Robustness testing and optimization of an adverse outcome pathway on cholestatic liver injury

Abstract

Adverse outcome pathways (AOPs) have been recently introduced as tools to map the mechanisms underlying toxic events relevant for chemical risk assessment. AOPs particularly depict the linkage between a molecular initiating event and an adverse outcome through a number of intermediate key events. An AOP has been previously introduced for cholestatic liver injury. The objective of this study was to test the robustness of this AOP for different types of cholestatic insult and the in vitro to in vivo extrapolation. For this purpose, in vitro samples from human hepatoma HepaRG cell cultures were exposed to cholestatic drugs (i.e. intrahepatic cholestasis), while in vivo samples were obtained from livers of cholestatic mice (i.e. extrahepatic cholestasis). The occurrence of cholestasis in vitro was confirmed through analysis of bile transporter functionality and bile acid analysis. Transcriptomic analysis revealed inflammation and oxidative stress as key events in both types of cholestatic liver injury. Major transcriptional differences between intrahepatic and extrahepatic cholestatic liver insults were observed at the level of cell death and metabolism. Novel key events identified by pathway analysis included endoplasmic reticulum stress in intrahepatic cholestasis, and autophagy and necroptosis in both intrahepatic as extrahepatic cholestasis. This study demonstrates that AOPs constitute dynamic tools that should be frequently updated with new input information.

Cholestasis Differentially Affects Liver Connexins

“…The presence of the concentrated mix of BAs in the in vitro system creates an environment more comparable to the in vivo situation, since cholestasis patients present with 30-50× increased concentrations of serum BA [21,56,57]. The addition of BAs seems to potentiate the effect of cholestatic drugs [31]. The sensitizing effect of BAs was also observed for Cx26, as mRNA levels were reduced following exposure of the human hepatoma HepaRG cells to cholestatic drugs and BAs.…”

“…In general, the outcome of cholestasis on Cx43 in vitro seems to depend on the nature of the drug. In this light, three triggering factors of DIC have been identified, namely transporter changes, hepatocellular changes and altered bile canaliculi dynamics [31], where all are differentially affected by ATV, CsA and NEF. These triggering factors result in BA accumulation, which in turn induces two cellular responses.…”

“…DIC is of high clinical concern and therefore constitutes a major focus of the current study. Our group recently introduced a new in vitro system to investigate DIC, relying on the treatment of human hepatoma HepaRG cell cultures for 3 days with a concentrated BA mixture and a cholestatic drug, namely atazanavir (ATV), cyclosporin A (CsA) or nefazodone (NEF) [31]. The present study combines this in vitro system with the BDL model, thus providing two experimental settings originating from two different species to study two different types of cholestasis.…”

Liver Adverse Outcome Pathways: What’s in for the Hepatologist?

“…The latter is critical in view of appropriate application. In this context, a recent study showed that the only available AOP on cholestatic liver injury seems fit to model the intrahepatic type of cholestasis, but less for the extrahepatic counterpart [23]. This suggests that more than one AOP is required to mechanistically describe a liver disorder with different etiology.…”

Neurotoxicity and underlying cellular changes of 21 mitochondrial respiratory chain inhibitors

“…This means that the evidence was collected from heterogeneous sources in the scientific literature. Such studies were not designed originally to support building an AOP, and there are only very few recent examples of AOPs that are based on dedicated research to build the AOP and to parameterize KERs (Browne et al 2015(Browne et al , 2017Gijbels et al 2020;Pistollato et al 2020;Spinu et al 2019).…”