Aop: 150

Title

Each AOP should be given a descriptive title that takes the form “MIE leading to AO”. For example, “Aromatase inhibition [MIE] leading to reproductive dysfunction [AO]” or “Thyroperoxidase inhibition [MIE] leading to decreased cognitive function [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 reduced VEGF

Short name
A short name should also be provided that succinctly summarises the information from the title. This name should not exceed 90 characters. More help
AHR activation to ELS mortality, via VEGF

Graphical Representation

A graphical summary of the AOP listing all the KEs in sequence, including the MIE (if known) and AO, and the pair-wise relationships (links or KERs) between those KEs should be provided. This is easily achieved using the standard box and arrow AOP diagram (see this page for example). The graphical summary is prepared and uploaded by the user (templates are available) and is often included as part of the proposal when AOP development projects are submitted to the OECD AOP Development Workplan. The graphical representation or AOP diagram provides a useful and concise overview of the KEs that are included in the AOP, and the sequence in which they are linked together. This can aid both the process of development, as well as review and use of the AOP (for more information please see page 19 of the Users' Handbook).If you already have a graphical representation of your AOP in electronic format, simple save it in a standard image format (e.g. jpeg, png) then click ‘Choose File’ under the “Graphical Representation” heading, which is part of the Summary of the AOP section, to select the file that you have just edited. Files must be in jpeg, jpg, gif, png, or bmp format. Click ‘Upload’ to upload the file. You should see the AOP page with the image displayed under the “Graphical Representation” heading. To remove a graphical representation file, click 'Remove' and then click 'OK.'  Your graphic should no longer be displayed on the AOP page. If you do not have a graphical representation of your AOP in electronic format, a template is available to assist you.  Under “Summary of the AOP”, under the “Graphical Representation” heading click on the link “Click to download template for graphical representation.” A Powerpoint template file should download via the default download mechanism for your browser. Click to open this file; it contains a Powerpoint template for an AOP diagram and instructions for editing and saving the diagram. Be sure to save the diagram as jpeg, jpg, gif, png, or bmp format. Once the diagram is edited to its final state, upload the image file as described above. More help

Authors

List the name and affiliation information of the individual(s)/organisation(s) that created/developed the AOP. In the context of the OECD AOP Development Workplan, this would typically be the individuals and organisation that submitted an AOP development proposal to the EAGMST. Significant contributors to the AOP should also be listed. A corresponding author with contact information may be provided here. This author does not need an account on the AOP-KB and can be distinct from the point of contact below. The list of authors will be included in any snapshot made from an AOP. More help

Authours: Amani Farhat1, and Sean W. Kennedy2

Environment and Climate Change Canada

Contact Information:

1) Amani_farhat@hotmail.com

2) sean.kennedy.ottawa@gmail.com

Point of Contact

Indicate the point of contact for the AOP-KB entry itself. This person is responsible for managing the AOP entry in the AOP-KB and controls write access to the page by defining the contributors as described below. Clicking on the name will allow any wiki user to correspond with the point of contact via the email address associated with their user profile in the AOP-KB. This person can be the same as the corresponding author listed in the authors section but isn’t required to be. In cases where the individuals are different, the corresponding author would be the appropriate person to contact for scientific issues whereas the point of contact would be the appropriate person to contact about technical issues with the AOP-KB entry itself. Corresponding authors and the point of contact are encouraged to monitor comments on their AOPs and develop or coordinate responses as appropriate.  More help
Arthur Author   (email point of contact)

Contributors

List user names of all  authors contributing to or revising pages in the AOP-KB that are linked to the AOP description. This information is mainly used to control write access to the AOP page and is controlled by the Point of Contact.  More help
  • Amani Farhat
  • Arthur Author

Status

The status section is used to provide AOP-KB users with information concerning how actively the AOP page is being developed, what type of use or input the authors feel comfortable with given the current level of development, and whether it is part of the OECD AOP Development Workplan and has been reviewed and/or endorsed. “Author Status” is an author defined field that is designated by selecting one of several options from a drop-down menu (Table 3). The “Author Status” field should be changed by the point of contact, as appropriate, as AOP development proceeds. See page 22 of the User Handbook for definitions of selection options. More help
Author status OECD status OECD project SAAOP status
Open for citation & comment TFHA/WNT Endorsed 1.7 Included in OECD Work Plan
This AOP was last modified on April 05, 2021 18:16
The date the AOP was last modified is automatically tracked by the AOP-KB. The date modified field can be used to evaluate how actively the page is under development and how recently the version within the AOP-Wiki has been updated compared to any snapshots that were generated. More help

Revision dates for related pages

Page Revision Date/Time
Activation, AhR March 22, 2018 14:00
dimerization, AHR/ARNT September 16, 2017 10:14
reduced dimerization, ARNT/HIF1-alpha March 23, 2018 13:11
Impairment, Endothelial network December 21, 2020 12:09
Altered, Cardiovascular development/function September 16, 2017 10:14
Increase, Early Life Stage Mortality March 22, 2018 10:23
reduced production, VEGF March 28, 2018 11:48
Activation, AhR leads to dimerization, AHR/ARNT March 22, 2018 11:02
dimerization, AHR/ARNT leads to reduced dimerization, ARNT/HIF1-alpha March 23, 2018 13:39
reduced dimerization, ARNT/HIF1-alpha leads to reduced production, VEGF March 23, 2018 13:53
reduced production, VEGF leads to Impairment, Endothelial network March 23, 2018 14:00
Impairment, Endothelial network leads to Altered, Cardiovascular development/function June 09, 2017 10:27
Altered, Cardiovascular development/function leads to Increase, Early Life Stage Mortality March 23, 2018 14:29
Activation, AhR leads to Increase, Early Life Stage Mortality April 14, 2019 15:17
Polychlorinated biphenyl November 29, 2016 18:42
Polychlorinated dibenzodioxins March 09, 2017 20:38
Polychlorinated dibenzofurans November 29, 2016 18:42

Abstract

In the abstract section, authors should provide a concise and informative summation of the AOP under development that can stand-alone from the AOP page. Abstracts should typically be 200-400 words in length (similar to an abstract for a journal article). Suggested content for the abstract includes the following: The background/purpose for initiation of the AOP’s development (if there was a specific intent) A brief description of the MIE, AO, and/or major KEs that define the pathway A short summation of the overall WoE supporting the AOP and identification of major knowledge gaps (if any) If a brief statement about how the AOP may be applied (optional). The aim is to capture the highlights of the AOP and its potential scientific and regulatory relevance More help

Interference with endogenous developmental processes that are regulated by the aryl hydrocarbon receptor (AHR), through sustained exogenous activation, causes molecular, structural, and functional cardiac abnormalities in avian, mammalian and piscine embryos; this cardiotoxicity ultimately leads to severe edema and embryo death in birds and fish and some strains of rat (Carney et al. 2006; Huuskonen et al. 1994; Kopf and Walker 2009). There have been numerous proposed mechanisms of action for this toxicity profile, many of which include the dysregulation of vascular endothelial growth factor (VEGF) as a key event, as it is essential for normal vasculogenesis and therefore cardiogenesis (Ivnitski-Steele and Walker 2005). This AOP describes the indirect suppression of VEGF expression through the sequestration of the aryl hydrocarbon receptor nuclear translocator (ARNT) by AHR. ARNT is common dimerization partner for both AHR and hypoxia inducible factor alpha (HIF-1α), which stimulates angiogenesis through the transcriptional regulation of VEGF (Ivnitski-Steele and Walker 2005).  There is considerable cross talk between these two signaling pathways (AHR and HIF-1α), leading to the hypothesis that AHR activation leads to sustained AHR/ARNT dimerization and reduced HIF-1α/ARNT dimerization, preventing the adequate transcription of essential angiogenic factors, such as VEGF. The suppression of VEGF thereby reduces cardiomyocyte and endothelial cell proliferation, altering cardiovascular morphology and reducing cardiac output, which ultimately leads to congestive heart failure and death (Lanham et al. 2014).

The biological plausibility of this AOP is strong, and there is significant evidence in the literature to support it; however, there exist some contradictory data regarding the effect of AHR on VEGF, which seem highly dependent on tissue type and life stage. There are also multiple targets of AHR activation, such as the COX-2 signaling pathway, that could potentially interact.Thus, other possible AOPs (ex. AOP 21) have also been proposed. These contradictions and alternate pathways are discussed below. The quantitative understanding of individual key even relationships (KERs) in this AOP is weak; however, there is a strong correlation between the molecular initiating event (MIE: AHR activation) and adverse outcome (AO: embryolethality), and a quantitative relationship is described for birds.

Background (optional)

This optional subsection should be 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. Examples of potential uses of the optional background section are listed on pages 24-25 of the User Handbook. More help

In 1957, millions of broiler chickens died due to a mysterious chick edema disease characterized by pericardial, subcutaneous and peritoneal edema (SCHMITTLE et al. 1958). This disease was later ascribed to the ingestion of feed contaminated with halogenated aromatic hydrocarbons (HAHs), including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Higginbotham et al. 1968; Metcalfe 1972). It has since become evident that TCDD is a prototypical agonist of the AHR: a transcription factor that modulates the expression of a vast array of genes involved in endogenous development and physiological responses to exogenous chemicals (Denison et al. 2011).  A general study in the 1980’s found that mothers exposed to herbicides during pregnancy had a 2.8-fold increase in risk of having a baby with congenital cardiovascular malformations (Loffredo et al. 2001). Epidemiological studies have correlated long-term TCDD exposure with ischemic heart disease (Bertazzi et al. 1998; Flesch-Janys et al. 1995); interestingly, and consistent with this AOP, sectioned and stained heart samples from patients with this disease lack epicardial cells (Di et al. 2010). Mammalian studies have confirmed that in utero exposure to TCDD increases susceptibility to cardiovascular dysfunction in adulthood (Aragon et al. 2008; Thackaberry et al. 2005b). The developing heart is highly dependent on oxygen saturation levels; somewhat counterintuitively, a state of hypoxia (relative to adult oxygen tension) drives normal formation and maturation. Deviation from this optimal oxygen level, either above or below normal, hinders myocardial and endothelial development, altering coronary artery connections, ventricle wall thickness and chamber formation (Patterson and Zhang 2010; Wikenheiser et al. 2009). Interestingly, AHR activation (by TCDD), inhibition, and knockdown significantly inhibited the formation of contractile cardiomyocyte nodes during spontaneous differentiation of embryonic stem cells into cardiomyocytes (in vitro) (Wang et al. 2013), indicating that AHR also has an optimal window of expression for normal cardiogenesis. TCDD significantly reduces the degree of myocardial hypoxia that normally occurs during myocyte proliferation and ventricular wall thickening in the developing embryo (Ivnitski-Steele et al. 2004; Lee et al. 2001). This reduction in hypoxia is associated with reduced expression of both HIF-1and the VEGF splice variant, VEGF166 mRNA, which is one of the primary VEGF variants required to mediate coronary vascularization (Ivnitski-Steele et al. 2004). Therefore, it is biologically plausible that sustained AHR activation sequesters ARNT from HIF-1α impairing hypoxia stimulated coronary angiogenesis.

Summary of the AOP

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

Events:

Molecular Initiating Events (MIE)
An MIE is a specialised KE that represents the beginning (point of interaction between a stressor and the biological system) of an AOP. More help
Key Events (KE)
This table summarises all of the KEs of the AOP. This table is populated in the AOP-Wiki as KEs are added to the AOP. Each table entry acts as a link to the individual KE description page.  More help
Adverse Outcomes (AO)
An AO is a specialised KE that represents the end (an adverse outcome of regulatory significance) of an AOP.  More help
Sequence Type Event ID Title Short name
1 MIE 18 Activation, AhR Activation, AhR
2 KE 944 dimerization, AHR/ARNT dimerization, AHR/ARNT
3 KE 945 reduced dimerization, ARNT/HIF1-alpha reduced dimerization, ARNT/HIF1-alpha
4 KE 948 reduced production, VEGF reduced production, VEGF
5 KE 110 Impairment, Endothelial network Impairment, Endothelial network
6 KE 317 Altered, Cardiovascular development/function Altered, Cardiovascular development/function
7 AO 947 Increase, Early Life Stage Mortality Increase, Early Life Stage Mortality

Relationships Between Two Key Events (Including MIEs and AOs)

TESTINGThis table summarises 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.To add a key event relationship click on either Add relationship: events adjacent in sequence or Add relationship: events non-adjacent in sequence.For example, if the intended sequence of KEs for the AOP is [KE1 > KE2 > KE3 > KE4]; relationships between KE1 and KE2; KE2 and KE3; and KE3 and KE4 would be defined using the add relationship: events adjacent in sequence button.  Relationships between KE1 and KE3; KE2 and KE4; or KE1 and KE4, for example, should be created using the add relationship: events non-adjacent button. This helps to both organize the table with regard to which KERs define the main sequence of KEs and those that provide additional supporting evidence and aids computational analysis of AOP networks, where non-adjacent KERs can result in artifacts (see Villeneuve et al. 2018; DOI: 10.1002/etc.4124).After clicking either option, the user will be brought to a new page entitled ‘Add Relationship to AOP.’ To create a new relationship, select an upstream event and a downstream event from the drop down menus. The KER will automatically be designated as either adjacent or non-adjacent depending on the button selected. The fields “Evidence” and “Quantitative understanding” can be selected from the drop-down options at the time of creation of the relationship, or can be added later. See the Users Handbook, page 52 (Assess Evidence Supporting All KERs for guiding questions, etc.).  Click ‘Create [adjacent/non-adjacent] relationship.’  The new relationship should be listed on the AOP page under the heading “Relationships Between Two Key Events (Including MIEs and AOs)”. To edit a key event relationship, click ‘Edit’ next to the name of the relationship you wish to edit. The user will be directed to an Editing Relationship page where they can edit the Evidence, and Quantitative Understanding fields using the drop down menus. Once finished editing, click ‘Update [adjacent/non-adjacent] relationship’ to update these fields and return to the AOP page.To remove a key event relationship to an AOP page, under Summary of the AOP, next to “Relationships Between Two Key Events (Including MIEs and AOs)” click ‘Remove’ The relationship should no longer be listed on the AOP page under the heading “Relationships Between Two Key Events (Including MIEs and AOs)”. More help

Network View

The AOP-Wiki automatically generates a network view of the AOP. This network graphic is based on the information provided in the MIE, KEs, AO, KERs and 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

Stressors

The stressor field is a structured data field that can be used to annotate an AOP with standardised terms identifying stressors known to trigger the MIE/AOP. Most often these are chemical names selected from established chemical ontologies. However, depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). It can also include non-chemical stressors such as genetic or environmental factors. Although AOPs themselves are not chemical or stressor-specific, linking to stressor terms known to be relevant to different AOPs can aid users in searching for AOPs that may be relevant to a given stressor. More help

Life Stage Applicability

Identify the life stage for which the KE is known to be applicable. More help
Life stage Evidence
Embryo 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 in relation to this KE. More help
Term Scientific Term Evidence Link
chicken Gallus gallus High NCBI
zebrafish Danio rerio High NCBI
mouse Mus musculus Low NCBI
Rattus norvegicus Rattus norvegicus Low NCBI

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help
Sex Evidence
Male High
Female High

Overall Assessment of the AOP

This section addresses the relevant biological domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and WoE for the overall AOP as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). The goal of the overall assessment is to provide a high level synthesis and overview of the relative confidence in the AOP and where the significant gaps or weaknesses are (if they exist). Users or readers can drill down into the finer details captured in the KE and KER descriptions, and/or associated summary tables, as appropriate to their needs.Assessment of the AOP is organised into a number of steps. Guidance on pages 59-62 of the User Handbook is available to facilitate assignment of categories of high, moderate, or low confidence for each consideration. While it is not necessary to repeat lengthy text that appears elsewhere in the AOP description (or related KE and KER descriptions), a brief explanation or rationale for the selection of high, moderate, or low confidence should be made. More help

Domain of Applicability

The relevant biological domain(s) of applicability in terms of sex, life-stage, taxa, and other aspects of biological context are defined in this section. Biological domain of applicability is informed by the “Description” and “Biological Domain of Applicability” sections of each KE and KER description (see sections 2G and 3E for details). In essence the taxa/life-stage/sex applicability is defined based on the groups of organisms for which the measurements represented by the KEs can feasibly be measured and the functional and regulatory relationships represented by the KERs are operative.The relevant biological domain of applicability of the AOP as a whole will nearly always be defined based on the most narrowly restricted of its KEs and KERs. For example, if most of the KEs apply to either sex, but one is relevant to females only, the biological domain of applicability of the AOP as a whole would be limited to females. While much of the detail defining the domain of applicability may be found in the individual KE and KER descriptions, the rationale for defining the relevant biological domain of applicability of the overall AOP should be briefly summarised on the AOP page. More help

Life Stage Applicability, Taxonomic Applicability, Sex Applicability Elaborate on the domains of applicability listed in the summary section above. Specifically, provide the literature supporting, or excluding, certain domains.

Life Stage Applicability: Exposure must occur early in embryo development in utero (mammals) or in ovo (birds and fish). Mammalian studies often dose between gestational days 14.5 and 17.5 as it represents a developmental window of cardiomyocyte proliferation (Kopf and Walker 2009). Cardiotoxicity has been observed in birds dosed on day zero or day 5 of incubation (Ivnitski-Steele et al. 2005; Walker et al. 1997). Zebrafish seem to have a particular sensitive window of cardio-development between 48 hours-post-fertilization (hpf) and 5 days pf, and become resistant to AHR-mediated cardiotoxicity if exposed after epicardium formation is complete (2 weeks pf) (Plavicki et al. 2013).

Taxonomic Applicability: Early embryonic exposure to AHR-agonists in mice causes cardiotoxicity that persists into adulthood, increasing susceptibility to heart disease (Thackaberry et al. 2005b) and can increased resorptions and late stage fetal death with edema in certain strains of rat (Huuskonen et al. 1994). AHR-agonists also cause cadivascular malformations in birds and fish, and the resulting reduction in cardiac output is fatal (Kopf and Walker 2009).

Therefore, this AOP is most strongly applicable to birds and fish.  Although strong AHR agonists cause foetal mortality in mice and rats (Kawakami et al. 2005; Hassoun et al. 1997; Sparschu et al. 1970; Debdas Mukerjee 1998), cardiac malformation is rarely cited as a cause of death. It appears that AHR-mediated effects on cardiaovascular development in mammals more frequently lead to long-term functional deficiencies rather than foetal death.   

Sex applicability: Embryonic dysfunction is equally robust in males and females, but adult abnormalities of mice exposed in utero are more prevalent in females (Carreira et al. 2015)

Essentiality of the Key Events

An important aspect of assessing an AOP is evaluating the essentiality of its KEs. 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.When assembling the support for essentiality of the KEs, authors should organise relevant data in a tabular format. The objective is to summarise briefly the nature and numbers of investigations in which the essentiality of KEs has been experimentally explored either directly or indirectly. See pages 50-51 in the User Handbook for further definitions and clarifications.  More help

Molecular Initiating Event Summary, Key Event Summary Provide an overall assessment of the essentiality for the key events in the AOP. Support calls for individual key events can be included in the molecular initiating event, key event, and adverse outcome tables above.

Molecular initiating event: AHR activation (Essentiality = Strong)

  • Zebrafish AHR2 morphants (transient knock-out of function) are protected against reduced blood flow, pericardial edema, erythrocyte maturation, and common cardinal vein migration (Bello et al. 2004; Carney et al. 2004; Prasch et al. 2003; Teraoka et al. 2003)
  • AHR2-/- zebrafish mutants were protected against TCDD toxicity, including pericardial edema and epicardium development (Goodale et al. 2012; Plavicki et al. 2013)
  • AHR activation specifically within cardiomyocytes accounts for heart failure (cardiac malformations, loss of circulation, pericardial edema) induced by TCDD as well as non-cardiac toxicity (swim bladder inflation and craniofacial defects) in zebrafish (Lanham et al. 2014).
  • AHR-null mice have impaired angiogenesis in vivo: endothelial cells failed to branch and form tube-like structures (Roman et al. 2009).
  • Ischemia-induced angiogenesis was markedly enhanced in AHR-null mice compared with that in wild-type animals (Ichihara et al. 2007)

Key Event 1: AHR/ARNT dimerization (Essentiality = Strong)

  • ARNT1 is essential for normal vascular and hematopoietic development (Abbott and Buckalew 2000; Kozak et al. 1997; Maltepe et al. 1997)
  • zfarnt2-/- mutation is larval lethal, and the mutants have enlarged heart ventricles and an increased incidence of cardiac arrhythmia (Hill et al. 2009)
  • ARNT1 morpholono knock-down protected against pericardial edema and reduced blood flow in zebrafish (Prasch et al. 2006)
  • ARNT overexpression rescued cells from the inhibitory effect of hypoxia on AHR-mediated luciferase reporter activity; therefore, the mechanism of interference of the signaling cross-talk between AHR and hypoxia pathways is at least partially dependent on ARNT availability (Vorrink et al. 2014).

Key Event 2: Reduced HIF1α/ARNT dimerization (Essentiality = Moderate)

  • Both ARNT–/– and HIF1α–/– mice display embryonic lethality with blocks in developmental angiogenesis and cardiovascular malformations (Iyer et al. 1998; Kozak et al. 1997; Maltepe et al. 1997; Ryan et al. 1998) demonstrating that signaling through the HIF-1 pathway is required for normal development of the cardiovascular system.
  • The myocardium exhibits a reduced oxygen status during the later stages of coronary vascular development in chick and mouse embryos (Ivnitski-Steele et al. 2004; Lee et al. 2001)
  • Rearing fish embryos in a hypoxic environment can modify cardiac activity, organ perfusion, and blood vessel formation (Pelster 2002)
  • TCDD toxicity in fish resembles defects in hypoxia sensing (Prasch et al. 2004)
  • Deviation in oxygen levels, below or above normal, during early chick embryogenesis results in abnormal coronary vasculature (Wikenheiser et al. 2009)
  • Hypoxia stimulates vasculogenesis and regulates VEGF transcription in vivo and in vitro (Goldberg and Schneider 1994; Levy et al. 1995; Liu et al. 1995) (Goldberg 1994; LEVY 1995A; Liu 1995)
  • Hypoxia stimulus can rescue TCDD inhibition of coronary vascular development in chick embryos (Ivnitski-Steele and Walker 2003)

Key Event 3: Reduced VEGF production (Essentiality = Moderate)

  • Loss of a single VEGF-A allele in mice results in defective vascularization and early embryonic lethality (Carmeliet et al. 1996; Ferrara et al. 1996).
  • Mice lacking VEGF isoforms 164 and 188 exhibit impaired myocardial angiogenesis and reduced contractility leading to ischemic cardiomyopathy (Carmeliet et al. 1999)
  • During vasculogenesis, angioblasts are stimulated to proliferate and differentiate into endothelial cells by VEGF-A (Ivnitski-Steele and Walker 2005)
  • Migration and assembly of epicardial angioblasts into coronary vessels is regulated by VEGF (Folkman 1992)
  • Cardiomyocyte-specific knockout of VEGF in mice results in phenotype similar to TCDD toxicity (thinner ventricular walls, ventricle cavity dilation, and contractile dysfunction) (Giordano et al. 2001; Ivnitski-Steele and Walker 2003)
  • Exogenous VEGF rescues the inhibitory effect of TCDD on vasculogenesis (Ivnitski-Steele and Walker 2003)

Key Event 4: Endothelial network impairment (Essentiality = Moderate)

  • The epicardium is the source of angioblasts, which penetrate into the myocardium, providing the endothelial and mural cell progenitor populations that eventually form the entire coronary vasculature (Viragh et al. 1993; Vrancken Peeters et al. 1999)
  • Sectioned and stained heart samples from patients with ischemic heart disease lack epicardial cells (Di et al. 2010)
  • Juvenile mice with induced cardiovascular disease show altered heart morphology and function, including epithelial dysfunction (Kopf et al. 2008)
  •   In zebrafish, cardiotoxicity coincides with epicardium formation. Cardiotoxicity begins at 48 hours post fertilization (hpf; start of pre-epicardium formation) and starts to decline at 5 days post fertilization, which is about the time the initial epicardial cell layer is complete. Cardiotoxicity disappears at 2 weeks, after epicardium formation is complete. TCDD prevented the formation of the epicardial cell layer when exposed 4hpf, and blocked epicardial expansion from the ventricle to the atrium following exposure at 96hpf. These effects ultimately result in valve malformation, reduced heart size, impaired development of the bulbus arteriosus, decreased cardiac output, reduced end diastolic volume, decreased peripheral blood flow, edema and death (Plavicki et al. 2013).
  • TCDD reduces human primary umbilical vein endothelial cells basal proliferation by 50% (Ivnitski-Steele and Walker 2005)
  • The phenotype observed in chick embryos following TCDD exposure on day zero of incubation resembles that observed in vertebrate models in which the epicardium fails to form (Ivnitski-Steele and Walker 2005)

Key Event 5: Altered cardiovascular development/ function (Essentiality = Strong)

  • The most common cause of infant death due to birth defects is congenital cardiovascular malformation (Kopf and Walker 2009)
  • A significant reduction in embryo survival was observed in 2,3,7,8-tetrachlorodibenzo-p-dioxin  (TCDD) exposed chick embryos, and was associated with heart failure resulting from altered heart morphology:
    • Increased heart width and weight, increased muscle mass, enlarged left ventricle, thinner left ventricle wall, and increased ventricular trabeculation and ventricular septal defects (Walker et al. 1997).
  • Changes in heart morphology consistent with dilated cardiomyopathy (decreased cardiac output and ventricular cavity expansion) were observed in chick embryos exposed to TCDD followed by progression to congestive heart failure edema (Walker and Catron 2000).
  • Changes in heart morphology and decreases in cardiac output and peripheral blood flow precede heart failure in Zebrafish (Antkiewicz et al. 2005; Belair et al. 2001; Henry et al. 1997; Plavicki et al. 2013)

Cardiotoxic effects of strong AHR-agonists

Zebrafish Embryo Chicken Embryo Mouse
  • Reduced extension of common cardinal vein
  • Reduced blood flow
  • Reduced heart rate
  • Disrupted erythropoiesis
  • Decreased heart volume
  • Pericardial edema
  • Overt heart malformations
  • Enlarged left ventricle
  • Increased heart rate
  • Increased myosin content
  • Reduced β-adrenergic responsiveness
  • Increased ANF mRNA
  • Arrhythmia
  • Increased apoptosis
  • Reduced myocyte proliferation
  • Pericardial edema
  • Overt heart malformations
Embryo/Fetus
  • Reduced heart-to-body weight
  • Reduced myocyte proliferation
  • Vascular remodeling

21 Days old

  • Increased heart-to-body weight
  • Increased left ventricle weight
  • Reduced heart rate
  • Cardiac hypertrophy
  • Increased ANF mRNA
  • Increased risk of heart disease

ANF= cardiac atrial natriuretic factor; an indicator of cardiac stress. Source: (Kopf and Walker 2009)

Evidence Assessment

The biological plausibility, empirical support, and quantitative understanding from each KER in an AOP are assessed together.  Biological plausibility of each of the KERs in the AOP is the most influential consideration in assessing WoE or degree of confidence in an overall hypothesised AOP for potential regulatory application (Meek et al., 2014; 2014a). Empirical support entails consideration of experimental data in terms of the associations between KEs – namely dose-response concordance and temporal relationships between and across multiple KEs. It is examined most often in studies of dose-response/incidence and temporal relationships for stressors that impact the pathway. While less influential than biological plausibility of the KERs and essentiality of the KEs, empirical support can increase confidence in the relationships included in an AOP. For clarification on how to rate the given empirical support for a KER, as well as examples, see pages 53- 55 of the User Handbook.  More help

Key Event Relationship

Weight of Evidence

Is there a mechanistic relationship between KEup and KEdown consistent with established biological knowledge?

Support for Biological Plausibility

Strong: Extensive understanding of the KER based on previous documentation and broad acceptance.

Moderate: KER is plausible based on analogy to accepted biological relationships, but scientific understanding is incomplete.

Weak: Empirical support for association between KEs, but the structural or functional relationship between them is not understood.

KER972: Activation, AhR leads to dimerization, AHR/ARNT

Strong

The mechanism of AHR-mediated transcriptional regulation is well understood (Fujii-Kuriyama and Kawajiri 2010). ARNT is a necessary dimerization partner for the transcriptional activation of AHR regulated genes (Hoffman et al. 1991; Poland et al. 1976).

KER973: dimerization, AHR/ARNT leads to reduced dimerization, ARNT/HIF1-alpha

Moderate

ARNT is common dimerization partner for both AHR and HIF-1α. Gel-shift and coimmunoprecipitation experiments have shown that the AHR and HIF1α compete for ARNT in vitro, with approximately equal dimerization efficiencies (Schmidt and Bradfield 1996). A number of studies have shown a reduced response to hypoxia following AHR activation (Chan et al. 1999, Seifert et al. 2008, Ivnitski-Steele et al. 2004), however this effect is highly tissue specific; in cells where ARNT is abundant, it does not deplete due to hypoxia or AHR activation (Chan et al. 1999; Pollenz et al. 1999)

KER974: reduced dimerization, ARNT/HIF1-alpha leads to reduced production, VEGF

Strong

The transcriptional control of VEGF by HIF-1 is well understood; The HIF-1 complex binds to the VEGF gene promoter, recruiting additional transcriptional factors and initiating VEGF transcription (Ahluwalia and Tarnawski 2012; Fong 2009)

KER975: reduced production, VEGF leads to Impairment, Endothelial network

Strong

The importance of VEGF for endothelial network formation and integrity is clear (Ivnitski-Steele and Walker 2005); loss of a single VEGF-A allele results in defective vascularization and early embryonic lethality (Carmeliet et al. 1996; Ferrara et al. 1996).

KER976: Impairment, Endothelial network leads to Altered, Cardiovascular development/function

Moderate

The importance of endothelial cell migration, proliferation and integrity in neovascularization and organogenesis is well documented. Development of vasculature into highly branched conduits needs to occur in numerous sites and in precise patterns to supply oxygen and nutrients to the rapidly expanding tissue of the embryo; aberrant regulation and coordination of angiogenic signals during development result in impaired organ development (Chung and Ferrara 2011; Ivnitski-Steele and Walker 2005). The extent to which the observed cardiovascular abnormalities are caused by deregulation of the underlying endothelial network remains unclear.

KER1567: Altered, Cardiovascular development/function leads to Increase, Early Life Stage Mortality

Strong

The connection between altered cardiovascular developement during embryogenesis, diminished cardiac output and embryonic death have been well studied (Thakur et al. 2013; Kopf and Walker 2009).

KER984: Activation, AhR leads to Increase, Early Life Stage Mortality

Strong

Differences in species sensitivity to dioxin-like compounds have been associated with differences in the AHR amino acid sequence in mammals, fish and birds; the identity of these amino acids in the AHR ligand binding domain affects DLC binding affinity, AHR transactivation and therefore toxicity (Farmahin et al. 2012; Head et al. 2008; Karchner et al. 2006; Mimura and Fujii-Kuriyama 2003; Wirgin et al. 2011). The predictive ability of an LRG assay measuring induction of AHR1-mediated gene expression was demonstrated by linear regression analysis comparing log-transformed LD50 values obtained from the literature to log-transformed PC20 values from the LRG assay (Farmahin et al. 2013; Manning et al. 2012)

Quantitative Understanding

Some proof of concept examples to address the WoE considerations for AOPs quantitatively have recently been developed, based on the rank ordering of the relevant Bradford Hill considerations (i.e., biological plausibility, essentiality and empirical support) (Becker et al., 2017; Becker et al, 2015; Collier et al., 2016). Suggested quantitation of the various elements is expert derived, without collective consideration currently of appropriate reporting templates or formal expert engagement. Though not essential, developers may wish to assign comparative quantitative values to the extent of the supporting data based on the three critical Bradford Hill considerations for AOPs, as a basis to contribute to collective experience.Specific attention is also given to how precisely and accurately one can potentially predict an impact on KEdownstream based on some measurement of KEupstream. This is captured in the form of quantitative understanding calls for each KER. See pages 55-56 of the User Handbook for a review of quantitative understanding for KER's. More help

Summary Table Provide an overall discussion of the quantitative information available for this AOP. Support calls for the individual relationships can be included in the Key Event Relationship table above.

The quantitative understanding of individual KERs in this AOP is weak; however, there is a strong correlation between the molecular initiating event (MIE: AHR activation) and adverse outcome (AO: embryolethality) in birds. This relationship is described in detail in KER984 ( Activation, AhR leads to Increase, Early Life Stage Mortality), found in the KER summary table. In brief, the AHR1 ligand binding domain (LBD) sequence alone could be used to predict DLC-induced embryolethality in a given bird species. The identity amino acids at two key positions within the LBD dictate the binding affinity of xenobiotics and therefore the strength of induction. AHR-mediated reporter gene induction can be measured using a luciferase reporter gene assay, the strength of which is correlated to the embryo-lethal dose of AHR aginists as shown below.

LRG Linear Regression Avian.jpg

Figure 1. Linear regression analysis comparing LD50 values with PC20 (logLD50 = 0.79logPC20 + 0.51) values derived from luciferase reporter gene (LRG) assay concentration-response curves. Open symbols represent LRG data from wild-type chicken, ring-necked pheasant or Japanese quail AHR1 expression vectors. Closed symbols represent LRG data from mutant AHR1 (Source: Manning, G. E. et al. (2012). Toxicol. Appl. Pharmacol. 263(3), 390-399.)

Uncertainties and Inconsistencies

Although crosstalk between AHR and HIF1α clearly exists, the nature of the relationship is still not clearly defined. It has been suggested that HIF1α and AHR do not competitively regulate each other for hetero-dimerization with ARNT, as ARNT is constitutively and abundantly expressed in cells and does not deplete due to hypoxia or AHR activation (Chan et al. 1999; Pollenz et al. 1999). In indirect support of this, a mutant zebrafish model (caAHR-dbd ) expressing an AHR that has lost DNA binding ability, but retains other functional aspects (such as dimerization and translocation) showed no signs of cardiotoxicity; this is in contrast to its counterpart (caAHR) in which sever cardiotoxicity was observed, having the same constitutive AHR expression level (Lanham et al. 2014). These results suggest that direct downstream transcription of AHR-regulated genes, not ARNT sequestration, is essential for cardiotoxicity. However, there is also considerable evidence demonstrating the inhibition of either AHR or HIF1α by activation of the other pathway. For example, TCDD inhibited the CoCl2 induction of a hypoxia response element (HRE) driven promoter and CoCl2 inhibited the TCDD induction of a dioxin response element (DRE) driven promoter, in Hep3B cells (Chan et al. 1999). TCDD also reduced HIF1α nuclear-localized staining in most areas of the heart in chick embryos (Wikenheiser et al. 2012), reduced the stabilization of HIF1α and HRE-mediated promoter activity in Hepa 1 cells and reduced hypoxia-mediated reporter gene activity in B-1 cells (Nie et al. 2001), whereas hypoxia inhibited AHR-mediated CYP1A1 induction in B-1 and Hepa 1 cells, but not H4IIE-luc (Nie et al. 2001). Some studies have shown that the effect of hypoxia on AHR mediated pathways is stronger than the reverse (Gassmann et al. 1997; Gradin et al. 1996; Nie et al. 2001; Prasch et al. 2004), which has been attributed to the stronger binding affinity of HIF1α to ARNT relative to AHR (Gradin et al. 1996). Contrary to this pattern, the combined exposure of juvenile orange spotted grouper to benzo[a]pyrine (BaP; an AHR agonist) and hypoxia, enhanced hypoxia-induced gene expression but did not alter BaP-induced gene expression (Yu et al. 2008). All in all, it appears the effect of cross-talk between AHR and HIF1α is highly dependent on tissue type and life stage, leading to seemingly contradictory results and making it difficult to elucidate a mechanism of action with high confidence.

There is significant evidence suggesting that sustained AHR activation during embryo development results in reduced cardiac VEGF expression (See KER pages for details); however, the opposite relationship has also been observed. In human microvascular endothelial cells, hexachlorobenzene (weak AHR agonist) exposure enhanced VEGF protein expression and secretion. TCDD induced VEGF-A transcription and production in retinal tissue of adult mice and in human retinal pigment epithelial cells (Takeuchi et al. 2009) and induced VEGF secretion from human bronchial epithelial cells (adult) (Tsai et al. 2015). It has been reported that the AHR/ARNT heterodimer binds to estrogen response elements, with mediation of the estrogen receptor (ER), and activates transcription of VEGF-A (Ohtake et al. 2003). The potential involvement of AHR in opposing regulatory cascades (directly inducing VEGF through ER and indirectly suppressing it by ARNT sequestration) helps explain the conflicting results found in the literature. Further complicating the picture is the potential for HIF-1-independent regulation of VEGF, as illustrated in an ARNT-deficient mutant cell line (Hepa1 C4) in which VEGF expression was only partially abrogated (Gassmann et al. 1997).

Alternate Pathways

Altered metabolism of the membrane lipid arachidonic acid (AA) by CYP1A enzymes is another potential mechanism of embryotoxicity. Induction of CYP1A is associated with increased production of AA epoxides that can lead to cytotoxicity and increased susceptibility to injury from oxidative stress due to increased production of oxygen radicals (Toraason et al. 1995). It has been suggested that cyclooxygenase 2 (COX-2) is essential in this toxic response as TCDD-induced morphological defects and edema in the heart were accompanied by COX-2 induction and were prevented with COX-2 inhibitors in fish (Dong et al. 2010; Teraoka et al. 2008). In chick embryos, TCDD-induced mortality, left ventricle enlargement and cardiac stress were prevented by selective COX-2 inhibition (Fujisawa et al. 2014). A non-genomic pathway (ie. ARNT-independent) was suggested as a mechanism for the AHR-mediated induction of COX-2 in which ligand-binding causes a rapid increase in intracellular Ca2+ concentration, activating cytosolic phospholipase A2, inducing COX-2 expression and resulting in an inflammatory response (Matsumura 2009). Interestingly, VEGF-A mRNA was up-regulated 2.7-fold by TCDD and was unaffected by COX-2 inhibition (Fujisawa et al. 2014) Studies investigating the role of CYP1A induction in mediating vascular toxicity have been contradictory, with some studies demonstrating that CYP1A mediates vascular toxicity (Cantrell et al. 1996; Dong et al. 2002; Teraoka et al. 2003), others demonstrating that it does not have an effect (Carney et al. 2004; Hornung et al. 1999), and some showing it to play a protective role (Billiard et al. 2006; Brown et al. 2015). Overall, cardiotoxicity is unlikely a downstream effect of CYP1A induction, but its generation of ROS and therefore oxidative stress likely contributes to the toxicity. Finally, since AHR has a role in heart development that is independent of exogenous ligand-mediated activation, it has been suggested that exogenous AHR ligands sequester it away from its endogenous function (Carreira et al. 2015). Cardiotoxicity may be mediated by Homeobox protein NKX2-5, an essential cardiogenesis transcription factor, as its expression was decreased in AHR-null mice.

Considerations for Potential Applications of the AOP (optional)

At their discretion, the developer may include in this section discussion of the 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. While it is challenging to foresee all potential regulatory application of AOPs and any application will ultimately lie within the purview of regulatory agencies, potential applications may be apparent as the AOP is being developed, particularly if it was initiated with a particular application in mind. This optional section is intended to provide the developer with an opportunity to suggest potential regulatory applications and describe his or her rationale.To edit the “Considerations for Potential Applications of the AOP” section, on an AOP page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Editing AOP.” Scroll down to the “Considerations for Potential Applications of the AOP” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update AOP’ to save your changes and return to the AOP page or 'Update and continue' to continue editing AOP text sections.  The new text should appear under the “Considerations for Potential Applications of the AOP” section on the AOP page. More help

This AOP was developed with the intended purpose of chemical screening as well as ecological risk assessment.  There has recently been significant advances in the understanding of differences in avian sensitivity to AHR agonists, and a similar effort is underway for fish.  Sequencing the AHR ligand binding domain of any bird species (and potentially fish species) allows for its classification as low, medium or high sensitivity, which aids in the chemical risk assessment of DLCs and other AHR agonists.  There is also potential use for this AOP in risk management, as minimum allowable environmental levels can be customized to the sensitivity of the native species in the area under consideration.

References

List the bibliographic references to original papers, books or other documents used to support the AOP. More help

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2. Antkiewicz, D. S., Burns, C. G., Carney, S. A., Peterson, R. E., and Heideman, W. (2005). Heart malformation is an early response to TCDD in embryonic zebrafish. Toxicol. Sci. 84(2), 368-377.

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4. Bello, S. M., Heideman, W., and Peterson, R. E. (2004). 2,3,7,8-Tetrachlorodibenzo-p-dioxin inhibits regression of the common cardinal vein in developing zebrafish. Toxicol. Sci. 78(2), 258-266.

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7. Brown, D. R., Clark, B. W., Garner, L. V., and Di Giulio, R. T. (2015). Zebrafish cardiotoxicity: the effects of CYP1A inhibition and AHR2 knockdown following exposure to weak aryl hydrocarbon receptor agonists. Environ Sci. Pollut. Res. Int. 22(11), 8329-8338.

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