Aop: 144


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

Endocytic lysosomal uptake leading to liver fibrosis

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
lysosomal uptake induced liver fibrosis

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


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

Marina Kuburic 

Kirsten Gerloff 

Brigitte Landesmann° 

° F3 Chemical Safety and Alternative Methods Unit incorporating EURL ECVAM

Directorate F – Health, Consumers and Reference Materials

Joint Research Centre, European Commission




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
Allie Always   (email point of contact)


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
  • Brigitte Landesmann
  • Marina Kuburic
  • Kirsten Gerloff
  • Allie Always


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 EAGMST Under Review 1.47 Included in OECD Work Plan
This AOP was last modified on May 08, 2022 11:33
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
N/A, Mitochondrial dysfunction 1 March 07, 2022 07:12
Cell injury/death September 11, 2020 08:27
Endocytotic lysosomal uptake December 05, 2018 08:00
Disruption, Lysosome November 12, 2018 08:23
Increased Pro-inflammatory mediators March 16, 2020 05:27
Leukocyte recruitment/activation December 01, 2017 09:33
Activation, Stellate cells November 10, 2019 05:25
Accumulation, Collagen May 04, 2022 12:47
N/A, Liver fibrosis December 05, 2018 08:29
endocytosis leads to Disruption, Lysosome November 12, 2018 10:41
Disruption, Lysosome leads to N/A, Mitochondrial dysfunction 1 November 12, 2018 10:47
N/A, Mitochondrial dysfunction 1 leads to Cell injury/death November 29, 2016 20:08
Cell injury/death leads to Increased pro-inflammatory mediators November 12, 2018 10:52
Increased pro-inflammatory mediators leads to Leukocyte recruitment/activation November 12, 2018 10:55
Leukocyte recruitment/activation leads to Activation, Stellate cells November 12, 2018 10:58
Activation, Stellate cells leads to Accumulation, Collagen December 05, 2018 08:51
Accumulation, Collagen leads to N/A, Liver fibrosis December 05, 2018 08:52
nanoparticles December 21, 2016 09:40
ROS December 21, 2016 09:40
o-methyl-serine dodecylamide hydrochloride (MSDH) December 21, 2016 09:43
3-aminopropanal December 21, 2016 09:44
artesunate December 21, 2016 09:44
Chloroquine bis(phosphate) December 04, 2018 04:25
Norfloxacin December 04, 2018 04:26
Ciprofloxacin December 04, 2018 04:26


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

Hepatotoxicity is known to be an important endpoint of regulatory concern; it has been one of the frequent reasons for pharmacovigilance safety reports and human health risk assessments. Liver fibrosis, in particular, is a health problem resulting from chronic or repeated-dose chemical exposure and it is considered as an adverse outcome of regulatory interest. Liver fibrosis is a long and complex process involving various hepatic cell types, molecular mediators, receptors and signaling pathways. It occurs as a result of the imbalance between collagen deposition and destruction but also changes in the extracellular matrix composition (ECM). Due to this complexity appropriate cell model is currently unavailable.

The current AOP links endocytic lysosomal uptake and the formation of liver fibrosis. The molecular initiating event (MIE) is endocytic lysosomal uptake of chemicals, leading to lysosomal disruption, the first key event (KE). Lysosomal disruption induces mitochondrial dysfunction, which leads to cell injury and both apoptosis and necrosis. Lysosomal disruption, mitochondrial dysfunction, and cell injury/death present KEs on the cellular level. Cell death releases damage-associated molecular patterns (DAMPs) which leads to increased production of pro-inflammatory mediators, the next KE along the path. Inflammatory mediators attract and activate leukocytes, which present the next KE. Activated leukocytes through molecular mediators activate hepatic stellate cells, which increases α-SMA in them. This KE increases the amount of collagen I and III, which causes its accumulation. Collagen accumulation presents KE at the tissue level and leads to adverse outcome (AO) - liver fibrosis, which changes the normal functioning of the whole organ. There is also an important on-going process present throughout the pathway, which is connected with different KEs- oxidative stress. It is not classified as individual KE, but described in KEs and KERs related to it.

The value of this AOP is that it might support chemical risk assessment by identifying upstream biomarkers for adverse outcome, even though the adequate cell model is not available. This systematic organization of existing knowledge, but also of present uncertainties can facilitate regulatory processes, but also indicate the need for the new testing methods.

The AOP endocytic lysosomal uptake to liver fibrosis has high biological plausibility, supported with empirical evidence. However, quantitative data and temporal sequences between KEs are currently lacking and further efforts are necessary for their provision, but where temporal sequences between KEs are available, they are presented.

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

Liver fibrosis is currently an important health problem potentially leading in its progressive form to cirrhosis and as such presents a significant economic burden (Lim and Kim, 2008). The only therapy for chronic liver failure is liver transplantation, with 5.500 liver transplantations in Europe on a yearly basis, costing up to €100.000 (approximately US$110.000) the first year (Safadi and Friedman, 2002) and estimated mean cost of US$163.438 in United States (van der Hilst et al., 2008). There are constant research attempts for new therapeutic strategies, but so far without success. AOP concept presents an alternative approach which organizing mechanistic toxicological knowledge can lead to the prevention of liver fibrosis.

The use and possible applications of nanomaterials (NMs) are in constant increase, for example in food, food-related products or cosmetics. Therefore, the safety of NMs systemically taken up in the body needs to be ensured. The liver is known to be one of the main target organs for ingested NMs, but inhaled particles can also reach the liver upon clearance from the lung (Johnston et al., 2010; Cui et al., 2011; Geraets et al., 2012). In vivo experiments on gavaged or injected (intraperitoneal or intravenously) TiO2 suggest a wide range of adverse effects on the liver: an increase in general serum markers for liver damage such as Alanine Aminotransferase or Aspartate Aminotransferase (Liu et al., 2009; Duan et al., 2010), an increase in inflammatory markers such as pro-inflammatory cytokines and/or infiltration of inflammatory cells (Ma et al., 2009; Cui et al., 2011; Kermanizadeh, 2012), an increase of markers for oxidative stress (Liu et al., 2010; Soliman et al., 2013), apoptosis, necrosis and also fibrosis (Chen et al., 2009; Alarifi et al., 2013). Oral NM administration appeared to induce overall milder adverse effects than systemic administration, most likely due to the typically limited absorption of NMs in the GI tract. Thus, it is important to keep in mind that the route of exposure and the size of the NM, play an important role whether these reach the liver, and to which extent they're accumulated (Kermanizadeh, 2014).

Once a chemical is taken up by a cell, it is transported into the lysosome. In the lysosome, the acidic environment can enhance the solubility of a NM, or it remains in the initial nano form.  Both situations can induce toxicity, causing lysosomal swelling, followed by lysosomal damage and the release of pro-apoptotic proteins (Wang et al., 2013; Cho et al., 2011; Cho et al., 2012). But not only NMs cause lysosomal damage: fluoroquinolones (Ouedraogo et al., 2000), lysosomotropic detergents such as o-methyl-serine dodecylamide hydrochloride (Villamil Giraldo et al., 2016), artesunate (Yang et al., 2014), chloroquine (Ashoor et al., 2013) can do the same, and Reactive Oxygen Species (ROS) such as H2O2 can amplify this effect (Repnik et al., 2012).  The amount of lysosomal enzymes released into the cytosol regulates the cell death pathway: controlled increased permeability of lysosomal membrane, caused by limited level of stress, plays a vital role in the induction of apoptosis, whereas massive lysosomal rupture, caused by high-stress levels, leads to necrosis (Bursch, 2001; Guicciardi et al., 2004). Lysosomes are known to trigger mitochondrial-mediated cell death by the release of cathepsins into the cytosol (Repnik et al., 2012). At the same time, however, lysosomes themselves are a source of ROS, which can lead to damage of the mitochondrial membrane (Wang et al., 2013; Kubota et al., 2010). Cell death further leads to inflammation (Faouzi et al., 2001), which activates hepatic stellate cells and induce them to secrete collagen (Casini et al., 1997). It is established that collagen accumulation is a prephase of liver fibrosis (Bataller and Brenner, 2005; Lee and Friedman, 2011).

Overall, the connection between lysosomal and mitochondrial damage with liver inflammation and further on with fibrosis, is well known, regardless if triggered by chemicals, proteins or NMs (reviewed in Malhi and Gores, 2008; Kong et al., 2014). Therefore, due to its high importance, it is described extensively in the current AOP.

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


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 1539 Endocytotic lysosomal uptake endocytosis
2 KE 898 Disruption, Lysosome Disruption, Lysosome
3 KE 177 N/A, Mitochondrial dysfunction 1 N/A, Mitochondrial dysfunction 1
4 KE 55 Cell injury/death Cell injury/death
5 KE 1493 Increased Pro-inflammatory mediators Increased pro-inflammatory mediators
6 KE 1494 Leukocyte recruitment/activation Leukocyte recruitment/activation
7 KE 265 Activation, Stellate cells Activation, Stellate cells
8 KE 68 Accumulation, Collagen Accumulation, Collagen
9 AO 344 N/A, Liver fibrosis N/A, Liver fibrosis

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


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
Not Otherwise Specified

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
human Homo sapiens High NCBI
mouse Mus musculus 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
Sex Evidence

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

Assessment of the Weight-of-Evidence supporting the AOP

Concordance of dose-response relationships

The present AOP is a purely qualitative description of the pathway. The present literature does not provide quantitative information on dose-response relationships.

Temporal concordance among the key events and adverse outcome

There is empirical evidence to support that a change in KEup leads to a change in the respective KEdown, leading to the AO.

Strength, consistency, and specificity of association of adverse outcome and initiating event

There is strong empirical evidence on the link between MIE and AO that has been described.

Biological plausibility, coherence, and consistency of the experimental evidence

There is high biological plausibility in the description of the AOP and its components. The available data describing the AOP are logic, coherent and consistent with current biological knowledge.

Uncertainties, inconsistencies and data gaps

The present AOP description is plausible but entirely qualitative, and the addition of quantitative data on dose-response relationship and temporal scale would improve its applicability. Also, though there is strong empirical evidence supporting this AOP, at certain KERs we found some inconsistencies that need to be further investigated. Existing uncertainties and inconsistencies are stated in each KER description, but here we will describe the most important ones.

Repnik and colleagues detected that after exposure to LLOMe, cathepsins remain in lysosomes and are being degraded there which is in contradiction with most of the previous studies (Repnik et al., 2017). There is strong empirical evidence that incubation of cathepsin B with mitochondria and cytosolic factors increase mitochondrial permeabilization, but in some studies pharmacological inhibition of cathepsins or knockout of genes coding for cathepsins failed to prevent mitochondrial membrane permeabilization and cell death, suggesting that other lysosomal proteases might be responsible for Bid cleavage (Reiners et al., 2002; Boya et al., 2003).

The histochemical analysis of the hippocampus of rats treated with domoic acid for 15 days has revealed no presence of apoptotic bodies and no Fluoro-Jade B positive cells (Schwarz et al., 2014).

The inflammatory role of HMGB-1 is still not completely clear. There are many studies that confirm its pro-inflammatory activity, but in some experiments, highly purified HMGB-1 had little pro-inflammatory activity (Rouhiainen et al., 2007), while in another injection of recombinant HMGB-1 into infarcted heart muscle in vivo stimulated regeneration and repair (Limana et al., 2005).

Lloyd and colleagues found that several chemokines can stimulate the adherence of peripheral blood lymphocytes to ICAM-1 coated slides (Loyd et al., 1996). However, by using a parallel plate flow chamber, another study failed to observe such an effect (Carr et al., 1996).

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

Life Stage Applicability

The described AOP is a general mechanism, that furthermore can be considered as not being limited to only the liver as the target organ. Also, to current knowledge, this AOP is not limited to a specific life stage.

Taxonomic Applicability

Most of the work performed to elaborate parts of this AOP was done using murine or human cells and cell lines, human blood samples or tissues, or mouse models. Examples include

mouse: Narumi et al., 1992; Fahy et al., 2001; Kagedal et al., 2001; Faouzi et al., 2001; Werneburg et al., 2002; Chen et al., 2007; Seki et al., 2007; Leung et al., 2008; Dalton et al., 2009; Lee et al., 2009 Gäbele et al., 2009; Nan et al., 2013; Pradere et al., 2013; McHedlidze et al., 2014; Chang et al., 2014;

rat: Rockey et al., 1992;George et al., 1999; Reeves et al., 2000; Luckey and Petersen, 2001; Duffield et al., 2005; Imamura et al., 2005; Natajaran et al., 2006; Liedtke et al., 2011; Li et al., 2012; Jung et al., 2015

human: Bleul et al., 1996; Miyamoto et al., 2000; Andersson et al., 2000; Yamada et al., 2001;  Scaffidi et al., 2002; Safadi and Friedman, 2002; Boya et al., 2003; Cirman et al., 2004; Bataller and Brenner, 2005; Bell et al., 2006; Rockey and Friedman, 2006; Friedman, 2008; Lim and Kim, 2008; Winklhofer and Haass, 2010; Clarke et al., 2010; Hamacher-Brady et al., 2011; Santibañez et al., 2011; Lee and Friedman, 2011; Wang et al., 2013; Loos et al., 2014; Decaris et al., 2015; Sun et al., 2015;

Sex Applicability

As described above, the AOP is widely applicable, therefore no specific sex applicability is known at this point.

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

The essentiality of almost all of the KEs in this AOP was rated high as there is much experimental evidence that the blocking of one KE prevents the next downstream KE and therefore the whole AOP. The essentiality of KE Mitochondrial dysfunction was rated as moderate, as there are two pathways to apoptosis (the next downstream KE), intrinsic and extrinsic, and only intrinsic pathway includes mitochondrial dysfunction (reviewed in Elmore, 2007).

Some of the evidence for the essentiality of KEs is listed below.

Exposure of cells to ammonium chloride prior to exposure to sphingosine resulted in the formation of NH3, which entered into lysosomes and became protonated and increased the pH of the organelle. This exposure prevented the accumulation of sphingosine in the lysosome and provided protection against its lysosomolytic and apoptosis-inducing effects (Kagedal et al., 2001).

Inhibition of lysosomal membrane permeabilization (LMP) with Baf A1 – that inhibits lysosomal vacuolar H+ ATPase, prevented mitochondrial membrane permeabilization (MMP) - the next downstream KE, while inhibition of MMP in Bax/Bak double knocks out cells didn't prevent LMP (Boya et al., 2003).

Faouzi and colleagues showed that the inhibition of apoptosis causes inhibition of the inflammatory response (Faouzi et al., 2001), while other study showed that an inhibitor of apoptosis is blocking the release of HMGB-1 mediator specifically (Bell et al., 2006).  

A human CXC chemokine antagonist, growth-related oncogene GROα(8-73), inhibited calcium mobilization induced by MIP-2, and the pretreatment of mice with this antagonist inhibited, in a dose-dependent manner, the influx of neutrophils induced by MIP-2, TNF-α, LPS and IL-1β (McColl and Clark-Lewis, 1999).

There is strong evidence that the blockade of TGF-β alone is sufficient to completely block experimental fibrogenesis in the liver (reviewed in Gressner et al., 2002). The activation of HSCs can be partially blocked by anti-TGF-β antibodies (Zimmermann et al., 2010) and blocked by overexpression of Smad7, a natural antagonist of TGF-β signaling (Dooley et al., 2003).  Macrophage depletion led to a significant reduction in the number of HSCs (Duffield et al., 2005).

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

Summary Table Provide an overall summary of the weight of evidence based on the evaluations of the individual linkages from the Key Event Relationship pages.

Support for essentiality of KEs

MIE Endocytic lysosomal uptake

Essentiality of the MIE is high.

Exposure of cells to NH3 prevented the accumulation of sphingosine, known lysosomotropic agent, in the lysosomes and provided protection against its lysosomolytic and apoptotic effects (Kagedal et al., 2001).

KE1 Lysosomal disruption

Essentiality of the KE1 is high.

Inhibition of LMP prevented MMP, the next downstream KE, and the entire pathway (Boya et al., 2003). The treatment of the cells with cathepsins inhibitors decreased MMP and prevented apoptosis (Roberg et al., 1999; Kagedal et al., 2001).

KE2 Mitochondrial dysfunction

Essentiality of the KE2 is moderate.

There are two apoptotic pathways, intrinsic and extrinsic, and only intrinsic pathway includes mitochondrial dysfunction (reviewed in Elmore, 2007).

KE3 Cell death/injury

Essentiality of the KE3 is high.

The inhibition of apoptosis causes blockage of the release of inflammatory mediators and inhibition of the inflammatory response (Faouzi et al., 2001; Bell et al., 2006).

KE4 Increased inflammatory mediators

Essentiality of the KE4 is high.

Chemokine antagonist can prevent the influx of neutrophils (McColl and Clark-Lewis, 1999).

KE5 Leukocyte recruitment/ activation

Essentiality of the KE5 is high.

Macrophage depletion leads to a significant reduction in the number of HSCs, the next downstream KE (Duffield et al., 2005). After bile duct ligation, Kupffer cell-depleted mice showed almost complete suppression of HSC activation and fibrosis (Seki et al., 2007).

KE6 HSC activation

Essentiality of the KE6 is high.

Experimental inhibition of HSC activation prevents fibrosis (Friedman, 2002; Anan et al., 2006; Kisseleva and Brenner, 2008; Son et al., 2009).

KE7 Collagen accumulation

Essentiality of the KE7 is high.

The continuing imbalance between the deposition and degradation of ECM is a pre-requisite of liver fibrosis (Lee and Friedman, 2011).

AO Liver fibrosis

It is generally accepted that any chronic and repeated liver injury can result in myofibroblast activation, leading to hepatic fibrosis and cirrhosis (Jaeschke, 2002; Lee William, 2003; Ramachandran and Kakar, 2009).

Support for Biological Plausibility of KERs


Biological plausibility of the KER between MIE and KE1 is high.

Trapping of lysosomotropic agents accumulates substances inside of the lysosomes, increases the volume of these organelles, and big lysosomes are more prone to rupture (Ono et al., 2003).


Biological plausibility of the KER between KE1 and KE2 is high.

In the last decade, there is a growing body of evidence about the strong functional link between lysosomes and mitochondria that play an important role in physiology and pathology (e.g. Todkar et al., 2017). The evidence also showed a link between lysosomal and mitochondrial damage, and that lysosomal damage precedes mitochondrial injury (e.g. Droga-Mazovec et al., 2008; Ghosh et al., 2011).


Biological plausibility of the KER between KE2 and KE3 is high.

There is functional mechanistic understanding supporting this relationship between KE2 and KE3, involving ROS formation, ATP depletion and apoptogenic factors (Richter et al., 1996; Leist et al., 1997; Nicotera et al., 1998; Brenner and Mak, 2009; Lu et al., 2014; Zhou et al., 2015).


Biological plausibility of the KER between KE3 and KE4 is high.

The dead cells can secrete inflammatory mediators that trigger the infiltration of immune cells. However, if this becomes chronic it has the potential to enhance tissue damage and ultimately induce fibrosis (Jaeschke, 2002; Cullen et al., 2013).


Biological plausibility of the KER between KE4 and KE5 is high.

There is much evidence that application of chemokines attracts leukocytes to a specific site in different species (Beck et al., 1997; Lee et al., 2000; Fahy et al., 2001; Nikiforou et al., 2016).


Biological plausibility of the KER between KE5 and KE6 is high.

The recruitment of immune cells from the circulation into the injured tissue is the key mechanism during fibrogenesis in the liver (Heymann and Tacke, 2016).


Biological plausibility of the KER between KE6 and KE7 is high.

The functional relationship between stellate cells activation and collagen accumulation is consistent with established biological knowledge (Milani et al., 1994; Benyon and Arthur; 2001; Safadi and Friedman, 2002; Kershenobich Stalnikowitz and Weisssbrod , 2003; Bataller and Brenner, 2005; Kolios et al., 2006; ; Guo and Friedman, 2007; Li, Jing-Ting et al., 2008; López-Novoa and Nieto, 2009; Lee und Friedman 2011).


Biological plausibility of the KER between KE7 and AO is high.

By definition, liver fibrosis is the excessive accumulation of ECM proteins that are produced by HSCs. The KER between this KE and the AO is undisputed (Poynard et al., 1997; Bataller and Brenner, 2005; Rockey and Friedman, 2006; Brancatelli et al., 2009; Lee and Friedman, 2011).

Empirical Support for KERs


Empirical support of the KER between MIE and KE1 is high.

Even the accumulation of substances that are physiologically present in lysosomes, such as pro-cathepsins, can lead to lysosomal dysfunction (Jung et al., 2015). Sphingosine is a lysosomotropic agent that accumulates within the lysosomes, where it permeabilizes the membrane via a detergent mechanism and provokes the release of lysosomal enzymes (Kagedal et al., 2001). NM captured in lysosomes can cause lysosomal swelling, disruption and the release of pro-apoptotic proteins (Cho et al., 2011; Cho et al., 2012; Wang et al., 2013).


Empirical support of the KER between KE1 and KE2 is high.

LMP is detected a couple of hours earlier than MMP, after exposure to ciprofloxacine, norfloxacine and hydroxychloroquine, and cells with MMP are sub-ensemble of the group of cells with LMP (Boya et al., 2003).

When isolated mitochondria are incubated with purified cathepsin B in the presence of cytolic extracts, a release of cytochrome c from mitochondria is detected (Guicciardi et al., 2000). The microinjection of cathepsin D to the cell causes cytochrome c release, caspases activation, and apoptosis (Roberg et al., 2002).

Bid protein needs to be cleaved in order to cause cytochrome c release (Luo et al., 1998; Gross et al., 1999; Stoka et al., 2001).

Incubation of Bax with isolated mitochondria resulted in cytochrome c release while Bcl-xl inhibits this release (Jurgensmeier et al., 1998).


Empirical support of the KER between KE2 and KE3 is moderate.

Mice injected intraperitoneally with DomA have shown an increase of the TUNEL positive cells in the hippocampus, decreased indicators of mitochondria function and elevated ROS levels (Lu et al., 2012, Wu et al., 2012). The incidence of downstream KE (cell death) is higher than the incidence of downstream KE (mitochondrial dysfunction).

In vitro studies (Giordano et al., 2007; 2009) suggest that both KEs are affected by the same dose of DomA and that the incidence of KE down (cell death) is higher than the incidence of KE up (mitochondrial dysfunction), with KE up happening earlier than KE down.


Empirical support of the KER between KE3 and KE4 is high.

During the apoptosis of Jurkat cells treated with various agents, HMGB-1 was released into the media (Bell et al., 2006), which was found to activate leukocytes and stimulate the production of pro-inflammatory mediators in vitro (Li et al., 2004; Zimmermann et al., 2004). ATP can also stimulate the production of pro-inflammatory cytokines from macrophages (Ferrari et al., 1997; Ferrari et al., 2006).

Neutralization or genetic deficiency of IL-1 inhibited inflammation responses to injected dead cells (Chen et al., 2007; Kono et al., 2010). Injection into mice of a variety of other dead cell types that genetically lack both IL-1α and IL- 1β stimulated an inflammatory response that was equivalent to that of wildtype necrotic cells (Kono et al., 2010). This implicates that IL-1 that is driving the sterile inflammatory response in many cases is not coming directly from the dead cell but is produced by cells in the host upon recognition of cell death.


Empirical support of the KER between KE4 and KE5 is high.

The exposure of mice to FliCind strain S. Typhimurium triggered a significant neutrophil influx in the spleen of wild-type mice, but not of Il1b−/−Il18−/− mice (Jorgensen et al., 2016).

It was shown that exposure of cells to IL-1β, TNF-α, and IFN-γ resulted in the induction of RANTES mRNA and protein (Ortiz et al., 1996; Miyamoto et al., 2000). Intradermal injection of RANTES induces a potent T-lymphocyte and eosinophils recruitment (Fahy et al., 2001; Beck et al., 1997).  Intradermal administration of MIP-1a resulted in the accumulation of monocytes, lymphocytes, eosinophils, and the recruitment of neutrophils (Lee et al., 2000).

The number of white blood cells, monocytes, and neutrophils were increased in cord blood after 6 days of IL-1α exposure (Nikiforou et al., 2016).


Empirical support of the KER between KE5 and KE6 is moderate.

Karlmark et al., 2009 found that intrahepatic CD11bF4/80 monocyte-derived cells and liver resident macrophages produce TGF-β1 and thereby directly activate HSCs. It was proven that the treatment of cultured hepatic cells with TGF-β1 increased type I pro-collagen mRNA levels (Czaja et al., 1989).


Empirical support of the KER between KE6 and KE7 is moderate.

It is difficult to stimulate sufficient collagen production and its incorporation into a pericellular matrix in vitro. Because of that analytical methods have focused on the measurement of pro-collagen secreted into culture medium or measurement of α-smooth muscle actin (α-SMA) expression, a marker of fibroblast activation. In primary culture, HSCs from normal liver begin to express α-SMA coincident with culture-induced activation (Rockey et al., 1992; Chen and Raghunath, 2009).


Empirical support of the KER between KE7 and AO is high.

There is a smooth transition from collagen accumulation to liver fibrosis without a definite threshold with plenty in vivo evidence (Poynard et al., 1997; Bataller and Brenner, 2005; Rockey and Friedman, 2006; Brancatelli et al., 2009; Lee and Friedman, 2011).

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

The quantitative understanding of the AOP is low, as there is a lack of quantitative data on the dose-response relationship. Further research efforts should be made in this direction to improve the quantitative understanding of the present AOP.

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


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

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