Key Event Overview
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AOPs Including This Key Event
|AOP Name||Event Type||Essentiality|
|Aryl hydrocarbon receptor activation leading to embryolethality via cardiotoxicty||KE||Strong|
Level of Biological Organization
How this Key Event works
Upon ligand binding, the aryl hydrocarbon receptor (AHR) migrates to the nucleus where it dissociates from the cytosolic complex and forms a heterodimer with AHR nuclear translocator (ARNT) (Mimura and Fujii-Kuriyama 2003). The AHR-ARNT complex then binds to a xenobiotic response element (XRE) found in the promoter of an AHR-regulated gene and recruits co-regulators such as CREB binding protein/p300, steroid receptor co-activator (SRC) 1, SRC-2, SRC-3 and nuclear receptor interacting protein 1, leading to induction or repression of gene expression (Fujii-Kuriyama and Kawajiri 2010). Expression levels of several genes, including phase I (e.g. cytochrome P450 (CYP) 1A, CYP1B, CYP2A) and phase II enzymes (e.g. uridine diphosphate glucuronosyl transferase (UDP-GT), glutathione S-transferases (GSTs)), as well as genes involved in cell proliferation (transforming growth factor-beta, interleukin-1 beta), cell cycle regulation (p27, jun-B) and apoptosis (Bax), are regulated through this mechanism (Fujii-Kuriyama and Kawajiri 2010; Giesy et al. 2006; Mimura and Fujii-Kuriyama 2003; Safe 1994).
How it is Measured or Detected
Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible?
The active AHR complexed with ARNT can be measured using protein-DNA interaction assays. Two methods are described in detail by Perez-Romero and Imperiale (Perez-Romero and Imperiale 2007). Chromatin immunoprecipitation measures the interaction of proteins with specific genomic regions in vivo. It involves the treatment of cells with formaldehyde to crosslink neighboring protein-protein and protein-DNA molecules. Nuclear fractions are isolated, the genomic DNA is sheared, and nuclear lysates are used in immunoprecipitations with an antibody against the protein of interest. After reversal of the crosslinking, the associated DNA fragments are sequenced. Enrichment of specific DNA sequences represents regions on the genome that the protein of interest is associated with in vivo. Electrophoretic mobility shift assay (EMSA) provides a rapid method to study DNA-binding protein interactions in vitro. This relies on the fact that complexes of protein and DNA migrate through a non-denaturing polyacrylamide gel more slowly than free DNA fragments. The protein-DNA complex components are then identified with appropriate antibodies. The EMSA assay was found to be consistent with the luciferase reporter gene assay (in chicken hepatoma cells dosed with dioxin-like compounds (Heid et al. 2001).
Evidence Supporting Taxonomic Applicability
This mechanism is conserved across species. Mammals possess a single AHR, whereas birds and fish express multiple isoforms, and all three express multiple ARNT isoforms. Not all of the isoforms identified are functionally active. For example, killifish AHR1 and AHR2 are active and display different transcription profiles, whereas zebrafish AHR2 and ARNT2 are active in mediating xenobiotic-mediated toxicity and AHR1 is inactive (Hahn et al. 2006; Prasch et al. 2006).
1. Fujii-Kuriyama, Y., and Kawajiri, K. (2010). Molecular mechanisms of the physiological functions of the aryl hydrocarbon (dioxin) receptor, a multifunctional regulator that senses and responds to environmental stimuli. Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 86(1), 40-53.
2. Giesy, J. P., Kannan, K., Blankenship, A. L., Jones, P. D., and Newsted, J. L. (2006). Toxicology of PCBs and related compounds. In Endocrine Disruption Biological Bases for Health Effects in Wildlife and Humans (D.O.Norris and J.A.Carr, Eds.), pp. 245-331. Oxford University Press, New York.
3. Hahn, M. E., Karchner, S. I., Evans, B. R., Franks, D. G., Merson, R. R., and Lapseritis, J. M. (2006). Unexpected diversity of aryl hydrocarbon receptors in non-mammalian vertebrates: insights from comparative genomics. J. Exp. Zool. A Comp Exp. Biol. 305(9), 693-706.
4. Heid, S. E., Walker, M. K., and Swanson, H. I. (2001). Correlation of cardiotoxicity mediated by halogenated aromatic hydrocarbons to aryl hydrocarbon receptor activation. Toxicol. Sci 61(1), 187-196.
5. 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(3), 263-268.
6. Perez-Romero, P., and Imperiale, M. J. (2007). Assaying protein-DNA interactions in vivo and in vitro using chromatin immunoprecipitation and electrophoretic mobility shift assays. Methods Mol. Med. 131, 123-139.
7. Prasch, A. L., Tanguay, R. L., Mehta, V., Heideman, W., and Peterson, R. E. (2006). Identification of zebrafish ARNT1 homologs: 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in the developing zebrafish requires ARNT1. Mol. Pharmacol. 69(3), 776-787.
8. Safe, S. (1994). Polychlorinated biphenyls (PCBs): Environmental impact, biochemical and toxic responses, and implications for risk assessment. Critical Reviews in Toxicology 24(2), 87-149.