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AOP: 456

Title

A descriptive phrase which references both the Molecular Initiating Event and Adverse Outcome.It should take the form “MIE leading to AO”. For example, “Aromatase inhibition leading to reproductive dysfunction” where Aromatase inhibition is the MIE and reproductive dysfunction the 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

Aryl hydrocarbon receptor activation leading to early life stage mortality via sox9 repression induced cardiovascular toxicity

Short name
A name that succinctly summarises the information from the title. This name should not exceed 90 characters. More help
Ahr mediated early stage mortality via cardiovascular toxicity
The current version of the Developer's Handbook will be automatically populated into the Handbook Version field when a new AOP page is created.Authors have the option to switch to a newer (but not older) Handbook version any time thereafter. More help
Handbook Version v2.0

Graphical Representation

A graphical representation of the AOP.This graphic should list all KEs in sequence, including the MIE (if known) and AO, and the pair-wise relationships (links or KERs) between those KEs. More help
Click to download graphical representation template Explore AOP in a Third Party Tool

Authors

The names and affiliations of the individual(s)/organisation(s) that created/developed the AOP. More help

Prarthana Shankar, Ph.D., US EPA Mid-Continent Ecology Division, Duluth, MN, USA (pshankar@usgs.gov)

Dan Villeneueve, Ph.D., US EPA Mid-Continent Ecology Division, Duluth, MN, USA (villeneuve.dan@epa.gov)

Point of Contact

The user responsible for managing the AOP entry in the AOP-KB and controlling write access to the page by defining the contributors as described in the next section.   More help
Prarthana Shankar   (email point of contact)

Contributors

Users with write access to the AOP page.  Entries in this field are controlled by the Point of Contact. More help
  • Prarthana Shankar

Coaches

This field is used to identify coaches who supported the development of the AOP.Each coach selected must be a registered author. More help

OECD Information Table

Provides users with information concerning how actively the AOP page is being developed and whether it is part of the OECD Workplan and has been reviewed and/or endorsed. OECD Project: Assigned upon acceptance onto OECD workplan. This project ID is managed and updated (if needed) by the OECD. OECD Status: For AOPs included on the OECD workplan, ‘OECD status’ tracks the level of review/endorsement of the AOP . This designation is managed and updated by the OECD. Journal-format Article: The OECD is developing co-operation with Scientific Journals for the review and publication of AOPs, via the signature of a Memorandum of Understanding. When the scientific review of an AOP is conducted by these Journals, the journal review panel will review the content of the Wiki. In addition, the Journal may ask the AOP authors to develop a separate manuscript (i.e. Journal Format Article) using a format determined by the Journal for Journal publication. In that case, the journal review panel will be required to review both the Wiki content and the Journal Format Article. The Journal will publish the AOP reviewed through the Journal Format Article. OECD iLibrary published version: OECD iLibrary is the online library of the OECD. The version of the AOP that is published there has been endorsed by the OECD. The purpose of publication on iLibrary is to provide a stable version over time, i.e. the version which has been reviewed and revised based on the outcome of the review. AOPs are viewed as living documents and may continue to evolve on the AOP-Wiki after their OECD endorsement and publication.   More help
OECD Project # OECD Status Reviewer's Reports Journal-format Article OECD iLibrary Published Version
Under Review
This AOP was last modified on June 16, 2023 12:57

Revision dates for related pages

Page Revision Date/Time
Activation, AhR February 28, 2024 05:12
dimerization, AHR/ARNT September 16, 2017 10:14
Decrease, sox9 expression August 11, 2022 15:09
Increase, slincR expression August 01, 2022 13:37
Altered, Cardiovascular development/function September 16, 2017 10:14
Increase, Early Life Stage Mortality March 22, 2018 10:23
Activation, AhR leads to dimerization, AHR/ARNT March 22, 2018 11:02
Activation, AhR leads to Decrease, sox9 expression April 13, 2023 12:57
dimerization, AHR/ARNT leads to Increase, slincR expression September 08, 2022 18:44
Increase, slincR expression leads to Altered, Cardiovascular development/function September 08, 2022 18:47
Increase, slincR expression leads to Decrease, sox9 expression September 08, 2022 18:45
Activation, AhR leads to Increase, Early Life Stage Mortality April 14, 2019 15:17
Decrease, sox9 expression leads to Altered, Cardiovascular development/function October 20, 2022 16:44
Activation, AhR leads to Altered, Cardiovascular development/function October 21, 2022 13:42
Altered, Cardiovascular development/function leads to Increase, Early Life Stage Mortality March 23, 2018 14:29
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) February 09, 2017 14:32

Abstract

A concise and informative summation of the AOP under development that can stand-alone from the AOP page. The aim is to capture the highlights of the AOP and its potential scientific and regulatory relevance. More help

The Aryl Hydrocarbon Receptors (Ahrs) are evolutionarily conserved ligand-dependent transcription factors that are activated by structurally diverse endogenous compounds as well as environmental chemicals such as polycyclic aromatic hydrocarbons and halogenated aromatic hydrocarbons . Ahr activation leads to several transcriptional changes that can cause developmental toxicity resulting in mortality. Evidence was assembled and evaluated for a novel adverse outcome pathway (AOP) which describes how Ahr activation (molecular initiating event; MIE) can lead to early-stage mortality (adverse outcome; AO), via SOX9-mediated cardiovascular toxicity. Using a key event relationship (KER)-by-KER approach, we collected evidence using both a narrative search, and through systematic review based on detailed search terms. Weight of evidence for each KER was assessed to inform overall confidence of the AOP. The AOP links to previous descriptions of Ahr activation (ex: AOPs 21 and 150), and connect them to two novel key events (KEs), increase in slincR expression, a newly characterized long non-coding RNA with regulatory functions, and suppression of SOX9, a critical transcription factor implicated in chondrogenesis and cardiac development. In general, confidence levels for KERs ranged between medium and strong, with few inconsistencies, as well as several opportunities for future research identified. While majority of the KEs have only been demonstrated in zebrafish with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as an Ahr activator, evidence suggests that the two AOPs likely apply to most vertebrates, and many Ahr activating chemicals. Addition of the AOP into the AOP-Wiki helps expand the growing Ahr-related AOP network to nineteen individual AOPs, of which six are endorsed or in progress, and the remaining 13 relatively underdeveloped.  

AOP Development Strategy

Context

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. More help

<<<<<The key events (KEs) associated with AOPs 455 and 456 are predominantly similar, with the exception of KE4 in each AOP. KE4 in AOP 455 is designated an AO and is Event 1559: “Facial cartilage structures are reduced in size and morphologically distorted”, and KE4 in AOP 456 is Event 317: “Altered, Cardiovascular development/function.” While AOP 456 may be of higher biologically relevance, both AOPs are ecologically important and contribute significantly to the growing network of AOPs beginning with the activation of the Aryl hydrocarbon receptor (Ahr). Since both AOPs have several overlapping KEs, some redundant text is to be expected in the individual AOP-Wiki pages.>>>>>

The Aryl Hydrocarbon Receptor (Ahrs) are evolutionarily conserved ligand-dependent transcription factors that can be activated by a wide range of structurally diverse compounds (Denison and Nagy 2003; Hahn et al. 2017). The Ahrs have critical physiological roles in normal development of both vertebrates and invertebrates, and several endogenous Ahr ligands, such as retinoic acid and metabolites of tryptophan, have been identified (Esteban et al. 2021; Nguyen and Bradfield 2008). In addition, Ahr activation by environmental pollutants including halogenated aromatic hydrocarbons (HAHs), polycyclic aromatic hydrocarbons (PAHs), and polychlorinated biphenyls (PCBs) can lead to a variety of adverse health effects, such as dysfunction to the immune, reproductive, and cardiovascular systems (Hansen et al. 2014; Hernandez-Ochoa et al. 2009; Stevens et al. 2009; Zhang 2011), as well as improper development and neurobehavior (Garcia et al. 2018a). Ahr activation is also associated with tumor promotion and carcinogenesis (Safe et al. 2013). Several studies in model organisms such as zebrafish and rodents have shown that Ahr-deficient animals in gene knock-out studies have either diminished or no harmful effects from exposure to Ahr activating environmental pollutants (Fernandez-Salguero et al. 1996; Garcia et al. 2018a; Goodale et al. 2015; Harrill et al. 2016), highlighting the significance of the receptors in mediating toxicity of Ahr-active chemicals.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a bioaccumulative and highly toxic HAH, is typically used as the prototypical molecular probe to investigate Ahr-related outcomes and is thus one of the most thoroughly investigated of the known Ahr agonists. One notable difference between dioxins such as TCDD, and labile PAHs for example, is that TCDD exposure leads to prolonged and continuous receptor activation, which is different from PAH-induced transient receptor activation that is generally considered an adaptive response. However, significant dioxin-like toxicity, including generation of oxidative stress, has been demonstrated in several organisms exposed to PAHs. Toxicity is generally attributed to the generation of harmful reactive metabolites, or from environmentally relevant chronic PAH exposures that can induce sustained Ahr activation (Billiard et al. 1999). Further, any differences in Ahr-dependent toxicity among species is likely because of the presence of multiple Ahr isoforms, in combination with their differential binding affinities to a specific chemical (Doering et al. 2013; Doering et al. 2018; Karchner et al. 2006). Regardless, it is widely accepted that upon activation of the Ahrs, a cascade of complex molecular events ensues, leading to crosstalk signaling and pathophysiological effects. While several possible lesser-understood signaling pathways exist (Sondermann et al. 2023; Wright et al. 2017), the most widely described and major signaling route is the canonical Ahr signaling pathway.

Canonical Ahr signaling involves the conversion of the inactive Ahr, which is present in the cytoplasm, to its active form that can translocate to the nucleus and dimerize with the Ahr nuclear translocator (ARNT) (Wright et al. 2017). The Ahr-ARNT heterodimer can consequently regulate transcription of several downstream genes either indirectly, or directly, which is the case for the cytochrome P450s (CYPs) that are induced via the direct binding of the heterodimer to the aryl hydrocarbon response elements (Ahres, or XREs or DREs) (Lo and Matthews 2012). To help organize the complexity of the concurrent regulation of 1000s of genes by the Ahr signaling pathway, as well as consequent toxicity effects, scientists have begun to organize existing evidence in the form of Adverse Outcome Pathways (AOPs) (Ankley et al. 2010) and AOP networks (Knapen et al. 2018). There are currently nineteen Ahr-related AOPs in the AOP-Wiki (as of April 10th, 2023; aopwiki.org), with six AOPs included in The Organization for Economic Co-operation and Development’s (OECD) Work Plan that are open for comments, and the remaining 13 relatively under developed. With the rapid rate at which new research on Ahr-mediated toxicity is being conducted, there is still extensive scope for assembly of existing and novel biological data into actionable knowledge that can support decision-making around Ahr-related environmental effects and disease outcomes.

Besides being highly relevant and important toxicity phenotypes in both humans and other vertebrates, both craniofacial malformations and cardiovascular toxicity are easily observable and measurable in zebrafish, and have been identified upon exposure to various Ahr activating environmental chemicals (Antkiewicz et al. 2005; Henry et al. 1997; Li et al. 2014). Importantly, developing zebrafish exposed to TCDD have severe heart and vasculature malformations, in addition to jaw structure impairments that occur secondarily to inhibited chondrogenesis (Carney et al. 2006). One of the genes whose expression is most reduced in the jaw upon TCDD exposure in zebrafish is sox9b, sry-box containing gene 9b (Xiong et al. 2008). This gene, one of two zebrafish paralogs of the SOX9 gene, is a critical transcription factor that has been implicated in several processes including chondrogenesis and cardiac development, in addition to skeletal development, male gonad genesis, and cancer progression (Lefebvre and Dvir-Ginzberg 2017; Panda et al. 2021). Based on current knowledge, primarily from developmental zebrafish studies, it is apparent that there are strong relationships between Ahr, SOX9, and craniofacial (AOP 455) or cardiovascular (AOP 456) malformations that can be causally linked in an AOP network.

The two AOPs also provide weight of evidence for the inclusion of a novel long non-coding RNA (lncRNA) as a key event. LncRNAs are transcripts longer than 200 nucleotides that do not encode functional proteins, but have their own promoters and the ability to be processed (spliced and polyadenylated) similar to mRNAs (Mattick et al. 2023). The nature of lncRNAs is such that they have diverse functions and can regulate gene expression at multiple levels, including by interacting with DNA, RNA, proteins, and altering transcription of both neighboring and distant genes (Statello et al. 2021). Importantly, there is growing recognition for the link between exposure to chemicals, differential expression profiles of lncRNAs, and consequent toxicity (Dempsey and Cui 2017). Specific to the proposed AOPs, evidence suggests an important role for the recently discovered lncRNA, “sox9b long intergenic non-coding RNA” (slincR) in the Ahr signaling toxicity pathway via its interaction with the transcription factor, SOX9 (Garcia et al. 2017). Thus, the ability of SOX9 to interact with Ahr signaling, paired with its functional versatility, implicates it as a critical player in the Ahr toxicity pathway, by mediating disruptions to both craniofacial and cardiovascular development.

Strategy

Provides a description of the approaches to the identification, screening and quality assessment of the data relevant to identification of the key events and key event relationships included in the AOP or AOP network.This information is important as a basis to support the objective/envisaged application of the AOP by the regulatory community and to facilitate the reuse of its components.  Suggested content includes a rationale for and description of the scope and focus of the data search and identification strategy/ies including the nature of preliminary scoping and/or expert input, the overall literature screening strategy and more focused literature surveys to identify additional information (including e.g., key search terms, databases and time period searched, any tools used). More help

A so called “KER-by-KER” approach (Svingen et al. 2021) was leveraged to gather different lines of evidence to support the two proposed AOPs. This basically describes a process in which evidence gathering is focused on the relationship between a specific pair of key events. Search terms relevant to the biological effects described in those events are employed, typically without regard for whether the stressors or experimental manipulations involved in the associated studies have relevance to the AOP as a whole. Such an approach can help keep focus on modular description of events and relationships in the AOP-Wiki (Villeneuve et al. 2014), and can serve as an effective way to approach collaborative AOP development. Both supporting and contradicting evidence was collected for the KERs and classified as evidence for “essentiality”, that is the upstream event must occur for the downstream event to happen (unless triggered via another intersecting pathway), “biological plausibility” of each KER, or “empirical evidence” supporting or negating each KER (Becker et al. 2015). The tools and sources utilized to assemble literature were dependent on the extent of information already included in the AOP-Wiki, and in general, weight of evidence was considered for both adjacent and non-adjacent KERs (Aop developers' handbook  2022). Of the seven adjacent KERs between the two AOPs, two have already been well-described and reviewed in the AOP-Wiki and will not be discussed in this report. These relationships are KER1 (Relationship 972: Activation, AhR leads to dimerization, AHR/ARNT) and KER5 of AOP 456 (Relationship 1567: Altered, Cardiovascular development/function leads to Increase, Early Life Stage Mortality). Additionally, evidence supporting the non-adjacent KER, Activation, AhR leads to Increase, Early Life Stage Mortality (Relationship 984) has also already been included in the AOP-Wiki, and provides strong support for the relationship between the molecular initiating event and the adverse outcome for both proposed AOPs.

Between the two novel  AOPs developed as part of this work, we introduce and describe a total of four new KERs in the AOP-Wiki that include “Increase, slincR expression” as either the upstream or the downstream key event: KER2 (Relationship 2683: dimerization, AHR/ARNT leads to Increase, slincR expression) and KER3 (Relationship 2684: Increase, slincR expression leads to Decrease, sox9 expression), as well as the non-adjacent KERs, Relationship 2690: Increase, slincR expression leads to Smaller and morphologically distorted facial cartilage structures (AOP 455), and Relationship 2727: Increase, slincR expression leads to Altered, Cardiovascular development/function (AOP 456). Present literature on slincR is relatively limited, therefore evidence pertaining to these four KERs was gathered primarily from two zebrafish studies that discovered and characterized slincR (Garcia et al. 2017; Garcia et al. 2018b). Additional supporting evidence for the regulatory role of lncRNAs and the complexity of the molecular signaling pathways leading up to the malformations, was obtained from the reference lists from the two Garcia studies, and other relevant literature associated with lncRNAs.   

Of the three remaining adjacent KERs, evidence for Relationship 2686: Smaller and morphologically distorted facial cartilage structures leads to Increase, Early Life Stage Mortality (AOP 455) was gathered from literature already identified from other literature searches, as well as upon request from known experts in the field. Evidence for the two relationships that include “Decrease, sox9 expression” as a key event, Relationship 2685: Decrease, sox9 expression leads to Smaller and morphologically distorted facial cartilage structures (AOP 455) and Relationship 2691: Decrease, sox9 expression leads to Altered, Cardiovascular development/function (AOP 456) were gathered using a systematic literature search. Evidence collection for the non-adjacent KERs, Relationship 2688: Activation, AhR leads to Decrease, sox9 expression, Relationship 2689: Activation, AhR leads to Smaller and morphologically distorted facial cartilage structures, and Relationship 2765: Activation, AhR leads to Altered, Cardiovascular development/function were all addressed in a similar manner.

We used Abstract Sifter (version 7) (Baker et al. 2017) to systematically collect evidence for three non-adjacent KERS, and the two adjacent KERs pertaining to SOX9 repression. The Microsoft Excel-based application enhances the existing functions of PubMed searches. Search terms for each of the KERs were used to identify a list of potentially relevant sources. Results were filtered via manual review based on the information in the titles and abstracts, such as relevant in vitro platforms, life-stage and toxicity endpoints measured, and the scientific context of the search results compared to that of the two AOPs. Non-relevant studies were excluded from further analysis. Our literature search methodology included two main drawbacks: 1. While our broad search terms captured several potentially relevant articles, there was a possibility of missing out on studies that did not, for example, include “AHR” or “aryl” in their abstracts, but were investigating one or more of the relevant KEs in this report using a known Ahr activator as the chemical. 2. Our search results were restricted to literature within PubMed, and thus did not capture studies outside of this database unless we happened to include them from reference lists of the selected manuscripts. Once literature from the search results was filtered, an evidence table organized in a manner that facilitates evaluation of Bradford Hill considerations such as dose-response concordance, temporal concordance, and incidence concordance (i.e., a concordance table; (Aop developers' handbook  2022; Becker et al. 2015) was built for each of the KERs 2685, 2691, 2688, 2689, and 2765 evaluating and classifying the experiments in the results based on the type of evidence they provide. The concordance tables can be found in the respective KER pages. Occasionally, additional references that were not present in the initial results’ lists were included from the references cited in studies identified via the original search. Of note, information present in the concordance tables is not comprehensive, and instead, is a summary of the relevant results obtained from our AbstractSifter searches and filters. 

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 prototypical stressor and the biological system) of an AOP. More help
Key Events (KE)
A measurable event within a specific biological level of organisation. More help
Adverse Outcomes (AO)
An AO is a specialized KE that represents the end (an adverse outcome of regulatory significance) of an AOP. More help
Type Event ID Title Short name
MIE 18 Activation, AhR Activation, AhR
KE 944 dimerization, AHR/ARNT dimerization, AHR/ARNT
KE 2021 Increase, slincR expression Increase, slincR expression
KE 2020 Decrease, sox9 expression Decrease, sox9 expression
KE 317 Altered, Cardiovascular development/function Altered, Cardiovascular development/function
AO 947 Increase, Early Life Stage Mortality Increase, Early Life Stage Mortality

Relationships Between Two Key Events (Including MIEs and AOs)

This table summarizes 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. More help

Network View

This network graphic is automatically generated based on the information provided in the MIE(s), KEs, AO(s), KERs and Weight of Evidence (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

Prototypical Stressors

A structured data field that can be used to identify one or more “prototypical” stressors that act through this AOP. Prototypical stressors are stressors for which responses at multiple key events have been well documented. More help

Life Stage Applicability

The life stage for which the AOP is known to be applicable. More help
Life stage Evidence
Embryo High
Development High

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected.In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available. More help
Term Scientific Term Evidence Link
zebrafish Danio rerio High NCBI
mouse Mus musculus Low NCBI
human Homo sapiens Low NCBI
chicken Gallus gallus Low NCBI

Sex Applicability

The sex for which the AOP is known to be applicable. More help
Sex Evidence
Unspecific High

Overall Assessment of the AOP

Addressess the relevant biological domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and Weight of Evidence (WoE) for the overall AOP as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). More help

See details below.

Domain of Applicability

Addressess the relevant biological domain(s) of applicability in terms of sex, life-stage, taxa, and other aspects of biological context. More help

Life Stage and Sex

The relationships between Ahr, Arnt, slincR and sox9b, and cardiac and craniofacial malformations have been well established in developing zebrafish, specifically as embryos, and thus sex is not a relevant parameter.

Taxonomic

Evidence gathered suggests that the domain of applicability covers most vertebrates, from fish to humans and other wildlife. It is important to highlight that while all the relationships within the AOP have been observed definitively in one species (Danio rerio), there is strong evidence for specific KERs in species other than zebrafish. For example, Ahr’s evolutionarily conserved role as a master regulator of toxicity of several environmental pollutants has been shown in animals including fish, birds, rodents, and humans (Hahn et al. 2017). Another example is SOX9’s highly conserved role as a critical transcriptional factor in craniofacial and cardiac development in animals such as fish, rodents, and amphibians (Garside et al. 2015; Lee and Saint-Jeannet 2011). While there is strong evidence for Ahr activation leading to sox9b repression in developing zebrafish, this relationship has been identified in other fish species such as white sturgeon and Atlantic salmon (Doering et al. 2016; Olufsen and Arukwe 2011). Additionally, while slincR has only been described in zebrafish so far, it is worth noting that putative mammalian orthologs have been identified (Garcia et al. 2018b), increasing the possibility that the zebrafish-specific results can be translated to other organisms. Cardiovascular toxicity is a significant complication when different vertebrates are exposed to Ahr-dependent chemicals such as TCDD and many PAHs. This cardiotoxicity can consequently lead to edema and embryonic death in both fish and birds (Elonen et al. 1998; Heid et al. 2001), suggesting the broad relevance of the cellular and molecular events between Ahr activation and defects to the cardiovascular system. Thus, the different lines of evidence suggest that the taxonomic domain of applicability for the two proposed AOPs can likely cover most vertebrates.

Essentiality of the Key Events

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. More help

Direct evidence for the essentiality of several of the key events in the AOP has been provided by gene modification and knockout studies of the Ahr, slincR, and sox9b (one of two orthologs of SOX9) genes in zebrafish. Highlights of the most important studies are provided here:

Event ID

Key Event

Evidence

Essentiality/Assessment

18

Activation, AhR

Strong

  1. Several studies in model organisms such as zebrafish and rodents have shown that Ahr-deficient animals in gene knock-out studies have either diminished or completely nonexistent harmful effects of both TCDD and several PAHs, including cardiovascular toxicity (Fernandez-Salguero et al. 1996; Garcia et al. 2018a; Goodale et al. 2015; Harrill et al. 2016).
  2. Ahr2 knock-out in zebrafish with 1ng/mL TCDD exposure had significantly diminished slincR expression at 48 hpf (Garcia et al. 2017).
  3. Ahr2 knockout zebrafish with 1ng/mL TCDD exposure did not have significantly reduced sox9b expression at 48 hpf (Garcia et al. 2018a).

944

Dimerization, AHR/ARNT

Strong

Canonical Ahr signaling involves the conversion of the inactive Ahr, which is present in the cytoplasm, to its active form that can translocate to the nucleus and dimerize with the Ahr nuclear translocator (ARNT) (Wright et al. 2017). Evidence suggests that the Ahr/ARNT heterodimer can consequently regulate gene expression within the Ahr signaling cascade.

2021

Increase, slincR expression

Strong

  1. When slincR expression is knocked down using a morpholino, normal sox9b expression levels and spatial pattern are altered during zebrafish development (Garcia et al. 2017). Specifically, in slincR morphants exposed to DMSO or TCDD, sox9b expression was significantly higher than in control morphant zebrafish.
  2. When slincR expression is knocked down using a morpholino, several downstream target genes of sox9b, such as, notch3, adamts3, fabp2, sfrp2, and fgfr3 were altered in their gene expression compared to control morphants (Garcia et al. 2017).
  3. slincR zebrafish morphants have a significantly lower percent incididence of blood hemorrhaging at 48 hpf  (Garcia et al. 2018b), suggesting slincR’s role in cardiovascular functional toxicity caused due to TCDD exposure.

2020

Decrease, sox9 expression

Strong

  1. Sox9b expression inhibited by a dominant negative specifically in the cardiomyocytes resulted in significant alterations to cardiovascular function (end diastolic volume, decrease in stroke volume, ejection fraction, and cardiac output were all altered) (Gawdzik et al. 2018). Additionally, epicardium formation was disrupted as well. The study also highlighted the significant decrease of expression of several cardiac development genes sych as nkx2.5, nkx2.7, myl7, and c-fos.
  2. Sox9b morpholino knockdown in zebrafish led to several structural and functional deformities including pericardial edema, elongated heart, and reduced blood circulation (Hofsteen et al. 2013).
  3. Mutations in the sox9 gene locus as well as in the gene regulatory region in humans have been associated with chronic and congenital heart diseases (Gong et al. 2022; Sanchez-Castro et al. 2013).
  4. Multiple studies investigating the impact of the loss of sox9 in mice, including one conditional inactivation study in endothelial cells and another using cre-lox generated sox9 mutants, show the significant effects on cardiovascular development and functioning in the absence of sox9 (Akiyama et al. 2004; Lincoln et al. 2007).

317

Altered, Cardiovascular development/function

Strong

Cardiovascular toxicity has been shown to cause early life stage mortality. This relationship has been discussed in depth in AOP 21 (aopwiki.org/aops/21) (peer-reviewed and endorsed).

947

Increase, Early Life Stage Mortality

N/A

This is the terminal key event in the AOP and hence its essentiality for downstream events cannot be evaluated.

Evidence Assessment

Addressess the biological plausibility, empirical support, and quantitative understanding from each KER in an AOP. More help

Biological Plausibility

  • Ahr – strong : Strongest Biological Plausibility evidence for AOPs 455 and 456 comes from our extensive understanding of the Ahr signaling pathway in multiple different organisms. The functional roles of Ahr and its binding partners, including ARNT, have been well-studied (Fujii-Kuriyama and Kawajiri 2010), and it is well known that the Ahr signaling pathway mediates a variety of physiological and toxicological functions (Larigot et al. 2018).
  • slincR and sox9 – strong: Strong evidence for the Biological Plausibility of slincR having a role in AOPs 455 and 456 comes from the nature of lncRNAs which is such that they have diverse functions and can regulate gene expression at multiple levels, including by interacting with DNA, RNA, proteins, and altering transcription of both neighboring and distant genes (Statello et al. 2021). Additionally, slincR (in situ hybridization) and sox9b (immunohistochemistry for sox9b-eGFP) are expressed in adjacent and overlapping tissues through multiple stages of zebrafish development, such as in the eye, otic vesicle, and in the lower jaw (Garcia et al. 2017) providing one line of evidence for slincR being able to regulate sox9b gene expression. Further, a capture hybridization analysis of RNA targets (CHART) experiment in both DMSO- and TCDD-exposed 48 hpf zebrafish identified enrichment of slincR in the 5’UTR of the sox9b locus (Garcia et al. 2018b) pointing to possible interaction between slincR and sox9b.
  • slincR and cardiovascular development - strong: Individual zebrafish exposures to the PAHs, retene, dibenzo[a,h]pyrene, and dibenzo[a,i]pyrene cause cyp1a vascular expression as well as a significant induction of slincR at 48 hours post fertilization (hpf) (Garcia et al. 2018b; Geier et al. 2018), suggesting the possibility of slincR involved in some aspect of cardiovascular function. Knockdown of slincR expression in developing zebrafish, alters expression of sox9b, as well as certain downstream targets of sox9, such as notch3, adamts3, fabp2, sfrp2, and fgfr3 (Garcia et al. 2017). These are different lines of Biological Plausibility evidence for slincR being a mediator between Ahr activation and craniofacial/cartilage malformations.
  • Sox9 and cardiovascular development - strong: Strong Biological Plausibility evidence for sox9’s role in cardiovascular dysfunction comes from one study where zebrafish exposed to 1ng/mL TCDD had significantly reduced sox9b expression in the heart of the developing animals (Hofsteen et al. 2013). Backing up this evidence, several studies in different organisms including rodents, chicken, frog, and fish have identified both sox9 mRNA and protein spatiotemporal expression in the developing hearts (please see KER page 2691 for references). In addition, a chip-seq experiment showed that the sox9 protein has been found to interact with the genomic regions of proliferation genes as well as important transcription factors involved in mouse heart development (Garside et al. 2015), making it conceivable that the loss of sox9 can have a significant impact on cardiovascular development.
  • Cardiovascular dysfunction and early-stage mortality - strong: Biological plausibility of this KER is considered high because the relationship between cardiovascular toxicity and early mortality has been demonstrated in several species including fish and birds (Kopf and Walker 2009). Note that this KER is already included in the AOP-Wiki as part of AOP 21 (peer-reviewed and endorsed; (Doering et al. 2019) with abundant evidence listed.

Dose Concordance

  • Ahr activation leading to early life stage mortality has been well-studied. The KER page (https://aopwiki.org/relationships/984) has examples in difference species for empirical evidence for this relationship.
  • Strongest evidence for dose concordance between Ahr activation, slincR induction, and sox9 repression comes from a developing zebrafish study that utilized TCDD as the Ahr activating chemical. The concentration-response experiment showed that cyp1a (biomarker for Ahr activation) and slincR expression increased in parallel as TCDD exposure concentration increased, and that cyp1a and slincR are induced at TCDD exposure concentrations lower than concentrations at which sox9b is repressed (Garcia et al. 2018b).
    • Both cyp1a and slincR were significantly induced starting at 0.0625 ng/mL TCDD exposure.
    • Significant cyp1a (~log2FC = 6) and slincR (~log2FC = 2) inductions were detected at 0.0625 ng/mL TCDD, while significant sox9b repression (~log2FC = -1) was detected only at 0.5ng/mL TCDD.
  • (Garcia et al. 2018b) also showed that with increasing concentrations of TCDD, the severity of overall developmental malformations, including pericardial edema (indicator of potential cardiotoxicity) and jaw malformations increased.
  • Strong dose concordance has been determined between cardiovascular malformations and early life stage mortality (please see KER page: https://aopwiki.org/relationships/1567), however, to the best of out knowledge, no systematic effort has been performed to identify  “dose concordance” evidence for the KER between craniofacial malformations and early life stage mortality.

Uncertainties, inconsistencies, data gaps

While we have listed out various possible uncertainties, inconsistencies, and data gaps in the respective KER pages, here we highlight the most important ones:

  1. One possible inconsistency in the literature is that not all ARNT isoforms in a particular species (for example, zebrafish) are important for mediating in vivo toxicity (Prasch et al. 2004), and future research could help clarify the relative influence of the different Ahr binding partners. The most well-studied Ahr binding partner is ARNT and it does appear to be important for TCDD toxicity – hence it is included as a KE in AOPs 455 and 456.
  2. While the relationships in AOPs 455 and 456 have been definitively shown with TCDD as the activating chemical, future research must investigate the KERs with other Ahr activators, such as PAHs and other HAHs. Similarly, future research in organisms other than zebrafish, will add significantly to the weight of evidence for AOPs 455 and 456.
  3. One inconsistency comes from a study exposing 16 individual PAHs to developing zebrafish where none were associated with a significant decrease in sox9b expression, despite six inducing both cyp1a and slincR expression (Garcia et al. 2018b). It is possible that the PAHs that are rapidly metabolized (unlike TCDD) induce different gene expression changes upon Ahr activation, or that the slincR/sox9b gene expression alterations are tissue-specific and are thus unable to be resolved consistently in whole animal transcriptomic studies.
  4. Morpholino knockdown of sox9b in zebrafish led to a significant increase in slincR expression suggesting that slincR and sox9b may share overlapping regulatory networks that is not fully understood (Garcia et al., 2018). 
  5. We note that slincR is not the only mechanism of regulation of sox9. Other studies have found evidence for different regulatory mechanisms of sox9, but the circumstances under which different pathways are turned on is still unknown (Dash et al., 2021; Fu et al., 2018).
  6. Impact of absence of slincR has only been studied with morpholino knockdown experiments (Garcia et al., 2017; Garcia et al., 2018), which have two relevant drawbacks: 1. Inability to maintain slincR repression by 72 hpf since morpholinos are transient in nature, and 2. Incomplete functional knockout which prevents us from understanding the true impact of the absence of slincR. Future studies using CRISPR-Cas-generated knockout lines, for example, will help overcome both limitations.
  7. Worth noting that not all chemicals that induce developmental cardiovascular toxicity induce sox9 expression. For example, developmental zebrafish exposed to the fungicide, procymidone, significantly increased sox9b expression despite the fish having significant pericardial edema (Wu et al., 2018).
  8. One study investigated sox9b expression (on a microarray) in heart tissue from zebrafish exposed to 1ng/mL TCDD and did not detect sox9b repression, despite the same study identifying sox9b repression in the zebrafish jaws (Xiong et al. 2008). The resolution of the microarray experiment might not have been good enough to detect sox9b repression which has been identified in other studies (Hofsteen et al., 2013).

Known Modulating Factors

Modulating factors (MFs) may alter the shape of the response-response function that describes the quantitative relationship between two KES, thus having an impact on the progression of the pathway or the severity of the AO.The evidence supporting the influence of various modulating factors is assembled within the individual KERs. More help
Modulating Factor (MF) Influence or Outcome KER(s) involved
     

Quantitative Understanding

Optional field to provide quantitative weight of evidence descriptors.  More help

Strongest quantitative understanding for the AOPs 455 and 456 is between the MIE (Activation, Ahr) and the AO (Increase, Early Life Stage Mortality) and is described in detail in the KER page (Event 984; https://aopwiki.org/relationships/984). Additionally, for the halogenated aromatic hydrocarbons (HAHs), we have a moderate quantitative understanding of the binding affinity of the different chemicals to the Ahr which partially led to the widespread use of the toxic equivalency factor (TEF) concept for humans, fish, and other wildlife risk assessment (Van den Berg et al. 1998). On the other hand, models that currently exist for chemicals such as the polycyclic aromatic hydrocarbons (PAHs) are often considered oversimplified due to the possible differences in receptor binding affinity and consequent differential metabolism and toxicity (Billiard et al. 2008). Nevertheless, we highlight that TCDD has been identified as the prototypical stressor for both AOPs 455 and 456, and the TEF concept could be leveraged to determine total toxic equivalencies (TEQs) for dioxin-like chemicals based on the known concentrations at which TCDD can induce different key events of the AOPs.

The presence of two measurable gene expression events (SOX9 and slincR) as well as easily observable zebrafish toxicity phenotypes in AOPs 455 and 456 has given opportunity for the beginning of our quantitative understanding of the pathways. Garcia et al (Garcia et al. 2018b) conducted a TCDD concentration-response experiment (0 – 1.0 ng/mL) in developing zebrafish and determined that after just 1 h of exposure at 6 hpf, the number of zebrafish with malformations in the developing jaw and pericardial edema was statistically significant at 0.25 ng/mL TCDD. The study also measured cyp1a, slincR, and sox9b expression, and showed significant cyp1a (a measure of Ahr activation) and slincR induction from 0.0625 ng/mL, and a trend for sox9b repression from 0.125 ng/mL which was significant from 0.5 ng/mL TCDD exposure compared to the DMSO vehicle control. Additionally, slincR morpholino knockdown which reduced slincR expression by 98% in control animals, and by 81% in TCDD-exposed zebrafish compared to their respective control morphants (Garcia et al. 2017) significantly altered sox9b spatial and quantitative expression (Garcia et al. 2017), as well as had impacts on both craniofacial development and the cardiovascular system of developing zebrafish (Garcia et al. 2018b). While this preliminary quantitative understanding between several of the relationships in the two AOPs is not available for other chemicals, taxonomic groups, or species, the TEF concept is still the most plausible and feasible method of risk assessment for dioxin-like chemicals even if they have broad species-specific responsiveness (Van den Berg et al. 1998).

Considerations for Potential Applications of the AOP (optional)

Addressess 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. More help

With the diversity of ligands that bind and activate the Ahrs, and the variety of biological and toxicological functions these receptors are involved in, AOPs describing different aspects of the Ahr signaling pathway could provide immense potential for cross-chemical and cross-taxa extrapolations. Additionally, the AOP networks can help prioritize the most relevant mechanistic data for regulatory decision making, while also identifying critical knowledge gaps for future research. Several in vitro and in silico assays are being leveraged to identify chemical structures that activate the Ahr (Larsson et al. 2018). A deeper understanding of the mechanisms of toxicity endpoints can not only help illuminate the specific conditions under which malformations might occur, but it can also provide phenotypic-specific genetic biomarkers, such as slincR and SOX9 as well as VEGF and COX2. These can be easily measured in short-term in vivo exposures as evidence for progression along an Ahr-mediated adverse outcome pathway. As such, both the current AOPs and the broader AOP network can support tiered and hypothesis directed testing strategies based on in vitro or in silico screening results. From an environmental monitoring standpoint, the novel AOPs provide one or more reliable effects-based indicators (ex: slincR or SOX9) that could serve as early warning signs before the onset of deformities or mortality. Assuming the biomarkers are conserved across species, which is likely the case for slincR and SOX9, gene expression measurements could also be used for predicting toxicant responses across a broad diversity of phylogenetic groups. Overall, the two proposed AOPs have the potential to:  1. Expand on the Ahr-related AOP network to gain a more comprehensive view of Ahr-related processes to support regulatory decisions, and 2. Integrate in vivo measures of gene expression response into the risk assessment paradigm for Ahr activating pollutants to enable extrapolations across both chemicals and taxa, while also identifying key differences between them.

References

List of the literature that was cited for this AOP. More help

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