Key Event Title
|Level of Biological Organization|
Key Event Components
|protein dimerization activity||hypoxia-inducible factor 1-alpha||decreased|
|protein dimerization activity||aryl hydrocarbon receptor nuclear translocator||decreased|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|AHR activation to embryolethality||KeyEvent|
|ahr/arnt affected by x||KeyEvent|
|Aryl hydrocarbon receptor (AhR) leads to Early life stage mortality||KeyEvent|
Key Event Description
The aryl hydrocarbon receptor nuclear translocator (ARNT; a.k.a HIF-1ß) serves as a dimerization partner for hypoxia inducible factor 1 alpha (HIF-1α), and this complex is involved in mediating physiological responses to hypoxia. HIF-1α abundance is negatively regulated by a subfamily of dioxygenases referred to as prolyl hydroxylase domain-containing proteins, which use oxygen as a substrate to hydroxylate HIF-1α subunits and hence tag them for rapid degradation. Under conditions of hypoxia, HIF-1α subunits accumulate due to reduced hydroxylation efficiency and form heterodimers (HIF-1) with ARNT. Dimerization between ARNT and HIF-1α forms a transcription factor complex (HIF-1) that binds to hypoxia response enhancer sequences on DNA to activate the expression of genes involved in angiogenesis, glucose metabolism, cell survival, and erythropoietin synthesis, among others[8-11].
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 HIF1- α 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.
Domain of Applicability
ARNT/HIF1-alpha dimerization and downstream gene regulation has been studies in chickens, mice, rats, and fish[14-16].
1. Forsythe, J. A., Jiang, B. H., Iyer, N. V., Agani, F., Leung, S. W., Koos, R. D., and Semenza, G. L. (1996). Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol. Cell Biol. 16(9), 4604-4613.
2. Goldberg, M. A., and Schneider, T. J. (1994). Similarities between the oxygen-sensing mechanisms regulating the expression of vascular endothelial growth factor and erythropoietin. J. Biol. Chem. 269(6), 4355-4359.
3. 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.
4. Jiang, B. H., Rue, E., Wang, G. L., Roe, R., and Semenza, G. L. (1996). Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1. J. Biol. Chem. 271(30), 17771-17778.
5. Maxwell, P. H., Dachs, G. U., Gleadle, J. M., Nicholls, L. G., Harris, A. L., Stratford, I. J., Hankinson, O., Pugh, C. W., and Ratcliffe, P. J. (1997). Hypoxia-inducible factor-1 modulates gene expression in solid tumors and influences both angiogenesis and tumor growth. Proc. Natl. Acad. Sci U. S. A 94(15), 8104-8109.
6. Shweiki, D., Itin, A., Soffer, D., and Keshet, E. (1992). Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 359(6398), 843-845.
7. Walker, M. K., Pollenz, R. S., and Smith, S. M. (1997). Expression of the aryl hydrocarbon receptor (AhR) and AhR nuclear translocator during chick cardiogenesis is consistent with 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced heart defects. Toxicol. Appl. Pharmacol. 143(2), 407-419.
8. Wikenheiser, J., Wolfram, J. A., Gargesha, M., Yang, K., Karunamuni, G., Wilson, D. L., Semenza, G. L., Agani, F., Fisher, S. A., Ward, N., and Watanabe, M. (2009). Altered hypoxia-inducible factor-1 alpha expression levels correlate with coronary vessel anomalies. Dev. Dyn. 238(10), 2688-2700.
9. Livingston DM, Shivdasani R. (2001). Toward mechanism-based cancer care. JAMA 285:588–593.
10. Semenza GL. (2003). Targeting HIF-1 for cancer therapy. Nat Rev Cancer 3: 721–732.
11. Dery M-A C, Michaud MD, Richard DE. (2005). Hypoxia-inducible factor 1: regulation by hypoxic and non-hypoxic activators. Int J Biochem Cell Biol 37: 535–540.
12. Jain S, Maltepe E, Lu MM, Simon C, and Bradfield CA (1998) Expression of ARNT, ARNT2, HIF1 alpha, HIF2 alpha and Ah receptor mRNAs in the developing mouse. Mech Dev 73:117–123.
13. Tipoe, G. L., and Fung, M. L. (2003). Expression of HIF-1alpha, VEGF and VEGF receptors in the carotid body of chronically hypoxic rat. Respir. Physiol Neurobiol. 138(2-3), 143-154.
14. Heise, K., Estevez, M.S., Puntarulo, S., Galleano, M., Nikinmaa, M., Portner, H.O., and Abele, D. (2007) Effects of seasonal and latitudinal cold on oxidative stress parameters and activation of hypoxia inducible factor (HIF-1) in zoarcid fish. Biochem. Syst. Environ. Physiol. 177: 765-77
15. Rissanen, E., Tranberg, H.K., Sollid, J., Nilsson, G.E., and Nikinmaa, M. (2006) Temperature regulates hypoxia-inducible factor-1 (HIF-1) in a poikilothermic vertebrate, crucian carp (Carassius carassius) J. Exp. Biol. 209 (6): 994-1003
16. Greenald, D., Jeyakani, J., Pelster, B., Sealy, I., Mathavan, S., and van Eeden, F.J. (2015) Genome-wide mapping of Hif-1 alpha binding sites in zebrafish. BMC Genomics. 16: 923