Aop: 27

Title

Each AOP should be given a descriptive title that takes the form “MIE leading to AO”. For example, “Aromatase inhibition [MIE] leading to reproductive dysfunction [AO]” or “Thyroperoxidase inhibition [MIE] leading to decreased cognitive function [AO]”. In cases where the MIE is unknown or undefined, the earliest known KE in the chain (i.e., furthest upstream) should be used in lieu of the MIE and it should be made clear that the stated event is a KE and not the MIE. More help

Cholestatic Liver Injury induced by Inhibition of the Bile Salt Export Pump (ABCB11)

Short name
A short name should also be provided that succinctly summarises the information from the title. This name should not exceed 90 characters. More help
Cholestatic Liver Injury induced by Inhibition of the Bile Salt Export Pump (ABCB11)

Graphical Representation

A graphical summary of the AOP listing all the KEs in sequence, including the MIE (if known) and AO, and the pair-wise relationships (links or KERs) between those KEs should be provided. This is easily achieved using the standard box and arrow AOP diagram (see this page for example). The graphical summary is prepared and uploaded by the user (templates are available) and is often included as part of the proposal when AOP development projects are submitted to the OECD AOP Development Workplan. The graphical representation or AOP diagram provides a useful and concise overview of the KEs that are included in the AOP, and the sequence in which they are linked together. This can aid both the process of development, as well as review and use of the AOP (for more information please see page 19 of the Users' Handbook).If you already have a graphical representation of your AOP in electronic format, simple save it in a standard image format (e.g. jpeg, png) then click ‘Choose File’ under the “Graphical Representation” heading, which is part of the Summary of the AOP section, to select the file that you have just edited. Files must be in jpeg, jpg, gif, png, or bmp format. Click ‘Upload’ to upload the file. You should see the AOP page with the image displayed under the “Graphical Representation” heading. To remove a graphical representation file, click 'Remove' and then click 'OK.'  Your graphic should no longer be displayed on the AOP page. If you do not have a graphical representation of your AOP in electronic format, a template is available to assist you.  Under “Summary of the AOP”, under the “Graphical Representation” heading click on the link “Click to download template for graphical representation.” A Powerpoint template file should download via the default download mechanism for your browser. Click to open this file; it contains a Powerpoint template for an AOP diagram and instructions for editing and saving the diagram. Be sure to save the diagram as jpeg, jpg, gif, png, or bmp format. Once the diagram is edited to its final state, upload the image file as described above. More help

Authors

List the name and affiliation information of the individual(s)/organisation(s) that created/developed the AOP. In the context of the OECD AOP Development Workplan, this would typically be the individuals and organisation that submitted an AOP development proposal to the EAGMST. Significant contributors to the AOP should also be listed. A corresponding author with contact information may be provided here. This author does not need an account on the AOP-KB and can be distinct from the point of contact below. The list of authors will be included in any snapshot made from an AOP. More help

Mathieu Vinken, Brigitte Landesmann, Marina Goumenou, Stefanie Vinken, Imran Shah, Hartmut Jaeschke, Catherine Willett, Maurice Whelan and Vera Rogiers.

Point of Contact

Indicate the point of contact for the AOP-KB entry itself. This person is responsible for managing the AOP entry in the AOP-KB and controls write access to the page by defining the contributors as described below. Clicking on the name will allow any wiki user to correspond with the point of contact via the email address associated with their user profile in the AOP-KB. This person can be the same as the corresponding author listed in the authors section but isn’t required to be. In cases where the individuals are different, the corresponding author would be the appropriate person to contact for scientific issues whereas the point of contact would be the appropriate person to contact about technical issues with the AOP-KB entry itself. Corresponding authors and the point of contact are encouraged to monitor comments on their AOPs and develop or coordinate responses as appropriate.  More help
Arthur Author   (email point of contact)

Contributors

List user names of all  authors contributing to or revising pages in the AOP-KB that are linked to the AOP description. This information is mainly used to control write access to the AOP page and is controlled by the Point of Contact.  More help
  • Mathieu Vinken
  • Arthur Author

Status

The status section is used to provide AOP-KB users with information concerning how actively the AOP page is being developed, what type of use or input the authors feel comfortable with given the current level of development, and whether it is part of the OECD AOP Development Workplan and has been reviewed and/or endorsed. “Author Status” is an author defined field that is designated by selecting one of several options from a drop-down menu (Table 3). The “Author Status” field should be changed by the point of contact, as appropriate, as AOP development proceeds. See page 22 of the User Handbook for definitions of selection options. More help
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 April 05, 2021 18:16
The date the AOP was last modified is automatically tracked by the AOP-KB. The date modified field can be used to evaluate how actively the page is under development and how recently the version within the AOP-Wiki has been updated compared to any snapshots that were generated. More help

Revision dates for related pages

Page Revision Date/Time
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 January 30, 2019 10:26
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

Abstract

In the abstract section, authors should provide a concise and informative summation of the AOP under development that can stand-alone from the AOP page. Abstracts should typically be 200-400 words in length (similar to an abstract for a journal article). Suggested content for the abstract includes the following: The background/purpose for initiation of the AOP’s development (if there was a specific intent) A brief description of the MIE, AO, and/or major KEs that define the pathway A short summation of the overall WoE supporting the AOP and identification of major knowledge gaps (if any) If a brief statement about how the AOP may be applied (optional). The aim is to capture the highlights of the AOP and its potential scientific and regulatory relevance More help

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.

Background (optional)

This optional subsection should be used to provide background information for AOP reviewers and users that is considered helpful in understanding the biology underlying the AOP and the motivation for its development. The background should NOT provide an overview of the AOP, its KEs or KERs, which are captured in more detail below. Examples of potential uses of the optional background section are listed on pages 24-25 of the User Handbook. More help

Summary of the AOP

This section is for information that describes the overall AOP. The information described in section 1 is entered on the upper portion of an AOP page within the AOP-Wiki. This is where some background information may be provided, the structure of the AOP is described, and the KEs and KERs are listed. More help

Events:

Molecular Initiating Events (MIE)
An MIE is a specialised KE that represents the beginning (point of interaction between a stressor and the biological system) of an AOP. More help
Key Events (KE)
This table summarises all of the KEs of the AOP. This table is populated in the AOP-Wiki as KEs are added to the AOP. Each table entry acts as a link to the individual KE description page.  More help
Adverse Outcomes (AO)
An AO is a specialised KE that represents the end (an adverse outcome of regulatory significance) of an AOP.  More help
Sequence Type Event ID Title Short name
1 MIE 41 Inhibition, Bile Salt Export Pump (ABCB11) Inhibition, Bile Salt Export Pump (ABCB11)
2 KE 288 Activation of specific nuclear receptors, Transcriptional change Activation of specific nuclear receptors, Transcriptional change
3 KE 214 Bile accumulation, Pathological condition Bile accumulation, Pathological condition
4 KE 87 Release, Cytokine Release, Cytokine
5 KE 149 Increase, Inflammation Increase, Inflammation
6 KE 249 Production, Reactive oxygen species Production, Reactive oxygen species
7 KE 209 Peptide Oxidation Peptide Oxidation
8 AO 357 Cholestasis, Pathology Cholestasis, Pathology

Relationships Between Two Key Events (Including MIEs and AOs)

TESTINGThis table summarises all of the KERs of the AOP and is populated in the AOP-Wiki as KERs are added to the AOP. Each table entry acts as a link to the individual KER description page.To add a key event relationship click on either Add relationship: events adjacent in sequence or Add relationship: events non-adjacent in sequence.For example, if the intended sequence of KEs for the AOP is [KE1 > KE2 > KE3 > KE4]; relationships between KE1 and KE2; KE2 and KE3; and KE3 and KE4 would be defined using the add relationship: events adjacent in sequence button.  Relationships between KE1 and KE3; KE2 and KE4; or KE1 and KE4, for example, should be created using the add relationship: events non-adjacent button. This helps to both organize the table with regard to which KERs define the main sequence of KEs and those that provide additional supporting evidence and aids computational analysis of AOP networks, where non-adjacent KERs can result in artifacts (see Villeneuve et al. 2018; DOI: 10.1002/etc.4124).After clicking either option, the user will be brought to a new page entitled ‘Add Relationship to AOP.’ To create a new relationship, select an upstream event and a downstream event from the drop down menus. The KER will automatically be designated as either adjacent or non-adjacent depending on the button selected. The fields “Evidence” and “Quantitative understanding” can be selected from the drop-down options at the time of creation of the relationship, or can be added later. See the Users Handbook, page 52 (Assess Evidence Supporting All KERs for guiding questions, etc.).  Click ‘Create [adjacent/non-adjacent] relationship.’  The new relationship should be listed on the AOP page under the heading “Relationships Between Two Key Events (Including MIEs and AOs)”. To edit a key event relationship, click ‘Edit’ next to the name of the relationship you wish to edit. The user will be directed to an Editing Relationship page where they can edit the Evidence, and Quantitative Understanding fields using the drop down menus. Once finished editing, click ‘Update [adjacent/non-adjacent] relationship’ to update these fields and return to the AOP page.To remove a key event relationship to an AOP page, under Summary of the AOP, next to “Relationships Between Two Key Events (Including MIEs and AOs)” click ‘Remove’ The relationship should no longer be listed on the AOP page under the heading “Relationships Between Two Key Events (Including MIEs and AOs)”. More help

Network View

The AOP-Wiki automatically generates a network view of the AOP. This network graphic is based on the information provided in the MIE, KEs, AO, KERs and WoE summary tables. The width of the edges representing the KERs is determined by its WoE confidence level, with thicker lines representing higher degrees of confidence. This network view also shows which KEs are shared with other AOPs. More help

Stressors

The stressor field is a structured data field that can be used to annotate an AOP with standardised terms identifying stressors known to trigger the MIE/AOP. Most often these are chemical names selected from established chemical ontologies. However, depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). It can also include non-chemical stressors such as genetic or environmental factors. Although AOPs themselves are not chemical or stressor-specific, linking to stressor terms known to be relevant to different AOPs can aid users in searching for AOPs that may be relevant to a given stressor. More help

Life Stage Applicability

Identify the life stage for which the KE is known to be applicable. More help
Life stage Evidence
Adult, reproductively mature High

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
humans Homo sapiens High NCBI

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help
Sex Evidence
Mixed High

Overall Assessment of the AOP

This section addresses the relevant biological domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and WoE for the overall AOP as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). The goal of the overall assessment is to provide a high level synthesis and overview of the relative confidence in the AOP and where the significant gaps or weaknesses are (if they exist). Users or readers can drill down into the finer details captured in the KE and KER descriptions, and/or associated summary tables, as appropriate to their needs.Assessment of the AOP is organised into a number of steps. Guidance on pages 59-62 of the User Handbook is available to facilitate assignment of categories of high, moderate, or low confidence for each consideration. While it is not necessary to repeat lengthy text that appears elsewhere in the AOP description (or related KE and KER descriptions), a brief explanation or rationale for the selection of high, moderate, or low confidence should be made. More help

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

The relevant biological domain(s) of applicability in terms of sex, life-stage, taxa, and other aspects of biological context are defined in this section. Biological domain of applicability is informed by the “Description” and “Biological Domain of Applicability” sections of each KE and KER description (see sections 2G and 3E for details). In essence the taxa/life-stage/sex applicability is defined based on the groups of organisms for which the measurements represented by the KEs can feasibly be measured and the functional and regulatory relationships represented by the KERs are operative.The relevant biological domain of applicability of the AOP as a whole will nearly always be defined based on the most narrowly restricted of its KEs and KERs. For example, if most of the KEs apply to either sex, but one is relevant to females only, the biological domain of applicability of the AOP as a whole would be limited to females. While much of the detail defining the domain of applicability may be found in the individual KE and KER descriptions, the rationale for defining the relevant biological domain of applicability of the overall AOP should be briefly summarised on the AOP page. More help

Essentiality of the Key Events

An important aspect of assessing an AOP is evaluating the essentiality of its KEs. The essentiality of KEs can only be assessed relative to the impact of manipulation of a given KE (e.g., experimentally blocking or exacerbating the event) on the downstream sequence of KEs defined for the AOP. Consequently evidence supporting essentiality is assembled on the AOP page, rather than on the independent KE pages that are meant to stand-alone as modular units without reference to other KEs in the sequence.The nature of experimental evidence that is relevant to assessing essentiality relates to the impact on downstream KEs and the AO if upstream KEs are prevented or modified. This includes: Direct evidence: directly measured experimental support that blocking or preventing a KE prevents or impacts downstream KEs in the pathway in the expected fashion. Indirect evidence: evidence that modulation or attenuation in the magnitude of impact on a specific KE (increased effect or decreased effect) is associated with corresponding changes (increases or decreases) in the magnitude or frequency of one or more downstream KEs.When assembling the support for essentiality of the KEs, authors should organise relevant data in a tabular format. The objective is to summarise briefly the nature and numbers of investigations in which the essentiality of KEs has been experimentally explored either directly or indirectly. See pages 50-51 in the User Handbook for further definitions and clarifications.  More help

Evidence Assessment

The biological plausibility, empirical support, and quantitative understanding from each KER in an AOP are assessed together.  Biological plausibility of each of the KERs in the AOP is the most influential consideration in assessing WoE or degree of confidence in an overall hypothesised AOP for potential regulatory application (Meek et al., 2014; 2014a). Empirical support entails consideration of experimental data in terms of the associations between KEs – namely dose-response concordance and temporal relationships between and across multiple KEs. It is examined most often in studies of dose-response/incidence and temporal relationships for stressors that impact the pathway. While less influential than biological plausibility of the KERs and essentiality of the KEs, empirical support can increase confidence in the relationships included in an AOP. For clarification on how to rate the given empirical support for a KER, as well as examples, see pages 53- 55 of the User Handbook.  More help

Quantitative Understanding

Some proof of concept examples to address the WoE considerations for AOPs quantitatively have recently been developed, based on the rank ordering of the relevant Bradford Hill considerations (i.e., biological plausibility, essentiality and empirical support) (Becker et al., 2017; Becker et al, 2015; Collier et al., 2016). Suggested quantitation of the various elements is expert derived, without collective consideration currently of appropriate reporting templates or formal expert engagement. Though not essential, developers may wish to assign comparative quantitative values to the extent of the supporting data based on the three critical Bradford Hill considerations for AOPs, as a basis to contribute to collective experience.Specific attention is also given to how precisely and accurately one can potentially predict an impact on KEdownstream based on some measurement of KEupstream. This is captured in the form of quantitative understanding calls for each KER. See pages 55-56 of the User Handbook for a review of quantitative understanding for KER's. More help

Considerations for Potential Applications of the AOP (optional)

At their discretion, the developer may include in this section discussion of the potential applications of an AOP to support regulatory decision-making. This may include, for example, possible utility for test guideline development or refinement, development of integrated testing and assessment approaches, development of (Q)SARs / or chemical profilers to facilitate the grouping of chemicals for subsequent read-across, screening level hazard assessments or even risk assessment. While it is challenging to foresee all potential regulatory application of AOPs and any application will ultimately lie within the purview of regulatory agencies, potential applications may be apparent as the AOP is being developed, particularly if it was initiated with a particular application in mind. This optional section is intended to provide the developer with an opportunity to suggest potential regulatory applications and describe his or her rationale.To edit the “Considerations for Potential Applications of the AOP” section, on an AOP page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Editing AOP.” Scroll down to the “Considerations for Potential Applications of the AOP” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update AOP’ to save your changes and return to the AOP page or 'Update and continue' to continue editing AOP text sections.  The new text should appear under the “Considerations for Potential Applications of the AOP” section on the AOP page. More help

References

List the bibliographic references to original papers, books or other documents used to support the AOP. More help

Allen, K., Jaeschke, H., Copple, B.L. (2011). Bile acids induce inflammatory genes in hepatocytes: a novel mechanism of inflammation during obstructive cholestasis. Am. J. Pathol. 178, 175-186.

Andersen, M.E., Clewell, R., Bhattacharya, S. (2012). Developing in vitro tools sufficient by themselves for 21st century risk assessment. In Towards the Replacement of In Vivo Repeated Dose Systemic Toxicity Testing Volume 2 (T. Gocht, M. Schwarz, Eds.), pp. 347-360. Imprimerie Mouzet, France.

Andersen, M.E., McMullen, P., Bhattacharya, S. (2013). Toxicogenomics for transcription factor-governed molecular pathways: moving on to roles beyond classification and prediction. Arch. Toxicol. 87, 7-11.

Ankley, G.T., Bennett, R.S., Erickson, R.J., Hoff, D.J., Hornung, M.W., Johnson, R.D., Mount, D.R., Nichols, J.W., Russom, C.L., Schmieder, P.K., Serrrano, J.A., Tietge, J.E., Villeneuve, D.L. (2010). Adverse outcome pathways: a conceptual framework to support ecotoxicology research and risk assessment. Environ. Toxicol. Chem. 29, 730-741.

Barbier, O., Torra, I.P., Sirvent, A., Claudel, T., Blanquart, C., Duran-Sandoval, D., Kuipers, F., Kosykh, V., Fruchart, J.C., Staels, B. (2003). FXR induces the UGT2B4 enzyme in hepatocytes: a potential mechanism of negative feedback control of FXR activity. Gastroenterology 124, 1926-1940.

Bernstein, H., Bernstein, C., Payne, C.M., Dvorakova, K., Garewal, H. (2005). Bile acids as carcinogens in human gastrointestinal cancers. Mutat. Res. 589, 47-65.

Bogdanffy, M.S., Daston, G., Faustman, E.M., Kimmel, C.A., Kimmel, G.L., Seed, J., Vu, V. (2001). Harmonization of cancer and noncancer risk assessment: proceedings of a consensus-building workshop. Toxicol. Sci. 61, 18-31.

Botla, R., Spivey, J.R., Aguilar, H., Bronk, S.F., Gores, G.J. (1995). Ursodeoxycholate (UDCA) inhibits the mitochondrial membrane permeability transition induced by glycochenodeoxycholate: a mechanism of UDCA cytoprotection. J. Pharmacol. Exp. Ther. 272, 930-938.

Boyer, J.L., Trauner, M., Mennone, A., Soroka, C.J., Cai, S.Y., Moustafa, T., Zollner, G., Lee, J.Y., Ballatori, N. (2006). Upregulation of a basolateral FXR-dependent bile acid efflux transporter OSTalpha-OSTbeta in cholestasis in humans and rodents. Am. J. Physiol. Gastrointest. Liver Physiol. 290, G1124-G1130.

Boyer, L.B. (2009). Adaptive regulation of hepatocyte transporters in cholestasis. In The Liver: Biology and Pathobiology 5th edition (I.M. Arias, H.J. Alter, J.L. Boyer, D.E. Cohen, N. Fausto, D.A. Shafritz, A.W. Wolkoff, Eds.), pp. 681-702. Wiley-Blackwell, Oxford.

Björnsson, E., Olsson, R. (2005). Outcome and prognostic markers in severe drug-induced liver disease. Hepatology 42, 481-489.

Blomme, E.A., Yang, Y., Waring, J.F. (2009). Use of toxicogenomics to understand mechanisms of drug-induced hepatotoxicity during drug discovery and development. Toxicol. Lett. 186, 22-31.

Chen, H.L., Chen, H.L., Liu, Y.J., Feng, C.H., Wu, C.Y., Shyu, M.K., Yuan, R.H., Chang, M.H. (2005). Developmental expression of canalicular transporter genes in human liver. J. Hepatol. 43, 472-477.

Cheng, X., Buckley, D., Klaassen, C.D. (2007). Regulation of hepatic bile acid transporters Ntcp and Bsep expression. Biochem. Pharmacol. 74, 1665-1676.

Cherrington, N.J., Slitt, A.L., Li, N., Klaassen, C.D. (2004). Lipopolysaccharide-mediated regulation of hepatic transporter mRNA levels in rats. Drug Metab. Dispos. 32, 734-741.

Dawson, S., Stahl, S., Paul, N., Barber, J., Kenna, J.G. (2011). In vitro inhibition of the bile salt export pump correlates with risk of cholestatic drug-induced liver injury in humans. Drug Metab. Dispos. 40, 130-138.

Denson, L.A., Sturm, E., Echevarria, W., Zimmerman, T.L., Makishima, M., Mangelsdorf, D.J., Karpen, S.J. (2001). The orphan nuclear receptor, shp, mediates bile acid-induced inhibition of the rat bile acid transporter, ntcp. Gastroenterology 121, 140-147.

Echchgadda, I., Song, C.S., Oh, T., Ahmed, M., De La Cruz, I.J., Chatterjee, B. (2007). The xenobiotic-sensing nuclear receptors pregnane X receptor, constitutive androstane receptor, and orphan nuclear receptor hepatocyte nuclear factor 4alpha in the regulation of human steroid-/bile acid-sulfotransferase. Mol. Endocrinol. 21, 2099-2111.

Eloranta, J.J., Kullak-Ublick, G.A. (2005). Coordinate transcriptional regulation of bile acid homeostasis and drug metabolism. Arch. Biochem. Biophys. 433, 397-412.

EU. (2003). Directive 2003/15/EC of the European parliament, of the council of 27 February amending council directive 76/768/EEC on the approximation of the laws of the member states relating to cosmetic products. OV. J. L. 066, 26-35.

Fattinger, K., Funk, C., Pantze, M., Weber, C., Reichen, J., Stieger, B., Meier, P.J. (2001). The endothelin antagonist bosentan inhibits the canalicular bile salt export pump: a potential mechanism for hepatic adverse reactions. Clin. Pharmacol. Ther. 69, 223-231.

Faucette, S.R., Sueyoshi, T., Smith, C.M., Negishi, M., Lecluyse, E.L., Wang, H. (2006). Differential regulation of hepatic CYP2B6 and CYP3A4 genes by constitutive androstane receptor but not pregnane X receptor. J. Pharmacol. Exp. Ther. 317, 1200-1209.

Feingold, K.R., Spady, D.K., Pollock, A.S., Moser, A.H., Grunfeld, C. (1996). Endotoxin, TNF, and IL-1 decrease cholesterol 7 alpha-hydroxylase mRNA levels and activity. J. Lipid Res. 37, 223-228.

Funk, C., Pantze, M., Jehle, L., Ponelle, C., Scheuermann, G., Lazendic, M., Gasser, R. (2001). Troglitazone-induced intrahepatic cholestasis by an interference with the hepatobiliary export of bile acids in male and female rats: correlation with the gender difference in troglitazone sulfate formation and the inhibition of the canalicular bile salt export pump (Bsep) by troglitazone and troglitazone sulfate. Toxicology 167, 83-98.

Gnerre, C., Blättler, S., Kaufmann, M.R., Looser, R., Meyer, U.A. (2004). Regulation of CYP3A4 by the bile acid receptor FXR: evidence for functional binding sites in the CYP3A4 gene. Pharmacogenetics 14, 635-645.

Gollamudi, R., Gupta, D., Goel, S., Mani, S. (2008). Novel orphan nuclear receptors-coregulator interactions controlling anti-cancer drug metabolism. Curr. Drug Metab. 9, 611-613.

Goodwin, B., Jones, S.A., Price, R.R., Watson, M.A., McKee, D.D., Moore, L.B., Galardi, C., Wilson, J.G., Lewis, M.C., Roth, M.E., Maloney, P.R., Willson, T.M., Kliewer, S.A. (2000). A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis. Mol. Cell 6, 517-526.

Gores, G.J., Miyoshi, H., Botla, R., Aguilar, H.I., Bronk, S.F. (1998). Induction of the mitochondrial permeability transition as a mechanism of liver injury during cholestasis: a potential role for mitochondrial proteases. Biochim. Biophys. Acta 1366, 167-175.

Green, R.M., Hoda, F., Ward, K.L. (2000). Molecular cloning and characterization of the murine bile salt export pump. Gene 241, 117-123.

Gu, X., Ke S., Liu, D., Sheng, T., Thomas, P.E., Rabson, A.B., Gallo, M.A., Xie ,W., Tian, Y. (2006). Role of NF-kappaB in regulation of PXR-mediated gene expression: a mechanism for the suppression of cytochrome P-450 3A4 by proinflammatory agents. J. Biol. Chem. 281, 17882-17889.

Gujral, J.S., Farhood, A., Bajt, M.L., Jaeschke, H. (2003). Neutrophils aggravate acute liver injury during obstructive cholestasis in bile duct-ligated mice. Hepatology 38, 355-363.

Gujral, J.S., Liu, J., Farhood, A., Hinson, J.A., Jaeschke, H. (2004). Functional importance of ICAM-1 in the mechanism of neutrophil-induced liver injury in bile duct-ligated mice. Am. J. Physiol. Gastrointest. Liver Physiol. 286, G499-G507.

Hill, A.B. (1965). The environment and disease: association or causation? Proc. R. Soc. Med. 58, 295-300.

Hofmann, A.F. (2009). Bile acids and the enterohepatic circulation. In The Liver: Biology and Pathobiology (I.M.; Arias, H.J., Alter, J.L., Boyer, D.E., Cohen, N., Fausto, D.A., Shafritz, A.W., Wolkoff Eds.), pp. 289-304. Wiley-Blackwell, Oxford.

http://caat.jhsph.edu/ (consulted June 2013).

http://www.seurat-1.eu/ (consulted June 2013).

Jaeschke, H., Hasegawa, T. (2006). Role of neutrophils in acute inflammatory liver injury. Liver Int. 26, 912-919.

Jemnitz, K., Veres, Z., Vereczkey, L. (2010). Contribution of high basolateral bile salt efflux to the lack of hepatotoxicity in rat in response to drugs inducing cholestasis in human. Toxicol. Sci. 115, 80-88.

Julien, E., Boobis, A.R., Olin, S.S. (2009). The key events dose-response framework: a cross-disciplinary mode-of-action based approach to examining dose-response and thresholds. Crit. Rev. Food Sci. Nutr. 49, 682-689.

Jung, D., Mangelsdorf, D.J., Meyer, U.A. (2006). Pregnane X receptor is a target of farnesoid X receptor. J. Biol. Chem. 281, 19081-19091.

Jung, D., Elferink, M.G., Stellaard, F., Groothuis, G.M. (2007). Analysis of bile acid-induced regulation of FXR target genes in human liver slices. Liver Int. 27, 137-144.

Kast, H.R., Goodwin, B., Tarr, P.T., Jones, S.A., Anisfeld, A.M., Stoltz, C.M., Tontonoz, P., Kliewer, S., Willson, T.M., Edwards, P.A. (2002). Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X-activated receptor, and constitutive androstane receptor. J. Biol. Chem. 277, 2908-2915.

Kim, M.S., Shigenaga, J., Moser, A., Grunfeld, C., Feingold, K.R. (2004). Suppression of DHEA sulfotransferase (Sult2A1) during the acute-phase response. Am. J. Physiol. Endocrinol. Metab. 287, E731-E738.

Kis, E., Ioja, E., Rajnai, Z., Jani, M., Méhn, D., Herédi-Szabó, K., Krajcsi, P. (2012). BSEP inhibition: in vitro screens to assess cholestatic potential of drugs. Toxicol. In Vitro 26, 1294-1299.

Knisely, A.S., Strautnieks, S.S., Meier, Y., Stieger, B., Byrne, J.A., Portmann, B.C., Bull, L.N., Pawlikowska, L., Bilezikçi, B., Ozçay, F., László, A., Tiszlavicz, L., Moore, L., Raftos, J., Arnell, H., Fischler, B., Németh, A., Papadogiannakis, N., Cielecka-Kuszyk, J., Jankowska, I. (2006). Hepatocellular carcinoma in ten children under five years of age with bile salt export pump deficiency. Hepatology 44, 478-486.

Kocarek, T.A., Shenoy, S.D., Mercer-Haines, N.A., Runge-Morris, M. (2002). Use of dominant negative nuclear receptors to study xenobiotic-inducible gene expression in primary cultured hepatocytes. J. Pharmacol. Toxicol. Methods 47, 177-187.

Kostrubsky, V.E., Vore, M., Kindt, E., Burliegh, J., Rogers, K., Peter, G., Altrogge, D., Sinz, M.W. (2001). The effect of troglitazone biliary excretion on metabolite distribution and cholestasis in transporter-deficient rats. Drug Metab. Dispos. 29, 1561-1566.

Krasowski, M.D., Yasuda, K., Hagey, L.R., Schuetz, E.G. (2005). Evolution of the pregnane X receptor: adaptation to cross-species differences in biliary bile salts. Mol. Endocrinol. 19, 1720-1739.

Kuntz, E., Kuntz, H.D. (2008). Cholestasis. In Hepatology: Textbook and Atlas : History, Morphology, Biochemistry, Diagnostics, Clinic, Therapy 3rd edition (E. Kuntz E., H.-D. Kuntz Eds.), pp. 235-250. Springer, Heidelberg.

Landesmann, B., Goumenou, M., Munn, S., Whelan, M. (2012). Description of prototype modes-of-action related to repeated dose toxicity. JRC Scientific and Policy Report 75689.

Lang, C., Meier, Y., Stieger, B., Beuers, U., Lang, T., Kerb, R., Kullak-Ublick, G.A., Meier, P.J., Pauli-Magnus, C. (2007). Mutations and polymorphisms in the bile salt export pump and the multidrug resistance protein 3 associated with drug-induced liver injury. Pharmacogenet. Genomics 17, 47-60.

Lang, T., Haberl, M., Jung, D., Drescher, A., Schlagenhaufer, R., Keil, A., Mornhinweg, E., Stieger, B., Kullak-Ublick, G.A., Kerb, R. (2006). Genetic variability, haplotype structures, and ethnic diversity of hepatic transporters MDR3 (ABCB4) and bile salt export pump (ABCB11). Drug Metab. Dispos. 34, 1582-1599.

Lecureur, V., Sun, D., Hargrove, P., Schuetz, E.G., Kim, R.B., Lan, L.B., Schuetz, J.D. (2000). Cloning and expression of murine sister of P-glycoprotein reveals a more discriminating transporter than MDR1/P-glycoprotein. Mol. Pharmacol. 57, 24-35.

Lee, J., Azzaroli, F., Wang, L., Soroka, C.J., Gigliozzi, A., Setchell, K.D., Kramer, W., Boyer, J.L. (2001). Adaptive regulation of bile salt transporters in kidney and liver in obstructive cholestasis in the rat. Gastroenterology 121, 1473-1484.

Li-Masters, T., Morgan, E.T. (2001). Effects of bacterial lipopolysaccharide on phenobarbital-induced CYP2B expression in mice. Drug Metab. Dispos. 29, 252-257.

Lucena, M.I., Andrade, R.J., Kaplowitz, N., García-Cortes, M., Fernández, M.C., Romero-Gomez, M., Bruguera, M., Hallal, H,. Robles-Diaz, M., Rodriguez-González, J.F., Navarro, J.M., Salmeron, J., Martinez-Odriozola, P., Pérez-Alvarez, R., Borraz, Y., Hidalgo, R. (2009). Phenotypic characterization of idiosyncratic drug-induced liver injury: the influence of age and sex. Hepatology 49, 2001-2009.

Maher, J.M., Cheng, X., Slitt, A.L., Dieter, M.Z., Klaassen, C.D. (2005). Induction of the multidrug resistance-associated protein family of transporters by chemical activators of receptor-mediated pathways in mouse liver. Drug Metab. Dispos. 33, 956-962.

Mazzon, E., Cuzzocrea, S. (2003). Role of iNOS in hepatocyte tight junction alteration in mouse model of experimental colitis. Cell. Mol. Biol. 49, 45-57.

Meek, M.E., Bucher, J.R., Cohen, S.M., Dellarco, V., Hill, R.N., Lehman-McKeeman, L.D., Longfellow, D.G., Pastoor, T., Seed, J., Patton, D.E. (2003). A framework for human relevance analysis of information on carcinogenic modes of action. Crit. Rev. Toxicol. 33, 591-653.

Meier, Y., Pauli-Magnus, C., Zanger, U.M., Klein, K., Schaeffeler, E., Nussler, A.K., Nussler, N., Eichelbaum, M., Meier, P.J., Stieger, B. (2006). Interindividual variability of canalicular ATP-binding-cassette (ABC)-transporter expression in human liver. Hepatology 44, 62-74.

Mennone, A., Soroka, C.J., Harry, K.M., Boyer, J.L. (2010). Role of breast cancer resistance protein in the adaptive response to cholestasis. Drug Metab. Dispos. 38, 1673-1678.

Morgan, R.E., Trauner, M., van Staden, C.J., Lee, P.H., Ramachandran, B., Eschenberg, M., Afshari, C.A., Qualls, C.W. Jr., Lightfoot-Dunn, R., Hamadeh, H.K. (2010). Interference with bile salt export pump function is a susceptibility factor for human liver injury in drug development. Toxicol. Sci. 118, 485-500.

NRC. (2007). Toxicity testing in the 21st century: a vision and a strategy. The National Academies Press, Washington DC.

OECD. (2011). Report of the workshop on using mechanistic information on forming chemical categories. Series on testing and assessment No. 138.

OECD. (2012a). Proposal for a template and guidance on developing and assessing the completeness of adverse outcome pathways.

OECD. (2012b). The adverse outcome pathway for skin sensitization initiated by covalent binding to proteins part I: scientific evidence. Series on testing and assessment No. 168.

Oude Elferink, R.P., Paulusma, C.C., Groen, A.K. (2006). Hepatocanalicular transport defects: pathophysiologic mechanisms of rare diseases. Gastroenterology 13, 908-925.

Ozer, J., Ratner, M., Shaw, M., Bailey, W., Schomaker, S. (2008). The current state of serum biomarkers of hepatotoxicity. Toxicology 245, 194-205.

Padda, M.S., Sanchez, M., Akhtar, A.J., Boyer, J.L. (2011). Drug-induced cholestasis. Hepatology 53, 1377-1387.

Pagani, R., Portolés, M.T., De La Viña, S., Melzner, I., Vergani, G. (2003). Alterations induced on cytoskeleton by Escherichia coli endotoxin in different types of rat liver cell cultures. Histol. Histopathol. 1, 837-848.

Pauli-Magnus, C., Meier, P.J. (2006). Hepatobiliary transporters and drug-induced cholestasis. Hepatology 44, 778-787.

Pauwels, M., Rogiers, V. (2010). Human health safety evaluation of cosmetics in the EU: a legally imposed challenge to science. Toxicol. Appl. Pharmacol. 243, 260-274.

Roman, I.D., Monte, M.J., Gonzalez-Buitrago, J.M., Esteller, A., Jimenez, R. (1990). Inhibition of hepatocytary vesicular transport by cyclosporin A in the rat: relationship with cholestasis and hyperbilirubinemia. Hepatology 12, 83-91.

Salgia, R., Becker, J.H., Sayeed, M.M. (1993). Altered membrane fluidity in rat hepatocytes during endotoxic shock. Mol. Cell. Biochem. 121, 143-148.

Schoemaker, M.H., Conde de la Rosa, L., Buist-Homan, M., Vrenken, T.E., Havinga, R., Poelstra, K., Haisma, H.J., Jansen, P.L., Moshage, H. (2004). Tauroursodeoxycholic acid protects rat hepatocytes from bile acid-induced apoptosis via activation of survival pathways. Hepatology 39, 1563-1573.

Seed, J., Carney, E.W., Corley, R.A., Crofton, K.M., DeSesso, J.M., Foster, P.M., Kavlock, R., Kimmel, G., Klaunig, J., Meek, M.E., Preston, R.J., Slikker, W. Jr., Tabacova, S., Williams, G.M., Wiltse, J., Zoeller, R.T., Fenner-Crisp, P., Patton, D.E. (2005). Overview: using mode of action and life stage information to evaluate the human relevance of animal toxicity data. Crit. Rev. Toxicol. 35, 664-672.

Sekine, S., Yano K.,, Saeki, J., Hashimoto, N., Fuwa, T., Horie, T. (2010). Oxidative stress is a triggering factor for LPS-induced Mrp2 internalization in the cryopreserved rat and human liver slices. Biochem. Biophys. Res. Commun. 399, 279-285.

Seok, J., Warren, H.S., Cuenca, A.G., Mindrinos, M.N., Baker, H.V., Xu, W., Richards, D.R., McDonald-Smith, G.P., Gao, H., Hennessy, L., Finnerty, C.C., López, C.M., Honari, S., Moore, E.E., Minei, J.P., Cuschieri, J., Bankey, P.E., Johnson, J.L., Sperry, J., Nathens, A.B., Billiar, T.R., West, M.A., Jeschke, M.G., Klein, M.B., Gamelli, R.L., Gibran, N.S., Brownstein, B.H., Miller-Graziano, C., Calvano, S.E., Mason, P.H., Cobb, J.P., Rahme, L.G., Lowry, S.F., Maier, R.V., Moldawer, L.L., Herndon, D.N., Davis, R.W., Xiao, W., Tompkin, R.G. (2013). Genomic responses in mouse models poorly mimic human inflammatory diseases. Proc. Natl. Acad. Sci. USA 110, 3507-3512.

Sokol, R.J., Dahl, R., Devereaux, M.W., Yerushalmi, B., Kobak, G.E., Gumpricht, E. (2005). Human hepatic mitochondria generate reactive oxygen species and undergo the permeability transition in response to hydrophobic bile acids. J. Pediatr. Gastroenterol. Nutr. 41, 235-243.

Trauner, M., Arrese, M., Lee, H., Boyer, J.L., Karpen, S.J. (1998a). Endotoxin downregulates rat hepatic ntcp gene expression via decreased activity of critical transcription factors. J. Clin. Invest. 101, 2092-2100.

Trauner, M., Meier, P.J., Boyer, J.L. (1998b). Molecular pathogenesis of cholestasis. N. Engl. J. Med. 339, 1217-1227.

US EPA. (2005). Guidelines for carcinogen risk assessment. Washington DC.

Vinken, M., Pauwels, M., Ates, G., Vivier, M., Vanhaecke, T., Rogiers, V. (2012). Screening of repeated dose toxicity data present in SCC(NF)P/SCCS safety evaluations of cosmetic ingredients. Arch. Toxicol. 86, 405-412.

Wagner, M., Zollner, G., Trauner, M. (2009). New molecular insights into the mechanisms of cholestasis. J. Hepatol. 51, 565-580.

Woolbright, B.L., Jaeschke, H. (2012). Novel insight into mechanisms of cholestatic liver injury. World. J. Gastroenterol. 18, 4985-4993.

Yasumiba, S., Tazuma, S., Ochi, H., Chayama, K., Kajiyama, G. (2001). Cyclosporin A reduces canalicular membrane fluidity and regulates transporter function in rats. Biochem. J. 354, 591-596.

Zhang, Y., Hong, J.Y., Rockwell, C.E., Copple, B.L., Jaeschke, H., Klaassen, C.D. (2012). Effect of bile duct ligation on bile acid composition in mouse serum and liver. Liver Int. 32, 58-69.

Zollner, G., Wagner, M., Moustafa, T., Fickert, P., Silbert, D., Gumhold, J., Fuchsbichler, A., Halilbasic, E., Denk, H., Marschall, H.U., Trauner, M. (2006). Coordinated induction of bile acid detoxification and alternative elimination in mice: role of FXR-regulated organic solute transporter-alpha/beta in the adaptive response to bile acids. J. Physiol. Gastrointest. Liver Physiol. 290, G923-G932.

Zollner, G., Trauner, M. (2006). Molecular mechanisms of cholestasis. Wien. Med. Wochenschr. 156, 380-385.

Zollner, G., Trauner, M. (2008). Mechanisms of cholestasis. Clin. Liver Dis. 12, 1-26.

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.