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AOP: 21


A descriptive phrase which references both the Molecular Initiating Event and Adverse Outcome.It should take the form “MIE leading to AO”. For example, “Aromatase inhibition leading to reproductive dysfunction” where Aromatase inhibition is the MIE and reproductive dysfunction the AO. In cases where the MIE is unknown or undefined, the earliest known KE in the chain (i.e., furthest upstream) should be used in lieu of the MIE and it should be made clear that the stated event is a KE and not the MIE.  More help

Aryl hydrocarbon receptor activation leading to early life stage mortality, via increased COX-2

Short name
A name that succinctly summarises the information from the title. This name should not exceed 90 characters. More help
AhR mediated mortality, via COX-2
The current version of the Developer's Handbook will be automatically populated into the Handbook Version field when a new AOP page is created.Authors have the option to switch to a newer (but not older) Handbook version any time thereafter. More help
Handbook Version v1.0

Graphical Representation

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


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

Jon Doering, Ph.D., National Research Council, US EPA Mid-Continent Ecology Division, Duluth, MN, USA, (doering.jonathon[at]

Prof. Markus Hecker, Ph.D. University of Saskatchewan, Saskatoon, Saskatchewan, Canada, (markus.hecker[at]

Dan Villeneuve, Ph.D., US EPA Mid-Continent Ecology Division, Duluth, MN, USA (villeneuve.dan[at]

Prof. Xiaowei Zhang, Ph.D., Nanjing University, School of the Environment, Nanjing, China (Zhangxw[at]

Point of Contact

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


Users with write access to the AOP page.  Entries in this field are controlled by the Point of Contact. More help
  • Markus Hecker
  • Jon Doering
  • Dan Villeneuve


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

OECD Information Table

Provides users with information concerning how actively the AOP page is being developed and whether it is part of the OECD Workplan and has been reviewed and/or endorsed. OECD Project: Assigned upon acceptance onto OECD workplan. This project ID is managed and updated (if needed) by the OECD. OECD Status: For AOPs included on the OECD workplan, ‘OECD status’ tracks the level of review/endorsement of the AOP . This designation is managed and updated by the OECD. Journal-format Article: The OECD is developing co-operation with Scientific Journals for the review and publication of AOPs, via the signature of a Memorandum of Understanding. When the scientific review of an AOP is conducted by these Journals, the journal review panel will review the content of the Wiki. In addition, the Journal may ask the AOP authors to develop a separate manuscript (i.e. Journal Format Article) using a format determined by the Journal for Journal publication. In that case, the journal review panel will be required to review both the Wiki content and the Journal Format Article. The Journal will publish the AOP reviewed through the Journal Format Article. OECD iLibrary published version: OECD iLibrary is the online library of the OECD. The version of the AOP that is published there has been endorsed by the OECD. The purpose of publication on iLibrary is to provide a stable version over time, i.e. the version which has been reviewed and revised based on the outcome of the review. AOPs are viewed as living documents and may continue to evolve on the AOP-Wiki after their OECD endorsement and publication.   More help
OECD Project # OECD Status Reviewer's Reports Journal-format Article OECD iLibrary Published Version
1.27 WPHA/WNT Endorsed Scientific Review iLibrary link
This AOP was last modified on April 29, 2023 16:02

Revision dates for related pages

Page Revision Date/Time
Activation, AhR February 28, 2024 05:12
dimerization, AHR/ARNT September 16, 2017 10:14
Altered, Cardiovascular development/function September 16, 2017 10:14
Increase, COX-2 expression March 20, 2018 16:33
Increase, Early Life Stage Mortality March 22, 2018 10:23
Activation, AhR leads to Increase, Early Life Stage Mortality April 14, 2019 15:17
Activation, AhR leads to dimerization, AHR/ARNT March 22, 2018 11:02
dimerization, AHR/ARNT leads to Increase, COX-2 expression May 09, 2017 16:30
Increase, COX-2 expression leads to Altered, Cardiovascular development/function March 28, 2018 13:24
Altered, Cardiovascular development/function leads to Increase, Early Life Stage Mortality March 23, 2018 14:29
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) February 09, 2017 14:32


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

This adverse outcome pathway details the linkage between activation of the aryl hydrocarbon receptor (AhR) and early life stage mortality in oviparous vertebrates. This AOP can be initiated by a range of planar aromatic hydrocarbons, but is best known as the target of dioxin-like compounds (DLCs). These planar compounds are able to bind to the AhR causing heterodimerization with the aryl hydrocarbon nuclear translocator (ARNT) and interaction with dioxin-responsive elements on the DNA causing an up-regulation in dioxin responsive genes. Hundreds to thousands of genes are regulated, either directly or indirectly, by the AhR. One dioxin-responsive gene is cyclooxygenase 2 (COX-2) which has roles in development of the cardiovascular system. Up-regulation in expression of COX-2 causes alteration in cardiovascular development and function which results in reduced heart pumping efficiency, reduced blood flow, and eventual cardiac collapse and death. Comparable apical manifestations of activation of the AhR have been recorded across freshwater and marine teleost and non-teleost fishes, as well as birds. Therefore, this AOP might be broadly applicable across oviparous vertebrate taxa. Despite conservation in the AOP across taxa, great differences in sensitivity to perturbation exist both among and within taxonomic groups. Therefore, this AOP has utility in support of application toward the mechanistic understanding of adverse effects of chemicals that act as agonists of the AhR, particularly with regard to cross-chemical, cross-species, and cross-taxa extrapolation. 

In general, biological plausibility of this AOP is strong based heavily on evidence collected from zebrafish (Danio rerio) through mechanistic investigations by use of targeted knockdown of AhR, ARNT, or COX-2 and through use of selective agonists and antagonists of COX-2. Quantitative understanding is largely limited to the indirect KER between AhR activation and early life stage mortality.

Activation of the AhR causes pleotropic responses, including interaction with multiple potential target genes such as CYP1A, Sox9b, and HIF1a/VEGF. Therefore, it is a challenge to elucidate the precise series of key events which link activation of the AhR to early life stage mortality. Because of this uncertainty, other AOPs, such as through the HIF1a/VEGF signalling pathway (AOP 150), have also been developed. These other AOPs likely occur simultaneously with COX-2 to cause altered cardiovascular development and function leading to early life stage mortality.

AOP Development Strategy


Used to provide background information for AOP reviewers and users that is considered helpful in understanding the biology underlying the AOP and the motivation for its development.The background should NOT provide an overview of the AOP, its KEs or KERs, which are captured in more detail below. More help
  • The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor in the basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) family of proteins (Okey 2007). The AhR is a highly conserved and ancient protein with homologs having been identified in most major animal groups, apart from the most ancient lineages, such as sponges (Porifera) (Hahn et al 2002).
  • 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; Lahvis and Bradfield 1998; Emmons et al 1999).
  • Activation of the AhR by anthropogenic pollutants that act as agonists can result in a range of adverse biological effects. These effects can include hepatotoxicity, histological lesions, hemopoiesis, suppression of immune responses and healing, impaired reproductive and endocrine processes, teratogenesis, carcinogenesis, wasting syndrome, and mortality (Kleeman et al 1988; Spitsbergen et al 1986; Walter et al 2000; Giesy et al 2002; Spitsbergen et al 1988a; 1988b).
  • Despite the AhR being a highly conserved protein, differences in relative sensitivity to adverse effects both among and within vertebrate taxa are greater than 1000-fold (Cohen-Barnhouse et al 2011; Doering et al 2013; Hengstler et al 1999; Korkalainen et al 2001).
  • Differences in binding affinity and transactivation of the AhR have been implicated as a key mechanism contributing to differences in sensitivity to agonists of the AhR among species and taxa. However, the precise mechanisms are not fully understood for all taxa.
  • High-throughput, next-generation ‘OMICs’ technologies have identified hundreds to thousands of different genes that are regulated, either directly or indirectly, by the AhR (Brinkmann et al 2016; Doering et al 2016; Huang et al 2014; Li et al 2013; Whitehead et al 2010). These genes include Phase I and Phase II biotransformation enzymes, such as cytochrome P450 1A (CYP1A). Expressions and activities of CYP1A are routinely used as biomarkers of exposure to anthropogenic pollutants that act as agonists of the AhR (Whyte et al 2008).
  • One gene which is regulated by AhR is cyclooxygenase-2 (COX-2) which is known to have roles in development of the heart in vertebrates (Dong et al 2010; Teraoka et al 2008; 2014). AhR-mediated dysregulation of COX-2 is associated with altered cardiovascular development, decreased blood flow, and cardiac failure causing mortality in early life stages of fish and birds (Dong et al 2010; Teraoka et al 2008; 2014).
  • Exposure to mixtures of agonists of the AhR during the 1950’s, 1960’s, and 1970’s has been implicated in early life stage mortality of Lake Ontario lake trout (Salvelinus namaycush) leading to population collapse (Cook et al 2003). However, populations of mummichog (Fundulus heteroclitus) and Atlantic tomcod (Microgadus tomcod) exposed to lethal concentrations of agonists of the AhR have evolved tolerance through several mechanisms which has protected against population collapse (Nacci et al 2010; Wirgin et al 2011).


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

Summary of the AOP

This section is for information that describes the overall AOP.The information described in section 1 is entered on the upper portion of an AOP page within the AOP-Wiki. This is where some background information may be provided, the structure of the AOP is described, and the KEs and KERs are listed. More help


Molecular Initiating Events (MIE)
An MIE is a specialised KE that represents the beginning (point of interaction between a prototypical stressor and the biological system) of an AOP. More help
Key Events (KE)
A measurable event within a specific biological level of organisation. More help
Adverse Outcomes (AO)
An AO is a specialized KE that represents the end (an adverse outcome of regulatory significance) of an AOP. More help
Type Event ID Title Short name
MIE 18 Activation, AhR Activation, AhR
KE 944 dimerization, AHR/ARNT dimerization, AHR/ARNT
KE 1269 Increase, COX-2 expression Increase, COX-2 expression
KE 317 Altered, Cardiovascular development/function Altered, Cardiovascular development/function
KE 947 Increase, Early Life Stage Mortality Increase, Early Life Stage Mortality

Relationships Between Two Key Events (Including MIEs and AOs)

This table summarizes all of the KERs of the AOP and is populated in the AOP-Wiki as KERs are added to the AOP.Each table entry acts as a link to the individual KER description page. More help

Network View

This network graphic is automatically generated based on the information provided in the MIE(s), KEs, AO(s), KERs and Weight of Evidence (WoE) summary tables. The width of the edges representing the KERs is determined by its WoE confidence level, with thicker lines representing higher degrees of confidence. This network view also shows which KEs are shared with other AOPs. More help

Prototypical Stressors

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

Life Stage Applicability

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

Taxonomic Applicability

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

Sex Applicability

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

Overall Assessment of the AOP

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

Assessment of WoE calls:

Activation, AhR leads to dimerization, AHR/ARNT: High

Rationale: The call of 'High' is based on overwhelming empirical evidence in numerous species of mammals, birds, amphibians, and fishes. Further, because of overwhelming evidence of essentiality based on targeted knockdown/knockout studies. No uncertainties or inconsistencies are known which affect the WoE call.

Dimerization, AHR/ARNT leads to increase, COX-2 expression: High

Rationale: The call of "High" is based on convincing empirical evidence in three species (two fish and one bird). Further, because of convincing biological plausibility based on identification of dioxin-response elements in the promoter region of COX-2. Uncertainties and inconsistencies are only related to lack of any information on species outside of the three model species that have been investigated.

Increase, COX-2 expression leads to altered, cardiovascular development/function: Moderate

Rationale: The call of "Moderate" is based on overwhelming empirical evidence and evidence of essentiality in three species (two fish and one bird) based on studies using targeted knockdown of genes and selective agonists/antagonists. However, a lack of information on the role of COX-2 in cardiovascular development/function makes biological plausibility questionable at this time. Further, there is some uncertainty associated with pleiotropic effects of AhR activation and the high probability of multiple mechanisms acting concurrently to cause altered cardiovascular development/function.

Altered, cardiovascular development/function leads to increase, early life stage mortality; High

Rationale: The call of "High" is based on overwhelming empirical evidence and biological plausibility in numerous species of mammals, birds, and fish. There are no known uncertainties or inconsistencies at this time.

Activation, AhR leads to increase, early life stage mortality: High

Rationale: The call of "High" is based on overwhelming empirical evidence and evidence of essentiality in numerous species of mammals, birds, amphibians, and fishes using regression analysis and targeted knockdown/knockout of AhR. There are no known uncertainties of inconsistences at this time.

Domain of Applicability

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

Sex: This AOP is only applicable to early life stages prior to sexual differentiation.

Life stages: This AOP is only applicable starting from embryonic development. In zebrafish, this critical window extends from fertilization to approximately 24 hours post fertilization (hpf) (Belair et al 2001; Goldstone & Stegeman 2008).

Taxonomic: The specific characteristics of altered cardiovascular development and function vary to some degree among taxonomic groups of vertebrates.

This AOP is applicable to:

  • All teleost and non-teleost fishes that have been investigated as embryos so far (Buckler et al 2015; Doering et al 2013; Elonen et al 1998; Johnson et al 1998; Park et al 2014; Tillitt et al 2016; Toomey et al 2001; Walker et al 1991; Yamauchi et al 2006; Zabel et al 1995).
  • All birds (Canga et al 1993; Cohen-Barnhouse et al 2011; Fujisawa et al 2014; Heid et al 2001; Ivnitski et al 2001; Walker & Catron 2000). However, some details of the AOP might be different in birds as cyclooxygenase-2 (COX-2) is believed to be up-regulated through non-genomic mechanisms in these taxa based on investigations in chicken (Gallus gallus) (Fujisawa et al 2014).
  • Amphibians and reptiles have insufficient mechanistic and early life stage mortality information to demonstrate applicability at this time. However, amphibians and reptiles express AhRs that are activated by agonists in a manner consistent with other vertebrates and express AhRs during embryonic development (Lavine et al 2005; Shoots et al 2015; Ohi et al 2003; Oka et al 2016). However, altered cardiovascular development and function and early life stage mortality have not been observed at any investigated concentration of DLC in amphibians studied to date (Jung et al 1997). This tolerance is believed to result from AhRs of amphibians having very low affinity for agonists (Lavine et al 2005; Shoots et al 2015). Therefore, it is acknowledged that this AOP is likely to be applicable to reptiles. However, it might not be applicable to amphibians due to their extreme tolerance to activation of the AhR.
  • Cartilaginous fishes (Chondrichthyes) have insufficient mechanistic and early life stage mortality information to demonstrate applicability at this time. However, sharks and rays are known to express AhRs that are structurally comparable to AhRs of teleost fishes (Hahn 2002). Sharks and rays have also been shown to respond to exposure to agonists of the AhR through responses that are comparable to teleost fishes, specifically through induction of CYP1A (Hahn et al 1998). Therefore, it is acknowledged that this AOP is likely to be applicable to Chondrichthyes.

This AOP is not applicable to:

  • Mammals because the cause of mortality of the young is primarily a result of wasting syndrome and not necessarily altered cardiovascular development and function (Kopf & Walker 2009). Further, studies of CYP1A1 and CYP1A2 null mice (Mus musculus) demonstrate that wasting syndrome and mortality are mediated by CYP1A1 in mammals (Uno et al 2004).
  • Invertebrates because AhRs of invertebrates have less diverse functionalities relative to vertebrates, AhRs of most invertebrates likely do not bind agonists that represent anthropogenic pollutants, and no AhR-mediated, critical adverse effects are known in invertebrates as a result of exposure to AhR agonists (Hahn 2002; Hahn et al 1994).
  • Jawless fishes, such as lamprey (Petromyzontiformes) and hagfish (Myxiniformes), because of a lack of measurable AhR-mediated responses (Hahn et al 1998). Although additional information is necessary for this taxa, it is currently acknowledged that this AOP is likely not applicable to jawless fishes.

Essentiality of the Key Events

The essentiality of KEs can only be assessed relative to the impact of manipulation of a given KE (e.g., experimentally blocking or exacerbating the event) on the downstream sequence of KEs defined for the AOP. Consequently, evidence supporting essentiality is assembled on the AOP page, rather than on the independent KE pages that are meant to stand-alone as modular units without reference to other KEs in the sequence. The nature of experimental evidence that is relevant to assessing essentiality relates to the impact on downstream KEs and the AO if upstream KEs are prevented or modified. This includes: Direct evidence: directly measured experimental support that blocking or preventing a KE prevents or impacts downstream KEs in the pathway in the expected fashion. Indirect evidence: evidence that modulation or attenuation in the magnitude of impact on a specific KE (increased effect or decreased effect) is associated with corresponding changes (increases or decreases) in the magnitude or frequency of one or more downstream KEs. More help

Support for essentiality for key events in the AOP was provided by a series of knockdown and targeted agonist and antagonist experiments. These investigations were conducted mainly with zebrafish as the model species and TCDD as the model agonist of the AhR.

 Rationale for essentiality calls:

  • AhR, activation: [Strong] Knockdown of AhR prevents TCDD induced alteration in cardiovascular development and function (Clark et al 2010; Hanno et al 2010; Karchner et al 1999; Prasch et al 2003; Van Tiem & Di Giulio 2011).
  • AhR/ARNT, dimerization: [Strong] Knockdown of ARNT prevents TCDD induced alteration in cardiovascular development and function (Antkiewicz et al 2006; Prasch et al 2004). Depletion of ARNT lessens or prevents TCDD induced alteration in cardiovascular development and function (Prasch et al 2004).
  • COX-2, increase: [Strong] Knockdown of COX-2 and selective antagonists of COX-2 prevent TCDD induced alteration in cardiovascular development and function (Dong et al 2010; Teraoka et al 2008; 2014). COX-2 inducers that are not agonists of the AhR cause altered cardiovascular development and function that is consistent with activation of the AhR (Huang et al 2007). Knockdown of and selective antagonists of thromboxane A synthase 1 (CYP5A), which is down-stream of COX-2 in the prostaglandin synthesis pathway, prevents TCDD induced alteration in cardiovascular development and function (Teraoka et al 2008). Exposure to the substrate for COX-2, arachidonic acid, causes an up-regulation in COX-2 and altered cardiovascular development and function that is consistent with exposure to TCDD (Dong et al 2010).
  • Cardiovascular development and function, altered: [Strong] Isosmotic rearing solution prevents yolk sac edema, but has no effect on TCDD induced alteration in cardiovascular development and function or mortality (Hill et al 2004). This indicates that mortality is not caused by yolk sac edema. Knockdown of cytochrome P450 1A (CYP1A) or injection with antioxidants decreases oxidative stress but has no effect on TCDD induced alteration in cardiovascular development and function or mortality (Carney et al 2004; Scott et al 2011). This is suggestive that mortality is not caused by oxidative stress. Exposure to agonists of the AhR post-heart development lessens or prevents alteration in cardiovascular development, decreased blood flow, and cardiac failure (Carney et al 2004; Lanham et al 2012). Exposure to agonists of the AhR post-heart development dramatically reduces mortality (Carney et al 2004; Lanham et al 2012). This suggests that mortality is caused by circulatory failure as a result of cardiovascular teratogenenesis. Concentrations of DLCs tested in amphibians studied to date were not sufficient to cause altered cardiovascular development or function and no increase in mortality was observed (Jung et al 1997).

Evidence Assessment

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

Biological Plausibility:

  • In general, the biological plausibility and coherence linking activation of the AhR through early life stage mortality from COX-2 induced alteration in cardiovascular development and function is strong.
  • The AhR is known to have critical roles in development of the heart and therefore dysregulation of these roles would be expected to result in altered cardiac development.
  • The prostaglandin synthesis pathway, of which COX-2 is a rate limiting step, is known to have roles in development of the heart (Delgado et al 2004; Gullestad & Aukrust 2005; Hocherl et al 2002; Huang et al 2007; Wong et al 1998; Dong et al 2010; Huang et al 2007; Teraoka et al 2008; 2014).
  • A properly functioning circulatory system is widely acknowledged to be crucial for survival of vertebrates (Kardong 2006). General dysfunction of the heart or associated vasculature is widely documented to have the potential to result in mortality through cardiac failure, regardless of the mechanism of dysfunction.

Concordance of dose-response relationships:

  • There is significant evidence showing concordance of dose-response for incidence and severity of alteration in cardiovascular development and function and subsequently mortality across at least 16 different species of fish (Buckler et al 2015; Elonen et al 1998; Huang et al 2012; Johnson et al 1998; Park et al 2014; Tillitt et al 2016; Toomey et al 2001; Walker et al 1991; Yamauchi et al 2006; Zabel et al 1995) and 8 different species of birds (Cohen-Barnhouse et al 2011; Brunstrom 1990; Brunstrom & Andersson 1988; Hoffman et al 1996; 1998; Powell et al 1998) for several PCDDs, PCDFs, planar PCBs, and PAHs. Concordance of dose-response has not been observed in amphibians studied to date because no elevated mortality or altered cardiovascular development and function was observed at any tested concentration of agonist (Jung et al 1997).
  • Less is known regarding concordance of dose-response relationships for COX-2. In Japanese medaka (Oryzias latipes), abundance of transcript of COX-2 is significantly greater than controls at concentrations of TCDD of 0.2 ppb and greater (Dong et al 2010). Likewise, incidence of cardiovascular development and heart area were both significantly different than controls at concentrations of TCDD of 0.2 ppb and greater (Dong et al 2010).

Temporal concordance among the key events and adverse effect:

  • Alterations in cardiovascular development or function is first observable in zebrafish around 48 hours post fertilization (hpf), while mortality does not begin to occur until around 86 hpf (Goldstone & Stegeman 2006).
  • AhR transcript and protein is first detectable in zebrafish around 24 hpf (Tanguay et al 1999).
  • COX-2 transcript is first detectable in zebrafish around 6 hpf (Teraoka et al 2008). Expression of COX-2 was investigated in zebrafish exposed to TCDD at 55 and 72 hpf (Teraoka et al 2014). At 55 hpf there was a trend towards up-regulation of COX-2 (~ 1.5-fold), while at 72 hpf there was a significant up-regulation of COX-2 (~ 4.5-fold) (Teraoka et al 2014).
  • Therefore, there is a general temporal concordance in this AOP.
  • However, there is some uncertainty in the early manifestations of altered cardiovascular development and up-regulation of COX-2. It is possible that the first manifestations of altered cardiovascular development result from mechanisms other than COX-2. For example, sex determining region Y-box-9b (Sox9b) is first expressed in zebrafish at around 24 hpf and has been known to cause some altered cardiovascular phenotypes (Hofsteen et al 2013; Li et al 2002). However, no studies have yet investigated temporal concordance of regulation of Sox9b by the AhR prior to 72 hpf (Hofsteen et al 2013). It is also possible that temporal concordance of early increases in COX-2 is obscured by the relatively little fold-changes observed for COX-2. Additional investigations into up-regulation of COX-2 by activation of the AhR across developmental stages is warranted.


  • There are no known AhR-mediated effects that occur at concentrations below those that cause alteration in cardiovascular development and function that result in early life stage mortality in fishes, amphibians, reptiles, or birds.
  • There are no studies in which COX-2 and altered cardiovascular development or function were co-investigated in which altered cardiovascular development or function occurred without an up-regulation in COX-2.
  • In TCDD exposure groups, some individuals do not manifest alterations in cardiovascular development or function (Dong et al 2010). TCDD exposed individuals of Japanese medaka that did not manifest alterations in cardiovascular development or function had expression of COX-2 that was not statistically different than controls, while individuals that did manifest alterations in cardiovascular development or function had increased expression of COX-2 (Dong et al 2010).
  • There is also consistency in the TCDD-induced alterations in cardiovascular phenotype between distantly related oviparous taxa, namely fish and birds (Teraoka et al 2008; Fujisawa et al 2014). Likewise, COX-2 is known to be up-regulated in both these taxa (Teraoka et al 2008; Fujisawa et al 2014). Cardiovascular development and function and COX-2 have not been investigated in amphibians or reptiles.

Uncertainties, inconsistencies, and data gaps:

  • There are several other pathways by which activation of the AhR could result in altered cardiovascular development and function in developing embryos. These include, but are not limited to, down-regulation in Sox9b, BMP-4, and genes in the cell cycle gene cluster (Hofsteen et al 2013; Jonsson et al 2007), oxidative stress (Goldstone & Stegeman 2008), and AhR cross-talk with hypoxia inducible factor 1α (HIF1α) causing reduced transcription of vascular endothelial growth factor (VEGF) (AOP 150).
  • Investigations of knockdown and null strains for Sox9b in zebrafish do not result in the complete phenotype of altered cardiovascular development recorded in embryos following exposure to planar aromatic hydrocarbons (Hofsteen et al 2013). Specifically, knockdown or knockout of Sox9b is associated with mild pericardial edema, unlooping, loss of proepicardium, and failure to form epicardium and endocardial cushions, but does not result in typical TCDD-mediated effects of a compacted ventricle or an elongated string-like atrium (Hofsteen et al 2013). Altered cardiovascular development as a result of complete knockout of Sox9b is not severe enough to cause complete cardiac failure and early life stage mortality in zebrafish (Hofsteen et al 2013). Injection of TCDD exposed embryos with Sox9b mRNA was able to prevent the Sox9b phenotype of cardiovascular development, however it did not prevent altered cardiovascular development altogether (Hofsteen et al 2013).Considering, TCDD is only able to decrease expression of Sox9b in the heart by up to about 50% and complete knockout of Sox9b expression is not lethal suggests that Sox9b is not essential to TCDD-mediated alteration in cardiovascular development and function (Hofsteen et al 2013).
  • Oxidative stress as a result of induction in CYP1A has commonly been proposed as the mechanism of altered cardiovascular development and function and CYP1A follows dose- and temporal concordance with mortality across numerous investigations in fishes and birds (Goldstone & Stegeman 2008). Early studies of CYP1A knockdown in zebrafish demonstrated protection against alteration in cardiovascular development and function induced by exposure to TCDD (Teraoka et al 2003). However, more recent investigations have observed no protection (Carney et al 2006). This inconsistency has been proposed to result from the earlier studies only recording alteration in cardiovascular development and function at early stages when adverse effects are difficult to accurately observe (Carney et al 2006). In birds, COX-2 inhibitors have no effect on expression of CYP1A but protect against TCDD induced alteration in cardiovascular development and function suggesting that CYP1A is not involved in toxicities (Fujisawa et al 2014).
  • For cross-species and cross-taxa extrapolation, there is uncertainty in whether COX-2 is up-regulated by AhR through genomic or non-genomic mechanisms. Specifically, there is no detailed analysis regarding how widespread COX-2 genes which contain DREs in the promoter region are among species and among taxa and whether non-genomic or genomic mechanisms of up-regulation in COX-2 are more ubiquitous.
  • All mechanistic investigations into mechanisms of AhR-mediated alteration in cardiovascular development and function have been conducted in zebrafish, Japanese medaka, and chicken. Therefore, no mechanistic information is available to conclude cross-species extrapolation outside of a shared phenotype of altered cardiovascular development and function. There is no information about AhR-mediated alteration in cardiovascular development or function or up-regulation of COX-2 in early fishes (Petromyzontiformes; Myxiniformes; Chondrichthyes), amphibians, or reptiles.
  • Despite these uncertainties, the strong, quantitative link between activation of the AhR and early life stage mortality means that elucidating the precise series of key events is less critical. Therefore, the evidence suggesting COX-2 as a primary mechanism might be all that is necessary, although multiple mechanisms acting together is the most likely true mechanism.

Known Modulating Factors

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

Quantitative Understanding

Optional field to provide quantitative weight of evidence descriptors.  More help
  • The majority of the quantitative understanding and the strongest quantitative understanding is for the indirect relationship between activation of the AhR and early life stage mortality.
  • There is a strong quantitative understanding between quantitative structure-activity relationship (QSAR) or binding affinity for the AhR and potency among PCDDs, PCDFs, and planar PCBs (Van den Berg et al 1998; 2006). Specifically, these studies have demonstrated that congeners with greater binding affinity have greater potency (Van den Berg et al 1998; 2006). This has partially contributed to the successful development of the toxic equivalency factor (TEF) methodology in risk assessment (Van den Berg et al 1998; 2006).
  • There is also a strong quantitative understanding of differences in binding affinity of the AhR among species of birds and differences in sensitivity to early life stage mortality (Karchner et al 2006; Farmahin et al 2012; 2013; Manning et al 2013). Specifically, these studies demonstrate that species of birds with AhRs with greater affinity for DLCs have greater sensitivity than species with AhRs with lesser affinity for DLCs (Karchner et al 2006). These differences in sensitivity range by more than 40-fold for TCDD (Cohen-Barnhouse et al 2011). However, a quantitative understanding between differences in binding affinity of the AhR among species and differences in sensitivity to early life stage mortality is not yet available for other taxa (Doering et al 2013).
  • There is some quantitative understanding between up-regulation of COX-2 and incidence of and severity of cardiac deformities for medakafish exposed to TCDD (Dong et al 2010). This quantitative understanding includes a strong linear relationship (R2 = 0.88) between abundance of COX-2 transcript and heart area (Dong et al 2010). However, this information is not available with regards to multiple investigations, species, taxonomic groups, or chemicals.
  • There is strong quantitative understanding between incidence of and severity of cardiovascular deformities and mortality. However, numerous different cardiovascular endpoints are investigated among studies making side-by-side comparisons difficult.

Considerations for Potential Applications of the AOP (optional)

Addressess potential applications of an AOP to support regulatory decision-making.This may include, for example, possible utility for test guideline development or refinement, development of integrated testing and assessment approaches, development of (Q)SARs / or chemical profilers to facilitate the grouping of chemicals for subsequent read-across, screening level hazard assessments or even risk assessment. More help

There are great differences in sensitivity to agonists of the AhR among species and among taxa and  great differences in potency among agonists of the AhR. Therefore, this AOP has utility towards the mechanistic understanding of adverse effects of agonists of the AhR with regard to cross-chemical, cross-species, and cross-taxa extrapolations. This utility has led to the development of a qAOP that has demonstrated utility in guiding more objective ecological risk assessments of native species to agonists of the AhR, particularly assessments of threatened or endangered species that often cannot be investigated in laboratory toxicity testing (Doering et al 2018).


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

Abnet, C.C.; Tanguay, R.L.; Heideman, W.; Peterson, R.E. 1999. Transactivation activity of human, zebrafish, and rainbow trout aryl hydrocarbon receptors expressed in COS-7 cells: Greater insight into species differences in toxic potency of polychlorinated dibenzo-p-dioxin, dibenzofuran, and biphenyl congeners. Toxicol. Appl. Pharmacol. 159, 41-51.

Antkiewicz, D.S.; Burns, C.G.; Carney, S.A.; Peterson, R.E.; Heideman, W. 2005. Heart malformation is an early response to TCDD in embryonic zebrafish. Toxicol. Sci. 84, 368-377.

Bak, S.M.; Lida, M.; Hirano, M.; Iwata, H.; Kim, E.Y. 2013. Potencies of red seabream AHR1- and AHR2-mediated transactivation by dioxins: implications of both AHRs in dioxin toxicity. Environ. Sci. Technol. 47 (6), 2877-2885.

Belair, C.D.; Peterson, R.E.; Heideman, W. (2001). Disruption of erythropoiesis by dioxin in the zebrafish. Dev. Dyn. 222 (4), 581-594.

Billiard, S.M.; Hahn, M.E.; Franks, D.G.; Peterson, R.E.; Bols, N.C.; Hodson, P.V. (2002). Binding of polycyclic aromatic hydrocarbons (PAHs) to teleost aryl hydrocarbon receptors (AHRs). Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 133 (1), 55-68.

Brunstrom, B. (1990). Mono-ortho-chlorinated chlorobiphenyls: toxicity and induction of 7-ethoxyresorufin O-deethylase (EROD) activity in chick embryos. Arch. Toxicol. 64, 188-192.

Brunstrom, B.; Andersson, L. (1988). Toxicity and 7-ethoxyresorufin O-deethylase-inducing potency of coplanar polychlorinated biphenyls (PCBs) in chick embryos. Arch. Toxicol. 62, 263-266.

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.

Canga, L., Paroli, L., Blanck, T. J., Silver, R. B., and Rifkind, A. B. (1993). 2,3,7,8-Tetrachlorodibenzo-p-dioxin increases cardiac myocyte intracellular calcium and progressively impairs ventricular contractile responses to isoproterenol and to calcium in chick embryo hearts. Mol. Pharmacol.  44, 1142–1151.

Carney, S.A.; Peterson, R.E.; Heideman, W. 2004. 2,3,7,8-tetrachlorodibenzo-p-dioxin activation of aryl hydrocarbon receptors/aryl hydrocarbon receptor nuclear translocator pathway causes developmental toxicity through a CYP1A-independent mechanism in zebrafish. Mol. Pharmacol. 66 (2), 512-521.

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.

Chen, G.; Bunce, N.J. (2003). Polybrominated diphenyl ethers as Ah receptor agonists and antagonists. Toxicol. Sci. 76 (2), 310-320.

Clark, B.W.; Matson, C.W.; Jung, D.; Di Giulio, R.T. 2010. AHR2 mediates cardiac teratogenesis of polycyclic aromatic hydrocarbons and PCB-126 in Atlantic killifish (Fundulus heteroclitus). Aquat. Toxicol. 99, 232-240.

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.

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