This AOP is licensed under a Creative Commons Attribution 4.0 International License.

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 cardiovascular toxicity

Short name

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Ahr mediated early stage mortality via cardiovascular toxicity

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

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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 (shankar.prarthana@epa.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

Status

Provides 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. OECD Status - Tracks the level of review/endorsement the AOP has been subjected to. OECD Project Number - Project number is designated and updated by the OECD. SAAOP Status - Status managed and updated by SAAOP curators. More help

Author status	OECD status	OECD project	SAAOP status
Under development: Not open for comment. Do not cite

This AOP was last modified on October 21, 2022 15:53

Revision dates for related pages

Page	Revision Date/Time
Activation, AhR	December 20, 2022 08:29
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	October 20, 2022 16:41
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 Receptor (Ahrs) are evolutionarily conserved ligand-dependent transcription factors that can be activated by structurally diverse compounds such as halogenated aromatic hydrocarbons, polycyclic aromatic hydrocarbons, and polychlorinated biphenyls. Ahr activation can cause developmental toxicity, including cardiovascular toxicity, that can lead to early-stage mortality. The current AOP describes a novel adverse outcome pathway (AOP) which describe how Ahr activation (molecular initiating event; MIE) can lead to early-stage mortality (adverse outcome; AO), via cardiovascular toxicity. The AOP includes two novel key events (KEs), increase in slincR expression, a newly characterized long non-coding RNA that has regulatory functions, and decrease in SOX9 expression, a critical transcription factor implicated in several processes including cardiovascular development. Using a key event relationship (KER)-by-KER approach, we collected evidence using both relevant zebrafish literature as well as through broader key word searches in the AbstractSifter tool. Weight of evidence for each KER was assessed to inform overall assessment of the two AOPs which is presented here. Overall confidence levels ranged between medium and strong, with few inconsistencies as well several opportunities for future research identified. While the AOP has only been demonstrated in zebrafish with TCDD as an Ahr activator, we provide lines of evidence for the AOP to apply to majority of animals, as well as many Ahr activating chemicals. The addition of the AOP into the AOP-Wiki helps expand the growing Ahr-related AOP network.

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 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).

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. 2018). Ahr activation is also associated with tumor promotion and carcinogenesis (Safe et al. 2013).

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. 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 (Fernandez-Salguero et al. 1996; Garcia et al. 2018; Goodale et al. 2015; Harrill et al. 2016), highlighting the significance of the Ahrs in mediating toxicological outcomes of the Ahr-active chemicals.

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 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) (Lo and Matthews 2012).

Upon exposure to various Ahr activating chemicals, craniofacial malformations and cardiovascular toxicity have been identified under various exposure paradigms (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. Evidence from studies conducted with other taxa provide further support and aid in evaluation of the domain of applicability as well as the breadth of the stressors that can cause the different relationships described in the two AOPs.

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 KER-by-KER approach was leveraged to gather different lines of evidence to support the two proposed AOPs, with a focus on adjacent relationships, and including non-adjacent KERs where applicable. Both supporting and contradicting evidence was collected for the KERs and classified as evidence for essentiality of the upstream KE to occur for the downstream KE to happen for each KER, biological plausibility of each KER, or empirical evidence supporting or negating each KER. The tools and sources utilized to assemble literature were dependent on the extent of information already included in the AOP-Wiki. 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 proposed AOPs, we discuss a total of four KERs 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, moderate biological plausibility evidence for Relationship 2686: Smaller and morphologically distorted facial cartilage structures leads to Increase, Early Life Stage Mortality (AOP 455) was identified. 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, a concordance table 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 by no means 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

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

947

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

Title	Adjacency	Evidence	Quantitative Understanding

Activation, AhR leads to dimerization, AHR/ARNT	adjacent	High	High
dimerization, AHR/ARNT leads to Increase, slincR expression	adjacent	High	High
Increase, slincR expression leads to Decrease, sox9 expression	adjacent	High	High
Decrease, sox9 expression leads to Altered, Cardiovascular development/function	adjacent	High
Altered, Cardiovascular development/function leads to Increase, Early Life Stage Mortality	adjacent	High	High

Activation, AhR leads to Decrease, sox9 expression	non-adjacent	High	High
Increase, slincR expression leads to Altered, Cardiovascular development/function	non-adjacent	Moderate	Moderate
Activation, AhR leads to Increase, Early Life Stage Mortality	non-adjacent	High	High
Activation, AhR leads to Altered, Cardiovascular development/function	non-adjacent	High

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

Name
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

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). 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	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). Ahr2 knock-out in zebrafish with 1ng/mL TCDD exposure had significantly diminished slincR expression at 48 hpf (Garcia et al. 2017). 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	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. 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). 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	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. 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). 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). 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	Moderate	While we are not aware of studies that specifically investigate the essentiality of cardiac dysfunction for early life stage mortality, it is conceivable that the cardiovascular system is important for survival and severe its dysfunction could lead to mortality.
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:

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.
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.
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.
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).
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).
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.
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).
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 strong 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).

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. 2018b) conducted a TCDD concentration-response experiment in developing zebrafish and determined that by 0.125 ng/mL TCDD exposure for one hour at around 6 hpf, zebrafish had malformations in the developing jaw and pericardial edema, which was statistically significant at 0.25 ng/mL TCDD exposure. 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). This preliminary quantitative understanding between several of the relationships in the two AOPs is not available for other chemicals, or taxonomic groups or species.

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), however, inclusion of toxicogenomics data in ecological risk assessment is the next major step that can help overcome some of the challenges that current ecotoxicologists face, while providing tools for a tiered hazard evaluation strategy. For example, 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, that can be easily measured in order to conduct reliable risk assessment. This approach can also be used for accurately predicting contaminants and their biological targets, and if 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 AOPs (AOP 455 and 456) 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 toxicogenomics 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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