Aryl hydrocarbon receptor activation leading to early life stage mortality via sox9 repression induced impeded craniofacial development

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)

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 craniofacial malformations. 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.

<<<<<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.

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

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

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

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

See details below.

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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