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: TCDD-BINDING_INH_RAD_GEL_3X%INH MCSRUN

Name: Late stage assay provider counterscreen for activators of Aryl hydrocarbon receptor (AHR): Radiometric [3H]TCDD Competitive Binding assay to identify compounds that inhibit binding of radiolabeled TCDD to AHR in cytosol isolated from guinea pig liver.

Description:

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).

References:

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.
17. Denison, M.S., Rogers, J.M., Rushing, S.R., Jones, C.L., Tetangco, S.C., and Heath-Pagliuso, S. (2002) Analysis of the aryl hydrocarbon receptor (AhR) signal transduction pathway, in Current Protocols in Toxicology (Morgan, K. S., Ed.), pp 4.8.1-4.8.45, John Wiley, New York.

Keywords:

TCDD, competition, TCDF, 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.

Assay Overview:

The purpose of this assay is to determine the ability of test compounds to reduce the specific binding of radiolabeled [3H]TCDD (2,3,7,8-Tetrachlorodibenzo-p-dioxin) to cytosolic AHR isolated from guinea pig liver. Cytosol is incubated with [3H]TCDD in the absence or presence the test compound or positive control competitor (TCDF: 2,3,7,8-tetrachlorodibenzofuran), the proteins bound to hydroxyapatite and the free/loosely-bound/unbound [3H]TCDD removed from the incubation by washing of the hydroxyapatite with Tween 20-containing buffer. The total amount of [3H]TCDD specific binding was obtained by subtracting the non-specific binding ([3H]TCDD and TCDF) from the total binding ([3H]TCDD). As designed, compounds that can bind to the AHR will competitively reduce [3H]TCDD specific binding and use of increasing concentrations of competitor can allow determination of its relative affinity of AHR binding. Compounds are tested in triplicate at a final nominal concentration of 10 uM.

Protocol Summary:

The hydroxyapatite (HAP) binding assay was performed to determine the ligand binding ability of test chemicals as described previously in detail (17). Guinea pig hepatic cytosol, was prepared in HEDG (25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.5, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiotreitol, 10% [v/v] glycerol) buffer as previously described, diluted to 2 mg protein/ml and ligand binding was performed using HAP (17). Aliquots of guinea pig cytosols (200 uL) were incubated at room temperature for 1 h with [3H]TCDD (2 nM) alone (total binding), [3H]TCDD (2 nM) and TCDF (200 nM, 100-fold excess of competitor, nonspecific binding) or [3H]TCDD (2 nM) in the presence of the test chemical (1 uM or 10 uM). All chemicals were dissolved in DMSO, in which DMSO content in reactions was adjusted to 2% (v/v) where necessary. Thereafter, hydroxyapatite suspension (250 uL) was added to the different reaction mixtures and incubated for an additional 30 min with gentle vortexing every 10 min. The reactions were washed three times with 1 mL of HEGT buffer (25 mM HEPES, pH 7.5, 1 mM EDTA, glycerol (10%, v/v), and Tween 80 (0.5%, v/v)). The HAP pellets were transferred to 4 mL scintillation vials, scintillation cocktail was added, and reactions were counted in a scintillation counter.

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 guinea pig cytosols (200 uL) were incubated at room temperature for 1 h with [3H]TCDD (2 nM) alone (total binding), [3H]TCDD (2 nM) and TCDF (200 nM, 100-fold excess of competitor, nonspecific binding) or [3H]TCDD (2 nM) in the presence of the test chemical (1 uM or 10 uM). All chemicals were dissolved in DMSO, in which DMSO content in reactions was adjusted to 2% (v/v) where necessary.

4. Thereafter, hydroxyapatite suspension (250 uL) was added to the different reaction mixtures and incubated for an additional 30 min with gentle vortexing every 10 min.

5. The HAP incubations were centrifuged at ~500xg and the pellets washed three times with 1 mL of HEGT buffer (25 mM HEPES, pH 7.5, 1 mM EDTA, glycerol (10%, v/v), and Tween 80 (0.5%, v/v)).

6. The HAP pellets were transferred to 4 mL scintillation vials, scintillation cocktail was added, and reactions were counted in a scintillation counter.

7. Specific binding of [3H]TCDD to the AhR was computed by subtracting the amount of [3H]-TCDD bound in the presence of TCDF (or test compoound) from the amount of [3H]TCDD bound in the absence of competitor.

PubChem Activity Outcome and Score:

While compounds that resulted in a reduction in [3H]TCDD specific that was statistically significant were considered AhR ligands, for screening purposes those that reduced specific binding by less than 10% were considered inactive. Compounds that reduced specific binding by greater than or equal to 10% were considered active.

The reported PubChem Activity Score has been normalized to 100% observed inhibition. Negative % inhibition values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-32, and for inactive compounds 9-9.

List of Reagents:

Hepatic cytosol from guinea pig liver in HEDG buffer
stock solution (200 uM) of [3H]TCDD in DMSO (specific activity 10-50 Ci/mmole, Chemsyn Laboratories; Terrachem)
stock solution (20 uM) of TCDF (Accustandard, Cambridge Isotope Laboratories or Wellington Laboratories) in DMSO
hydroxyapatite (HAP) (BioRad Biogel HTP)
Tween 80
Bio-Rad protein reagent
Scintillation counting cocktail

This assay was run by the assay provider. Possible artifacts of this assay can include, but are not limited to: solubility of the test compounds, compounds that modulate ligand binding or result in denaturation of the AhR; fragmentation of the HAP with too vigorous vortexing or high centrifugation speeds; loss of HAP during transfer to scintillation vials. 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.

BAO: version: 1.4b1090

BAO: bioassay specification: assay stage: confirmatory

BAO: bioassay specification: assay biosafety level: bsl1

BAO: assay format: cell-free format: subcellular: cytosol format

BAO: bioassay specification: assay measurement type: endpoint assay

BAO: bioassay specification: assay readout content: assay readout method: regular screening

BAO: bioassay specification: assay readout content: content readout type: single readout

BAO: meta target: molecular target: protein target: receptor: nuclear receptor

BAO: meta target: biological process target: regulation of molecular function

BAO: meta target detail: binding reporter specification: interaction: protein-small molecule

BAO: assay design: conformation reporter: protein

BAO: detection technology: radiometry: scintillation counting: filter assay

Grant Number: 1-X01-DA026558-01