Mathieu Vinken, Brigitte Landesmann, Marina Goumenou, Stefanie Vinken, Imran Shah, Hartmut Jaeschke, Catherine Willett, Maurice Whelan and Vera Rogiers.
Point of Contact
- Mathieu Vinken
|Author status||OECD status||OECD project||SAAOP status|
|Under development: Not open for comment. Do not cite||Under Development||1.19||Included in OECD Work Plan|
This AOP was last modified on December 03, 2016 16:37
|Inhibition, Bile Salt Export Pump (ABCB11)||September 16, 2017 10:14|
|Activation of specific nuclear receptors, Transcriptional change||September 16, 2017 10:14|
|Bile accumulation, Pathological condition||September 16, 2017 10:14|
|Release, Cytokine||September 16, 2017 10:14|
|Increase, Inflammation||September 16, 2017 10:14|
|Production, Reactive oxygen species||December 03, 2016 16:37|
|Peptide Oxidation||November 13, 2017 10:22|
|Cholestasis, Pathology||December 03, 2016 16:33|
|Bile accumulation, Pathological condition leads to Activation of specific nuclear receptors, Transcriptional change||December 03, 2016 16:37|
|Activation of specific nuclear receptors, Transcriptional change leads to Cholestasis, Pathology||December 03, 2016 16:37|
|Release, Cytokine leads to Increase, Inflammation||December 03, 2016 16:37|
|Production, Reactive oxygen species leads to Peptide Oxidation||December 03, 2016 16:37|
|Inhibition, Bile Salt Export Pump (ABCB11) leads to Bile accumulation, Pathological condition||December 03, 2016 16:38|
|Bile accumulation, Pathological condition leads to Release, Cytokine||December 03, 2016 16:38|
|Increase, Inflammation leads to Cholestasis, Pathology||December 03, 2016 16:38|
|Bile accumulation, Pathological condition leads to Production, Reactive oxygen species||December 03, 2016 16:38|
|Peptide Oxidation leads to Cholestasis, Pathology||December 03, 2016 16:38|
Adverse outcome pathways (AOPs) have been recently introduced in human risk assessment as pragmatic tools with multiple applications. As such, AOPs intend to provide a clear-cut mechanistic representation of pertinent toxicological effects. AOPs are typically composed of a molecular initiating event, a series of intermediate steps and key events, and an adverse outcome. In the current study, an AOP framework is proposed for cholestasis triggered by drug-mediated inhibition of the bile salt export pump transporter protein. For this purpose, an in-depth survey of relevant scientific literature was carried out in order to identify intermediate steps and key events. The latter include bile accumulation, the induction of oxidative stress and inflammation, and the activation of specific nuclear receptors. Collectively, these mechanisms drive both a deteriorative cellular response, which underlies directly caused cholestatic injury, and an adaptive cellular response, which is aimed at counteracting cholestatic insults. AOP development was performed according to OECD guidance, including critical consideration of the Bradford Hill criteria for weight of evidence assessment and the OECD key questions for evaluating AOP confidence. The postulated AOP is expected to serve as the basis for the development of new in vitro tests and the characterization of novel biomarkers of drug-induced cholestasis.
Summary of the AOP
Molecular Initiating Event
|Inhibition, Bile Salt Export Pump (ABCB11)||Inhibition, Bile Salt Export Pump (ABCB11)|
|Activation of specific nuclear receptors, Transcriptional change||Activation of specific nuclear receptors, Transcriptional change|
|Bile accumulation, Pathological condition||Bile accumulation, Pathological condition|
|Release, Cytokine||Release, Cytokine|
|Increase, Inflammation||Increase, Inflammation|
|Production, Reactive oxygen species||Production, Reactive oxygen species|
|Peptide Oxidation||Peptide Oxidation|
|Cholestasis, Pathology||Cholestasis, Pathology|
Relationships Between Two Key Events (Including MIEs and AOs)
|Bile accumulation, Pathological condition leads to Activation of specific nuclear receptors, Transcriptional change||Directly leads to||Strong|
|Activation of specific nuclear receptors, Transcriptional change leads to Cholestasis, Pathology||Directly leads to||Strong|
|Release, Cytokine leads to Increase, Inflammation||Directly leads to||Strong|
|Production, Reactive oxygen species leads to Peptide Oxidation||Directly leads to||Strong|
|Inhibition, Bile Salt Export Pump (ABCB11) leads to Bile accumulation, Pathological condition||Directly leads to||Strong|
|Bile accumulation, Pathological condition leads to Release, Cytokine||Directly leads to||Strong|
|Increase, Inflammation leads to Cholestasis, Pathology||Directly leads to||Moderate|
|Bile accumulation, Pathological condition leads to Production, Reactive oxygen species||Directly leads to||Strong|
|Peptide Oxidation leads to Cholestasis, Pathology||Directly leads to||Moderate|
Life Stage Applicability
|Adult, reproductively mature||Strong|
Graphical RepresentationClick to download graphical representation template
Overall Assessment of the AOP
1. Concordance of dose-response relationships: Morgan and colleagues investigated the potential of more than 200 benchmark drugs to inhibit BSEP. As much as 16% of the tested drugs displayed high potency of BSEP inhibition (IC50 ≤ 25 µM), the majority of which are associated with liver liabilities in humans (Morgan et al., 2010). Likewise, 17 of 85 pharmaceuticals tested by Dawson and coworkers inhibited BSEP (IC50 ≤ 100 µM), all which are known to cause DILI (Dawson et al., 2011). Furthermore, several of the BSEP-inhibiting drugs cause cholestastic liver injury in a dose-dependent way, such as is the case for troglitazone and bosentan in rats (Funk et al., 2001) and humans (Fattinger et al., 2001), respectively. Thus, there is a clear relationship between the IC50 of BSEP inhibition and the occurrence of (cholestatic) DILI.
2. Temporal concordance among the key events and adverse effect: Inhibition of BSEP activity and the resulting accumulation of bile acids primarily triggers a direct cellular response, which is associated with deteriorative processes, such as inflammation, oxidative stress and cell death. It also causes a secondary and rather indirect cellular response, which is adaptive in nature. Indeed, a well-orchestrated network of mechanisms is activated, all of which are targeted towards the elimination of bile from the liver (Boyer, 2009; Zollner and Trauner, 2006 and 2008; Wagner et al., 2009). The temporal concordance between these cellular responses is not clear. It is, however, conceivable to assume that the adaptive response becomes manifested at a somewhat later stage when compared to the primary events, especially since this secondary response highly depends on transcriptional regulation. Nonetheless, it is clear that the graphical linear representation of cholestatic DILI resulting from BSEP inhibition as a sequence of events is an oversimplification of a probably very complex network of entangled consecutive and parallel reactions.
3. Strength, consistency, and specificity of association of adverse effect and initiating event: BSEP is considered as the major apical transporter protein that pumps bile salts from hepatocytes into bile canaliculi. As a part of this pivotal task, BSEP has a very narrow substrate specificity with only a few known non-bile substrates (Dawson et al., 2011; Kis et al., 2012; Morgan et al., 2010). Defects in BSEP expression or function therefore can be anticipated to have drastic consequences with respect to bile homeostasis. Indeed, a plethora of studies has demonstrated that BSEP inhibition or impairment is causally linked to the induction of cholestasis in a dose-dependent way, both in experimental animals and in humans (Zollner and Trauner, 2006 and 2008; Wagner et al., 2009). Thus, there is a well-established, direct, specific and quantitative association between BSEP inhibition and the onset of cholestatic DILI.
4. Biological plausibility, coherence, and consistency of the experimental evidence: In essence, BSEP inhibition (i.e. the MIE) activates a number of mechanisms that drive a deteriorative cellular response, which underlies directly caused cholestatic injury, as well as an adaptive cellular response, which is aimed at counteracting cholestatic insults. Both these responses contribute to the clinical manifestation of cholestasis (i.e. the AO). Serum concentrations of ALT, AST, ALP, GGT and 5’-NT indeed increase because of bile acid-induced membrane damage of hepatocytes and cholangiocytes (Hofmann, 2009; Kuntz and Kuntz, 2008; Padda et al., 2011). At the same time, elevated concentrations of bilirubin in serum and urine are observed, reflecting the compensatory response of the organism to counteract bile acid accumulation. Hyperbilirubinemia causes jaundice, while the increased presence of bile acids in serum is thought to induce pruritus (Hofmann, 2009; Kuntz and Kuntz, 2008; Padda et al., 2011; Zollner and Trauner, 2008).
5. Alternative mechanisms that logically present themselves and the extent to which they may distract from the postulated AOP. It should be noted that alternative mechanisms of action, if supported, require a separate AOP: Although predominant, BSEP inhibition is not the sole MIE in cholestatic DILI, as depicted in the established AOP. In this regard, cyclosporine A not only inhibits BSEP (Dawnson et al., 2011; Kis et al., 2012; Morgan et al., 2010), but also induces cholestasis by inhibition of intrahepatic vesicle transport (Roman et al., 1990) and by affecting canalicular membrane fluidity (Yasumiba et al., 2001). Several of these events, in particular the cytoskeletal changes, might be considered as secondary and non-specific phenomena (Trauner et al., 1998b). Nevertheless, separate AOPs could be drafted for each of these alternative mechanisms in cholestatic DILI.
6. Uncertainties, inconsistencies and data gaps: Although a clear causal and dose-dependent relationship has been established between BSEP inhibition and the clinical onset of cholestasis (Zollner and Trauner, 2006 and 2008; Wagner et al., 2009), several mechanisms of the intermediate steps and key events as well as their linkage are not fully understood. A prominent discussion in this respect relates to the nature of the cell death mode, namely apoptosis or necrosis, associated with cholestasis (Woolbright and Jaeschke, 2012). High concentrations of hydrophobic bile acids induce apoptotic cell death in cultures of primary hepatocytes (Gores et al., 1998; Botla et al., 1995; Schoemaker et al., 2004), yet such concentrations are not achieved in vivo (Zhang et al., 2012). It has therefore been suggested that the main mechanism of cell death in cholestasis in vivo is necrosis (Woolbright and Jaeschke, 2012). In fact, this seems to be a general consideration of the AOP, as several other constituting data also have been derived from in vitro experimentation and need to be substantiated in vivo. On the other hand, a number of data are still lacking, including the full identification of FXR, PXR and CAR target genes, which may additionally contribute to the adaptive response to BSEP inhibition. Furthermore, ongoing research regarding the regulation of these nuclear receptors in cholestatic DILI might add complexity to the AOP. It is known that they act, at least in part, by recruiting co-activators and co-repressors (Gollamudi et al., 2008; Wagner et al., 2009). Moreover, compelling evidence suggests that nuclear receptors are regulated epigenetically, which might necessitate inclusion in the AOP (Elloranta and Kullak-Ublick, 2005; Wagner et al., 2009). Additional uncertainties, inconsistencies and data gaps associated with the established AOP relate to the temporal concordance of the intermediate steps and key events, the consistency of the available experimental data, alternative mechanisms involved, interspecies and intraspecies differences.
Domain of Applicability
Essentiality of the Key Events
Weight of Evidence Summary
Considerations for Potential Applications of the AOP (optional)
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Confidence in the AOP
Information from this section should be moved to the Key Event Relationship pages!
1. How well characterized is the AOP? Drug-induced cholestasis is a well understood AO that is causally and dose-dependently linked to BSEP inhibition, being the predominant MIE (Dawson et al., 2011; Kis et al., 2012; Morgan et al., 2010). Furthermore, the critical role of the key events, namely the accumulation of bile, the induction of inflammation and oxidative stress and the activation of specific nuclear receptors, as well as the different intermediate steps in the AOP, is supported by a wealth of experimental data. Thus, despite a number of limitations in scientific evidence, as will be discussed further, the established general structure and components of the AOP can be considered as being well-characterized.
2. How well are the initiating and other key events causally linked to the outcome? It has been demonstrated on numerous occasions that BSEP inhibition is causally linked to the induction of cholestasis in a dose-dependent way, both in experimental animals and in humans (Zollner and Trauner, 2006 and 2008; Wagner et al., 2009). BSEP inhibition directly leads to bile accumulation, which subsequently activates an inflammatory reaction as well as the occurrence of oxidative stress (Gujral et al., 2003 and 2004; Jaeschke and Hasegawa, 2006; Woolbright and Jaeschke, 2012). The resulting cell death and associated bile acid-induced membrane damage of hepatocytes and cholangiocytes underlie the increased serum concentrations of ALT, AST, ALP, GGT and 5’-NT, being a prominent clinical hallmark of cholestasis (Hofmann, 2009; Kuntz and Kuntz, 2008; Padda et al., 2011). To compensate for the cholestatic insults, an adaptive response is induced, which is initiated by nuclear receptor activation and that is targeted towards the elimination of bile from the organism (Boyer, 2009; Zollner and Trauner, 2006 and 2008; Wagner et al., 2009). Consequently, elevated concentrations of bilirubin in serum and urine are observed. The former causes jaundice, while the increased presence of bile acids in serum is thought to induce pruritus (Hofmann, 2009; Kuntz and Kuntz, 2008; Padda et al., 2011; Zollner and Trauner, 2008). Thus, there is a direct, causal and, at least in some cases, quantitative association between the MIE, the key events and the AO in the established AOP.
3. What are the limitations in the evidence in support of the AOP? There are a number of limitations in the scientific evidence in support of the AOP in relation to temporal concordance of the intermediate steps and key events, the consistency of the available experimental data, alternative mechanisms involved, interspecies and intraspecies differences, uncertainties, inconsistencies and data gaps. These shortcomings are addressed in previous sections.
4. Is the AOP specific to certain tissues, life stages or age classes? Although the entire process of drug-induced cholestasis mainly takes place in the liver, more specifically in hepatocytes, the adaptive changes to this insult also occur in other tissues, including the kidney and the intestine. In this context, expression of MRP2 is induced in renal tubular cells in experimental models of cholestasis (Lee et al., 2001). Alterations in transporter protein expression during cholestasis also occur in other tissues, such as the ileum (Mennone et al., 2010). Like in the liver, the overall goal of these alterations is to increase elimination of bile salts via the urine and feces (Zollner and Trauner, 2006 and 2008; Wagner et al., 2009). Regarding age-specificity, no significant quantitative differences in BSEP expression between fetal and human liver have been detected (Chen et al., 2005). Hepatocellular accumulation of bile acids causes giant cell hepatitis and progressive liver damage in children (Oude Elferink et al., 2006), which may burgeon into hepatocellular carcinoma (Bernstein et al., 2005; Knisely et al., 2006). On the other hand, it is well established that age over 50 years poses an increased risk to develop drug-induced hepatic damage (Pauli-Magnus and Meier, 2006) and that DILI in elder people is of cholestatic rather than of hepatocelluar nature (Lucena et al., 2009). In general, women are more susceptible for developing DILI than men (Pauli-Magnus and Meier, 2006), yet no gender differences exist in liver BSEP expression in humans (Cheng et al., 2007). In contrast, male rats are more prone to troglitazone-induced cholestasis than female rats because of higher rates of troglitazone sulphate formation (Funk et al., 2001; Kostrubsky et al., 2001). At the population level, genetic variability in the BSEP gene, leading to its decreased expression, may predispose different ethnic populations to drug-induced cholestasis (Lang et al., 2006 and 2007; Meier et al., 2006).
5. Are the initiating and key events expected to be conserved across taxa? Standard animal studies conducted during drug development, using mainly rodents, usually pick up about half of all human hepatotoxic compounds because of interspecies differences (Blomme et al., 2009; Ozer et al., 2008). In the case of BSEP inhibition, however, interspecies differences are mostly of quantitative nature. This could be due to the fact that there is a high amino acid similarity between human BSEP and its rodent counterparts, namely 80% in mouse and 82% in rat (Green et al., 2000; Lecureur et al., 2000). Accordingly, while IC50 values for BSEP inhibition differ only minimally between human and mouse for troglitazone, they differ by almost an order of magnitude for glibenclamide (Kis et al., 2012). Other reasons for the absence of hepatotoxicity induced by human-relevant cholestatic drugs in rats include higher rates of basolateral bile salt efflux, which could represent an additional protective mechanism against cholestasis (Jemnitz et al., 2010). In addition to BSEP inhibition, the key events of the proposed AOP are expected to be generally well conserved among taxa. Nevertheless, a recent report showed that considerable differences exist in inflammatory responses between human and mouse (Seok et al., 2013). Despite the occurrence of interspecies differences in their expression or ligand binding, such as shown for PXR (Krasowski et al., 2005), activation of nuclear receptors is a critical event in different animal models of cholestasis (Wagner et al., 2009; Zollner and Trauner, 2006 and 2008). It remains to be established whether data included in the AOP can be extrapolated from animals to humans and vice versa.