Aop: 362

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

Immune mediated hepatitis

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
Hepatitis

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

Jung-Hwa Oh, Korea Institute of Toxicology

Juergen Borlak, Hannover Medical School

Seokjoo Yoon, Korea Insitute of Toxicology

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
Jung-Hwa Oh   (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
  • Jung-Hwa Oh

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
This AOP was last modified on June 04, 2021 17:09
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
benzoquinone imine and acylglucuronide metabolites November 02, 2020 07:03
Mitochondrial dysfunction November 02, 2020 07:11
Apoptotic cell death November 02, 2020 07:11
Immune cell activation November 02, 2020 07:12
IFNγ signaling November 02, 2020 07:13
Increase in inflammation May 03, 2019 14:27
Immune mediated hepatitis November 02, 2020 07:14
Activation of ER stress November 02, 2020 07:09
Reactive Metabolite leads to Mitochondrial dysfunction November 02, 2020 07:14
Mitochondrial dysfunction leads to Apoptotic cell death November 02, 2020 07:15
Reactive Metabolite leads to Immune cell activation November 02, 2020 07:16
Mitochondrial dysfunction leads to ER stress November 02, 2020 07:18
Immune cell activation leads to IFNγ signaling November 02, 2020 07:18
IFNγ signaling leads to Increase in inflammation November 02, 2020 07:19
Apoptotic cell death leads to Increase in inflammation November 02, 2020 07:19
Increase in inflammation leads to hepatitis November 02, 2020 07:20
ER stress leads to Apoptotic cell death November 02, 2020 07:20

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

Many drugs have the potential to cause drug induced liver injury (DILI); however underlying mechanisms are diverse. We report an AOP for immune mediated and allergic hepatitis as a case study with diclofenac and the findings of this NSAID can be extended to other ones. By considering genomic, histo- and clinical pathology data from mice and dogs we developed AOPs specific for immune mediated and drug hypersensitivity/allergic hepatitis. We define reactive metabolites including quinone imine catalysed by CYP monooxygenase and myeloperoxidases of Kupffer cells as well as acylglucuronide as molecular initiating events (MIE). The reactive metabolites bind to proteins and act as neo-antigen and involve antigen presenting cells to elicit B-and T-cell responses. Given the different immune system between mice and dogs 6 different key events (KE) at the cellular and up to 4 KEs at the organ level are defined with mechanistic plausibility for the onset and progression of liver inflammation. With mice, cellular stress response, interferon gamma-, adipocytokine- and chemokine signaling provide a rational for the AOP of immune-mediated hepatitis. With dogs, an erroneous programming of the innate and adaptive immune response results in mast cell activation; their infiltration into liver parenchyma and the shift to M2 polarized Kupffer cells signifies allergic hepatitis and the occurrence of granulomas of the liver. Taken together, diclofenac induces divergent immune response among two important preclinical animal species and the injury pattern seen among clinical cases confirms the relevance of the developed AOP for immune-mediated hepatitis.

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
2 KE 1816 Mitochondrial dysfunction Mitochondrial dysfunction
7 KE 1815 Activation of ER stress ER stress
3 KE 1817 Apoptotic cell death Apoptotic cell death
4 KE 1818 Immune cell activation Immune cell activation
5 KE 1819 IFNγ signaling IFNγ signaling
6 KE 1633 Increase in inflammation Increase in inflammation
8 AO 1820 Immune mediated hepatitis hepatitis

Relationships Between Two Key Events (Including MIEs and AOs)

This 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

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
Homo sapiens Homo sapiens High NCBI
rat Rattus norvegicus High NCBI

Sex Applicability

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

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

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

MIE

Drug induced hepatitis is a multistep process and frequently involves reactive metabolites which subsequently form drug-protein adducts. These are sensed by antigen presenting cells to elicit innate and adaptive immune responses (Adams et al. 2010). Diclofenac is extensively metabolized by CYP monooxygenases and myeloperoxidases (MPO) of neutrophils and Kupffer cells into reactive metabolites notably quinoneimine intermediates. The metabolism of diclofenac to reactive metabolites causes organ toxicity and this defines the MIE. A further example for a benzoquinone imine reactive metabolite causing liver injury is the metabolism of paracetamol to NAPQI. Correspondingly, NAPQI is the initial cause of hepatotoxicity and once again defines the MIE. Indeed, for a wide range of drugs quinoneimines are implicated in liver injury. Diclofenac caused significant regulation of CYP monooxygenases after single and repeated treatment of mice (Lee et al. 2016). Independent investigations also support the key role of the acyl glucuronide produced by uridine diphosphoglucuronosyl transferase as a molecular initiation event (Oda et al. 2017; Seitz and Boelsterli 1998) and among NSAIDs the salicyl acyl glucuronide derived from aspirin is a further example. Thus, reactive metabolites are formed by hepatocytes, neutrophils and Kupffer cells with benzoquinone imine intermediates and acyl glucuronides being particularly harmful. If not sufficiently detoxified the reactive metabolites damage organelles, proteins and membrane lipids and eventually trigger programmed cell death. Specifically, the accumulation of the reactive metabolite triggers oxidative stress and mitochondrial permeability transitions, i.e. mitochondrial toxicity by inhibiting ATP synthesis that leads to hepatocellular damage (Syed et al. 2016). Next to direct effects the reactive metabolites can covalently bind to proteins to form adducts. These function as neoantigens and are sensed and phagocytosed by APCs. Through interaction with the major histocompatibility complex APCs elicit B and T cell responses (Aithal 2011; Boelsterli 2003). Additionally, diclofenac acyl glucuronide inhibit the Mrp2 transport. This results in intrahepatic cholestasis and damage of the biliary epithelium (Boelsterli 2003; Lagas et al. 2010; Seitz and Boelsterli 1998). Similar to diclofenac the NSAIDs lumiracoxib and indomethacin produce quinoneimine reactive intermediates and next to ibuprofen and naproxen a wider range of carboxylic acid containing drugs are associated with allergic reactions (Stepan et al. 2011). The reactivity of acyl glucuronides derived from carboxylic acid containing drugs and the evidence for its toxicological concerns was recently summarized (Darnell et al. 2015; Van Vleet et al. 2017). Note, the covalent binding of acyl glucuronides to proteins constitutes a mechanism of toxicity and the safety assessment of acyl glucuronides was the subject of a recent commentary with zomepirac being a prominent example for NSAID toxicity (Smith et al. 2018). Importantly, inhibition of MPO ameliorate adverse effects of MPO-derived oxidants (Malle et al. 2007) and MPO ko mice are an excellent system to study the importance of MPO in systemic inflammatory reactions. Alike, amelioration of diclofenac induced toxicity was observed with cytochrome P450 reductase (CPR) null mice (Zhu and Zhang 2012) and multidrug resistance-associated protein 3 plays an important role in protection against acute toxicity of diclofenac acyl glucuronide as evidenced in Mrp3-null (KO) mice (Scialis et al. 2015). Moreover, NSAIDs are known to produce reactive oxygen species that result in cardiovascular disease (Ghosh et al. 2015) and a structural alert/reactive metabolite concept of 200 common drugs producing a wide range of different reactive metabolites was reported (Stepan et al. 2011). Collectively, the structural alerts quinoneimine and acyl glucuronides function as MIEs in immune-mediated and allergic hepatitis. Notwithstanding, defining an unequivocal MIE in the AOP framework can be complex as discussed in the seminal paper of Allen and coworkers (Allen et al. 2014). 

KEs related to immune mediated hepatitis

The biochemical and genomic data revealed diclofenac treatment to induce complex immune mediated inflammatory signalling particularly from resident and migratory cells of the sinusoidal and space of Disse. Within hepatocytes, the reactive metabolites elicit cellular stress responses including oxidative stress, mitochondrial dysfunction, apoptosis and ER stress/unfolded protein responses. In repeated dose studies with mice, diclofenac treatment induced expression of the plasma membrane cysteine carrier (Slc3a1) to imply adaptive responses to oxidative stress. Furthermore, cysteine is an essential building block for the hepatic synthesis of reduced GSH and therefore of fundamental importance in alleviating oxidative stresses.

KE1: Mitochondrial dysfunction. Diclofenac caused mitochondrial dysfunction through an inhibition of ATP synthesis (Boelsterli 2003, Kang, et al. 2016, Syed, et al. 2016). The significant repression of mitochondrial membrane transport proteins and key members of the oxidative phosphorylation pathway is testimony of an impaired mitochondrial respiration and ATP synthesis (Lee, et al. 2016). Conversely, the plasma-membrane Mg2+ transporter is strongly induced to increase intracellular Mg2+ concentration. Note, increased Mg2+ uptake counteracts the detrimental effects of diclofenac treatment to alleviate mitochondrial stress and the opening of the Ca2+ dependent permeability transition pore to dampen apoptotic signaling.

KE2: Apoptotic cell death. Reactive metabolites of diclofenac can directly or indirectly induce apoptotic cell death by activating several pro- and anti-apoptotic factors notably toll like receptors, cytokine signaling inducible factors such as S100 calcium binding proteins and pro-inflammatory adipokines to augment ER stress-induced apoptosis (Lee, et al. 2016, Sawa, et al. 2009). Damaged hepatocytes send alarm signals like the damage-associated molecular patterns (DAMPs) that induce the immune and inflammatory response by activating immune cells. Although, several DAMP molecules including S100 proteins were upregulated after repeated dosing of mouse study, the major components of the inflammasome are not regulated at the transcript level. Meanwhile, the danger hypothesis proposed that DAMPs can also be influenced by immune or inflammatory response. Our previous study demonstrated that diclofenac treatment induced the expression of inflammatory proteins which are released from macrophages/Kupffer cells. The subsequent inflammatory responses can reinforce the cellular damage of hepatocyte and in a vicious cycle strengthen inflammation (Lee, et al. 2016).

KE3: ER stress/unfolded protein response. Genes involved in ER stress and unfolded protein response (UPR) were significantly regulated in the liver of diclofenac treated mice. Independent studies evidenced diclofenac to trigger ER stress and UPR by PERK and ATF6 pathways as well as eIF2α phosphorylation (Foufelle and Fromenty 2016, Franceschelli, et al. 2011, Fredriksson, et al. 2014). However, the prolonged activation of PERK/eIF2α pathway induces apoptosis by activating the pro-apoptotic factor CHOP (Franceschelli, et al. 2011, Fredriksson, et al. 2014). In addition, ER stress can alter the lipid metabolism by UPR and leads to dyslipidemia (Basseri and Austin 2012). As reported by us, diclofenac caused hepatic cholersterolosis in mice with significant alteration in the transcription of genes coding for fatty acid and cholesterol metabolism (Lee, et al. 2016). On the other hand, the repeated diclofenac treatment induced Cyp7a1 expression which catalyzes the hydroxylation of cholesterol into bile acids (Lee, et al. 2016). Alike, the induction of the apical sodium–bile acid transporter (Slc10a2) evidences changes in the transcellular transport of bile acids across the biliary epithelium to support the enterohepatic cycling of bile acids (Lee, et al. 2016). Moreover, the organic anion transporter Slc10a6 was induced and functions on taurolithocholic acid-3-sulfate (TCA-3S). Note TCA-3S excretion into urine is about 90-fold higher in patients diagnosed with intrahepatic cholestasis of pregnancy thus highlighting its potential as a biomarker of hepatic cholestasis (Lee, et al. 2016).

KE4: Immune cell activation. Diclofenac adducts are sensed by APC and other phagotysing cells and trigger immune responses. The immune-mediated hepatitis is the result of complex interplay of innate and adaptive immune responses and involves the regulation of various cytokines/chemokines and their receptors (Lee, et al. 2016). Specifically, the released chemokines recruit the neutrophils, leukocytes, and B lymphocytes to the sinusoidal space or to harmed hepatocytes, while cytokines endorse differentiation of myeloid and cytotoxic CD8+ T-cells (Saiman and Friedman 2012, Sawa, et al. 2009). Increased expressions of interleukins modulate the activation and proliferation of T and/or NK cell responses (Hammerich and Tacke 2014, Zwirner and Domaica 2010) while members of the interleukin-1 superfamily stimulate the production of type 2 cytokines by T-helper cells (Miller 2011). Thus, diclofenac treatment resulted in an activation of several cytokines to affect T cell differentiation. Collectively, diclofenac stimulated an activation of diverse immune cells including monocytes, Kupffer cells and APC (Lee, et al. 2016).

KE5: IFNγ signaling. Increased expression of IFNγ hallmarks innate and adaptive immune responses. IFNγ plays a pivotal role in host defence in response to infections and mediating the inflammation by producing the pro-inflammatory cytokines (Muhl and Pfeilschifter 2003). Genomic analysis revealed Ptpn2 (Protein tyrosine phosphatase non-receptor type 2) to be significantly regulated in mice after diclofenac treatment and Ptpn2 plays a critical role in modulating IFNγ signaling (Lee, et al. 2016, Scharl, et al. 2010). Together, diclofenac treatment induced expression of inflammatory cytokines including IFNγ, interleukins and TNFα (Dutta, et al. 2008, Yano, et al. 2012).

KE6: Inflammation. Our computational studies defined key master regulatory molecules and their associated networks. Based on independent RT-qPCR studies induced expression of suppressor of cytokine signaling (Socs), leptin, growth hormone receptor (Ghr), and Ptpn proteins could be confirmed and these function in IFNγ, Jak/Stat, pro- and anti-inflammatory signaling pathways. Stat3 is one of the major transcription factors activated by cytokines and growth factors and influences pro-inflammatory (Mapk, p38, Jnk, and IκB kinase) and anti-inflammatory signaling (Pi3k-Akt) events Among individual animals marked induction of this protein was observed which controls transcription of Socs and inhibits Jak/Stat3 signaling (Kong, et al. 2002). In addition, leptin and other adipokine signaling molecules play a major role in energy intake to influence monocyte and macrophage activity during inflammation (Fantuzzi and Faggioni 2000). Next to its role in the control of energy intake leptin and its receptor modulates Jak/Stat, Erk 1/2 and Pi3k signaling (Bjorbaek and Kahn 2004, Cottrell and Mercer 2012, Paz-Filho, et al. 2012) and therefore play decisive role in inflammation.  Similarly, the growth hormone receptor is influenced by multiple intracellular signaling cascades (Jak-Stat and chemokine signaling) and functions in liver regeneration. Diclofenac treatment reinforced Ghr degradation as evidenced by immunoblotting to suppress hepatic Ghr signaling (Takahashi 2017). Diclofenac also induced expression of the endothelial-leukocyte adhesion molecule selectin which recruits leukocytes to the inflammatory site and the computational analysis defined selectin as a master regulator (Lee, et al. 2016, Ley 2003). Furthermore, the highly significant induction of lipocalin-2 (LCN2) signifies sterile inflammation and neutralization of LCN2 controls neutrophilic inflammation as had been summarized (Moschen, et al. 2017). Taken together, the IFNγ, Jak/Stat, adipocytokine and chemokine signaling pathways provide a rational for the AOP of immune-mediated hepatitis observed in mice in response to diclofenac treatment.

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

A long-term prospective clinical trial involving 17,289 arthritis patients who were randomly assigned to diclofenac (150 mg daily) or etoricoxib (60 or 90 mg daily) evidenced diclofenac to cause common aminotransferase elevations (Laine et al. 2009). Alike, a systematic review of randomized clinical trial data of 3 NSAIDs revealed diclofenac to be top ranking for hepatotoxic events (Sriuttha et al. 2018). Earlier studies already reported cases of acute hepatitis induced by diclofenac (Helfgott et al. 1990; Iveson et al. 1990; Purcell et al. 1991; Sallie 1990) and liver biopsy findings defined a histological injury pattern of granulomatous hepatitis. Note our studies with dogs also demonstrated granulomatous hepatitis as a key finding (see above KE4) and the granulomas are composed of inflammatory cells and histocytes (Ramachandran and Kakar 2009). Moreover, a current study compared 30 DILI cases among 8 NSAIDs and found diclofenac to be the most frequently implicated NSAID. The cases are characterized by hepatocellular injury, prolonged hospitalization and included a patient with fatal Stevens-Johnson syndrome (Schmeltzer et al. 2015). In their study 38% of diclofenac DILI cases presented fever, rash, and eosinophilia and the findings are consistent with the clinical features of drug hypersensitivity reactions (Schmeltzer et al. 2015). Altogether, the proposed AOP of immunemediated and allergic hepatitis is relevant for human DILI induced by NSAIDs.

References

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