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Environment and Climate Change Canada
Point of Contact
Amani Farhat (email point of contact)
- Amani Farhat
|Author status||OECD status||OECD project||SAAOP status|
|Open for comment. Do not cite||EAGMST Approved||1.7||Included in OECD Work Plan|
This AOP was last modified on May 03, 2019 13:37
|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||February 13, 2018 15:56|
|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|
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.
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
Events: Molecular Initiating Events (MIE)
|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)
|Activation, AhR leads to dimerization, AHR/ARNT||adjacent||High||Moderate|
|dimerization, AHR/ARNT leads to reduced dimerization, ARNT/HIF1-alpha||adjacent||Moderate||Low|
|reduced dimerization, ARNT/HIF1-alpha leads to reduced production, VEGF||adjacent||Moderate||Moderate|
|reduced production, VEGF leads to Impairment, Endothelial network||adjacent||High||Low|
|Impairment, Endothelial network leads to Altered, Cardiovascular development/function||adjacent||Moderate||Low|
|Altered, Cardiovascular development/function leads to Increase, Early Life Stage Mortality||adjacent||High||Low|
|Activation, AhR leads to Increase, Early Life Stage Mortality||non-adjacent||High||Moderate|
Life Stage Applicability
|Rattus norvegicus||Rattus norvegicus||Low||NCBI|
Overall Assessment of the AOP
Domain of Applicability
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
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|
21 Days old
ANF= cardiac atrial natriuretic factor; an indicator of cardiac stress. Source: (Kopf and Walker 2009)
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
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
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
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
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
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
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
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)
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.
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).
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)
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.
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