57UJ) and resistant (AKR/J) inbred mice, The aim of this study was to investigatethe identity of Lithl and Abcb11 by dissecting the structure of the gene and conducting a molecular comparison of the mouse strains. Methods: Adaptor ligated PCRwas usedto generateunknown 5' flanking sequenceof the Abcb11gene. Bacterial Artificial Chromosomes (BAC) containing the Abcbll gene were identified by screening a genomic BAC library from the gallstone-susceptibleinbred mouse strain C57BU6J (RPCI23) and characterizedby pulse-field gel electmphoresis. A single BAC clone was submitted to the Trans-NIH Mouse Initiative for complete sequencing. Genomic DNA from strains C57L andAKR was comparedby SingleStrand ConformationPolymorphism(SSCP)analysis,which employsgel electrophoresisto detect nucleotidesequencepolymorphismsthat alter secondary structures of single-strandedDNA,and direct sequencingof PCRfragments. Results:Screening of the BAC library resulted in 8 Abcb11positive clones.A clone with a 180 kb insert containing both the 5' flanking region and the 3' end of the gene was chosen for sequencing.Alignment of the BAC sequence and the Abcbll cDNA revealed that the gene spans 66 kb and is comprised of 28 exons and introos ranging from 91 to 15285 bp. Sequencecomparison of the gallstone-susceptiblestrains C57L and C57BU6 and the resistant strain AKR showed no difference in the coding sequenceor the minimal promoter. However,a single polymorphism (C-T transition) was detected in the 5' flanking region of the gene. Conclusions: (1) The complete genestructure of the murine Abcb11 gene provides a basic framework for the study of molecular regulation of the bile salt export pump in geneticallydefined mouse models. (2) The detected polymorphism between the gallstone-susceptibleand resistant inbred strains bears a putative regulatory element for gene transcription of Abcb11, which could result in both differential Abcb11 expression and gallstone susceptibility. Supportedby Trans-NIH Mouse Inib'ative
3 UrsodeoxycholicAcid Feeding Aggravates Liver Injury in Bile Duct-ligated Mice Peter Fickert, Gernot Zollner, Andrea Fuchsbichler, Cenny Stumptner, KarI-FranzensUniv, Graz Austria; Frank Lammert, Univ of Aachen, Aachen Germany; Kurt Zatloukal, Helmut Denk, Michael Trauner, KarI-FranzensUniv, Graz Austria BACKGROUND& OBJECTIVES:Ursodeoxycholicacid (UDCA) has beneficialeffects in various cholestatic liver diseases(e.g., pimary biliary cirrhosis, primary sclerosing cholangitis, intrahepatic cholastasis of pregnancy, cystic fibrosis, etc.). However, it is unclear whether UDCA also improves liver injury caused by biliary obstruction. Therefore, it was the aim of this study to determine the effects of UDCA in obstructive cholestasis in mice. METHODS:Mice were fed a UDCA (0.5% w/w) supplementeddiet or control diet for 7 days. Thereafter mice were subjected to common bile duct ligation (CBDL; n=24)or selective bile duct ligation (SBDL; n = f 2)and continuedon UDCAor control diet for further 3 days. In anotherexperimental series, mice were fed UDCA only after CBDL (n=6), Mortality rates, serum liver enzymes, total serum bile acid levels, bile composition, and liver histology were investigated.RESULTS: CBDLand SBDL(ligated lobes) in control diet-fed mice resulted in disseminatedhepatocellular necrosis, bile infarct, and periductular fibrosis, whereas non-ligated lobes (SBDL) remained unchanged. CBDL in UDCA-preted mice caused more severe hepatocellular necrosis and significantly increasedmortality rates comparedwith CBDL in control mice receivingstandard diet (7/12 vs. 1/12 in controls; p
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