AOP ID and Title:

Short Title: Ahr mediated early stage mortality via cardiovascular toxicity

Authors

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

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

Status

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

Background

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

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

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

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

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

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

Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Key Event Relationships

Stressors

Overall Assessment of the AOP

See details below.

Domain of Applicability

Life Stage Applicability Taxonomic Applicability Sex Applicability

Life Stage and Sex

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

Taxonomic

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

Essentiality of the Key Events

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	Strong	Cardiovascular toxicity has been shown to cause early life stage mortality. This relationship has been discussed in depth in AOP 21 (aopwiki.org/aops/21) (peer-reviewed and endorsed).
947	Increase, Early Life Stage Mortality	N/A	This is the terminal key event in the AOP and hence its essentiality for downstream events cannot be evaluated.

Weight of Evidence Summary

Biological Plausibility

• Ahr – strong : Strongest Biological Plausibility evidence for AOPs 455 and 456 comes from our extensive understanding of the Ahr signaling pathway in multiple different organisms. The functional roles of Ahr and its binding partners, including ARNT, have been well-studied (Fujii-Kuriyama and Kawajiri 2010), and it is well known that the Ahr signaling pathway mediates a variety of physiological and toxicological functions (Larigot et al. 2018).

• slincR and sox9 – strong: Strong evidence for the Biological Plausibility of slincR having a role in AOPs 455 and 456 comes from the nature of lncRNAs which is such that they have diverse functions and can regulate gene expression at multiple levels, including by interacting with DNA, RNA, proteins, and altering transcription of both neighboring and distant genes (Statello et al. 2021). Additionally, slincR (in situ hybridization) and sox9b (immunohistochemistry for sox9b-eGFP) are expressed in adjacent and overlapping tissues through multiple stages of zebrafish development, such as in the eye, otic vesicle, and in the lower jaw (Garcia et al. 2017) providing one line of evidence for slincR being able to regulate sox9b gene expression. Further, a capture hybridization analysis of RNA targets (CHART) experiment in both DMSO- and TCDD-exposed 48 hpf zebrafish identified enrichment of slincR in the 5’UTR of the sox9b locus (Garcia et al. 2018b) pointing to possible interaction between slincR and sox9b.

• slincR and cardiovascular development - strong: Individual zebrafish exposures to the PAHs, retene, dibenzo[a,h]pyrene, and dibenzo[a,i]pyrene cause cyp1a vascular expression as well as a significant induction of slincR at 48 hours post fertilization (hpf) (Garcia et al. 2018b; Geier et al. 2018), suggesting the possibility of slincR involved in some aspect of cardiovascular function. Knockdown of slincR expression in developing zebrafish, alters expression of sox9b, as well as certain downstream targets of sox9, such as notch3, adamts3, fabp2, sfrp2, and fgfr3 (Garcia et al. 2017). These are different lines of Biological Plausibility evidence for slincR being a mediator between Ahr activation and craniofacial/cartilage malformations.

• Sox9 and cardiovascular development - strong: Strong Biological Plausibility evidence for sox9’s role in cardiovascular dysfunction comes from one study where zebrafish exposed to 1ng/mL TCDD had significantly reduced sox9b expression in the heart of the developing animals (Hofsteen et al. 2013). Backing up this evidence, several studies in different organisms including rodents, chicken, frog, and fish have identified both sox9 mRNA and protein spatiotemporal expression in the developing hearts (please see KER page 2691 for references). In addition, a chip-seq experiment showed that the sox9 protein has been found to interact with the genomic regions of proliferation genes as well as important transcription factors involved in mouse heart development (Garside et al. 2015), making it conceivable that the loss of sox9 can have a significant impact on cardiovascular development.

• Cardiovascular dysfunction and early-stage mortality - strong: Biological plausibility of this KER is considered high because the relationship between cardiovascular toxicity and early mortality has been demonstrated in several species including fish and birds (Kopf and Walker 2009). Note that this KER is already included in the AOP-Wiki as part of AOP 21 (peer-reviewed and endorsed; (Doering et al. 2019) with abundant evidence listed.

 

Dose Concordance

• Ahr activation leading to early life stage mortality has been well-studied. The KER page (https://aopwiki.org/relationships/984) has examples in difference species for empirical evidence for this relationship.

• Strongest evidence for dose concordance between Ahr activation, slincR induction, and sox9 repression comes from a developing zebrafish study that utilized TCDD as the Ahr activating chemical. The concentration-response experiment showed that cyp1a (biomarker for Ahr activation) and slincR expression increased in parallel as TCDD exposure concentration increased, and that cyp1a and slincR are induced at TCDD exposure concentrations lower than concentrations at which sox9b is repressed (Garcia et al. 2018b).
  • Both cyp1a and slincR were significantly induced starting at 0.0625 ng/mL TCDD exposure.
  • Significant cyp1a (~log2FC = 6) and slincR (~log2FC = 2) inductions were detected at 0.0625 ng/mL TCDD, while significant sox9b repression (~log2FC = -1) was detected only at 0.5ng/mL TCDD.

• (Garcia et al. 2018b) also showed that with increasing concentrations of TCDD, the severity of overall developmental malformations, including pericardial edema (indicator of potential cardiotoxicity) and jaw malformations increased.

• Strong dose concordance has been determined between cardiovascular malformations and early life stage mortality (please see KER page: https://aopwiki.org/relationships/1567), however, to the best of out knowledge, no systematic effort has been performed to identify  “dose concordance” evidence for the KER between craniofacial malformations and early life stage mortality.

Uncertainties, inconsistencies, data gaps

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

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

Quantitative Consideration

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

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

Considerations for Potential Applications of the AOP (optional)

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

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Appendix 1

List of MIEs in this AOP

Event: 18: Activation, AhR

Short Name: Activation, AhR

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

The AHR structure has been shown to contribute to differences in species sensitivity to DLCs in several animal models. In 1976, a 10-fold difference was reported between two strains of mice (non-responsive DBA/2 mouse, and responsive C57BL/6 14 mouse) in CYP1A induction, lethality and teratogenicity following TCDD exposure^[3]. This difference in dioxin sensitivity was later attributed to a single nucleotide polymorphism at position 375 (the equivalent position of amino acid residue 380 in chicken) in the AHR LBD^[30][19][31]. Several other studies reported the importance of this amino acid in birds and mammals^{[32][30][22][33][34][35][31][36]}. It has also been shown that the amino acid at position 319 (equivalent to 324 in chicken) plays an important role in ligand-binding affinity to the AHR and transactivation ability of the AHR, due to its involvement in LBD cavity volume and its steric effect^[35]. Mutation at position 319 in the mouse eliminated AHR DNA binding^[35].

The first study that attempted to elucidate the role of avian AHR1 domains and key amino acids within avian AHR1 in avian differential sensitivity was performed by Karchner et al.^[22]. Using chimeric AHR1 constructs combining three AHR1 domains (DBD, LBD and TAD) from the chicken (highly sensitive to DLC toxicity) and common tern (resistant to DLC toxicity), Karchner and colleagues^[22], showed that amino acid differences within the LBD were responsible for differences in TCDD sensitivity between the chicken and common tern. More specifically, the amino acid residues found at positions 324 and 380 in the AHR1 LBD were associated with differences in TCDD binding affinity and transactivation between the chicken (Ile324_Ser380) and common tern (Val324_Ala380) receptors^[22]. Since the Karchner et al. (2006) study was conducted, the predicted AHR1 LBD amino acid sequences were been obtained for over 85 species of birds and 6 amino acid residues differed among species^[14][37] . However, only the amino acids at positions 324 and 380 in the AHR1 LBD were associated with differences in DLC toxicity in ovo and AHR1-mediated gene expression in vitro^[14][37][16]. These results indicate that avian species can be divided into one of three AHR1 types based on the amino acids found at positions 324 and 380 of the AHR1 LBD: type 1 (Ile324_Ser380), type 2 (Ile324_Ala380) and type 3 (Val324_Ala380)^[14][37][16].

• Little is known about differences in binding affinity of AhRs and how this relates to sensitivity in non-avian taxa.
• Low binding affinity for DLCs of AhR1s of African clawed frog (Xenopus laevis) and axolotl (Ambystoma mexicanum) has been suggested as a mechanism for tolerance of these amphibians to DLCs (Lavine et al 2005; Shoots et al 2015).
• Among reptiles, only AhRs of American alligator (Alligator mississippiensis) have been investigated and little is known about the sensitivity of American alligator or other reptiles to DLCs (Oka et al 2016).
• Among fishes, great differences in sensitivity to DLCs are known both for AhRs and for embryos among species that have been tested (Doering et al 2013; 2014).
• Differences in binding affinity of the AhR2 have been demonstrated to explain differences in sensitivity to DLCs between sensitive and tolerant populations of Atlantic Tomcod (Microgadus tomcod) (Wirgin et al 2011).
  • This was attributed to the rapid evolution of populations in highly contaminated areas of the Hudson River, resulting in a 6-base pair deletion in the AHR sequence (outside the LBD) and reduced ligand binding affinity, due to reduces AHR protein stability.
• Information is not yet available regarding whether differences in binding affinity of AhRs of fishes are predictive of differences in sensitivity of embryos, juveniles, or adults (Doering et al 2013).

The AhR is a very conserved and ancient protein (95) and the AhR is present  in human and mice (96–98). The AhR is present in human physiology and pathology. The AhR is highly expressed at several important physiological barriers such as the placenta, lung, gastrointestinal system, and liver in human (Wakx, Marinelli, Watanabe).  In these tissues, the AhR is involved in both detoxication processes involving xenobiotic metabolizing enzymes such as cytochromes P450, and in immune functions translating chemical signals into immune defence pathways (Marinelli, Stobbe). Moreover, it has a regulatory role in human dendritic cells and myelination (Kado, Shackleford). The lung constitutes another barrier exposed to components of air pollution such as particles and hydrocarbons (air pollution, cigarette smoke). The AhR detects such hydrocarbons and protects the pulmonary cells from their deleterious effects through metabolization. The regulatory effect on blood cells of the AhR, balancing different related cell types, can be extended to the megakaryocytes and their precursors; indeed, StemRegenin 1 (SR1), an antagonist of the AhR increases the human population of CD34+CD41low cells, a fraction of very efficient precursors of proplatelets (Bock). The occurrence of a nystagmus has been subsequently diagnosed in humans bearing a AhR mutation (Borovok).

In human cancer, the AhR has either a pro or con tumor effect depending on the tissue, the ligand, and the duration of the activation (Zudaire, Chang, Litzenburg, Gramatzki, Lin, Wang). In human breast cancer, the AhR is thoughts to be responsible of its progression (Goode, Kanno, Optiz, Novikov, Hall, Subramaniam, Barhoover). In human mammary benign cells, Brooks et al. noted that a high level of AhR was associated with a modified cell cycle (with a 50% increase in population doubling time in cells expressing the AhR by more than 3-fold) and EMT including increased cell migration. Narasimnhan et al. found that suppression of the AhR pathway had a pro-tumorigenic effect in vitro (EMT, tumor migration) in triple negative breast cancer.

Many endogenous and exogenous ligands are present for the AhR in human (Optiz, Adachi, Schroeder, Rothhammer). Indoles, such as indole-3-carbinol or one of its secondary metabolites, 3-3'- Diindolylmethane, are degradation products found in cruciferous vegetables and characterized as AhR ligands (Ema, Kall, Miller) they are also inducers of the human and rat CYP1A1 (Optiz). FICZ is the most potent AhR ligand known to date: it has a stronger affinity than TCDD for the human AhR (TCDD Kd=0.48 nM/FICZ Kd=0.07 nM) (Coumoul).

 

 

Key Event Description

The AHR Receptor

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that belongs to the basic helix-loop-helix Per-ARNT-Sim (bHLH-PAS) superfamily and consists of three domains: the DNA-binding domain (DBD), ligand binding domain (LBD) and transactivation domain (TAD)^[1]. Other members of this superfamily include the AHR nuclear translocator (ARNT), which acts as a dimerization partner of the AHR ^[2][3]; Per, a circadian transcription factor; and Sim, the “single-minded” protein involved in neuronal development ^[4][5]. This group of proteins shares a highly conserved PAS domain and is involved in the detection of and adaptation to environmental change^[4].

Investigations of invertebrates possessing early homologs of the AhR suggest that the AhR evolutionarily functioned in regulation of the cell cycle, cellular proliferation and differentiation, and cell-to-cell communications (Hahn et al 2002). However, critical functions in angiogenesis, regulation of the immune system, neuronal processes, metabolism, development of the heart and other organ systems, and detoxification have emerged sometime in early vertebrate evolution (Duncan et al., 1998; Emmons et al., 1999; Lahvis and Bradfield, 1998).

The molecular Initiating Event

The molecular mechanism for AHR-mediated activation of gene expression is presented in Figure 1. In its unliganded form, the AHR is part of a cytosolic complex containing heat shock protein 90 (HSP90), the HSP90 co-chaperone p23 and AHR-interacting protein (AIP)^[6]. Upon ligand binding, the AHR migrates to the nucleus where it dissociates from the cytosolic complex and forms a heterodimer with ARNT^[7]. The AHR-ARNT complex then binds to a xenobiotic response element (XRE) found in the promoter of an AHR-regulated gene and recruits co-regulators such as CREB binding protein/p300, steroid receptor co-activator (SRC) 1, SRC-2, SRC-3 and nuclear receptor interacting protein 1, leading to induction or repression of gene expression^[6]. Expression levels of several genes, including phase I (e.g. cytochrome P450 (CYP) 1A, CYP1B, CYP2A) and phase II enzymes (e.g. uridine diphosphate glucuronosyl transferase (UDP-GT), glutathione S-transferases (GSTs)), as well as genes involved in cell proliferation (transforming growth factor-beta, interleukin-1 beta), cell cycle regulation (p27, jun-B) and apoptosis (Bax), are regulated through this mechanism ^[6][8][7][9].

AHR Isoforms

• Over time the AhR has undergone gene duplication and diversification in vertebrates, which has resulted in multiple clades of AhR, namely AhR1, AhR2, and AhR3 (Hahn 2002).
• Fishes and birds express AhR1s and AhR2s, while mammals express a single AhR that is homologous to the AhR1 (Hahn 2002; Hahn et al 2006).
• The AhR3 is poorly understood and known only from some cartilaginous fishes (Hahn 2002).
• Little is known about diversity of AhRs in reptiles and amphibians (Hahn et al 2002).
• In some taxa, subsequent genome duplication events have further led to multiple isoforms of AhRs in some species, with up to four isoforms of the AhR (α, β, δ, γ) having been identified in Atlantic salmon (Salmo salar) (Hansson et al 2004).
• Although homologs of the AhR have been identified in some invertebrates, compared to vertebrates these AhRs have differences in binding of ligands in the species investigated to date (Hahn 2002; Hahn et al 1994).

 

Roles of isoforms in birds:

Two AHR isoforms (AHR1 and AHR2) have been identified in the black-footed albatross (Phoebastria nigripes), great cormorant (Phalacrocorax carbo) and domestic chicken (Gallus gallus domesticus)^[10]. AHR1 mRNA levels were similar in the kidney, heart, lung, spleen, brain, gonad and intestine from the great cormorant but were lower in muscle and pancreas. AHR2 expression was mainly observed in the liver, but was also detected in gonad, brain and intestine. AHR1 levels represented a greater proportion (80%) of total AHR levels than AHR2 in the cormorant liver^[10], and while both AHR isoforms bound to TCDD, AHR2 was less effective at inducing TCDD-dependent transactivation compared to AHR1 in black-footed albatross, great cormorant and domestic chicken^[11][10].

• AhR1 and AhR2 both bind and are activated by TCDD in vitro (Yasui et al 2007).
• AhR1 has greater binding affinity and sensitivity to activation by TCDD relative to AhR2 (Yasui et al 2007).
• AhR1 is believed to mediate toxicities of DLCs, while AhR2 has no known role in toxicities (Farmahin et al 2012; Farmahin et al 2013; Manning et al 2012).

Roles of isoforms in fishes:

• AhR1 and AhR2 both bind and are activated by TCDD in vitro (Bak et al 2013; Doering et al 2014; 2015; Karchner et al 1999; 2005).
• AhR1 has greater sensitivity to activation by TCDD than AhR2 in red seabream (Pagrus major), white sturgeon (Acipenser transmontanus), and lake sturgeon (Acipenser fulvescens) (Bak et al 2013; Doering et al 2014; 2015)
• AhR2 has greater binding affinity or activation by TCDD than AhR1 in zebrafish (Danio rerio) and mummichog (Fundulus heteroclitus) (Karchner et al 1999; 2005).
• AhR2 is believed to mediate toxicities in fishes, while AhR1 has no known role in toxicities. Specifically, knockdown of AhR2 protects against toxicities of dioxin-like compounds (DLCs) and polycyclic aromatic hydrocarbons (PAHs) in zebrafish (Danio rerio) and mummichog (Fundulus heteroclitus), while knockdown of AhR1 offers no protection (Clark et al 2010; Prasch et al 2003; Van Tiem & Di Giulio 2011).

Roles of isoforms in amphibians and reptiles:

• Less is known about AhRs of amphibians or reptiles.
• AhR1 is believed to mediate toxicities in amphibians (Hahn 2002; Lavine et al 2005; Oka et al 2016; Shoots et al 2015). However, all AhRs of amphibians that have been investigated have very low affinity for TCDD (Hahn 2002; Lavine et al 2005; Oka et al 2016; Shoots et al 2015).
• Both AhR1s and AhR2 of American alligator (Alligator mississippiensis) are activated by agonists with comparable sensitivities (Oka et al 2016). AhRs of no other reptiles have been investigated.

Role in mammals

AhR expression is essentially ubiquitous in mammals consistent with a broad-spectrum homeostatic role, however expression levels varying widely across tissues with the liver, thymus, lung, kidney, spleen, and placenta exhibiting greatest expression (Harper PA). Additionally, AhR expression is developmentally regulated, and more recent evidence indicates a role for the AhR in developmental process affecting hematopoiesis, immune system biology, neural differentiation, and liver architecture (Wright E J) . AHR is involved in regulating the rate of apoptosis of oocytes in germ cell nests during embryonic life and in regulating survival of oocytes in the fetal and neonatal ovary. Specifically, studies have shown that ovaries obtained from AHRKO mice on ED13.5 and cultured for 72 h in the absence of hormonal support with the aim of inducing apoptosis, contained higher numbers of non-apoptotic germ cells compared to wild-type (WT) ovaries cultured in the same conditions (Hernández-Ochoa)

 

How it is Measured or Detected

Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible?

Transactivation Reporter Gene Assays (recommended approach)

Transient transfection transactivation

Transient transfection transactivation is the most common method for evaluating nuclear receptor activation^[12]. Full-length AHR cDNAs are cloned into an expression vector along with a reporter gene construct (chimeric luciferase, P-lactamase or CAT reporter vectors containing the appropriate response elements for the gene of interest). There are a number of commercially available cell lines that can serve as recipients for these vectors (CV-1, HuH7, FLC-7, LS174T, LS180 MCF-7, HEC1, LLC-PK1, HEK293, HepG2, and Caco-2 cells)^[12]. The greatest advantage of using transfected cells, rather than primary cell cultures, is the assurance that the nuclear receptor of interest is responsible for the observed induction. This would not be possible in a primary cell culture due to the co-regulation of different receptors for the same target genes. This model makes it easy to compare the responsiveness of the AHR across multiple species under the same conditions simply by switching out the AHR clone. One disadvantage to the transient transfection assay is the inherent variability associated with transfection efficiency, leading to a movement towards the use of stable cell lines containing the nuclear receptor and reporter gene linked to the appropriate response elements^[12].

Luciferase reporter gene (LRG) assay

The described luciferase reporter gene (LRG) assays have been used to investigate activation of AhRs of:

• Humans (Homo sapiens) (Abnet et al 1999) 
• Species of birds, namely chicken (Gallus gallus), ring-necked pheasant (Phasianus colchicus), Japanese quail (Coturnix japonica), and common tern (Sterna hirundo) (Farmahin et al 2012; Manning et al 2013), Mutant AhR1s with ligand binding domains resembling those of at least 86 avian species have also been investigated (Farmahin et al 2013). AhR2s of birds have only been investigated in black-footed albatross (Phoebastria nigripes) and common cormorant (Phalacrocorax carbo) (Yasio et al 2007).
• American alligator (Alligator mississippiensis) is the only reptile for which AhR activation has been investigated (Oka et al 2016), AhR1A, AhR1B, and AhR2 of American alligator were assayed (Oka et al 2016).
• AhR1 of two amphibians have been investigated, namely African clawed frog (Xenopus laevis) and salamander (Ambystoma mexicanum) (Lavine et al 2005; Shoots et al 2015; Ohi et al 2003),
• AhR1s and AhR2s of several species of fish have been investigated, namely Atlantic salmon (Salmo salar), Atlantic tomcod (Microgadus tomcod), white sturgeon (Acipenser transmontanus), rainbow trout (Onchorhynchys mykiss), red seabream (Pagrus major), lake sturgeon (Acipenser fulvescens), and zebrafish (Danio rerio) (Andreasen et al 2002; Abnet et al 1999; Bak et al 2013; Doering et al 2014; 2015; Evans et al 2005; Hansson & Hahn 2008; Karchner et al 1999; Tanguay et al 1999; Wirgin et al 2011).

For demonstrative purposes, a luciferase reporter gene assay used to measure AHR1-mediated transactivation for avian species is described here. However, comparable assays are utilized for investigating AHR1s and AHR2s of all taxa. A monkey kidney cell line (Cos-7) that has low endogenous AHR1 expression was transfected with the appropriate avian AHR1 clone, cormorant ARNT1, a CYP1A5 firefly luciferase reporter construct and a Renilla luciferase vector to control for transfection efficiency. After seeding, the cells were exposed to DLC and luciferase activity was measured using a luminometer. Luminescence, which is proportional to the extent of AHR activation, is expressed as the ratio of firefly luciferase units to Renilla luciferase units ^[13]. This particular assay was modified from its original version to increase throughput efficiency; (a) cells were seeded in 96-well plates rather than Petri dishes or 48- well plates, (b) DLCs were added directly to the wells without changing the cell culture medium, and (c) the same 96-well plates were used to measure luminescence without lysing the cells and transferring to another plate. Similar reporter gene assays have been used to measure AHR1 activation in domestic and wild species of birds, including the chicken, ring-necked pheasant (Phasianus colchicus), Japanese quail (Coturnix japonica), great cormorant, black-footed albatross and peregrine falcon (Falco peregrinus).^{[14][13][15][11][16][17]}

Transactivation in stable cell lines

Stable cell lines have been developed and purified to the extent that each cell contains both the nuclear receptor and appropriate reporter vector, eliminating the variability associated with transfection ^[12]. A stable human cell line containing a luciferase reporter driven by multiple dioxin response elements has been developed that is useful in identifying AhR agonists and antagonists^[18]. An added benefit of this model is the potential to multiplex 3 assays in a single well: receptor activation, cell viability and enzyme activity^[12]. Such assays are used extensively in drug discovery due to their high throughput efficiency, and may serve just as useful for risk assessment purposes.

Ligand-Binding Assays

Ligand binding assays measure the ability of a test compound to compete with a labeled, high-affinity reference ligand for the LBD of a nuclear receptor. It is important to note that ligand binding does not necessitate receptor activation and therefore cannot distinguish between agonists and antagonists; however, binding affinities of AHR ligands are highly correlated with chemical potencies^[19] and can explain differences in species sensitivities to DLCs^[20][21][22]; they are therefore worth mentioning. Binding affinity and efficacy have been used to develop structure-activity relationships for AHR disruption^[20][23] that are potentially useful in risk-assessment. There has been tremendous progress in the development of ligand-binding assays for nuclear receptors that use homogenous assay formats (no wash steps) allowing for the detection of low-affinity ligands, many of which do not require a radiolabel and are amenable to high throughput screening^[24][12]. This author however was unable to find specific examples of such assays in the context of AHR binding and therefore some classic radioligand assays are described instead.

Hydroxyapatite (HAP) binding assay

The HAP binding assay makes use of an in vitro transcription/translation method to synthesize the AHR protein, which is then incubated with radiolabeled TDCPP and a HAP pellet. The occupied protein adsorbs to the HAP and the radioactivity is measured to determine saturation binding. An additional ligand can also be included in the mixture in order to determine its binding affinity relative to TCDD (competitive binding)^[25][22]. This assay is simple, repeatable and reproducible; however, it is insensitive to weak ligand-receptor interactions^[22][21][26].

Whole cell filtration binding assay

Dold and Greenlee^[27] developed a method to detect specific binding of TCDD to whole mammalian cells in culture and was later modified by Farmahin et al.^[21] for avian species. The cultured cells are incubated with radiolabeled TCDD with or without the presence of a competing ligand and filtered. The occupied protein adsorbs onto the filter and the radioactivity is measured to determine saturation binging and/or competitive binding. This assay is able to detect weak ligand-receptor interactions that are below the detection limit of the HAP assay^[21].

Protein-DNA Interaction Assays

The active AHR complexed with ARNT can be measured using protein-DNA interaction assays. Two methods are described in detail by Perez-Romero and Imperiale^[28]. Chromatin immunoprecipitation measures the interaction of proteins with specific genomic regions in vivo. It involves the treatment of cells with formaldehyde to crosslink neighboring protein-protein and protein-DNA molecules. Nuclear fractions are isolated, the genomic DNA is sheared, and nuclear lysates are used in immunoprecipitations with an antibody against the protein of interest. After reversal of the crosslinking, the associated DNA fragments are sequenced. Enrichment of specific DNA sequences represents regions on the genome that the protein of interest is associated with in vivo. Electrophoretic mobility shift assay (EMSA) provides a rapid method to study DNA-binding protein interactions in vitro. This relies on the fact that complexes of protein and DNA migrate through a nondenaturing polyacrylamide gel more slowly than free DNA fragments. The protein-DNA complex components are then identified with appropriate antibodies. The EMSA assay was found to be consistent with the LRG assay in chicken hepatoma cells dosed with dioxin-like compounds^[29].

In silico Approaches

In silico homology modeling of the ligand binding domain of the AHR in combination with molecular docking simulations can provide valuable insight into the transactivation-potential of a diverse array of AHR ligands.  Such models have been developed for multiple AHR isoforms and ligands (high/low affinity, endogenous and synthetic, agonists and antagonists), and can accurately predict ligand potency based on their structure and physicochemical properties (Bonati et al 2017; Hirano et al 2015; Sovadinova et al 2006).

References

1. ↑ ^{1.0 1.1} Okey, A. B. (2007). An aryl hydrocarbon receptor odyssey to the shores of toxicology: the Deichmann Lecture, International Congress of Toxicology-XI. Toxicol.Sci. 98, 5-38.
2. ↑ Hoffman, E. C., Reyes, H., Chu, F. F., Sander, F., Conley, L. H., Brooks, B. A., and Hankinson, O. (1991). Cloning of a factor required for activity of the Ah (dioxin) receptor. Science 252, 954-958.
3. ↑ ^{3.0 3.1} Poland, A., Glover, E., and Kende, A. S. (1976). Stereospecific, high affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin by hepatic cytosol. Evidence that the binding species is receptor for induction of aryl hydrocarbon hydroxylase. J.Biol.Chem. 251, 4936-4946.
4. ↑ ^{4.0 4.1} Gu, Y. Z., Hogenesch, J. B., and Bradfield, C. A. (2000). The PAS superfamily: sensors of environmental and developmental signals. Annu.Rev.Pharmacol.Toxicol. 40, 519-561.
5. ↑ Kewley, R. J., Whitelaw, M. L., and Chapman-Smith, A. (2004). The mammalian basic helix-loop-helix/PAS family of transcriptional regulators. Int.J.Biochem.Cell Biol. 36, 189-204.
6. ↑ ^{6.0 6.1 6.2 6.3} Fujii-Kuriyama, Y., and Kawajiri, K. (2010). Molecular mechanisms of the physiological functions of the aryl hydrocarbon (dioxin) receptor, a multifunctional regulator that senses and responds to environmental stimuli. Proc.Jpn.Acad.Ser.B Phys.Biol.Sci. 86, 40-53.
7. ↑ ^{7.0 7.1} Mimura, J., and Fujii-Kuriyama, Y. (2003). Functional role of AhR in the expression of toxic effects by TCDD. Biochimica et Biophysica Acta - General Subjects 1619, 263-268.
8. ↑ Giesy, J. P., Kannan, K., Blankenship, A. L., Jones, P. D., and Newsted, J. L. (2006). Toxicology of PCBs and related compounds. In Endocrine Disruption Biological Bases for Health Effects in Wildlife and Humans (D. O. Norris, and J. A. Carr, Eds.), pp. 245-331. Oxford University Press, New York.
9. ↑ ^{9.0 9.1 9.2} Safe, S. (1994). Polychlorinated biphenyls (PCBs): Environmental impact, biochemical and toxic responses, and implications for risk assessment. Critical Reviews in Toxicology 24, 87-149.
10. ↑ ^{10.0 10.1 10.2} Yasui, T., Kim, E. Y., Iwata, H., Franks, D. G., Karchner, S. I., Hahn, M. E., and Tanabe, S. (2007). Functional characterization and evolutionary history of two aryl hydrocarbon receptor isoforms (AhR1 and AhR2) from avian species. Toxicol.Sci. 99, 101-117.
11. ↑ ^{11.0 11.1} Lee, J. S., Kim, E. Y., and Iwata, H. (2009). Dioxin activation of CYP1A5 promoter/enhancer regions from two avian species, common cormorant (Phalacrocorax carbo) and chicken (Gallus gallus): association with aryl hydrocarbon receptor 1 and 2 isoforms. Toxicol.Appl.Pharmacol. 234, 1-13.
12. ↑ ^{12.0 12.1 12.2 12.3 12.4 12.5} Raucy, J. L., and Lasker, J. M. (2010). Current in vitro high throughput screening approaches to assess nuclear receptor activation. Curr. Drug Metab 11 (9), 806-814.
13. ↑ ^{13.0 13.1 13.2} Farmahin, R., Wu, D., Crump, D., Hervé, J. C., Jones, S. P., Hahn, M. E., Karchner, S. I., Giesy, J. P., Bursian, S. J., Zwiernik, M. J., and Kennedy, S. W. (2012). Sequence and in vitro function of chicken, ring-necked pheasant, and Japanese quail AHR1 predict in vivo sensitivity to dioxins. Environ.Sci.Technol. 46, 2967-2975.
14. ↑ ^{14.0 14.1 14.2 14.3} Farmahin, R., Manning, G. E., Crump, D., Wu, D., Mundy, L. J., Jones, S. P., Hahn, M. E., Karchner, S. I., Giesy, J. P., Bursian, S. J., Zwiernik, M. J., Fredricks, T. B., and Kennedy, S. W. (2013b). Amino acid sequence of the ligand binding domain of the aryl hydrocarbon receptor 1 (AHR1) predicts sensitivity of wild birds to effects of dioxin-like compounds. Toxicol.Sci. 131, 139-152.
15. ↑ Fujisawa, N., Ikenaka, Y., Kim, E. Y., Lee, J. S., Iwata, H., and Ishizuka, M. (2012). Molecular evidence predicts aryl hydrocarbon receptor ligand insensitivity in the peregrine falcon (Falco peregrines). European Journal of Wildlife Research 58, 167-175.
16. ↑ ^{16.0 16.1 16.2} Manning, G. E., Farmahin, R., Crump, D., Jones, S. P., Klein, J., Konstantinov, A., Potter, D., and Kennedy, S. W. (2012). A luciferase reporter gene assay and aryl hydrocarbon receptor 1 genotype predict the embryolethality of polychlorinated biphenyls in avian species. Toxicol.Appl.Pharmacol. 263, 390-399.
17. ↑ Mol, T. L., Kim, E. Y., Ishibashi, H., and Iwata, H. (2012). In vitro transactivation potencies of black-footed albatross (Phoebastria nigripes) AHR1 and AHR2 by dioxins to predict CYP1A expression in the wild population. Environ.Sci.Technol. 46, 525-533.
18. ↑ Yueh, M. F., Kawahara, M., and Raucy, J. (2005). Cell-based high-throughput bioassays to assess induction and inhibition of CYP1A enzymes. Toxicol. In Vitro 19 (2), 275-287.
19. ↑ ^{19.0 19.1} Poland, A., and Knutson, J. C. (1982). 2,3,7,8-tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. Annu. Rev. Pharmacol. Toxicol. 22, 517-554.
20. ↑ ^{20.0 20.1} Hestermann, E. V., Stegeman, J. J., and Hahn, M. E. (2000). Relative contributions of affinity and intrinsic efficacy to aryl hydrocarbon receptor ligand potency. Toxicol. Appl. Pharmacol 168 (2), 160-172.
21. ↑ ^{21.0 21.1 21.2 21.3 21.4} Farmahin, R., Jones, S. P., Crump, D., Hahn, M. E., Giesy, J. P., Zwiernik, M. J., Bursian, S. J., and Kennedy, S. W. (2014). Species-specific relative AHR1 binding affinities of 2,3,4,7,8-pentachlorodibenzofuran explain avian species differences in its relative potency. Comp Biochem. Physiol C. Toxicol. Pharmacol. 161C, 21-25.
22. ↑ ^{22.0 22.1 22.2 22.3 22.4 22.5 22.6} Karchner, S. I., Franks, D. G., Kennedy, S. W., and Hahn, M. E. (2006). The molecular basis for differential dioxin sensitivity in birds: Role of the aryl hydrocarbon receptor. Proc. Natl. Acad. Sci. U. S. A 103 (16), 6252-6257.
23. ↑ Lee, S., Shin, W. H., Hong, S., Kang, H., Jung, D., Yim, U. H., Shim, W. J., Khim, J. S., Seok, C., Giesy, J. P., and Choi, K. (2015). Measured and predicted affinities of binding and relative potencies to activate the AhR of PAHs and their alkylated analogues. Chemosphere 139, 23-29.
24. ↑ Jones, S. A., Parks, D. J., and Kliewer, S. A. (2003). Cell-free ligand binding assays for nuclear receptors. Methods Enzymol. 364, 53-71.
25. ↑ Gasiewicz, T. A., and Neal, R. A. (1982). The examination and quantitation of tissue cytosolic receptors for 2,3,7,8-tetrachlorodibenzo-p-dioxin using hydroxylapatite. Anal. Biochem. 124 (1), 1-11.
26. ↑ Nakai, J. S., and Bunce, N. J. (1995). Characterization of the Ah receptor from human placental tissue. J Biochem. Toxicol. 10 (3), 151-159.
27. ↑ Dold, K. M., and Greenlee, W. F. (1990). Filtration assay for quantitation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) specific binding to whole cells in culture. Anal. Biochem. 184 (1), 67-73.
28. ↑ Perez-Romero, P., and Imperiale, M. J. (2007). Assaying protein-DNA interactions in vivo and in vitro using chromatin immunoprecipitation and electrophoretic mobility shift assays. Methods Mol. Med. 131, 123-139.
29. ↑ Heid, S. E., Walker, M. K., and Swanson, H. I. (2001). Correlation of cardiotoxicity mediated by halogenated aromatic hydrocarbons to aryl hydrocarbon receptor activation. Toxicol. Sci 61 (1), 187-196.
30. ↑ ^{30.0 30.1} Ema, M., Ohe, N., Suzuki, M., Mimura, J., Sogawa, K., Ikawa, S., and Fujii-Kuriyama, Y. (1994). Dioxin binding activities of polymorphic forms of mouse and human arylhydrocarbon receptors. J.Biol.Chem. 269, 27337-27343.
31. ↑ ^{31.0 31.1} Poland, A., Palen, D., and Glover, E. (1994). Analysis of the four alleles of the murine aryl hydrocarbon receptor. Mol.Pharmacol. 46, 915-921.
32. ↑ Backlund, M., and Ingelman-Sundberg, M. (2004). Different structural requirements of the ligand binding domain of the aryl hydrocarbon receptor for high- and low-affinity ligand binding and receptor activation. Mol.Pharmacol. 65, 416-425.
33. ↑ Murray, I. A., Reen, R. K., Leathery, N., Ramadoss, P., Bonati, L., Gonzalez, F. J., Peters, J. M., and Perdew, G. H. (2005). Evidence that ligand binding is a key determinant of Ah receptor-mediated transcriptional activity. Arch.Biochem.Biophys. 442, 59-71.
34. ↑ Pandini, A., Denison, M. S., Song, Y., Soshilov, A. A., and Bonati, L. (2007). Structural and functional characterization of the aryl hydrocarbon receptor ligand binding domain by homology modeling and mutational analysis. Biochemistry 46, 696-708.
35. ↑ ^{35.0 35.1 35.2} Pandini, A., Soshilov, A. A., Song, Y., Zhao, J., Bonati, L., and Denison, M. S. (2009). Detection of the TCDD binding-fingerprint within the Ah receptor ligand binding domain by structurally driven mutagenesis and functional analysis. Biochemistry 48, 5972-5983.
36. ↑ Ramadoss, P., and Perdew, G. H. (2004). Use of 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin as a probe to determine the relative ligand affinity of human versus mouse aryl hydrocarbon receptor in cultured cells. Mol.Pharmacol. 66, 129-136.
37. ↑ ^{37.0 37.1 37.2} Head, J. A., Hahn, M. E., and Kennedy, S. W. (2008). Key amino acids in the aryl hydrocarbon receptor predict dioxin sensitivity in avian species. Environ.Sci.Technol. 42, 7535-7541.
38. ↑ ^{38.0 38.1} Denison, M. S., Soshilov, A. A., He, G., DeGroot, D. E., and Zhao, B. (2011). Exactly the same but different: promiscuity and diversity in the molecular mechanisms of action of the aryl hydrocarbon (dioxin) receptor. Toxicol.Sci. 124, 1-22.
39. ↑ van den Berg, M., Birnbaum, L. S., Bosveld, A. T., Brunström, B., Cook, P., Feeley, M., Giesy, J. P., Hanberg, A., Hasegawa, R., Kennedy, S. W., Kubiak, T. J., Larsen, J. C., Van Leeuwen, F. X. R., Liem, A. K. D., Nolt, C., Peterson, R. E., Poellinger, L., Safe, S., Schrenk, D., Tillitt, D. E., Tysklind, M., Younes, M., Wærn, F., and Zacharewski, T. R. (1998). Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife. Environ.Health Perspect. 106, 775-792.
40. ↑ Cohen-Barnhouse, A. M., Zwiernik, M. J., Link, J. E., Fitzgerald, S. D., Kennedy, S. W., Hervé, J. C., Giesy, J. P., Wiseman, S. B., Yang, Y., Jones, P. D., Wan, Y., Collins, B., Newsted, J. L., Kay, D. P., and Bursian, S. J. (2011b). Sensitivity of Japanese quail (Coturnix japonica), Common pheasant (Phasianus colchicus), and White Leghorn chicken (Gallus gallus domesticus) embryos to in ovo exposure to TCDD, PeCDF, and TCDF. Toxicol.Sci. 119, 93-103.
41. ↑ Farmahin, R., Crump, D., Jones, S. P., Mundy, L. J., and Kennedy, S. W. (2013a). Cytochrome P4501A induction in primary cultures of embryonic European starling hepatocytes exposed to TCDD, PeCDF and TCDF. Ecotoxicology 22(4), 731-739.
42. ↑ Hervé, J. C., Crump, D., Jones, S. P., Mundy, L. J., Giesy, J. P., Zwiernik, M. J., Bursian, S. J., Jones, P. D., Wiseman, S. B., Wan, Y., and Kennedy, S. W. (2010a). Cytochrome P4501A induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin and two chlorinated dibenzofurans in primary hepatocyte cultures of three avian species. Toxicol. Sci. 113(2), 380-391.
43. ↑ Hervé, J. C., Crump, D. L., McLaren, K. K., Giesy, J. P., Zwiernik, M. J., Bursian, S. J., and Kennedy, S. W. (2010b). 2,3,4,7,8-pentachlorodibenzofuran is a more potent cytochrome P4501A inducer than 2,3,7,8-tetrachlorodibenzo-p-dioxin in herring gull hepatocyte cultures. Environ. Toxicol. Chem. 29(9), 2088-2095.
44. ↑ Poland, A., and Glover, E. (1973). Studies on the mechanism of toxicity of the chlorinated dibenzo-p-dioxins. Environ.Health Perspect. 5, 245-251.
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46. ↑ ^{46.0 46.1} Omiecinski, C. J., Vanden Heuvel, J. P., Perdew, G. H., and Peters, J. M. (2011). Xenobiotic metabolism, disposition, and regulation by receptors: from biochemical phenomenon to predictors of major toxicities. Toxicol.Sci. 120 Suppl 1, S49-S75.
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48. ↑ Diani-Moore, S., Ma, Y., Labitzke, E., Tao, H., David, W. J., Anderson, J., Chen, Q., Gross, S. S., and Rifkind, A. B. (2011). Discovery and biological characterization of 1-(1H-indol-3-yl)-9H-pyrido[3,4-b]indole as an aryl hydrocarbon receptor activator generated by photoactivation of tryptophan by sunlight. Chem. Biol. Interact. 193(2), 119-128.
49. ↑ Wincent, E., Bengtsson, J., Mohammadi, B. A., Alsberg, T., Luecke, S., Rannug, U., and Rannug, A. (2012). Inhibition of cytochrome P4501-dependent clearance of the endogenous agonist FICZ as a mechanism for activation of the aryl hydrocarbon receptor. Proc. Natl. Acad. Sci. U. S. A 109(12), 4479-4484.
50. ↑ Baba, T., Mimura, J., Nakamura, N., Harada, N., Yamamoto, M., Morohashi, K., and Fujii-Kuriyama, Y. (2005). Intrinsic function of the aryl hydrocarbon (dioxin) receptor as a key factor in female reproduction. Mol.Cell Biol. 25, 10040-10051.
51. ↑ Fernandez-Salguero, P. M., Pineau, T., Hilbert, D. M., McPhail, T., Lee, S. S., Kimura, S., Nebert, D. W., Rudikoff, S., Ward, J. M., and Gonzalez, F. J. (1995). Immune system impairment and hepatic fibrosis in mice lacking the dioxin-binding Ah receptor. Science 268, 722-726.
52. ↑ Ichihara, S., Yamada, Y., Ichihara, G., Nakajima, T., Li, P., Kondo, T., Gonzalez, F. J., and Murohara, T. (2007). A role for the aryl hydrocarbon receptor in regulation of ischemia-induced angiogenesis. Arterioscler.Thromb.Vasc.Biol. 27, 1297-1304.
53. ↑ Lahvis, G. P., Lindell, S. L., Thomas, R. S., McCuskey, R. S., Murphy, C., Glover, E., Bentz, M., Southard, J., and Bradfield, C. A. (2000). Portosystemic shunting and persistent fetal vascular structures in aryl hydrocarbon receptor-deficient mice. Proc.Natl.Acad.Sci U.S.A 97, 10442-10447.
54. ↑ Mimura, J., Yamashita, K., Nakamura, K., Morita, M., Takagi, T. N., Nakao, K., Ema, M., Sogawa, K., Yasuda, M., Katsuki, M., and Fujii-Kuriyama, Y. (1997). Loss of teratogenic response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice lacking the Ah (dioxin) receptor. Genes Cells 2, 645-654.
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List of Key Events in the AOP

Event: 944: dimerization, AHR/ARNT

Short Name: dimerization, AHR/ARNT

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Taxonomic Presence of ARNT genes:

• ARNTs have been identified in all tetrapods investigated to date (Drutel et al 1996; Hirose et al 1996; Hoffman et al 1991; Lee et al 2007; Lee et al 2011).
• ARNTs have been identified in a great phylogenetic diversity of fishes, including early fishes (Doering et al 2014; 2016).
• ARNT has been identified in investigated invertebrates (Powell-Coffman et al 1998).

Taxonomic Applicability of Heterodimerization of ARNT isoforms with AhR isoforms:

•  In mouse (Mus mus) and chicken (Gallus gallus) both the ARNT1 and ARNT2 were capable of heterdimerizing with AHR and interacting with dioxin-responsive elements on the DNA in vitro (Hirose et al 1996; Lee et al 2007; Lee et al 2011; Prasch et al 2004). However, no studies have yet confirmed involvement of both ARNT1 and ARNT2 in vivo.
• In zebrafish, all adverse effects of DLCs so far examined in vivo are mediated solely by ARNT1 based on knockdown studies, although ARNT2 is capable of heterodimerizing with AHR2 and interacting with dioxin-responsive elements on the DNA in vitro (Prasch et al 2004; Prasch et al 2006). In addition to AHRs of zebrafish, AHRs of Atlantic salmon (Salmo salar), Atlantic tomcod (Microgadus tomcod), mummichog, rainbow trout, and red seabream (Pagrus major) have been demonstrated to heterodimerize with ARNT1 in vitro (Abnet et al 1999; Bak et al 2013; Hansson & Hahn 2008; Karchner et al 1999; Wirgin et al 2011), while AHRs of white sturgeon (Acipenser transmontanus), and lake sturgeon (Acipenser fulvescens) have been demonstrated to heterodimerize with ARNT2 in vitro (Doering et al 2014b; 2015b; Prasch et al 2004; 2006). 

This mechanism is conserved across species. Mammals possess a single AHR, whereas birds and fish express multiple isoforms, and all three express multiple ARNT isoforms. Not all of the isoforms identified are functionally active. For example, killifish AHR1 and AHR2 are active and display different transcription profiles, whereas zebrafish AHR2 and ARNT2 are active in mediating xenobiotic-mediated toxicity and AHR1 is inactive (Hahn et al. 2006; Prasch et al. 2006).

Key Event Description

Structure and Function of ARNT

• The aryl hydrocarbon receptor nuclear translocator (ARNT) is a member of the Per-Arnt-Sim (PAS) family of proteins (Gu et al 2000).
• PAS proteins share highly conserved PAS domains (Gu et al 2000).
• PAS proteins act as transcriptional regulators in response to environmental and physiological cues (Gu et al 2000).
• ARNTs have numerous key roles in vertebrates related to responses to developmental and environmental cues.

Isoforms of ARNT:

• Over time ARNT has undergone gene duplication and diversification in vertebrates, which has resulted in three clades of ARNT, namely ARNT1, ARNT2, and ARNT3.
• Each clade can include multiple isoforms and splice variants (Hill et al 2009; Lee et al 2007; Lee et al 2011; Powel & Hahn 2000; Tanguay et al 2000).
• ARNT1s have been demonstrated to function predominantly through heterodimerization with the aryl hydrocarbon receptor (AhR) and hypoxia inducible factor 1 α (HIF1α) (Prasch et al 2004; 2006; Wang et al 1995).
• ARNT2s are believed to function predominantly through heterodimerization with Single Minded (SIM) (Hirose et al 1996).
• ARNT3s, which are also known as ARNT-like (ARNTL), Brain and Muscle ARNT-like-1 (BMAL1), or Morphine Preference 3 (MOP3), are believed to function predominantly through heterodimerization with Circadian Locomotor Output Cycles Kaput (CLOCK) (Gekakis et al 1998).

Roles of ARNTs in mammals:

• ARNT1 functions in normal vascular and hematopoietic development (Kozak et al 1997; Maltepe et al 1997; Abbott & Buckalew 2000).
• ARNT2 functions in development of the hypothalamus and nervous system (Hosoya et al 2001; Keith et al 2001).
• ARNT3 functions in biological rhythms (Gekakis et al 1998).

• Several isoforms of ARNT have recently been identified in mammalian and aquatic species based on their sequence identity to ARNT. They are grouped into four ARNT types that include: ARNT (HIF-1), ARNT2, BMAL1 (ARNT3, MOP3, JAP3, ARNTL1, TIC), and BMAL2 (ARNT4, ARNTL2, MOP9) (Dougherty et al 2010). 

• Coexpression of ARNT with AhR is crucial for steroid synthesis, secretion, and cellular functions during non-pregnancy, pregnancy, and pseudopregnancy. In ovarian follicles, AhR and ARNT are expressed in the follicular epithelia of primordial and growing follicles, contributing to oocyte maturation and follicular development starting from the primary follicle stage. ARNT also regulates ovarian steroid hormones, such as estradiol and progesterone, impacting ovarian physiology. Knockout studies in mice show that ARNT is essential for embryonic development, with embryos failing to survive beyond day 9.5 due to growth retardation, such findings highlight ARNT's vital role in reproduction and development (Hasan et al 2003, Khorram et al 2002).

Roles of ARNTs in other taxa:

• ARNTs have been demonstrated to have roles in development of the heart, brain, liver, and possibly the peripheral nervous system in zebrafish (Danio rerio) (Hill et al 2009).
• Roles of ARNTs in other taxa have not been sufficiently investigated to date.

Interaction with AHR

• Both ARNT1s and ARNT2s are able to heterodimerize with AhR and interact with dioxin-responsive elements on the DNA in in vitro systems (Hirose et al 1996; Lee et al 2007; Lee et al 2011; Prasch et al 2004).
• Selective knockdown of ARNTs in zebrafish (Danio rerio) demonstrates that ARNT1s, but not ARNT2s, are required for activation of the AhR in vivo (Prasch et al 2004; 2006).
• In limited investigations ARNT3 has not been demonstrated to interact with the AHR either in vivo or in vitro (Jain et al 1998). 

Upon ligand binding, the aryl hydrocarbon receptor (AHR) migrates to the nucleus where it dissociates from the cytosolic complex and forms a heterodimer with AHR nuclear translocator (ARNT) (Mimura and Fujii-Kuriyama 2003). The AHR-ARNT complex then binds to a xenobiotic response element (XRE) found in the promoter of an AHR-regulated gene and recruits co-regulators such as CREB binding protein/p300, steroid receptor co-activator (SRC) 1, SRC-2, SRC-3 and nuclear receptor interacting protein 1, leading to induction or repression of gene expression (Fujii-Kuriyama and Kawajiri 2010). Expression levels of several genes, including phase I (e.g. cytochrome P450 (CYP) 1A, CYP1B, CYP2A) and phase II enzymes (e.g. uridine diphosphate glucuronosyl transferase (UDP-GT), glutathione S-transferases (GSTs)), as well as genes involved in cell proliferation (transforming growth factor-beta, interleukin-1 beta), cell cycle regulation (p27, jun-B) and apoptosis (Bax), are regulated through this mechanism (Fujii-Kuriyama and Kawajiri 2010; Giesy et al. 2006; Mimura and Fujii-Kuriyama 2003; Safe 1994).

How it is Measured or Detected

AhR/ARNT heterodimerization can be measured in several ways:

1) The active AHR complexed with ARNT can be measured using protein-DNA interaction assays. Two methods are described in detail by Perez-Romero and Imperiale (Perez-Romero and Imperiale 2007). Chromatin immunoprecipitation measures the interaction of proteins with specific genomic regions in vivo. It involves the treatment of cells with formaldehyde to crosslink neighboring protein-protein and protein-DNA molecules. Nuclear fractions are isolated, the genomic DNA is sheared, and nuclear lysates are used in immunoprecipitations with an antibody against the protein of interest. After reversal of the crosslinking, the associated DNA fragments are sequenced. Enrichment of specific DNA sequences represents regions on the genome that the protein of interest is associated with in vivo. Electrophoretic mobility shift assay (EMSA) provides a rapid method to study DNA-binding protein interactions in vitro. This relies on the fact that complexes of protein and DNA migrate through a non-denaturing polyacrylamide gel more slowly than free DNA fragments. The protein-DNA complex components are then identified with appropriate antibodies. The EMSA assay was found to be consistent with the luciferase reporter gene assay (in chicken hepatoma cells dosed with dioxin-like compounds (Heid et al. 2001).

2) Species-specific differences in dimerization and differences in dimerization between ARNT isoform and AhR isoform combinations have been assessed through luciferase reporter gene (LRG) assays utilizing COS-7 cells transfected with expression constructs of AhR and ARNT isoforms of mammals, birds, and fishes (Abnet et al 1999; Bak et al 2013; Doering et al 2014; 2015; Hansson & Hahn 2008; Hirose et al 1996; Karchner et al 1999; Lee et al 2007; Lee et al 2011; Prasch et al 2004; Wirgin et al 2011). However, this method is indirect as it also includes binding of a ligand to the AhR, and interaction of the AhR/ARNT heterodimer with dioxin-responsive elements on the DNA.

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Prasch, A.L.; Tanguay, R.L.; Mehta, V.; Heideman, W.; Peterson, R.E. (2006). Identification of zebrafish ARNT1 homologs: 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in the developing zebrafish requires ARNT1. Mol. Pharmacol. 69 (3), 776-787.

 

Prasch, A.L.; Teraoka, H.; Carney, S.A.; Dong, W.; Hiraga, T.; Stegeman, J.J.; Heideman, W.; Peterson, R.E. 2003. Toxicol. Sci. Aryl hydrocarbon receptor 2 mediated 2,3,7,8-tetrachlorodibenzo-p-dioxin developmental toxicity in zebrafish. 76 (1), 138-150.

 

Shoots, J.; Fraccalvieri, D.; Franks, D.G.; Denison, M.S.; Hahn, M.E.; Bonati, L.; Powell, W.H. 2015. An aryl hydrocarbon receptor from the salamander Ambystoma mexicanum exhibits low sensitivity to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Enviro. Sci. Technol. 49, 6993-7001.

 

Tanguay, R.L.; Abnett, C.C.; Heideman, W.; Peterson, R.E. 1999. Cloning and characterization of the zebrafish (Danio rerio) aryl hydrocarbon receptor. Biochem. Biophys. Acta. 1444, 35-48.

 

Tanguay, R.L.; Andreasen, E.; Heideman, W.; Peterson, R.E. (2000). Identification and expression of alternatively spliced aryl hydrocarbon nuclear translocator 2 (ARNT2) cDNAs from zebrafish with distinct functions. BBA. 1494 (1-2), 117-128.

 

Van den Berg, M.; Birnbaum, L.; Bosveld, A.T.C.; Brunstrom, B.; Cook, P.; Feeley, M.; Giesy, J.P.; Hanberg, A.; Hasegawa, R.; Kennedy, S.W.; Kubiak, T.; Larsen, J.C.; van Leeuwen, R.X.R.; Liem, A.K.D.; Nolt, C.; Peterson, R.E.; Poellinger, L.; Safe, S.; Schrenk, D.; Tillitt, D.; Tysklind, M.; Younes, M.; Waern, F.; Zacharewski, T. Toxic equivalency factors (TEFs) for PCBs, PCDDs, PECDFs for human and wildlife. Enviro. Hlth. Persp. 1998, 106, 775-792.

 

Van den Berg, M.; Birnbaum, L.S.; Dension, M.; De Vito, M.; Farland, W.; Feeley, M.; Fiedler, H.; Hakansson, H.; Hanberg, A.; Haws, L.; Rose, M.; Safe, S.; Schrenk, D.; Tohyama, C.; Tritscher, A.; Tuomisto, J.; Tysklind, M.; Walker, N.; Peterson, R.E. 2006. The 2005 World Health Organization reevaluation of human and mammalian toxic equivalency factors for dioxins and dioxin-like compounds. Toxicol. Sci. 93 (2), 223-241.

 

Waller, C.L.; McKinney, J.D. (1992). Comparative molecular field analysis of polyhalogenated dibenzo-p-dioixns, dibenzofurans, and biphenyls. J. Med. Chem. 35, 3660-2666.

 

Waller, C.; McKinney, J. (1995). Three-dimensional quantitative structure-activity relationships of dioxins and dioxin-like compounds: model validation and Ah receptor characterization. Chem. Res. Toxicol. 8, 847-858.

 

Wang, G.L., Jiang, B.H., Rue, E.A., Semenza, G.L. 1995. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc. Natl. Acad. Sci. U.S.A. 92, 5510-5514.

 

Whitlock, J.P.; Okino, S.T.; Dong, L.Q.; Ko, H.S.P.; Clarke Katzenberg, R.; Qiang, M.; Li, W. 1996. Induction of cytochrome P4501A1: a model for analyzing mammalian gene transcription. Faseb. J. 10, 809-818.

 

Whyte, J.J.; Jung, R.E.; Schmitt, C.J.; Tillitt, D.E. (2008). Ethoxyresorufin-O-deethylase (EROD) activity in fish as a biomarker of chemical exposure. Crit. Rev. Toxicol. 30 (4), 347-570.

 

Wirgin, I.; Roy, N.K.; Loftus, M.; Chambers, R.C.; Franks, D.G.; Hahn, M.E. 2011. Mechanistic basis of resistance to PCBs in Atlantic tomcod from the Hudson River. Science. 331, 1322-1324.

Jain, S.; Maltepe, E.; Lu, M.M.; Simon, C.; Bradfield, C.A. 1998. Expression of ARNT, ARNT2, HIF1 alpha, HIF2 alpha, and Ah receptor mRNAs in the developing mouse. Mech. Dev. 73, 117-123.

Event: 2021: Increase, slincR expression

Short Name: Increase, slincR expression

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

• slincR was discovered and characterized in developing zebrafish (Garcia et al., 2017; Garcia et al., 2018).

• Additionally, putative mammalian orthologs have also been identified using the slncky Evolution Browser (Garcia et al., 2018). The mouse ortholog was identified from an unpublished RNA sequencing dataset from male and female mouse urogenital epithelial tissue exposed to TCDD using a combination of proximity to the sox9 locus and TCDD-induced gene expression. Only one lncRNA (2610035D17Rik) matched the criteria. The human ortholog (LINC00673) of the mouse lncRNA was identified using slncky. Expression of both the mouse and human lncRNA orthologs from NCBI were consistent with zebrafish slincR expression.

Key Event Description

Descriptions of the KE comes from two studies that discovered and described slincR in zebrafish (Garcia et al., 2017; Garcia et al., 2018).

• The sox9b long intergenic non-coding RNA or slincR is a novel long non-coding RNA (lncRNA) that was recently discovered in developing zebrafish 

• slincR gene expression is dependent on Aryl hydrocarbon receptor (Ahr) activation, with slincR induced up to ~log₂FC=5 in whole-animal zebrafish exposed to the potent Ahr ligand, TCDD. This induction takes place only in the presence of a functional Ahr protein. SlincR is also induced by multiple other Ahr ligands. 

• slincR is located approximately 40,000 bp upstream and antisense of the sox9b gene locus in zebrafish. sox9b is one of the most reduced transcripts in the jaw when zebrafish are exposed to TCDD (Xiong et al., 2008), and is one of two zebrafish paralogs of sox9, 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 (Panda et al., 2021; Lefebvre et al., 2017).

• slincR was found to be enriched in the 5'UTR of the sox9b gene, suggesting possible interactions between slincR and sox9b. A slincR morpholino experiment demonstrated that slincR is required for sox9b repression. 

• Morpholino knockdown of slincR showed slincR's ability to regulate cartilage development, and play a role in TCDD-induced hemorrhaging, both via whole-animal transcriptomics and phenotypic analyses. 

How it is Measured or Detected

slincR gene expression can be measured by quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) and has been measured in embryonic zebrafish at 48 and 96 hours post fertilization (hpf) (Garcia et al., 2017). 

slincR tissue localization of expression can be measured by in situ hybridization and has been measured in embryonic zebrafish at 24, 36, 48, 60, and 72 hpf (Garcia et al., 2017). 

slincR molecular localization can be measured by capture hybridization analysis of RNA targets (CHART) and was measured in 48 hpf zebrafish embryos (Garcia et al., 2018).

References

Garcia GR, Goodale BC, Wiley MW, La Du JK, Hendrix DA, Tanguay RL. 2017. In vivo characterization of an ahr-dependent long noncoding rna required for proper sox9b expression. Mol Pharmacol. 91(6):609-619.

Garcia GR, Shankar P, Dunham CL, Garcia A, La Du JK, Truong L, Tilton SC, Tanguay RL. 2018. Signaling events downstream of ahr activation that contribute to toxic responses: The functional role of an ahr-dependent long noncoding rna (slincr) using the zebrafish model. Environ Health Perspect. 126(11):117002.

Lefebvre V, Dvir-Ginzberg M. 2017. Sox9 and the many facets of its regulation in the chondrocyte lineage. Connect Tissue Res. 58(1):2-14.

Panda M, Tripathi SK, Biswal BK. 2021. Sox9: An emerging driving factor from cancer progression to drug resistance. Biochim Biophys Acta Rev Cancer. 1875(2):188517.

Xiong KM, Peterson RE, Heideman W. 2008. Aryl hydrocarbon receptor-mediated down-regulation of sox9b causes jaw malformation in zebrafish embryos. Mol Pharmacol. 74(6):1544-1553.

Event: 2020: Decrease, sox9 expression

Short Name: Decrease, sox9 expression

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Key Event Description

• The sox family of proteins are a group of highly conserved transcriptional regulators that are present in most groups of animals from invertebrates and unicellular organisms (Phochanukul and Russell 2010) to the more complex vertebrates.

• Sox proteins are characterized by containing the highly conserved high mobility group (HMG) domain, and around 20 different sox proteins have been discovered in mice and humans to date (Jo et al., 2014).

• Sox9, which is part of the soxE subgroup, was initially discovered as the gene underlying campomelic dysplasia (CD), a haplosufficiency disorder characterized by abnormal chondrogenesis, as well as autosomal XY sex reversal from males to females (Wagner et al., 1994).

• Since then, sox9 has been implicated in several functions such as in chondrogenesis, skeletal development, male gonad genesis, development of mesodermal tissues such as cardiac valves and septa, and pyloric sphincter, in ectoderm development (neural stem cells, gliogenesis, and neural stem cells), in hair follicle stem cells, retinal progenitor cells, and the otic placode, and during endoderm development impacting the pancreas, liver, intestine, and lungs. The developmental functions of sox9 have been comprehensively reviewed (Jo et al., 2014; Kawaguchi 2013; Lee and Saint-Jeannet 2011; Lefebvre and Dvir-Ginzberg 2017).

• Several of sox9’s functions are hypothesized to take place as a result of its role as a repressor of the Wnt/B-catenin signaling pathway. Of note, the canonical Wnt signaling pathway promotes chondrocyte differentiation in a sox9-dependent manner (Yano et al., 2005).

• Sox9b (one of two paralogs of the sox9 gene in zebrafish) is one of the most reduced transcripts in the jaw upon TCDD exposure in zebrafish which causes severe lower jaw defects (Xiong et al., 2008), supporting role of sox9’s repression in craniofacial defects.

How it is Measured or Detected

The studies cited in the KE Description used common methods such as RT-qPCR, to measure sox9 expression. It should be noted that the starting level of sox9 expression can be important to determine the magnitude of its repression that is needed to have a significant effect on craniofacial effects (for example), but there still exists a knowledge gap regarding this aspect. 

Event: 317: Altered, Cardiovascular development/function

Short Name: Altered, Cardiovascular development/function

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

• Some form of cardiovascular system is present in members of the clade Bilateria (Bishopric 2005). This clade includes most animal phyla, except for sponges (Porifera), jellyfishes and corals (Cnidaria), placozoans (Placozoa), and comb jellies (Ctenophora).
• Differences in cardiovascular systems are present among taxa. Vertebrates have closed circulatory systems, while some invertebrate taxa have open circulatory systems (Kardong 2006).

Key Event Description

This key event applies to the disruption of cardiogenesis early enough in embryogenesis to result in gross morphological alterations leading to reduced cardiac function.

How it is Measured or Detected

Altered cardiovascular development/function can be measured in numerous ways:

1) As blood flow in the mesencephalic vein by use of time-lapse recording using a digital video camera (Teraoka et al 2008; 2014). Blood flow is measured as the number of red blood cells passing the mesencephalic vein per second (Teraoka et al 2008; 2014). This method is described in detail by Teraoka et al (2002). However, some studies have assessed blood flow through visualized scoring techniques by use of a microscope as (1) same rate as control, (2) slower rate than control, or (3) no flow (Henry et al 1997).

2) As heart area, pericardial edema area, or yolk sac edema area quantified with area analysis by use of a microscope linked digital camera and conventional image software (Dong et al 2010; Teraoka et al 2008; 2014; Yamauchi et al 2006). Images at the same magnification are used to obtain the area measured as number of pixels (Teraoka et al 2008; 2014). This method can use either live individuals or histologic samples. This method is described in detail by Teraoka et al (2003).

3) As basic physical measurements such as heart weight, heart aspect ratio (horizontal length versus vertical length), heart weight to body weight ratio (Fujisawa et al 2014).

4) As incidence of malformation measured as percent occurrence among individuals (Buckler et al 2015; Dong et al 2010; Park et al 2014; Yamauchi et al 2006).This method is described in detail by Dong et al (2010).

5) As heartbeat rate measured by direct observation by use of a microscope (Park et al 2014). This method is described in detail by Park et al (2014).

References

1. Carro, T., Dean, K., and Ottinger, M. A. (2013a). Effects of an environmentally relevant polychlorinated biphenyl (PCB) mixture on embryonic survival and cardiac development in the domestic chicken. Environ. Toxicol. Chem. 23(6), 1325-1331.

2. Carro, T., Taneyhill, L. A., and Ottinger, M. A. (2013b). The effects of an environmentally relevant 58 congener polychlorinated biphenyl (PCB) mixture on cardiac development in the chick embryo. Environ. Toxicol. Chem.

3. DeWitt, J. C., Millsap, D. S., Yeager, R. L., Heise, S. S., Sparks, D. W., and Henshel, D. S. (2006). External heart deformities in passerine birds exposed to environmental mixtures of polychlorinated biphenyls during development. Environ. Toxicol. Chem. 25(2), 541-551.

4. Heid, S. E., Walker, M. K., and Swanson, H. I. (2001). Correlation of cardiotoxicity mediated by halogenated aromatic hydrocarbons to aryl hydrocarbon receptor activation. Toxicol. Sci 61(1), 187-196.

5. Walker, M. K., and Catron, T. F. (2000). Characterization of cardiotoxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related chemicals during early chick embryo development. Toxicol. Appl. Pharmacol. 167(3), 210-221.

6. Walker, M. K., Pollenz, R. S., and Smith, S. M. (1997). Expression of the aryl hydrocarbon receptor (AhR) and AhR nuclear translocator during chick cardiogenesis is consistent with 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced heart defects. Toxicol. Appl. Pharmacol. 143(2), 407-419.

7. Kopf, P. G., and Walker, M. K. (2009). Overview of developmental heart defects by dioxins, PCBs, and pesticides. J. Environ. Sci. Health C. Environ. Carcinog. Ecotoxicol. Rev. 27(4), 276-285.

Bishopric, N.H. (2005). Evolution of the heart from bacteria to man. Ann. N. Y. Acad. Sci. 1047, 13-29.

 

Buckler J.; Candrl, J.S.; McKee, M.J.; Papoulias, D.M.; Tillitt, D.E.; Galat, D.L. Sensitivity of shovelnose sturgeon (Scaphirhynchus platorynchus) and pallid sturgeon (S. albus) early life stages to PCB-126 and 2,3,7,8-TCDD exposure. Enviro. Toxicol. Chem. 2015, 34(6), 1417-1424.

 

Carney, S.A.; Prasch, A.L.; Heideman, W.; Peterson, R.E. 2006. Understanding dioxin developmental toxicity using the zebrafish model. Birth Defects Research. A. 76, 7-18.

 

Cohen-Barnhouse, A.M.; Zwiernik, M.J.; Link, J.E.; Fitzgerald, S.D.; Kennedy, S.W.; Herve, J.C.; Giesy, J.P.; Wiseman, S.; Yang, Y.; Jones, P.D.; Yi, W.; Collins, B.; Newsted, J.L.; Kay, D.; Bursian, S.J. 2011. Sensitivity of Japanese quail (Coturnix japonica), common pheasant (Phasianus colchicus), and white leghorn chicken (Gallus gallus domesticus) embryos to in ovo exposure to TCDD, PeCDF, and TCDF. Toxicol. Sci. 119, 93-102.

 

Elonen, G.E.; Spehar, R.L.; Holcombe, G.W.; Johnson, R.D.; Fernandez, J.D.; Erickson, R.J.; Tietge, J.E.; Cook, P.M. Comparative toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin to seven freshwater fish species during early life-stage development. Enviro. Toxico. Chem. 1998, 17, 472-483.

 

Goldstone, H.M.H.; Stegeman, J.J. (2008). Molecular mechanisms of 2,3,7,8-tetrachlorodibenzo-p-dioxin cardiovascular embryotoxicity. Drug. Metab. Rev. 38, 261-289.

 

Heid, S.E.; Walker, M.K.; Swanson, H.I. (2001). Correlation of cardiotoxicity mediated by halogenated aromatic hydrocarbons to aryl hydrocarbon receptor activation. Toxicol. Sci. 61 (1), 187-196.

 

Huang, L.; Wang, C.; Zhang, Y.; Li, J.; Zhong, Y.; Zhou, Y.; Chen, Y.; Zuo, Z. (2012). Benzo[a]pyrene exposure influences the cardiac development and the expression of cardiovascular relative genes in zebrafish (Daniorerio) embryos. Chemosphere. 87 (4), 369-375.

 

Johnson, R.D.; Tietge, J.E.; Jensen, K.M.; Fernandez, J.D.; Linnum, A.L.; Lothenbach, D.B.; Holcombe, G.W.; Cook, P.M.; Christ, S.A.; Lattier, D.L.; Gordon, D.A. Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin to early life stage brooke trout (Salvelinus fontinalis) following parental dietary exposure. Enviro. Toxicol. Chem. 1998, 17 (12), 2408-2421.

 

Kardong, K.V. (2006). Vertebrates: comparative anatomy, function, evolution. McGraw-Hill Higher Eduction. Boston, USA.

 

Lemly, A.D. (2002). Symptoms and implications of selenium toxicity in fish: the Belews Lake case example. Aquat. Toxicol. 57 (1-2), 39-49.

 

Park, Y.J.; Lee, M.J.; Kim, H.R.; Chung, K.H.; Oh, S.M. Developmental toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in artificially fertilized crucian carp (Carassius auratus) embryo. Sci. Totl. Enviro. 2014, 491-492, 271-278.

Teraoka, H.; Dong, W.; Hiraga, T. (2003). Zebrafish as a novel experimental model for development toxicology. Congenit. Anom. 43, 123-132.

 

Teraoka, T.; Dong, W.; Ogawa, S.; Tsukiyama, S.; Okuhara, Y.; Niiyama, M.; Ueno, N.; Peterson, R.E. (2002). 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in the zebrafish embryo: Altered regional blood flow and impaired lower jaw development. Toxicol. Sci. 65, 192-199.

 

Tillitt, D.E.; Buckler, J.A.; Nicks, D.K.; Candrl, J.S.; Claunch, R.A.; Gale, R.W.; Puglis, H.J.; Little, E.E.; Linbo, T.L.; Baker, M. Sensitivity of lake sturgeon (Acipenser fulvescens) early life stages to 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3,3’,4,4’,5-pentachlorobiphenyl. 2015. Enviro. Toxicol. Chem. DOI: 10.1002/etc.3614.

 

Toomey, B.H.; Bello, S.; Hahn, M.E.; Cantrell, S.; Wright, P.; Tillitt, D.; Di Giulio, R.T. TCDD induces apoptotic cell death and cytochrome P4501A expression in developing Fundulus heteroclitus embryos. Aquat. Toxicol. 2001, 53, 127-138.

 

Walker, M.K.; Spitsbergen, J.M.; Olson, J.R.; Peterson, R.E. 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) toxicity during early life stage development of lake trout (Salvelinus namaycush). Canad. J. Fisheries Aqua. Sci. 1991, 48, 875-883.

 

Yamauchi, M.; Kim, E.Y.; Iwata, H.; Shima, Y.; Tanabe, S. Toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in developing red seabream (Pagrus major) embryos: an association of morphological deformities with AHR1, AHR2 and CYP1A expressions. Aquat. Toxicol. 2006, 16, 166-179.

 

Zabel, E.W; Cook, P.M.; Peterson, R.E. Toxic equivalency factors of polychlorinated dibenzo-p-dioxin, dibenzofuran and biphenyl congeners based on early-life stage mortality in rainbow trout (Oncorhynchus mykiss). Aquat Toxicol. 1995. 31, 315-328.

List of Adverse Outcomes in this AOP

Event: 947: Increase, Early Life Stage Mortality

Short Name: Increase, Early Life Stage Mortality

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

All members of the subphylum vertebrata are susceptible to early life stage death (Weinstein 1999).

Key Event Description

Increased early life stage mortality refers to an increase in the number of individuals dying in an experimental replicate group or in a population over a specific period of time.

In Birds:

Early life stage mortality occurs at any stage in development prior to birth/hatch and is considered embryolethal.

In Fishes:

Early Life Stage Mortality refers to death prior to yolk sac adsorption and swim-up.

How it is Measured or Detected

In birds it may be identified as failure to hatch or lack of movement within the egg when candled; heartbeat monitors are available for identifying viable avian and reptillian eggs (ex. Avitronic's Buddy monitor). In mammals, stillborn or mummified offspring, or an increased rate of resorptions early in pregnancy are all considered embryolethal, and can be detected using ultra-high frequency ultrasound (30-70 MHz; a.k.a. ultrasound biomicroscopy) (Flores et al. 2014). In fishes, mortality is typically measured by observation. Lack of any heart beat, gill movement, and body movement are typical signs of death used in the evaluation of mortality.

Regulatory Significance of the AO

Poor early life stage survival is an endpoint of major relevance to environmental regulators, as it is likely to lead to population decline.  Early-life stage, acute and chronic test guidelines have been established by the Organisation for Economic Co-operation and Development (OECD), U.S. Environmental Protection Agency (EPA) and Environment and Climate Change Canada (ECCC), and are currently used in risk assessments to set limits for safe exposures.  Aquatic test guidlines are most prevalent and include OECD210, OECD229, EPA850.1400 and ECCC  EPS 1/RM/28 for fish and OECD241 for frogs.

References

1. Flores, L.E., Hildebrandt, T.B., Kuhl, A.A., and Drews, B. (2014) Early detection and staging of spontaneous embryo resorption by ultrasound biomicroscopy in murine pregnancy. Reproductive Biology and Endocrinology 12(38). DOI: 10.1186/1477-7827-12-38

2. Weinstein, B. M. (1999). What guides early embryonic blood vessel formation? Dev. Dyn. 215(1), 2-11.

Doering, J.A.; Giesy, J.P.; Wiseman S.; Hecker, M. (2013). Predicting the sensivity of fishes to dioxin-like compounds: possible role of the aryl hydrocarbon receptor (AhR) ligand binding domain. Environmental Science and Pollution Research. 20 (3), 1219-1224.

Appendix 2

List of Key Event Relationships in the AOP