Aop:150

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AOP Title

Aryl hydrocarbon receptor activation leading to embryolethality via cardiotoxicty
Short name: AHR activation to embryolethality

Authors

Amani Farhat

Status

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Under development: Do not distribute or cite.

OECD Project 1.7: The Adverse Outcome Pathways for Sustained Activation of the Aryl Hydrocarbon Receptor.

This AOP page was last modified on 12/11/2016.

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Abstract

Interference with endogenous developmental functions of the aryl hydrocarbon receptor (AHR) by sustained exogenous activation causes structural, molecular and functional cardiac abnormalities and altered heart physiology 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 the two nuclear receptors, 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. 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)

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, and other AHR agonists, disrupt the normal development and function of the heart. 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. 2010), 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

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Molecular Initiating Event

Molecular Initiating Event Support for Essentiality
AHR, Activation Strong

Key Events

Event Support for Essentiality
AHR/ARNT, dimerization Strong
ARNT/HIF1-alpha, reduced dimerization Moderate
Endothelial network, Impairment Moderate
Cardiovascular development/function, Altered Strong
Pericardial edema, Increase Strong
VEGF, reduced production Moderate

Adverse Outcome

Adverse Outcome
Embryolethality, Increase

Relationships Among Key Events and the Adverse Outcome

Event Description Triggers Weight of Evidence Quantitative Understanding
AHR, Activation Directly Leads to AHR/ARNT, dimerization Strong Strong
ARNT/HIF1-alpha, reduced dimerization Directly Leads to VEGF, reduced production Strong Moderate
VEGF, reduced production Directly Leads to Endothelial network, Impairment Strong Weak
Endothelial network, Impairment Directly Leads to Cardiovascular development/function, Altered Moderate Weak
Cardiovascular development/function, Altered Directly Leads to Pericardial edema, Increase Moderate Weak
Pericardial edema, Increase Directly Leads to Embryolethality, Increase Moderate Weak
AHR/ARNT, dimerization Directly Leads to ARNT/HIF1-alpha, reduced dimerization Moderate Weak
AHR, Activation Indirectly Leads to Embryolethality, Increase Strong Moderate

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Life Stage Applicability

Life Stage Evidence Links
Embryo Strong

Taxonomic Applicability

Name Scientific Name Evidence Links
chicken Gallus gallus Strong NCBI
zebrafish Danio rerio Strong NCBI
mouse Mus musculus Strong NCBI
Rattus norvegicus NCBI

Sex Applicability

Sex Evidence Links
Male Strong
Female Strong

Graphical Representation

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Overall Assessment of the AOP

AHR cardiotoxicity.png

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). The resulting cardiac malformations and edema in birds and fish are fatal (Kopf and Walker 2009).

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)
    • TCDD prevents the formation and migration of the epicardial cell layer in zebrafish (Plavicki et al. 2013)
  • TCDD exposed chick, zebrafish and mouse embryos have reduced number of cardiac myocytes, which is due to decreased cardiomyocyte proliferation in the chick and mouse (Antkiewicz et al. 2005; Ivnitski et al. 2001; Thackaberry et al. 2005b)
    • Note that myocardial migration is dependent on epithelial integrity (Trinh and Stainier 2004)
  • Endothelial tube length (40%±1.7%) and number (36%±3%) were significantly reduced in TDCC treated chick explants (cell culture derive from treated embryos) (Ivnitski-Steele and Walker 2003)
  • TCDD reduced coronary artery number in chick embryos (by 53±8%) and reduced tube outgrowth and endothelial cell responsiveness to angiogenic stimuli in chick explants (Ivnitski-Steele et al. 2005)
  • 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)
  • Epicardial cells are missing from the surface of human hearts with ischemic cardiomyopathy (Di et al. 2010)

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)
  • The most common heart abnormality observed in nestlings exposed to dioxin-like compounds is thinning of the ventricular wall (Carro et al. 2013); thinning was attributed to reduced cardiomyocyte proliferation in TCDD exposed chick and mouse embryos (Ivnitski et al. 2001; Thackaberry et al. 2005a)
  • TCDD reduces blood flow and circulatory function in essentially all fish species studied, including medaka, lake trout, rainbow trout, brook trout, and zebrafish (Ivnitski-Steele and Walker 2005).

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)

Key Event 6: Increased pericardial edema (Essentiality = Strong)

  • Edema is a hallmark sign of developmental toxicity in fish, chick, and mammalian species exposed to strong AHR agonists early in embryogenesis (Carney et al. 2006)
    • Note that it presents as pericardial and yolk sac edema in fish, pericardial, peritoneal and subcutaneous edema on chicks, and peritoneal and subcutaneous edema in mice.
  • Edema and hemorrhage are common developmental effects among species exposed to AHR-agonists prior to death; however, edema is a secondary effect rather than a primary target, as cardiotoxicity is observed prior to, or even without, the onset of edema (Walker et al. 1997; Walker and Catron 2000).
  • Edema is not observed at sub-lethal doses of TCDD (Walker et al. 1997)

Weight of Evidence Summary

Summary Table
Provide an overall summary of the weight of evidence based on the evaluations of the individual linkages from the Key Event Relationship pages.

Support for Biological Plausibility of KERs Defining Question High (Strong) Moderate Low (Weak)
Is there a mechanistic relationship between KEup and KEdown consistent with established biological knowledge? Extensive understanding of the KER based on previous documentation and broad acceptance. KER is plausible based on analogy to accepted biological relationships, but scientific understanding is incomplete. Empirical support for association between KEs, but the structural or functional relationship between them is not understood.
MIE => KE1: 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).
KE1 => KE2: 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)
KE2 => KE3: 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)
KE3 => KE4: 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).
KE4 => KE5: 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.
KE5 => KE6: Moderate Severe cardiac dysfunction can result in congestive fetal heart failure leading to fluid build-up in tissues and cavities (edema and effusion, respectively)(Thakur et al. 2013). Pericardial edema is secondary to heart failure in zebrafish and chicken embryos as changes in heart morphology and decreases in cardiac output precede congestive heart failure (Henshel et al. 1993; Cheung et al. 1981; Canga et al. 1993; Walker et al. 1997; Antkiewicz et al. 2005; Belair et al. 2001; Henry et al. 1997; Plavicki et al. 2013). Furthermore, when mannitol is used as a protective agent against chemical-induced edema in zebrafish, cardiotoxic effects are still observed (Antkiewicz et al. 2005; Plavicki et al. 2013).
KE6 => AO: Moderate The connection between edema and diminished cardiac output is well understood in fish, birds and mammals (Antkiewicz et al. 2005; Thakur et al. 2013; Walker 1998; Walker et al. 1997). Fluid buildup exerts a positive pressure on fetal cardiac chambers, further limiting the diastolic ventricular filling reserve, potentiating the diminished cardiac output and leading to fetal death (Thakur et al. 2013). Although pericardial edema consistently precedes embryo death in many fish species (Kopf and Walker 2009; Ivnitski-Steele and Walker 2005), the causal linkage is less clear in avian species. A number of studies report heart malformations leading to embryo death without the observation of pericardial edema (Cheung et al. 1981, Walker et al. 1997, Carro et al. 2013, Wikenheiser et al. 2012); in fact, subcutaneous edema is more often reported in chicken ebryos exposed to AHR agonists (Cheung et al. 1981, Brunstrom 1988, Brunstrom and Anderson 1988, Walker and Catron 2000).
MIE => AO: 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 Considerations

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 the KER: AHR activation leads to embryolethality, 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)

References


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8. Cantrell, S. M., Lutz, L. H., Tillitt, D. E., and Hannink, M. (1996). Embryotoxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD): the embryonic vasculature is a physiological target for TCDD-induced DNA damage and apoptotic cell death in Medaka (Orizias latipes). Toxicol. Appl. Pharmacol. 141(1), 23-34.

9. Carmeliet, P., Ferreira, V., Breier, G., Pollefeyt, S., Kieckens, L., Gertsenstein, M., Fahrig, M., Vandenhoeck, A., Harpal, K., Eberhardt, C., Declercq, C., Pawling, J., Moons, L., Collen, D., Risau, W., and Nagy, A. (1996). Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380(6573), 435-439.

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