Effect of bile duct ligation on bile acid composition in mouse serum and liver

Abstract

Background Cholestatic liver diseases can be caused by genetic defects, drug toxicities, hepatobiliary malignancies or obstruction of the biliary tract. Cholestasis leads to accumulation of bile acids (BAs) in hepatocytes. Direct toxicity of BAs is currently the most accepted hypothesis for cholestatic liver injury. However, information on which bile acids are actually accumulating during cholestasis is limited. Aims Assess BA composition in liver and serum after bile duct ligation (BDL) in male C57Bl/6 mice between 6 h and 14 days and evaluate toxicity of most abundant BAs. Results BA concentrations increased in liver (27-fold) and serum (1400-fold) within 6 h after surgery and remained elevated up to 14 days. BAs in livers of BDL mice became more hydrophilic than sham controls, mainly due to increased 6β-hydroxylation and taurine conjugation. Among the 8 unconjugated and 16 conjugated BAs identified in serum and liver, only taurocholic acid (TCA), β-muricholic acid (βMCA) and TβMCA were substantially elevated representing >95% of these BAs over the entire time course. Although glycochenodeoxycholic acid and other conjugated BAs increased in BDL animals, the changes were several orders of magnitude lower compared to TCA, βMCA and TβMCA. A mixture of these BAs did not cause apoptosis or necrosis but induced inflammatory gene expression in cultured murine hepatocytes. Conclusion The concentrations of cytotoxic BAs are insufficient to cause hepatocellular injury. In contrast, TCA, βMCA and TβMCA are able to induce pro-inflammatory mediators in hepatocytes. Thus, BAs act as inflammagens and not as cytotoxic mediators after BDL in mice.

Bile acids initiate cholestatic liver injury by triggering a hepatocyte-specific inflammatory response

“…Recent studies suggest that BA may induce liver injury via an inflammatory response, as neutrophil infiltration correlated with the injury in cholestatic rodent models (9,10). This notion is further strengthened by the observation that BA stimulated the expression of inflammatory cytokines in cultured mouse hepatocytes (6,11). This induction is partially dependent on the transcription factor early growth response 1 (Egr1), although it is not clear how BAs activate Egr1.…”

“…sera from cholestatic patients as well as sera of mice after 14-day BDL (6,23). Normally, once BAs get into hepatocytes, they are rapidly excreted into the lumen of the bile canaliculus.…”

“…This induction is partially dependent on the transcription factor early growth response 1 (Egr1), although it is not clear how BAs activate Egr1. Elevated levels of cytokines such as Ccl2, Cxcl2, and IL-8 were also detected in the serum and liver of patients with cholestasis and in cholestatic animal models (6,(11)(12)(13). Antagonizing inflammatory cytokines reduced liver injury in BDL mice (14)(15)(16), further supporting the notion of the role of the inflammatory response in Mechanisms of bile acid-induced (BA-induced) liver injury in cholestasis are controversial, limiting development of new therapies.…”

“…This assumption is based primarily on in vitro observations from cultured cells that were exposed to high concentrations of toxic BAs (3,4). However, such high concentrations are rarely achieved in the serum of cholestatic patients and animal models (5,6). Most importantly, apoptosis of hepatocytes has not been detected in vivo in the liver of bile duct-ligated (BDL) mice or in vitro in BA-treated human hepatocytes (7,8), suggesting that BA-induced liver injury must occur by alternative mechanisms.…”

Chimeric TK-NOG Mice: A Predictive Model for Cholestatic Human Liver Toxicity

“…Bosentan's effect on bile transport resembles (in some ways) that of bile duct ligation, and humans and rodents exhibit very different responses to biliary obstruction. In mice (Zhang et al, 2012) and rats (Takita et al, 1988;Naito et al, 1996), bile duct ligation causes a rapid increase in the synthesis of mostly (95%) water-soluble muricholic acids that are excreted in the urine, which provides rodents with a route for elimination of hepatotoxic bile acids (Dueland et al, 1991;Naito et al, 1996). In contrast, human liver produces a very different set of hepatotoxic bile acids (chenodeoxycholic acid, cholic acid, and their glycine conjugates) (Russell, 2003), which could be more difficult to eliminate when BSEP is inhibited.…”