Aop: 34

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

LXR activation leading to hepatic steatosis

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
LXR Activation to Liver Steatosis

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

Marina Goumenou

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
Marina Goumenou   (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
  • Marina Goumenou

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
Not under active development Under Development
This AOP was last modified on June 15, 2020 16:18
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
Activation, LXR September 16, 2017 10:14
Up Regulation, CD36 September 16, 2017 10:14
Increase, FA Influx September 16, 2017 10:14
Activation, ChREBP September 16, 2017 10:14
Activation, SREBP-1c September 16, 2017 10:14
Activation, FAS September 16, 2017 10:14
Activation, SCD-1 September 16, 2017 10:14
Synthesis, De Novo FA September 16, 2017 10:14
Accumulation, Triglyceride September 16, 2017 10:14
Damaging, Mitochondria September 27, 2017 16:05
N/A, Liver Steatosis September 16, 2017 10:14
peroxisome proliferator activated receptor promoter demethylation September 16, 2017 10:14
Activation, LXR leads to Up Regulation, CD36 December 03, 2016 16:37
Up Regulation, CD36 leads to Increase, FA Influx December 03, 2016 16:37
Activation, LXR leads to Activation, ChREBP December 03, 2016 16:37
Activation, LXR leads to Activation, SREBP-1c December 03, 2016 16:37
Activation, LXR leads to Activation, FAS December 03, 2016 16:37
Activation, LXR leads to Activation, SCD-1 December 03, 2016 16:37
Activation, SCD-1 leads to Synthesis, De Novo FA December 03, 2016 16:37
Activation, ChREBP leads to Synthesis, De Novo FA December 03, 2016 16:37
Activation, SREBP-1c leads to Synthesis, De Novo FA December 03, 2016 16:37
Activation, FAS leads to Synthesis, De Novo FA December 03, 2016 16:37
Synthesis, De Novo FA leads to Accumulation, Triglyceride December 03, 2016 16:37
Increase, FA Influx leads to Accumulation, Triglyceride December 03, 2016 16:37
Accumulation, Triglyceride leads to Damaging, Mitochondria December 03, 2016 16:37
demethylation, PPARg promoter leads to Up Regulation, CD36 November 29, 2016 20:10

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

Liver steatosis (fatty liver) is considered as one of the first manifestations of possible hepatotoxicity, however it is not regarded as an adverse effect per se and usually can be reversible. The importance of steatosis is highlighted from the fact that it is a prerequisite for the development of non-alcoholic fatty liver disease (NAFLD). NAFLD is a clinicopathological condition that comprises a wide spectrum of liver damage, ranging from steatosis alone to steatohepatitis, advanced fibrosis and cirrhosis. Non-alcoholic steatohepatitis (NASH) represents only a stage in the spectrum of NAFLD and is defined pathologically by the presence of steatosis together with necro-inflammatory activity. The clinical implications of NAFLD are derived mostly from its potential to progress to end-stage liver disease, whereas simple uncomplicated steatosis follows a relatively benign course in most patients. Steatosis is the output of the disturbance on the homeostasis of hepatic lipids which depends on the dynamic balance of several pathways including fatty acid (FA) uptake, de novo FA synthesis, β-oxidation, and very low-density lipoprotein (VLDL) secretion. It is characterized by the accumulation of lipid droplets (mainly triglycerides) in the hepatocytes. This AOP describes the linkage between hepatic steatosis triggered by nuclear receptors activation (PPAR gamma and LXR) through modulation of genes responsible for lipid homeostasis [the carbohydrate response element binding protein (ChREBP), the sterol response element binding protein 1c (SREBP-1c), the free fatty acid uptake transporter FAT/CD36, the fatty acid synthase (FAS), the stearoyl-CoA desaturase 1 (SCD1)] which subsequently leasds to in rease of de novo fatty acids/triglycerides synthesis and fat influx from the peripheral tissues to liver. The accumulation of lipid in the hepatocytes can cause cytoplasm displacement, nucleus distortion, mitochondrial toxicity and eventually necrosis and/or apoptosis. The progression of this condition can lead to tissue inflammation (steatohepatitis) and fibrosis with the involvement of other cells of the hepatic tissue like the Kupffer (inflammation) and the stellate (fibrosis) cells. This purely qualitative AOP description is plausible, the scientific data supporting the AOP are logic, coherent and consistent and there is temporal agreement between the individual KEs. Quantitative data on dose-response-relationships and temporal sequences between key events are still lacking; the provision of quantitative data will strengthen the weight of evidence and make the AOP applicable for chemical risk assessment purposes.

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 167 Activation, LXR Activation, LXR
2 MIE 228 peroxisome proliferator activated receptor promoter demethylation demethylation, PPARg promoter
3 KE 54 Up Regulation, CD36 Up Regulation, CD36
4 KE 115 Increase, FA Influx Increase, FA Influx
5 KE 66 Activation, ChREBP Activation, ChREBP
6 KE 264 Activation, SREBP-1c Activation, SREBP-1c
7 KE 116 Activation, FAS Activation, FAS
8 KE 258 Activation, SCD-1 Activation, SCD-1
9 KE 89 Synthesis, De Novo FA Synthesis, De Novo FA
10 KE 291 Accumulation, Triglyceride Accumulation, Triglyceride
11 KE 176 Damaging, Mitochondria Damaging, Mitochondria
12 AO 345 N/A, Liver Steatosis N/A, Liver Steatosis

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 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

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

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

Consider the following criteria (may include references to KE Relationship pages): 1. concordance of dose-response relationships; 2. temporal concordance among the key events and adverse effect; 3. strength, consistency, and specificity of association of adverse effect and initiating event; 4. biological plausibility, coherence, and consistency of the experimental evidence; 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; 6. uncertainties, inconsistencies and data gaps.

Concordance of dose-response relationships

The existing studies do not provide dose-response curves. However it may be possible in some cases to construct curves from the given numerical data and to relate the dose response for LXR activation with the dose response for TG accumulation in vitro and in vivo in a second more quantitative iteration as the next step of this AOP development.

Temporal concordance among the key events and adverse outcome

According to the available information the sequence of the events is in strong agreement and consequently the presented MoA could be considered as qualitatively accurate.

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

The scientific evidence is presented in Scientific Evidence in support of the MoA.

Biological plausibility, coherence, and consistency of the experimental evidence

The steatogenic effect of chemicals like LXR ligands is well established in the literature (Peet 1998, Schultz et al. 2000, Horton et al. 2002) and it is well correlated with the expression of the receptor (Moya et al. 2010) the binding to it. In addition it is believed that LXR acts as a cholesterol sensor. Consistent with this role, it has been proposed that LXR induces SREBP-1c in order to generate fatty acids needed for the formation of cholesterol esters, which buffer the free cholesterol concentration (Ferré & Foufelle 2007). Further analysis of the logic, coherence and consistency along with the experimental data has already been presented in Chapter Scientific Evidence in support of the MoA.

Alternative mechanism(s) that logically present themselves and the extent to which they may distract from the postulated AOP.

As already mentioned abovem there are many other possible MoAs of a chemical in the development of steatosis including MoAs involving the inhibition of β-oxidation, the inhibition of oxidative phosphorylation (leading to a decrease of ATP needed for β-oxidation) and the malfunction of the mechanisms of the excretion of TG from the cell. These pathways are not covered in the presented MoA as they are not directly linked with the activation of LXR. Furthermore, as already explained LXR is not the only receptor which has been identified to be involved in fatty acid metabolism and steatogenesis. Exogenous chemicals acting as ligands for any of the following Nuclear Receptors (AhR, PXR, PPARa, PPARγ and ER) may play a role in the development of steatosis (grey elements on the AOP flow diagram, Fig. 3). There also known interactions or cross-talk between the NRs. Examples of possible interactions are related with fact that LXR is also regulated by the PPARa, the FAT/36 up-regulation from the AhR, PXR and PPARγ, the inhibition of β-oxidation from PPARa and indirectly from the ER.

It may be possible from existing literature, or further experimental work to develop MoAs taking binding to each of these receptors as the molecular initiating event and describing the converging pathways leading to steatosis. In fact this work is in progress and indicated as the grey elements on the AOP flow diagram above.

The biology of LXR function has been studied using the high affinity synthetic ligands T0901317. According to the study of Mitro (2007), T0901317 binds and activates hPXR and hLXRβ with similar affinity, and can regulate multiple PXR target genes in human cells and mice (like CD36) with similar efficacy to established PXR ligands, but significantly greater potency (Mitro 2007). The author suggested that some of the effects observed with T0901317 such as the more deleterious increase in lipogenesis and hepatic lipid accumulation (in comparison to the LXR-selective GW3965) that have been ascribed to LXR activation maybe the result of simultaneous stimulation of PXR and LXR activity and that the assumption that T0901317 behaves as an LXR-selective agonist may have led to some inaccurate conclusions regarding the effects of LXR activation in vivo.

From the data of this study it is evident that SREBP-1c, FAS and SCD-1, which are LXR but not PXR regulated genes, were significantly up-regulated by T0901317. In contrast GW3965 up-regulates less effectively the SREBP-1c, marginally the SCD-1 and not at all the FAS despite the fact that it is considered as a selective LXR agonist (Mitro 2007). The CD36 gene is considered also as a liver specific target of LXR activation (Zhou 2008). However, in the study of Mitro (2007), GW3965 did not up-regulate CD36. These findings could be explained by the lower affinity of this synthetic LXR agonist (EC50 of 0.19 and 0.03 μM for hLXRα and hLXRβ) in relation to the T0901317 (EC50 of 0.02-0.05 μM for both isoforms). Interestingly and despite the low up-regulating activity, GW3965 increases FA and TG accumulation in rat and primary human hepatocytes (Kotokorpi et al. 2007). Based on this information, it could be possible that T0901317 binding on PXR could enhance its steatogenic activity with the proposed MoA still being plausible. This plausibility, however, is clearly related with quantitative aspects.

In conclusion, the MoA described can be considered very well supported by the available scientific evidence and it is biologically plausible.

Uncertainties, inconsistencies and data gaps

The information used for the development of the present pathway is based on in-vitro and in-vivo studies. In the in-vitro studies several cell lines have been used. The expression of the LXR, the SREBP-1c and other elements on these cell lines is a key factor for the plausibility of the pathway in human. According to the study from Moya et al 2010, LXR expression (as measured from mRNA using RT-PCR) in human hepatocytes, HepG2 and HeLa cells was approximately 70%, 70% and 50% in relation to the level of expression in human liver. In addition the expression of SREBP-1c was significantly down-regulated (to less than 25% of normal levels of expression in the liver) in all 3 cell lines. Consequently positive results in relation to fat accumulation after LXR activation from studies using these cell lines may under-estimate the magnitude of effect on human liver while negative results could be interpreted as inconclusive. The assessment of the relative expression of these receptors in other cell lines would be of great importance in order to evaluate the relevance of each in vitro study result.

In relation to the in vivo studies which have be made mainly (if not exclusively) in rodents the relevance for humans should be addressed. LXR expression is considered adequately conserved from rodents to humans. In addition it is well known that all the other elements of the pathway are present in human liver. A good example of this is that the well-defined pathogenic role of FAT/CD36 in hepatic steatosis in rodents is also confirmed by the up-regulation in humans of the FAT/CD36 in cases of NASH, NAFLD, insulin resistance, hyperinsulinaemia, HCV and morbidly obese patients (Zhu et al. 2011, Love-Gregory & Abmurad 2011, Miquilena-Colina et al. 2011, Bechmann et al. 2010). However, there is some speculation in relation to the extent that adverse side effects observed in rodents will occur in higher species, including humans. These speculations are raised due to the different behaviour of the LXR agonist GW3965 in in vitro systems which although markedly stimulating lipogenic gene expression in primary human hepatocytes leading to significant TG accumulation at all 3 dose levels after 48 hr, produced only a very modest increase in the triglyceride content in rat cells (Kotokorpi et al. 2007), demonstrating that the use of this rat cell line could underestimate the effect in humans. FA increase was reported in both cell lines.

Another interesting finding is that in humans, total CD36 deficiency is relatively common (3–5%) in persons of African and Asian descents (Su & Abmurad 2009). Consequently the presented MoA could be affected mainly quantitatively among humans of different origin.

Induction of lipogenic enzymes from the SREBP-1c is evidenced in adult mice but not during the fetal life indicating a different role of the SREBP-1c between these two stages (Liang et al. 2002). This finding gives a strong indication that the presented pathway may be altered in other than adult life stage.

Another finding is that of the study of Hu et al. 2005 according to which administration of T0901317 in PPAR-null mice promoted a dose-dependent increase in the rate of peroxisomal β-oxidation in the liver and in relation only to the LXRα. The author suggests that this induction may serve as a counter regulatory mechanism for responding to the hypertriglyceridemia and liver steatosis that is promoted by potent LXR agonists in vivo.

T090137 was shown to up-regulate hepatic expression and plasma activity of PLTP in mice in addition to angiopoietin-like protein 3 (Angptl3), playing a critical role in LXR-induced hypertriglyceridemia. However it should be noted that hypertriglyceridemic effect of LXR agonists is usually transient and limited to the first few days of the treatment likely due to enhanced VLDL-triglyceride hydrolysis resulting from increased expression of hepatic LPL (Baranowski 2008).

Some studies have demonstrated absence of triglyceride accumulation on SREBP-1c (-/-) mice suggesting that SREBP-1c is a crucial element of the present MoA (Liang et al. 2002, Schultz et al. 2000, Horton et al. 2002, Shimano et al. 1999). In another study in FAT/CD36 knockout mice the effect of LXR agonists on increasing hepatic and circulating levels of triglycerides and free fatty acids (FFAs) was largely abolished suggesting that intact expression and/or activation of FAT/CD36 is required for the steatotic effect of LXR agonists (Febbraio et al. 1999). These two findings together and considering that they are constant and not related with specific experimental conditions could lead one to the hypothesis that both SREBP-1c and CD36 are imperative elements for the cause of steatosis. This hypothesis could be further examined.

The present MoA could also be affected by factors related to the formation of steatosis such as trends in adipose tissue (AT) deposition, the total body fat, the visceral AT and the subcutaneous AT which vary among different life stages such as childhood, puberty and adolescence, between sexes and among humans of different origin (Staiano 2012).

Assessment of the quantitative understanding of the AOP

In the present study only qualitative assessment of the proposed MoA was performed. In the studies used there are numerical data mainly to support the expression and up-regulation of the different elements of the pathway. However, further analysis of these numerical data is suggested in following steps.

Interestingly, the existence of many network motifs along the pathways was noted during the analysis of the literature, e.g. the positive feed forward LXR up-regulation. This information could be used in the future for the quantitative interpretation of dose response curves and the development of quantitative prediction models of the adverse outcome following the activation of the LXR.

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

Confidence in the AOP

Information from this section should be moved to the Key Event Relationship pages! Elaborate on the domains of applicability listed in the summary section above. Specifically, provide the literature supporting, or excluding, certain domains.

How well characterised is the AOP?

Liver steatosis is a well understood adverse outcome. A great number of publications from in vitro, in vivo, mechanistic, clinical and epidemiological studies exist for the qualitative assessment of steatosis. However, the quantitative analysis of the role of a specific exogenous chemical in an adverse outcome in human is a very challenging task due to the involvement of a large number of inter-related factors following the MIE. In fact one chemical may bind to more than one receptor and consequently have different impacts either quantitatively or qualitatively on the downstream events.

How well are the initiating and other key events causally linked to the outcome?

LXR agonists such T0901317 have been shown to produce LXR activation, as well as triglyceride accumulation, which has been demonstrated in rodent (mouse and rat) and human liver cell lines in vitro. The same chemicals shown to be LXR agonists in the in vitro assays have shown triglyceride accumulation in the liver leading to steatosis in animals and humans through steps of the reported MoA.

What are the limitations in the evidence in support of the AOP?

Disagreement in the scientific evidence supporting the presented AOP was not found. In relation to data gaps in addition to lack of quantitative information as discussed above there is also lack of specific information in relation to the role of other target genes expressed after the LXR activation.

Is the AOP specific to certain tissues, life stages / age classes?

There is evidence of different levels of expression of CD36 in different ethnic groups which may be expected to alter the sensitivity to development of steatosis. There may also be differences in expression and role of the same proteins/enzymes in foetal life but this has not been fully elucidated. Further information can be found in the Chapter Uncertainties, inconsistencies and data gaps.

Are the initiating and key events expected to be conserved across taxa?

From the analysis of the available information from experimental studies using rodents the elements of the MoA appeared to be well conserved between mice and rats. Some concerns in relation to the relevance of the in vivo studies to human are raised mainly due to the different behaviour of the LXR agonist GW3965 which while stimulating lipogenic gene expression in human hepatocytes, causes only a slight increase in TGs in rats (Kotokorpi et al. 2007). Some more differences were also reported between hamsters and monkeys in relation to hypertriglyceridemia (Groot et al. 2005).

Scientific Evidence to Support AOP

Information from this section should be moved to the Key Event Relationship pages! Detailed Description

Old format, potemtially to be migrated to table above and underlying articles

Events Scientific Support Strength of Evidence
LXR binding and activation

(Molecular initiating event) receptor

[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]

Very Strong
Binding in the LXREs [11] Very Strong
Target genes transcription [12] Very Strong
Auto-regulation of the LXRa

[13] [14] [15] [16]

Very Strong
Increase in expression and activity of the carbohydrate response element-binding protein (ChREBP)

[17] [18]

Strong

According to the study of Denechaud increase in the glucose flux in the cell is a prerequisite for ChREBP activation from T0901317 in mice

Increase in expression of the SREBP-1c from LXR

[19] [3] [20] [21] [22]

Very Strong

However, there are many studies supporting a different behaviour between LXRα and LXRβ, suggesting that SREBP-1c up-regulation is only due to LXRα

Increase in expression of the SREBP-1c from the ChREBP [23] Well established
Induction of lipogenic enzymes from the SREBP-1c (FAS, ACC, GK)

[24] [25] [26] [27] [3] [28] [29] [30] [31]

Very Strong

However there is evidence that this effect is not induced in the embryonic state indicating a different role of the SREBP-1c between embryonic and adult life (Liang et al. 2002). It is also suggested that for lipogenic genes, SREBP-1c acts together with ChREBP (Ishii et al. 2004).

Direct induction of the fatty acid synthase (FAS)

[3] [32] [33]

Very Strong
Direct induction of the stearoyl-CoA desaturase 1 (SCD1)

[34] [35] [3] [36]

Very Strong
Up-regulation of the free fatty acid uptake transporter FAT/CD36

[37] [38] [39] [40] [41] [42]

Very Strong
De novo fatty acids and triglyceride synthesis [43] Very Strong
Fat influx from the peripheral tissues

[11] [44] [45]

Very Strong
Steatosis

[45] [46]

Very Strong

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