Upstream eventActivation, AhR
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding|
|Aryl hydrocarbon receptor activation leading to embryolethality via cardiotoxicity||non-adjacent||High||Moderate|
|Japanese quail||Coturnix japonica||High||NCBI|
|Ring-necked pheasant||Phasianus colchicus||High||NCBI|
|bobwhite quail||Colinus virginianus||High||NCBI|
|American kestrel||Falco sparverius||High||NCBI|
|Double-crested cormorant||Double-crested cormorant||High||NCBI|
|Eastern bluebird||Eastern bluebird||High||NCBI|
Life Stage Applicability
Key Event Relationship Description
Stong aryl hydrocarbon receptor (AHR) agonists that cause sustained AHR activation interfere with the receptor's endogenouse role in embryogenesis; this disruption causes numerouse develpemental abnormalities and ultimetly leads to emryonic death (Kopf and Walker 2009; Carreira et al 2015)
It's inportant to note that his relationship only applies to AHR agonists that cause sustained AHR activation. Strong AHR agonists that are rapidly metabolized, such as polycyclic aromatic hydrocarbons, only cause transient AHR activation leading to an alternate mode of toxicity.
Evidence Supporting this KER
AHR Ligand Binding Domain
- Mammalian and avian sensitivity to DLCs ultimately comes down to the identity of two particular amino acids in the ligand binding domain (LBD) of the AHR: positions 375 and 319 in mice and 380 and 324 in birds.
- A 10-fold difference between two strains of mice (non-responsive DBA/2 mouse, and responsive C57BL/6 14 mouse) in CYP1A induction, lethality and teratogenicity following TCDD exposure (Poland et al. 1976), was attributed to a single nucleotide polymorphism at position 375 (Ema et al. 1994; Poland et al. 1994; Poland and Knutson 1982).
- Several other studies reported the importance of this amino acid in birds and mammals (Backlund and Ingelman-Sundberg 2004; Ema et al. 1994; Karchner et al. 2006; Murray et al. 2005; Pandini et al. 2007; Pandini et al. 2009; Poland et al. 1994; Ramadoss and Perdew 2004).
- The amino acid at position 319 plays an important role in ligand-binding affinity to the AHR and transactivation ability of the AHR, due to its involvement in LBD cavity volume and its steric effect (Pandini et al. 2009).
- Mutation at position 319 in the mouse eliminated AHR DNA binding (Pandini et al. 2009).
Using AHR LBD Constructs to Determine Avian Sensitivity
- Using chimeric AHR1 constructs combining three AHR1 domains (DBD, LBD and TAD) from the chicken (highly sensitive to DLC toxicity) and common tern (resistant to DLC toxicity), Karchner and colleagues (2006), showed that amino acid differences within the LBD were responsible for differences in TCDD sensitivity between the chicken and common tern.
- They specifically attributed positions 324 and 380 with differences in TCDD binding affinity and transactivation between the chicken (Ile324_Ser380) and common tern (Val324_Ala380) receptors.
- The LBD of over 85 bird species have since been analyzed to find that 6 amino acid residues differed among species (Farmahin et al. 2013; Head et al. 2008), but only positions 324 and 380 in the AHR1 LBD were associated with differences in DLC toxicity in ovo and AHR1-mediated gene expression in vitro (Farmahin et al. 2013; Head et al. 2008; Manning et al. 2012).
- Based on these results, avian species can be divided into one of three AHR1 types based on the amino acids found at positions 324 and 380 of the AHR1 LBD: type 1 (Ile324_Ser380; most sensitive), type 2 (Ile324_Ala380; moderately sensitive) and type 3 (Val324_Ala380; least sensitive) (Farmahin et al. 2013; Head et al. 2008; Manning et al. 2012).
- A sampling of bird species and their AHR LBD category is described in table 1. A list of 86 species and their subtype can be found in Farmahin et al. (2013).
Include consideration of temporal concordance here
Binding of dioxin-like compounds (DLCs) to avian AHR1 (Farmahin et al. 2014; Karchner et al. 2006) and AHR1-mediated transactivation measured using luciferase reporter gene (LRG) assays have been demonstrated in domestic and wild species of birds (Farmahin et al. 2012; Farmahin et al. 2013b; Fujisawa et al. 2012; Lee et al. 2009; Manning et al. 2012; Mol et al. 2012), and binding affinity was found to be strongly correlated with embryotoxicity (Manning et al. 2012) .
Uncertainties and Inconsistencies
Interestingly, interference with endogenous AHR functions, either by knock-out or by agonist exposure during early development,
causes similar cardiac abnormalities (Carierra et al 2015). Although this is counterintuitive, it demonstrates that the AHR has an optimal window of activity, and deviation either above or below this range results in toxicity.
Quantitative Understanding of the Linkage
Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships?
The predictive ability of an LRG assay measuring induction of AHR1-mediated gene expression in cells transfected with different avian AHR1 expression vectors 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. 2013b; Manning et al. 2012). PC20 values represent the concentration of DLC that elicited 20% of the TCDD maximal response, and were calculated according to the procedure described in OECD guideline 455 (OECD 2009). LD50 values used in regression analyses were obtained from the literature. As shown in the linear regression analysis (Figure 1), logLD50 values were associated with logPC20 and a significant relationship (R2 = 0.93, p < 0.0001) was observed. Thus, to predict the in ovo LD50 for a given species and DLC, one could use the species’ AHR1 LBD sequence to design an AHR1 expression vector, measure the PC20 of the DLC in the LRG assay, and use the regression to obtain an LD50 value.
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.)
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
- The correlation between AHR-mediated reporter gene activity and embryo death has been demonstrated in avian species as described above.
- Little is known about differences in binding affinity of AhRs and how this relates to sensitivity in non-avian taxa.
- Low binding affinity for DLCs of AhR1s of African clawed frog (Xenopus laevis) and axolotl (Ambystoma mexicanum) has been suggested as a mechanism for tolerance of these amphibians to DLCs (Lavine et al 2005; Shoots et al 2015).
- Among reptiles, only AhRs of American alligator (Alligator mississippiensis) have been investigated and little is known about the sensitivity of American alligator or other reptiles to DLCs (Oka et al 2016).
- Among fishes, great differences in sensitivity to DLCs are known both for AhRs and for embryos among species that have been tested (Doering et al 2013; 2014).
- Differences in binding affinity of the AhR2 have been demonstrated to explain differences in sensitivity to DLCs between sensitive and tolerant populations of Atlantic Tomcod (Microgadus tomcod) (Wirgin et al 2011).
- Information is not yet available regarding whether differences in binding affinity of AhRs of fishes are predictive of differences in sensitivity of embryos, juveniles, or adults (Doering et al 2013).
1. Backlund, M., and Ingelman-Sundberg, M. (2004). Different structural requirements of the ligand binding domain of the aryl hydrocarbon receptor for high- and low-affinity ligand binding and receptor activation. Mol. Pharmacol. 65(2), 416-425.
2. Ema, M., Ohe, N., Suzuki, M., Mimura, J., Sogawa, K., Ikawa, S., and Fujii-Kuriyama, Y. (1994). Dioxin binding activities of polymorphic forms of mouse and human arylhydrocarbon receptors. J. Biol. Chem. 269(44), 27337-27343.
3. Farmahin, R., Manning, G. E., Crump, D., Wu, D., Mundy, L. J., Jones, S. P., Hahn, M. E., Karchner, S. I., Giesy, J. P., Bursian, S. J., Zwiernik, M. J., Fredricks, T. B., and Kennedy, S. W. (2013). Amino acid sequence of the ligand-binding domain of the aryl hydrocarbon receptor 1 predicts sensitivity of wild birds to effects of dioxin-like compounds. Toxicol. Sci. 131(1), 139-152.
4. Head, J. A., Hahn, M. E., and Kennedy, S. W. (2008). Key amino acids in the aryl hydrocarbon receptor predict dioxin sensitivity in avian species. Environ. Sci. Technol. 42(19), 7535-7541.
5. Karchner, S. I., Franks, D. G., Kennedy, S. W., and Hahn, M. E. (2006). The molecular basis for differential dioxin sensitivity in birds: Role of the aryl hydrocarbon receptor. Proc. Natl. Acad. Sci. U. S. A 103(16), 6252-6257.
6. Manning, G. E., Farmahin, R., Crump, D., Jones, S. P., Klein, J., Konstantinov, A., Potter, D., and Kennedy, S. W. (2012). A luciferase reporter gene assay and aryl hydrocarbon receptor 1 genotype predict the embryolethality of polychlorinated biphenyls in avian species. Toxicol. Appl. Pharmacol. 263(3), 390-399.
7. Murray, I. A., Reen, R. K., Leathery, N., Ramadoss, P., Bonati, L., Gonzalez, F. J., Peters, J. M., and Perdew, G. H. (2005). Evidence that ligand binding is a key determinant of Ah receptor-mediated transcriptional activity. Arch. Biochem. Biophys. 442(1), 59-71.
8. Pandini, A., Denison, M. S., Song, Y., Soshilov, A. A., and Bonati, L. (2007). Structural and functional characterization of the aryl hydrocarbon receptor ligand binding domain by homology modeling and mutational analysis. Biochemistry 46(3), 696-708.
9. Pandini, A., Soshilov, A. A., Song, Y., Zhao, J., Bonati, L., and Denison, M. S. (2009). Detection of the TCDD binding-fingerprint within the Ah receptor ligand binding domain by structurally driven mutagenesis and functional analysis. Biochemistry 48(25), 5972-5983.
10. Poland, A., Glover, E., and Kende, A. S. (1976). Stereospecific, high affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin by hepatic cytosol. Evidence that the binding species is receptor for induction of aryl hydrocarbon hydroxylase. J. Biol. Chem. 251(16), 4936-4946.
11. Poland, A., and Knutson, J. C. (1982). 2,3,7,8-tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. Annu. Rev. Pharmacol. Toxicol. 22, 517-554. 12. Poland, A., Palen, D., and Glover, E. (1994). Analysis of the four alleles of the murine aryl hydrocarbon receptor. Mol. Pharmacol. 46(5), 915-921.
13. Ramadoss, P., and Perdew, G. H. (2004). Use of 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin as a probe to determine the relative ligand affinity of human versus mouse aryl hydrocarbon receptor in cultured cells. Mol. Pharmacol. 66(1), 129-136.
14. Farmahin, R., Wu, D., Crump, D., Hervé, J.C., Jones, S.P., Hahn, M.E., Karchner, S.I., Giesy, J.P., Bursian, S.J., Zwiernik, M.J., Kennedy, S.W. (2012) Sequence and in vitro function of chicken, ring-necked pheasant, and Japanese quail AHR1 predict in vivo sensitivity to dioxins. Environ Sci Technol. 46(5), 2967-75.
15. Mimura, J., and Fujii-Kuriyama, Y. (2003). Functional role of AhR in the expression of toxic effects by TCDD. Biochimica et Biophysica Acta - General Subjects 1619, 263-268.
16. Wirgin, I., Roy, N. K., Loftus, M., Chambers, R. C., Franks, D. G., and Hahn, M. E. (2011). Mechanistic basis of resistance to PCBs in Atlantic tomcod from the Hudson River. Science 331, 1322-1325
17. Kopf, P. G., and Walker, M. K. (2009). Overview of developmental heart defects by dioxins, PCBs, and pesticides. J. Environ. Sci. Health C. Environ. Carcinog. Ecotoxicol. Rev. 27(4), 276-285.
18. Lavine, J.A.; Rowatt, A.J.; Klimova, T.; Whitington, A.J.; Dengler, E.; Beck, C.; Powell, W.H. 2005. Aryl hydrocarbon receptors in the frog Xenopus laevis: two AhR1 paralogs exhibit low affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Toxicol. Sci. 88 (1), 60-72.
19. Shoots, J.; Fraccalvieri, D.; Franks, D.G.; Denison, M.S.; Hahn, M.E.; Bonati, L.; Powell, W.H. 2015. An aryl hydrocarbon receptor from the salamander Ambystoma mexicanum exhibits low sensitivity to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Enviro. Sci. Technol. 49, 6993-7001.
20. Oka, K.; Kohno, S.; Ohta, Y.; Guillette, L.J.; Iguchi, T.; Katsu, Y. (2016). Molecular cloning and characterization of the aryl hydrocarbon receptors and aryl hydrocarbon receptor nuclear translocators in the American alligator. Gen. Comp. Endo. 238, 13-22.
21. Doering, J.A.; Giesy, J.P.; Wiseman, S.; Hecker, M. Predicting the sensitivity of fishes to dioxin-like compounds: possible role of the aryl hydrocarbon receptor (AhR) ligand binding domain. Environ. Sci. Pollut. Res. Int. 2013, 20(3), 1219-1224.
22. Doering, J.A.; Farmahin, R.; Wiseman, S.; Kennedy, S.; Giesy J.P.; Hecker, M. 2014. Functionality of aryl hydrocarbon receptors (AhR1 and AhR2) of white sturgeon (Acipenser transmontanus) and implications for the risk assessment of dioxin-like compounds. Enviro. Sci. Technol. 48, 8219-8226.