Late stage assay provider dose-response counterscreen for activators of Aryl hydrocarbon receptor (AhR): Radiometric electrophoretic mobility shift assay (EMSA) to identify compounds that stimulate AhR transformation and binding to its specific DNA recognition site in vitro
Late stage assay provider dose-response counterscreen for activators of Aryl hydrocarbon receptor (AhR): Radiometric electrophoretic mobility shift assay (EMSA) to identify compounds that stimulate AhR transformation and binding to its specific DNA recognition site in vitro. ..more
BioActive Compounds: 10
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Michael Denison, University of California, Davis
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1-X01-DA026558-01
Grant Proposal PI: Michael Denison
External Assay ID: AHR-TCDD-EMSA_INH_RAD_GEL_3X%INH MCSRUN
Late stage assay provider dose-response counterscreen for activators of Aryl hydrocarbon receptor (AhR): Radiometric electrophoretic mobility shift assay (EMSA) to identify compounds that stimulate AhR transformation and binding to its specific DNA recognition site in vitro.
Transcription factors are critical regulators of gene expression (1). Under conditions such as environmental stress and exposure to endogenous toxins, transcription factors can rapidly modulate the transcription of genes whose products regulate cell proliferation and metabolism. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor of the basic helix-loop-helix protein superfamily involved in the biological response to aromatic hydrocarbons, and regulates the expression of xenobiotic-metabolizing enzymes such as cytochrome P450, aldehyde dehydrogenase, quinone reductase, and other phase I and phase II detoxification genes (2, 3). In response to various compounds, including the environmental pollutants dioxins, benzo(a)pyrene, dietary contaminants, grapefruit juice, endogenous toxins, and plant products such as carotinoids, nicotine and caffeine (2, 4-6), cytosolic AHR complexes with chaperones hsp90, p23, and XAP2, translocates to the nucleus where it dimerizes with the AHR nuclear translocator (ARNT) to influence target gene transcription (7, 8). Gain-of-function studies in mice reveal the oncogenic potential of AHR (9), while other reports show roles for AHR in diverse biologic events such as organ development (10, 11), immune function and allergy (12), and estrogen responsiveness (13). The identification of agonists of AHR will provide useful tools to elucidate the roles of this receptor in cell metabolism, transcriptional control, and tumor formation (14-16).
1. Ptashne, M., Regulation of transcription: from lambda to eukaryotes. Trends Biochem Sci, 2005. 30(6): p. 275-9.
2. McMillan, B.J. and Bradfield, C.A., The aryl hydrocarbon receptor sans xenobiotics: endogenous function in genetic model systems. Mol Pharmacol, 2007. 72(3): p. 487-98.
3. Puga, A., Tomlinson, C.R., and Xia, Y., Ah receptor signals cross-talk with multiple developmental pathways. Biochem Pharmacol, 2005. 69(2): p. 199-207.
4. Bock, K.W. and Kohle, C., Ah receptor: dioxin-mediated toxic responses as hints to deregulated physiologic functions. Biochem Pharmacol, 2006. 72(4): p. 393-404.
5. Denison, M.S. and Nagy, S.R., Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals. Annu Rev Pharmacol Toxicol, 2003. 43: p. 309-34.
6. de Waard, P.W., Peijnenburg, A.A., Baykus, H., Aarts, J.M., Hoogenboom, R.L., van Schooten, F.J., and de Kok, T.M., A human intervention study with foods containing natural Ah-receptor agonists does not significantly show AhR-mediated effects as measured in blood cells and urine. Chem Biol Interact, 2008.
7. Hankinson, O., The aryl hydrocarbon receptor complex. Annu Rev Pharmacol Toxicol, 1995. 35: p. 307-40.
8. Petrulis, J.R. and Perdew, G.H., The role of chaperone proteins in the aryl hydrocarbon receptor core complex. Chem Biol Interact, 2002. 141(1-2): p. 25-40.
9. Andersson, P., McGuire, J., Rubio, C., Gradin, K., Whitelaw, M.L., Pettersson, S., Hanberg, A., and Poellinger, L., A constitutively active dioxin/aryl hydrocarbon receptor induces stomach tumors. Proc Natl Acad Sci U S A, 2002. 99(15): p. 9990-5.
10. Ramos, K.S., Transcriptional profiling and functional genomics reveal a role for AHR transcription factor in nephrogenesis. Ann N Y Acad Sci, 2006. 1076: p. 728-35.
11. Walisser, J.A., Glover, E., Pande, K., Liss, A.L., and Bradfield, C.A., Aryl hydrocarbon receptor-dependent liver development and hepatotoxicity are mediated by different cell types. Proc Natl Acad Sci U S A, 2005. 102(49): p. 17858-63.
12. Lawrence, B.P., Denison, M.S., Novak, H., Vorderstrasse, B.A., Harrer, N., Neruda, W., Reichel, C., and Woisetschlager, M., Activation of the aryl hydrocarbon receptor is essential for mediating the anti-inflammatory effects of a novel low-molecular-weight compound. Blood, 2008. 112(4): p. 1158-65.
13. Ohtake, F., Takeyama, K., Matsumoto, T., Kitagawa, H., Yamamoto, Y., Nohara, K., Tohyama, C., Krust, A., Mimura, J., Chambon, P., Yanagisawa, J., Fujii-Kuriyama, Y., and Kato, S., Modulation of oestrogen receptor signalling by association with the activated dioxin receptor. Nature, 2003. 423(6939): p. 545-50.
14. Zhao, B., Baston, D.S., Hammock, B., and Denison, M.S., Interaction of diuron and related substituted phenylureas with the Ah receptor pathway. J Biochem Mol Toxicol, 2006. 20(3): p. 103-13.
15. Garrison, P.M., Tullis, K., Aarts, J.M., Brouwer, A., Giesy, J.P., and Denison, M.S., Species-specific recombinant cell lines as bioassay systems for the detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin-like chemicals. Fundam Appl Toxicol, 1996. 30(2): p. 194-203.
16. Han, D., Nagy, S.R., and Denison, M.S., Comparison of recombinant cell bioassays for the detection of Ah receptor agonists. Biofactors, 2004. 20(1): p. 11-22.
late stage, powders, purchased, synthesized, AHR, EMSA, gel shift, polyacrylamide, oligonucleotide, radioactivity, electrophoresis, binding, guinea pig, DNA, DRE, dioxin response element, aryl hydrocarbon receptor, receptor, transcription factor, triplicate, dose response, counterscreen, assay provider, cell, extract, cytosol, liver, activator, agonist, activation, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
§ Panel component ID.
The purpose of this assay is to determine the ability of test compounds to stimulate transformation (ligand-dependent dimerization of the AHR with ARNT) and DNA (i.e. DRE) binding of the AHR in vitro. Guinea pig hepatic cytosol is incubated with test compound, followed by incubation of an aliquot of the reaction mixture with poly[dIdC] and [32P]-DRE. AhR:DRE complexes arre resolved by through a non-denaturing polyacrylamide gel and AhR:[32P]-DRE complexes visualized and quantified by phosphorimager analysis of the dried gels (28). As designed, compounds that act as AHR agonists will bind to and activate the AHR, leading to an increased amount of AhR:[32P]-DRE complex and the amount of complex is directly proportional to the amount of AHR activation and agonist concentration. Compounds are tested in triplicate at a final nominal concentration of 10 uM. Details of this protocol can be found in Denison et al 2002 (17).
1. Male Hartley guinea pigs (400 g) were obtained from Charles River Laboratories (Wilmington, MA). All animals were exposed to 12 h of light:12 h of dark daily and given free access to food and water.
2. Hepatic cytosol was prepared in HEDG (25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.5, 1 mM ethylenediaminetetraacetic acid, 1 mM dithiotreitol, 10% [v/v] glycerol) buffer as described in detail by Denison et al. (2002) and protein concentrations determined by Bio-Rad Protein Assay (Bio-Rad). Aliquots of cytosol were stored at -80 C until use.
3. Aliquots of diluted cytosol (8 mg protein/ml HEDG) were incubated in the presence of 20 nM TCDD, 10 uM of test compound or 1% (v/v) solvent control DMSO for 2.5 h at room temperature in the final reaction volume of 10 ul (with three replicate reactions for each condition).
4. An aliquot (11 ul) of dIdC-HEDGK solution (37.5 ng of dIdC [Roche] in 0.218 M KCl HEDG buffer) was added to each tube and incubated for 15 minutes at room temperature.
5. An aliquot (4 ul) of [32P]-labeled DRE oligonucleotide (~100,000 dpm, diluted in HEDG) was added to each tube and incubated for 15 min.
7. Gels were transferred to 3MM paper, dried, exposed overnight on an imaging plate and radiolabeled DNA visualized by FLA-9000 analysis (Fujifilm). Gel images were quantitated in MultiGauge (Fujifilm). Specific band densities were adjusted to the background levels (non-band areas of the gel).
8. The amount of induced AhR:DRE complex is normalized to that obtained with a maximal inducing concentration of TCDD (20 nM).
PubChem Activity Outcome and Score:
The following applies to each panel:
Compounds that induced a change in binding activity less than 10% were considered inactive. Compounds wthat induced a change in binding activity equal to or greater than 10% were considered active.
The reported PubChem Activity Score has been normalized to 100% of observed average % inhibition.
Exp 1: The PubChem Activity Score range for active compounds is 100-22, and for inactive compounds 18-8.
Exp 2: The PubChem Activity Score range for active compounds is 100-24, and for inactive compounds 22-6.
Overall Outcome and Score:
The overall outcome was active if the compound was active in all panels, inactive otherwise.
The overall score is 0 if the compound was inactive and 100 if the compound was active.
The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 0-0.
List of Reagents:
Hepatic cytosol from guinea pig liver
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Accustandard, product D404N)
non-denaturing 4% polyacrylamide gels
[32P]-Labeled double-stranded wild-type DRE-oligonucleotide (5'-GATCTGGCTCTTCTCACGCAACTCCG-3' and 5'-GATCCGGAGTTGCGTGAGAAGAGCCA-3')
poly dI*dC (Roche, product 108812)
This assay was run by the assay provider. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the polyacrylamide gel, compounds that modulate DNA binding. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided.
Categorized Comment - additional comments and annotations
** Test Concentration. § Panel component ID.