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. Author manuscript; available in PMC: 2024 Oct 1.
Published in final edited form as: Environ Toxicol Chem. 2023 Aug 2;42(10):2063–2077. doi: 10.1002/etc.5699

AOP Report: Aryl Hydrocarbon Receptor (Ahr) Activation Leads to Early Life Stage Mortality via Sox9 Repression Induced Craniofacial and Cardiac Malformations

Prarthana Shankar a,b, Daniel L Villeneuve a
PMCID: PMC10772968  NIHMSID: NIHMS1953611  PMID: 37341548

Abstract

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 two novel adverse outcome pathways (AOPs) which describe how Ahr activation (molecular initiating event; MIE) can lead to early life stage mortality (adverse outcome; AO), via either SOX9-mediated craniofacial malformations (AOP 455) or cardiovascular toxicity (AOP 456). 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 AOPs. The AOPs link to previous descriptions of Ahr activation, 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 AOPs into the AOP-Wiki (https://aopwiki.org/) 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.

1. Introduction and Background

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 classes of endogenous Ahr ligands, such as, indoles, tetrapyroles, metabolites of arachidonic acid, heme and tryptophan, as well as retinoids and carotenoids, have been identified (Denison and Nagy 2003; Nguyen and Bradfield 2008). In addition, Ahr activation by environmental pollutants including polycyclic aromatic hydrocarbons (PAHs), and halogenated aromatic hydrocarbons (HAHs) comprising dioxins and dioxin-like chemicals 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 (for example, Ahr1a, Ahr1b, and Ahr2 in zebrafish (Shankar et al. 2020), or Ahr1 and Ahr2 in birds such as the common cormorant and black-footed albatross (Yasui et al. 2007)), 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, endocrine and metabolic alterations, 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 (Figure 1) 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 (Ahres1) (Lo and Matthews 2012). To help organize this 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 listed Ahr-related AOPs in the AOP-Wiki (as of April 10th, 2023; https://aopwiki.org/), with six listed 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 underdeveloped. With the rapid rate at which new research on Ahr-mediated toxicity is being conducted, there is 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.

Figure 1.

Figure 1.

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Graphic of the canonical Ahr signaling pathway. An example downstream target gene is cyp1a, member of the cytochrome P450 family of proteins involved in metabolism of numerous endogenous molecules as well as synthetic chemicals. Hsp = heat shock protein, p23 = co-chaperone protein, ARNT = aryl hydrocarbon nuclear translocator, AHRE = ayrl hydrocarbon response element.

Here we report on the development of two AOPs beginning with the molecular initiating event (MIE) of Ahr activation and progressing towards the adverse outcome (AO) of early life stage mortality via either one of two key events (KE), craniofacial malformations (AOP 455) or cardiovascular toxicity (AOP 456). (Please refer (Aop developers’ handbook 2022) for details on AOP terminology). 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. In this report, we also present evidence from studies conducted with other taxa to 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.

It should be emphasized that cardiac and craniofacial malformations are not the only Ahr-dependent toxicity outcomes that could lead to early life stage mortality, and are just the KEs of the AOPs presented in this report. Additionally, craniofacial malformations have already been described as a consequence of histone deacetylase inhibition in AOP 274 (https://aopwiki.org/aops/274) demonstrating that Ahr activation is only one possible route via which craniofacial structures can be disrupted. Likewise, while there is compelling evidence for SOX9 repression being a player in Ahr-mediated cardiac and craniofacial toxicity, SOX9 is likely not the only KE mediating Ahr activation and the two endpoints. Two other AOPs that were previously peer reviewed and endorsed via the OECD identify gene expression events connecting Ahr activation and cardiovascular toxicity, leading to early life stage mortality as the AO. AOP 21 discusses increased cyclooxygenase 2 (COX-2) expression (https://aopwiki.org/aops/21; (Doering et al. 2019)), and AOP 150 describes the suppression of the vascular endothelial growth factor (VEGF) (https://aopwiki.org/aops/150; (Farhat and Kennedy 2019)). While both the increase of COX-2 expression and the decrease of VEGF production lead to reduction of cardiac output followed by cardiac failure and death, there is some uncertainty on whether COX-2 expression is induced by the direct or indirect interaction with the Ahr. On the other hand, VEGF is indirectly suppressed through sequestration of ARNT by Ahr, ARNT being a common dimerization partner for Ahr and hypoxia inducible factor alpha (HIF-1alpha), the transcriptional regulator of VEGF. Overall, AOPs, 21 and 150, provide evidence for two different Ahr activation-dependent molecular signaling events that can lead to cardiovascular toxicity, and AOP 456 will include an additional KE to the Ahr-related cardiovascular development AOP network. Thus, the two new AOPs described here add to a growing network of AOPs documenting the potential adverse effects associated with Ahr activation as well as contribute to a more comprehensive understanding of the molecular signaling pathways that can lead to specific Ahr-related hazards.

2. AOP Description

The proposed AOPs link activation of the Ahr (MIE) to the same AO of early life stage mortality, specifically via a decrease of SOX9 gene expression, and two distinct KEs, craniofacial malformations (AOP 455), and cardiovascular toxicity (AOP 456) (Figure 2). Based on our interpretation of the gathered evidence (described later), we identified craniofacial malformations as an alternative AO in AOP 455 and cardiovascular toxicity as a KE within AOP 456. The AOPs also include the KEs (numbered linearly from KE1-KE4 to help readers follow the progression of each AOP), dimerization of Ahr/ARNT (KE1), and increased expression of the long non-coding RNA (lncRNA), slincR (KE2), that precede SOX9 repression (KE3) and the tissue- or organ-level toxicity outcomes (AO in AOP 455 and KE4 in AOP 456). The two novel KEs of slincR induction and SOX9 repression differentiate AOP 456 from the already endorsed AOPs 21 and 150. Summaries of AOPs 455 and 456 showing each of their individual KEs, and the associated AOP-Wiki links with the KE identification (ID) numbers that are automatically assigned in the AOP-Wiki, are provided in AOP ID Boxes 1 and 2.

Figure 2.

Figure 2.

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Graphical representation of the two adverse outcome pathways (AOPs) described in this report. MIE= Molecular Initiating Event, KE = Key Event, KER = Key Event Relationship, AO = Adverse Outcome. Numbers in parentheses indicate the event IDs (KE) or the relationship IDs (KER) automatically assigned in the AOP-Wiki. Adjacent KERs are indicated with solid arrows, and non-adjacent KERs are the dotted lines. Bolded IDs are new additions to the AOP-Wiki, and un-bolded IDs correspond to KEs and KERs that were described previously.

BOX 1: AOP ID Box.

BOX 2: AOP ID Box.

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.

The two AOPs are intentionally reported on together because of the overlap in the majority of their KEs. Additionally, even though most of the strongest lines of evidence comes from zebrafish literature, the proposed KEs were purposely generalized to include a range of organisms and Ahr activating stressors, since there is convincing evidence from multiple vertebrates and chemical types for individual KERs. While the proposed AOPs describe one potential molecular mechanism through which the two phenotypic hazards develop before resulting in early life stage mortality, the AOPs also expand on the network around craniofacial malformations and cardiovascular dysfunction, two KEs that have already been described in the AOP-Wiki.

The MIE, AhR activation (Event 18), along with the consequent dimerization of Ahr-ARNT (KE1, Event 944), is a known evolutionarily conserved process that is part of other OECD-endorsed AOPs, including AOPs 21 and 150 that relate Ahr activation to altered cardiovascular development and early life stage mortality. This MIE encompasses activation of all Ahr isoforms that may be found in various species, for example, Ahr1a, Ahr1b, and Ahr2 in zebrafish (Shankar et al. 2020). Whenever relevant, we discuss the specific Ahr types based on available evidence to acknowledge potential functional differences between the isoforms. However, all isoforms fall under the KE of AhR activation since research is still in the early stages of examining functions of Ahr paralogs in most species. Commonly used methods to evaluate Ahr activation are reporter gene and ligand binding assays; however, CYP1A gene expression and associated enzyme activity (measured using the EROD assay) are generally measured in vivo as biomarkers for Ahr activation and are used as evidence for the MIE several times within this report. CYP1A is part of the cytochrome P450 family of metabolizing enzymes, is a direct downstream gene target of the activated Ahr, and is a commonly used indicator for environmental contamination by Ahr activating pollutants, including the dioxins (Goksoyr 1995). While CYP1A is a sensitive and specific adaptive response of organisms exposed to Ahr activating chemicals, we note that induction of the gene does not always indicate observance of toxicity (Scott et al. 2011).

The second KE, “increase, slincR expression,” (KE2, Event 2021) represents the measurable increase in expression of a functional long non-coding RNA (lncRNA) named “sox9b long intergenic non-coding RNA” (slincR). slincR is the first lncRNA to be included in the AOP-Wiki, and is an important addition because of the possibility of slincR to not only regulate SOX9 expression as described in the current report, but to also be integrated into other AOPs as new functions of slincR are discovered. For example, a recent study generated a slincR zebrafish knockout line and identified slincR’s possible role in fin regeneration (Dasgupta et al. 2023). “Inhibition, Fin regeneration” is already a KE (Event 1761) within AOP 334 (https://aopwiki.org/aops/334) in the AOP-Wiki.

The third KE, “decrease, sox9 expression,” (KE3, Event 2020) is the second measurable gene expression change that is described in the report. This KE is generalized to include all isoforms of SOX9 in the different taxa, for example sox9a and sox9b in teleosts such as stickleback and zebrafish (Cresko et al. 2003). While our proposed AOPs highlight only a couple of the processes (craniofacial and cardiovascular development) that this important transcription factor is involved in, and one mechanism (Ahr via slincR) by which its expression is altered, future work can expand on the AOP network around SOX9 repression to include other SOX9 molecular interactions, as well as associated phenotypic outcomes.

The fourth KE in the two AOPs relate to either craniofacial malformations (AO, Event 1559; AOP 455) or cardiovascular toxicity (KE4, Event 317; AOP 456), two measurable toxicity endpoints that are potential consequences of Ahr activation followed by slincR induction and SOX9 repression. Both Events 1559 and 317 were already described in the AOP-Wiki. However, they were intentionally generalized in a way to apply to a range of different organisms, thereby facilitating their use in developing AOP networks. For example, in humans, deletions in the region upstream of the SOX9 gene has been associated with Pierre Robin sequence (PRS) which is characterized by underdeveloped jaw and cleft palate (Gordon et al. 2014). On the other hand, in a study in rockfish embryos (Sebastiscus marmoratus), exposure to the PAH, pyrene, not only resulted in a decrease in sox9a expression, but also led to significant alterations to the Meckel’s cartilage (lower jaw), palatoquadrate (upper jaw), and ceratobranchial (branchial arches) (Shi et al. 2012). These taxon-specific manifestations of a perturbed developmental process can reasonably be captured under the same Event (1559), titled “Facial cartilage structures are reduced in size and morphologically distorted.” Similarly, Event 317 of AOP 456, “Altered, Cardiovascular development/function”, accommodates differences in the cardiovascular phenotype between organisms. These can range from chronic and congenital heart diseases in humans with mutations in their SOX9 gene locus (Gong et al. 2022; Sanchez-Castro et al. 2013) to pericardial edema observed in developing zebrafish and salmon exposed to Ahr activating chemicals that also disrupt sox9 expression (Garcia et al. 2018b; Olufsen and Arukwe 2011a). Both are encompassed by the Event description in the AOP-Wiki (Event 1559).

The final KE is the AO of early life stage mortality (Event 947) included in both AOPs 455 and 456 as the potential higher level AO linked to cardiac and craniofacial malformations. The connection between cardiac toxicity and early life stage mortality were previously documented as part of AOPs 21 and 150, and Relationship 1567 contained therein. However, the relationship between craniofacial malformations and early life stage mortality warranted further development of empirical support and is included in the current AOP report.

3. Overview of AOP development approach

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 approach basically describes a process in which evidence gathering is focused on the relationship between a specific pair of KEs. 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 for the KERs were collected; each line of evidence was 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, that is, the consistency of the relationship between the upstream and downstream events with established biological knowledge, or empirical evidence, that is, support for a change in the upstream KE leading to an appropriate change in the downstream KE (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 following guidance provided in the AOP Developers’ Handbook (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 MIE and the AO 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 KE: 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 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), and one recent study that generated a slincR knockout zebrafish line (Dasgupta et al. 2023). 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 KE, 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 (Table 1). 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.

Table 1.

Summary of search terms used in AbstractSifter, and total number of results for each search are presented in the table.

AOP KER Adjacent or Non-adjacent KER KER title Search terms Relevant/total results Pertinent notes
455 2685 Adjacent Decrease, sox9 expression leads to Smaller and morphologically distorted facial cartilage structures “sox9 AND (jaw OR cranio)” 42/106 Additional three studies from reference lists used for biological plausibility evidence.
456 2691 Adjacent Decrease, sox9 expression leads to Altered, Cardiovascular development/function “sox9 AND (heart OR cardi)” 21/156 Additional 17 studies with tangential evidence used for biological plausibility evidence.
455, 456 2688 Non-adjacent Activation, AhR leads to Decrease, sox9 expression “(AHR OR aryl) AND sox9” 10/16 Other relevant studies included from reference lists.
455 2689 Non-adjacent Activation, AhR leads to Smaller and morphologically distorted facial cartilage structures “(AHR OR aryl) AND (cranio OR jaw) 25/63 Other relevant studies included from reference lists.
456 2765 Non-adjacent Activation, AhR leads to Altered, Cardiovascular development/function “(aryl hydrocarbon receptor) AND (heart)” 24/40 Due to the large number of results (>300), results were filtered for abstracts containing the term “development”, ordered by frequency of occurrence, and only the first 40 results were reviewed.
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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 (Table 1). 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.

4. Summary of Scientific Evidence Assessment

4.1. Overview of key evidence supporting the AOP

This section provides important highlights of the scientific evidence that was gathered to evaluate if the KEs in each of the proposed AOPs are causally related to each other based on evidence for biological plausibility and empirical support for KERs. Additionally, essentiality of KEs from knockout, morpholino knockdown, and agonist and antagonist experiments was evaluated. These studies were primarily conducted in developing zebrafish with TCDD as the prototypical Ahr activator, but occasionally, other kinds of studies support essentiality of the KEs in the two proposed AOPs. All evidence gathered was collectively leveraged to determine if the strength of the KEs and their relationships to one another is “high”, “moderate”, or “low,” based on the OECD’s Guidance Document for Developing and Assessing AOPs (Organisation for Economic Cooperation and Development [OECD] 2017) and the associated AOP Developers’ Handbook (Aop developers’ handbook 2022). While an in-depth assessment of each KER is included in the AOP-Wiki, this section of the report provides a more wholistic representation highlighting the overall strengths and weaknesses of the AOPs.

4.1.1. Evidence linking Ahr activation (MIE) with each KE within AOPs 455 and 456

The relationships within the two AOPs with the strongest lines of evidence is KER1 (Relationship 972: Activation, AhR leads to dimerization, AHR/ARNT) and the non-adjacent indirect KER linking Ahr activation and the AO (Relationship 984: Activation, AhR leads to Increase, Early Life Stage Mortality). Overwhelming evidence comes from several taxa around canonical Ahr signaling which describes Ahr activation leading to translocation into the nucleus and Ahr-ARNT dimerization prior to transcriptional regulation. Additionally, during development, sustained Ahr activation in particular, can interfere with the receptor’s endogenous signaling leading to early life stage mortality. KER1 has already been reviewed and included in four other AOPs, while Relationship 984 has been included in two others (as of April 10th, 2023; https://aopwiki.org/) in the AOP-Wiki. Below, we highlight evidence showing compelling empirical support as well as strong evidence for the essentiality of both the MIE (Event 18: Activation, AhR) and KE1 (Event 944: dimerization, AHR/ARNT) as they relate to each of the other KEs in the AOPs.

A plethora of evidence showing the link between Ahr activation by environmental contaminants, and cardiotoxicity or craniofacial malformations comes from laboratory studies in multiple taxa with a variety of stressors investigating Ahr-related toxicity outcomes. Exposure to HAHs and PAHs in fish (Incardona et al. 2014b; Le Bihanic et al. 2014; Staal et al. 2018), birds (Brunström 1990; Canga et al. 1993; Cohen-Barnhouse et al. 2011), and rodents and other mammals (Perkins et al. 2016; Yoshioka and Tohyama 2019) is associated with some level of craniofacial malformations and/or cardiotoxicity. Several comprehensive hazard assessment reports have also been published for different Ahr activating environmental chemicals, especially the dioxins and dioxin-like chemicals (EFSA 2018; US EPA 2012). Additionally, Ahr knockout (Fernandez-Salguero et al. 1996; Garcia et al. 2018a; Goodale et al. 2015; Harrill et al. 2016) and morpholino knockdown (Massarsky et al. 2016) studies in zebrafish and rodents provide strong evidence for the Ahr-dependent toxicities, with lack of at least one of the Ahr orthologs leading to a reduction or complete absence of toxicity induced by Ahr ligands such as TCDD and PAHs. There is also compelling evidence demonstrating that ARNT is required for TCDD-induced toxicity, with particular emphasis on cardiovascular toxicity in zebrafish (Antkiewicz et al. 2006; Prasch et al. 2006). Homozygosity for ARNT deficiency in rodents is embryonic lethal, with animals displaying defective angiogenesis before dying (Kozak et al. 1997; Maltepe et al. 1997). 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. While the prevalence of Ahr-dependent craniofacial and cardiac toxicities during the critical window of development of many organisms is evident from the literature, the remainder of this AOP report focuses on the more novel KERs associated with the two proposed Ahr-related AOPs.

In addition to the Ahr-dependent phenotypic effects of cardiotoxicity and craniofacial malformations, both the proposed AOPs include two gene expression events, KE2 (Event 2021: Increase, slincR expression) and KE3 (Event 2020: Decrease, sox9 expression). slincR is a lncRNA that was recently discovered and described in the zebrafish model system (Garcia et al. 2017). slincR’s expression is Ahr2 dependent (Ahr2 is one of the zebrafish orthologs of Ahr), increasing in expression with TCDD exposure only in the presence of a functional Ahr2 protein (Garcia et al. 2017). Putative AHREs are found in multiple locations in the zebrafish slincR promoter (Garcia et al. 2017), but no experiment to date has been conducted to confirm the functional roles of the putative binding locations of AHR/ARNT, reducing the strength of the biological plausibility of KER2 (Relationship 2683: dimerization, AHR/ARNT leads to Increase, slincR expression). The overall strength of the relationship is assigned as “moderate” since so far, convincing evidence for Ahr activation-induced slincR expression comes from only developing zebrafish studies.

Likewise, in the absence of Ahr2, expression of sox9b (one of two zebrafish paralogs of the SOX9 gene) is no longer significantly reduced even when zebrafish are exposed to TCDD (Garcia et al. 2018a). The strong evidence for essentiality of Ahr activation for sox9 repression is further supported by key experiments demonstrating strong dose concordance between Ahr2 activation, indicated by induction of cyp1a, and sox9b repression in zebrafish (Garcia et al. 2018b). Temporal concordance between the upstream event of Ahr activation (indicated by cyp1a induction) and repression of sox9b was shown for isolated jaw tissue from zebrafish larvae exposed to TCDD (Xiong et al. 2008) and evaluated at multiple timepoints until 12 hours post TCDD exposure. Supporting evidence for the relationship between Ahr activation and SOX9 repression comes from one transcriptomic study showing significant repression of the sox9 transcript in livers of white sturgeon exposed to equipotent concentrations of TCDD, PCB-77, and the PAH, benzo[a]pyrene (Doering et al. 2016), as well as one study demonstrating sox9 repression in whole Atlantic salmon (Salmo salar) larvae exposed to PCB-77 (Olufsen and Arukwe 2011b). The overall strength of the relationship is rated as “high” since in addition to essentiality evidence, support was identified in multiple species, with different Ahr activators.

4.1.2. Evidence linking craniofacial toxicity with early life stage mortality

While there is strong support for the KER between cardiovascular toxicity and early life stage mortality (https://aopwiki.org/relationships/1567) which is already included in the AOP-Wiki as part of AOPs 21 and 150 (peer-reviewed and endorsed; (Doering et al. 2019; Farhat and Kennedy 2019)), we discuss here the new KER between craniofacial malformations and early life stage mortality (Relationship 2686). It is reasonable to infer that malformed jaw structure of animals in the wild could impact their feeding success, leading to reduced growth and possible early mortality. Impacts on animals to capture prey can also lead to population-wide changes to both the predators and prey (Weis et al. 2001), constraining foraging patterns and thus recruitment success. Few studies have demonstrated the relationships between jaw malformations, reduced feeding, and mortality in fish (Noble et al. 2012). Specifically, studies in developing zebrafish and mummichog (Fundulus heteroclitus) have found that low concentrations of TCDD or PCB126 exposure can lead to subtle malformations in the lower jaw, in addition to reduced feeding capabilities of the fish (Couillard et al. 2011; King Heiden et al. 2009). However, both studies observed a reduction in feeding even in the fish that did not display jaw malformations, consequently, reduced feeding was not directly linked to an inability to capture prey due to the craniofacial deformity. Overall, it is likely that a combination of different malformations (ex: effects on both the heart and jaw) contribute to Ahr activation-induced mortality. These results are corroborated by an evaluation of TCDD toxicity in seven fish species, where despite the observation of craniofacial malformations in all species, TCDD toxicity, including mortality, decreased once exogenous feeding began suggesting the lack of a strong causal link between craniofacial malformations and poor survival (Elonen et al. 1998). An important caveat to the conclusions based on laboratory studies is that the relative abundance of food in an experimental test system relative to the natural environment may hide minor impacts on feeding efficiency that could be more impactful in nature. However, field-based investigations of the effect of craniofacial malformations on prey capture, feeding, and overall ecological fitness are difficult to address due to inherent complexities of the ecosystem. This is true in avian species as well; several studies have determined an association between dioxin and dioxin-like chemical exposure and beak deformities (Gilbertson et al. 1991), but only few have shown a possible relationship between bill defects (specifically, the cross-bill syndrome) and mortality (Ludwig et al. 1996). It is also worth emphasizing that cleft-palate in mammals which is homologous to cross-bill syndrome is not only induced by TCDD exposure (Pratt et al. 1984), but this congenital disorder can also cause death in humans (GBD 2018)

Even if the link between craniofacial malformations and early life stage mortality is weak, and largely based on plausibility, the occurrence of craniofacial malformations in fish and wildlife is still regarded as an AO in several regulatory contexts. For example, animal craniofacial deformities, specifically in birds and fish, are a commonly cited Beneficial Use Impairment at Great Lakes Areas of Concern (IJC 1991). Significant reductions in the occurrence and prevalence of deformities at these sites is a criterion for delisting. Consequently, craniofacial malformation is an important phenotype for risk assessment of Ahr activating chemicals, even if it does not directly lead to reduced survival. Thus, we have characterized craniofacial malformations as an AO within AOP 455, preceding the AO of early life stage mortality.

4.1.3. Evidence linking SOX9 repression with craniofacial and cardiovascular toxicities

While the relationships between Ahr-Arnt and sox9b has been well documented in the zebrafish literature, majority of evidence for SOX9’s role in craniofacial and cardiovascular development comes from biological plausibility evidence, as well as SOX9’s functional role from various mammalian studies. There is overwhelming evidence for SOX9’s evolutionarily conserved role in craniofacial development specifically by being a master regulator of cartilage development (Lee and Saint-Jeannet 2011). SOX9 has been shown to control expression of several genes involved in chondrocyte differentiation, and is also expressed abundantly in the craniofacial structure through development of multiple organisms including fish, frogs, opossum, and rodents (Li et al. 2002; Spokony et al. 2002; Wakamatsu et al. 2014; Wright et al. 1995). Similar to SOX9’s role in regulating craniofacial development, there is also strong evidence from several species including fish and rodents for SOX9’s role in cardiac morphology and functioning (Garside et al. 2015; Gawdzik et al. 2018). Thus, the biological plausibility of KER4 of AOP 455 (Relationship 2685: Decrease, sox9 expression leads to Smaller and morphologically distorted facial cartilage structures) and of AOP 456 (Relationship 2691: Decrease, sox9 expression leads to Altered, Cardiovascular development/function) is rated “high” across multiple taxa. More compelling evidence for the role of SOX9 is provided by two independent studies demonstrating essentiality of KE3 (Event 2020: Decrease, sox9 expression): sox9b morpholino knockdown led to zebrafish embryos with severe craniofacial malformations phenocopying TCDD exposure, and rescue of jaw malformations was obtained with injection of sox9 mRNA (Xiong et al. 2008). Similarly, sox9b morpholino knockdown in zebrafish also led to TCDD-like heart malformations (Hofsteen et al. 2013). Additionally, a few studies provide strong essentiality evidence for the relationship between mutations in the SOX9 gene in mammals, and some form of impact to chondrogenesis in the craniofacial region or cardiovascular dysfunction (Gong et al. 2022; Gordon et al. 2014; He et al. 2020). There is also more empirical evidence in other organisms showing the connection between SOX9 repression and the malformations (please see respective KER pages for more specific lines of evidence), however, we note that future work investigating if these relationships persist with Ahr activation (non-adjacent KER) in other developing vertebrates would help support existing KERs within AOPs 455 and 456. Overall, the strength of evidence for the relationship between SOX9 repression and craniofacial malformations is rated as “high”, while that of SOX9 repression and cardiovascular toxicity is “moderate” because of other known genes (ex: COX-2 and VEGF) that are also involved in causing the phenotype.

4.1.4. Evidence linking slincR induction with craniofacial and cardiovascular toxicities, and SOX9 repression

One question that remained unanswered was the possible mechanisms by which Ahr was regulating SOX9 expression. Even though there are eight putative AHREs near the sox9b promoter, its repression in zebrafish does not occur immediately after TCDD exposure (Xiong et al. 2008). The current AOP report provides evidence from the zebrafish literature supporting the intermediary role of a lncRNA named slincR. Essentiality of KE2 (Event 2021: Increase, slincR expression) is demonstrated by the fact that sox9b relative expression, as well as several targets known to be regulated by sox9b, is higher in zebrafish with morpholino knockdown of slincR, (Garcia et al. 2017). Importantly, slincR knockdown produces changes to zebrafish angiogenesis and vascular development (Garcia et al. 2018b), and both slincR knockdown and knockout result in abnormal cartilage structures (Dasgupta et al. 2023; Garcia et al. 2018b). To support the importance of slincR induction, significant empirical evidence (dose concordance) comes from a concentration-response experiment showing that slincR is induced at TCDD exposure concentrations lower than concentrations at which sox9b is repressed (Garcia et al. 2018b). Biological plausibility of KER3 (Relationship 2684: Increase, slincR expression leads to Decrease, sox9 expression) is supported by literature showing that lncRNAs have key roles in regulation of gene expression (Long et al. 2017; Marchese et al. 2017; Statello et al. 2021). Importantly, one study provided evidence for slincR and sox9b being expressed in adjacent and overlapping tissues through multiple stages of zebrafish development (Garcia et al. 2017). A follow-up study identified slincR being enriched just upstream of the sox9b locus of zebrafish suggesting a molecular interaction between slincR and the sox9b promoter, and importantly, the potential regulatory mechanism of slincR (Garcia et al. 2018b). Future studies showing the direct interaction between slincR and sox9b using techniques such as cross-linking and immunoprecipitation (CLIP) or chromatin isolation by RNA purification (ChIRP) (Cao et al. 2019) would increase the strength of evidence for KER’s biological plausibility. It should be noted that slincR is likely not the only mechanism of regulation of SOX9. Other molecules such as A disintegrin and metalloprotease (ADAM) 10 (Fu et al. 2018) and Mediator complex subunit (Med23) (Dash et al. 2021) have been shown to modulate SOX9. This uncertainty is further corroborated by evidence for the ability of retinoic acid to suppress SOX9 expression (Sekiya et al. 2001), as well as Ahr activation impacting retinoic acid metabolism (Esteban et al. 2021; Herlin et al. 2021). We have rated the relationships between slincR induction and, SOX9 suppression, craniofacial malformations, and cardiovascular toxicities as “moderate” because of the combination of the relatively few number of studies (two Garcia et al. studies, one Dasgupta et al. study) the convincing evidence comes from, in addition to possible other mechanisms of regulation of SOX9 expression.

4.2. Domain of applicability and known stressors

Evidence gathered suggests that the two AOPs have similar domains of applicability covering most vertebrates, from fish to humans and other wildlife. The relationships between Ahr, Arnt, slincR and sox9b, and cardiac and craniofacial malformations have been well established in developing zebrafish, specifically as embryos (i.e., prior to sexual differentiation), and thus sex is not a relevant parameter. It is important to highlight that while all the relationships (except the KER between craniofacial malformations and mortality) 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). 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 2011a). Additionally, while slincR has only been described in zebrafish so far, it is worth noting that putative human and mouse orthologs have been identified (Garcia et al. 2018b), increasing the possibility that the zebrafish-specific results can be translated to other organisms. The formation of craniofacial structures is a predominantly evolutionarily conserved dynamic and complex process that begins early in embryonic development (Helms et al. 2005; Kuratani 2005). Consequently, craniofacial development can be thought of as a potential target of disruption in early embryos, and an AOP network around this KE would be highly relevant to both humans and general wildlife. Similarly, cardiovascular toxicity is a significant complication when different vertebrates are exposed to Ahr-dependent chemicals such as TCDD, dioxin-like chemicals, 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.

Evidence for the AOPs primarily comes from TCDD as a chemical stressor. However, it is possible that other Ahr activating chemicals such as other HAHs and PAHs, that lead to craniofacial malformations, sometimes altering jaw formation in animals such as fish and mink (Hornung et al. 1999; Render et al. 2000), or both structural and functional cardiotoxicity (Heid et al. 2001; Incardona et al. 2009; Incardona et al. 2014b) could be stressors whose toxicities are mediated by the slincR-SOX9 interaction. One example is from a study that exposed Atlantic salmon (Salmo salar) to two concentrations of the dioxin-like PCB-77 and identified both the reduction of sox9 mRNA expression and eventual cardiac toxicity (Olufsen and Arukwe 2011a). With the increase in non-targeted transcriptomic studies in the toxicology field, future research could begin to identify SOX9 and slincR gene expression changes as possible indicators of craniofacial and/or cardiac toxicities. An inconsistency comes from a study exposing developing zebrafish to 16 individual PAHs to 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. Despite the disparity, it remains conceivable that the progression of the KEs laid out in these AOPs is not unique to TCDD as a strong Ahr activating chemical, and testing of other chemicals would confirm the chemical applicability domain.

4.3. Quantitative understanding

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). We highlight that TCDD has been identified as the prototypical stressor for both AOPs 455 and 456, and the toxic equivalency factor (TEF) concept could be leveraged to determine total toxic equivalencies (TEQs) for a defined set of dioxin-like chemicals based on the known concentrations at which TCDD can induce different KEs of the AOPs (US EPA 2003; Van den Berg et al. 1998). 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 that can be leveraged for risk assessment of dioxin-like chemicals even if they have broad species-specific responsiveness (Van den Berg et al. 1998).

Additionally, for other 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 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).

4.4. Relevance at environmentally realistic exposure concentrations

The AOPs in the current report are considered environmentally relevant primarily because the AO of early life stage mortality has been shown to occur in several vertebrate species under exposure to environmentally realistic concentrations of Ahr activating chemicals. Strong evidence from several key studies for the non-adjacent KER between Ahr activation and early life stage mortality has been described in the AOP-Wiki in relationship 984 (https://aopwiki.org/relationships/984). In addition, reviews by (Cook 1993) and (Walker and Peterson 1994), for example, provide compelling lines of evidence for lethal and sublethal aquatic toxicity associated with TCDD and other dioxin-like chemicals. Another example is the Great Lakes ecosystem from the 1940s, when significant effects were detected in both avian population (Gilbertson et al. 1991; Ludwig et al. 1996) as well as several species of fish, particularly in the sensitive lake trout (Tillitt 2008).

There are also several examples of cardiovascular toxicity (Carls et al. 1999), sometimes in combination with craniofacial malformations (lower jaw malformations in fish, or bill defects in birds) (Incardona et al. 2014a) occurring under environmental realistic concentrations of pollutant mixtures, such as crude oil, increasing the overall environmental relevance of each of AOPs 455 and 456. Crude oil consists of several different types of PAHs including their persistent derivatives (Wells et al. 1995), and oil spills such as the Exxon Valdez (1989) and Deepwater Horizon (2010) are sources of chronic exposures to these chemicals (Barron et al. 2020). Many of the PAHs from oil spills can activate the Ahr signaling pathway in a variety of organisms as measured by CYP1A gene and protein expression inductions (Dubansky et al. 2017). Along the same lines, of particular importance for the applicability of the two new AOPs for risk assessment is the inclusion of quantifiable gene expression events. Significant alteration in gene expression of slincR, and reduction in sox9b’s expression in developing zebrafish occurred at a TCDD exposure concentration (0.0625 ng/mL, 1 h exposure) much lower than when apparent phenotypic defects were visible (Garcia et al. 2018b). It is possible that the gene expression events when translated to other organisms and chemical exposures could be detected at environmentally relevant scenarios, especially since in general, dioxin developmental toxicity in zebrafish is consistent with toxicity endpoints observed in several types of wild fish (King-Heiden et al. 2012).

When considering the AOPs together, evidence, primarily from laboratory fish studies, suggests that upon Ahr activation at environmentally relevant concentrations, developmental cardiovascular toxicity is more likely to result in early life stage mortality even if fish are displaying craniofacial malformations (Couillard et al. 2011; King Heiden et al. 2009). Consequently, AOP 456, potentially in combination with AOPs 21 and 150 are likely to represent the more “critical path” (Knapen et al. 2018) linking Ahr activation to mortality rather than AOP 455. While the serious implications of craniofacial defects in mammals, as well as the possible deterioration in prey capture and thus survival in other organisms such as birds and fish should not be ignored, under the context of sustained Ahr activation, AOP 456 is considered more environmentally realistic.

5. Potential Applications

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 AOP. 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 responses into the risk assessment of the various Ahr activating pollutants (Kramer et al. 2011; US EPA 2008) to enable extrapolations across both chemicals and taxa, while also identifying key differences between them.

Supplementary Material

Supplement1
Supplement2

Acknowledgements

The present study was funded by a US EPA Cooperative Training Partnership in Aquatic Toxicology and Ecosystem Research award to the University of Wisconsin-Madison Aquatic Sciences Center (reference award number 83940101). We would also like to thank Jennifer H. Olker and Thomas B. Knudsen for providing important critical feedback on an earlier version of the manuscript.

6. Abbreviation List

Ahr

Aryl Hydrocarbon Receptor

AO

Adverse Outcome

AOP

Adverse Outcome Pathway

Ahre

aryl hydrocarbon response element

ARNT

Ahr nuclear translocator

ChIRP

chromatin isolation by RNA purification

CLIP

cross-linking and immunoprecipitation

COX-2

cyclooxygenase 2

CYP

cytochrome P450

EROD

Ethoxyresorufin-O-deethylase

HAH

halogenated aromatic hydrocarbons

ID

identification

KE

Key Event

KER

Key Event Relationship

lncRNA

long non-coding RNA

MIE

Molecular Initiating Event

OECD

Organization for Economic Co-operation and Development

PAH

polycyclic aromatic hydrocarbons

PCB

polychlorinated biphenyls

PRS

Pierre Robin sequence

slincR

sox9b long intergenic non-coding RNA

SOX9(b)

sry-box containing gene 9(b)

TCDD

2,3,7,8-Tetrachlorodibenzo-p-dioxin

TEF

toxic equivalency factor

TEQ

the toxic equivalency

VEGF

vascular endothelial growth factor

Footnotes

1

Ahres is sometimes used interchangeably in the literature with xenobiotic response elements (XREs) or dioxin response elements (DREs)

Conflict of Interest

The authors declare no conflict of interest.

Disclaimer

The viewpoints expressed are those of the authors and do not necessarily reflect opinions/policies of the US Environmental Protection Agency (US EPA). Mention of trade names and commercial products does not constitute endorsement or recommendation for use. This paper has been reviewed and approved for publication in accordance with US EPA guidelines.

AOP Editors and Reviewers

Dries Knapen was the handling editor on this AOP Pathways and Predictions. Sean Kennedy (Research Scientist, Environment Canada), Jon Doering (Assistant Professor, Department of Environmental Sciences, Louisiana State University), and Helen Håkansson (Professor, Environmental Medicine, Karolinska Institutet) reviewed the submission and supplied feedback. The reviewers declared no conflict of interest. The review process was carried out following the principles in the OECD Guidance Document for the scientific review of AOPs (Organisation for Economic Co-operation and Development [OECD] 2021).

Data Availability Statement

No new data were generated for this review. All review reports can be accessed at https://aopwiki.org/aops/455/comments and https://aopwiki.org/aops/456/comments. The final snapshot pdfs of these AOPs can be accessed at https://aopwiki.org/aopwiki/snapshot/html_file/455-2023-06-06T21:12:42+00:00.html and https://aopwiki.org/aopwiki/snapshot/html_file/456-2023-06-06T21:12:59+00:00.html. The snapshot pdfs that were used during the review process can be found at https://aopwiki.org/aopwiki/snapshot/html_file/455-2022-10-21T19:58:01+00:00.html and https://aopwiki.org/aopwiki/snapshot/html_file/456-2022-10-21T19:59:04+00:00.html.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplement1
NIHMS1953611-supplement-Supplement1.pdf (646.7KB, pdf)
Supplement2
NIHMS1953611-supplement-Supplement2.pdf (646KB, pdf)

Data Availability Statement

No new data were generated for this review. All review reports can be accessed at https://aopwiki.org/aops/455/comments and https://aopwiki.org/aops/456/comments. The final snapshot pdfs of these AOPs can be accessed at https://aopwiki.org/aopwiki/snapshot/html_file/455-2023-06-06T21:12:42+00:00.html and https://aopwiki.org/aopwiki/snapshot/html_file/456-2023-06-06T21:12:59+00:00.html. The snapshot pdfs that were used during the review process can be found at https://aopwiki.org/aopwiki/snapshot/html_file/455-2022-10-21T19:58:01+00:00.html and https://aopwiki.org/aopwiki/snapshot/html_file/456-2022-10-21T19:59:04+00:00.html.

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