Bile Acid-Induced Mallory Body Formation in Drug-Primed Mouse Liver

Abstract

Chronic cholestasis is associated with retention of bile acids and profound cytoskeletal alterations in hepatocytes including Mallory body (MB) formation. The mechanisms responsible for MB formation in cholestatic liver diseases are unclear. The aim of our study was to determine the relevance of cholestasis and bile acids for MB formation. For this purpose mice received a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-supplemented diet for 2.5 months to induce MB formation. After recovery from DDC intoxication for 4 weeks followed by disappearance of MBs, these drug-primed mice were subjected to DDC refeeding, common bile duct ligation (CBDL), and feeding of a cholic acid (CA)-supplemented diet for 7 days, respectively. Cytokeratin (CK) 8 and CK 18 expression was studied by competitive reverse transcriptase-polymerase chain reaction and Western blot analysis. Cytoskeletal alterations of hepatocytes and MB formation were monitored by immunofluorescence microscopy and immunohistochemistry using CK-, ubiquitin-, and MB-specific antibodies. Like DDC refeeding, both CBDL and CA feeding of drug-primed mice significantly increased CK 8 and CK 18 mRNA and protein levels (with excess of CK 8) and resulted in ubiquitination and abnormal phosphorylation of CKs. Furthermore, CBDL and CA feeding resulted in rapid neoformation of MBs in drug-primed mice. It is concluded that MB formation in cholestatic liver diseases may be triggered by the action of potentially toxic bile acids. 1 The CK subfamily has more than 20 members forming heteropolymers of type I and type II CKs.1 CK 8 and CK 18 are subunits of the IFs of hepatocytes and were also identified as major components of Mallory bodies (MBs) associated with certain human liver diseases and related mouse models.2 MBs are characteristic cytoplasmic hyaline inclusions in hepatocytes reflecting a peculiar morphological manifestation of chronic liver cell injury.2,3 Their appearance is related to alterations of the CK-IF cytoskeleton including overexpression and posttranslational modifications of CKs (eg, cross-linking, abnormal phosphorylation, ubiquitination).2,4 -10 In humans, MBs are typically associated with alcoholic and nonalcoholic steatohepatitis, but are also found in chronic cholestatic conditions such as primary biliary cirrhosis and primary sclerosing cholangitis. 2,8,11 A common denominator of these etiologically different liver diseases is their association with cholestasis and elevated serum bile acid levels. In mice, MBs can be induced by chronic griseofulvin (GF) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) intoxication.12-15 Administration of these porphyrinogenic agents also induces cholestasis in mice indicated by elevated serum bile acid levels that may at least partly result from the formation of protoporphyrin plugs and stones obstructing the bile drainage system. 16,17 MB formation requires prolonged intoxication (ϳ2.5 months) with GF or DDC.2,4 Interestingly, after recovery from intoxication with disappearance of MBs, which takes ϳ4 weeks (primed...

Role of nuclear receptors and hepatocyte-enriched transcription factors for Ntcp repression in biliary obstruction in mouse liver

“…Controls underwent a sham operation with exposure, but without ligation, of the common bile duct and without removal of the gallbladder. To prevent eventual rupture of the gallbladder as a result of increased biliary pressure and subsequent biliary peritonitis, prophylactic cholecystectomy was performed as described previously (11,13,47,56). The livers were excised 3 and 7 days after CBDL under general anesthesia with tribromoethanol (Avertin, Sigma-Aldrich, Steinheim, Germany; 10 mg ip) and immediately snap frozen and stored in liquid nitrogen until RNA extraction.…”

Animal models of chronic liver diseases

“…DDC diet. 3,5-Diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding in mice is a well-established model to study MalloryDenk body formation, which is specifically associated with alcoholic and nonalcoholic steatohepatitis, metabolic liver diseases, and chronic cholestatic liver diseases (44,225). Recently, the DDC diet was also suggested to be used as a model for sclerosing cholangitis and biliary fibrosis.…”

Genetic background effects of keratin 8 and 18 in a DDC-induced hepatotoxicity and Mallory-Denk body formation mouse model

“…A macro-command served for automatic identification and measurement of the total area of OPN þ cells within a given portal field. All portal fields (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31) in each sample were measured.…”

“…27 Quantitative Real-Time PCR Routine protocols were performed. Primers and probes (synthesized by MWG-Biotech AG, Germany) are listed in Table 1.…”