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BioAssay: AID 602171

Late stage assay provider counterscreen for the probe development effort to identify activators of the Aryl Hydrocarbon Receptor (AHR): Luminescence-based Human Hepatoma (HG2L7.5c1) Cell-based assay to identify activators of AhR

Name: Late stage assay provider counterscreen for the probe development effort to identify activators of the Aryl Hydrocarbon Receptor (AHR): Luminescence-based Human Hepatoma (HG2L7.5c1) Cell-based assay to identify activators of AhR. ..more
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 Tested Compounds
 Tested Compounds
All(33)
 
 
Active(33)
 
 
 Tested Substances
 Tested Substances
All(33)
 
 
Active(33)
 
 
AID: 602171
Data Source: The Scripps Research Institute Molecular Screening Center (AHR_ACT_LUMI_0096_3X%ACT MCSRUN)
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2012-01-05
Hold-until Date: 2013-01-03
Modify Date: 2013-01-03

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 33
Depositor Specified Assays
Show more
AIDNameTypeComment
2796Luminescence-based primary cell-based high throughput screening assay to identify activators of the Aryl Hydrocarbon Receptor (AHR)screeningPrimary screen (AHR activators in singlicate)
2804Summary of probe development efforts to identify activators of the Aryl Hydrocarbon Receptor (AHR)summarySummary (AHR activators)
2845Luminescence-based cell-based high throughput confirmation assay for activators of the Aryl Hydrocarbon Receptor (AHR)screeningConfirmation (AHR activators in triplicate)
434939Counterscreen for activators of the Aryl Hydrocarbon Receptor (AHR): luminescence-based cell-based high throughput screening assay to identify activators of the Pregnane X Receptor (PXR)screeningCounterscreen (PXR activators in triplicate)
463086Luminescence-based counterscreen for activators of the Aryl Hydrocarbon Receptor (AHR): cell-based high throughput dose response screening assay for activators of the Pregnane X Receptor (PXR)confirmatoryDose response counterscreen (PXR activators in triplicate)
463088Luminescence-based cell-based high throughput dose response assay for activators of the Aryl Hydrocarbon Receptor (AHR)confirmatoryDose response (AHR activators in triplicate)
602226Late 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 liverother
624397Late stage assay provider counterscreen for activators of Aryl hydrocarbon receptor (AHR): Absorbance-based cell-based assay to identify compounds that modulate proliferation of ER-positive breast cancer cells (MCF7), Set 2confirmatory
624398Late stage assay provider counterscreen for the probe development effort to identify activators of the Aryl Hydrocarbon Receptor (AHR): Luminescence-based Human Ovarian Carcinoma (BG1Luc4E2) Cell-based assay to identify inhibitors of Estrogen Receptor-Dependent Gene Expression, Set 2other
624399Late stage assay provider counterscreen for the probe development effort to identify activators of the Aryl Hydrocarbon Receptor (AHR): Luminescence-based Human Ovarian Carcinoma (BG1Luc4E2) Cell-based assay to identify activators of Estrogen Receptor-Dependent Gene Expressionother
624400Late 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, Set 2other
624401Late stage assay provider counterscreen for the probe development effort to identify activators of the Aryl Hydrocarbon Receptor (AHR): Luminescence-based Human Hepatoma (HG2L7.5c1) Cell-based assay to identify activators of AhR, Set 2confirmatory
624402Late 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 vitroother
Description:
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_ACT_LUMI_0096_3X%ACT MCSRUN

Name: Late stage assay provider counterscreen for the probe development effort to identify activators of the Aryl Hydrocarbon Receptor (AHR): Luminescence-based Human Hepatoma (HG2L7.5c1) Cell-based assay to identify activators of AhR.

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. He G, Tsutsumi T, Zhao B, Baston DS, Zhao J, Heath-Pagliuso S, Denison MS. Third-generation Ah receptor-responsive luciferase reporter plasmids: amplification of dioxin-responsive elements dramatically increases CALUX bioassay sensitivity and responsiveness. Toxicol Sci. 2011 Oct;123(2):511-22.

Keywords:

late stage, powders, purchased, synthesized, AHR, Human Hepatoma, HG2L7.5c1, aryl hydrocarbon receptor, receptor, transcription factor, triplicate, dose response, counterscreen, stably transfected, stable, assay provider, 96, well, cell, liver, reporter, plasmid, activator, agonist, activation, luciferase, luminescence, reporter, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Assay Overview:

The purpose of this assay is to detemine the AhR agonist potency of powder samples of compounds identified as possible agonist probe candidates of the human ligand activated transcription factor, aryl hydrocarbon receptor (AHR). This cell-based assay measures the ability of compounds to activate AHR signaling. The assay employs human hepatoma (HepG2) cells stably transfected with the AHR-dependent pGudLuc7.5-DRE plasmid (HG2L7.5c1 cell line), which expresses the firefly luciferase reporter gene under control of the mouse MMTV promoter and 20 dioxin response elements (DREs) from concatenated fragments of the upstream dioxin responsive domain of the murine CYP1A1 gene. Cells are incubated with test compounds for 24 hours, followed by cell lysis and detection of well luminescence using a commercially available luciferase reagent. As designed, compounds that act as AHR agonists will stimulate the AHR, leading to nuclear translocation, DRE binding, increased transcription of the luciferase reporter gene, and increased well luminescence in the presence of substrate. Compounds are tested in triplicate at a final nominal concentration of 10 uM. This assay was modified from He et al (17).

Protocol Summary:

General maintenance: HG2L7.5c1 Cells are maintained in alpha-MEM (Invitrogen, 12000-063) containing 10% premium fetal bovine serum (Atlanta Biologicals, S11150) with 400 mg/L of G418. Cells should not exceed 90% confluency before passaging.

Protocol for plating cells into 96-well plate:

1. Double rinse cells with PBS (5 mL per plate per rinse), trypsinize and transfer into 50 mL sterile tubes.

2. Fill all tubes to 50 mL with media (alpha-MEM with 10% FBS without G418) and centrifuge centrifuge at room temperature for 5 minutes at 1,100 rpm.

3. In tissue culture hood, carefully aspirate media from centrifuged tubes. Add 10 mL media to tube and gently resuspend cells.

4. Remove a 10 uL aliquot of resuspended cells for cell counting. For the bioassay, the optimal cell density for HG2L7.5c1 cells in the 96-well plate format is 750,000 cells/mL. A 100 microliters aliquot of cells is added per well using a cell trough and multichannel pipette. Cells are incubated at 37 C for 12-24 hours before use.

Protocol for treating HG2L7.5c1 cells:

In tissue culture hood, prepare treatments with 1000 uL pipette and sterilized tips in 7 mL glass tubes using a 1:100 ratio of chemical (or sample) to media (i.e. 10 uL chemical diluted in 990 microliters media); a maximal inducing concentration of TCDD (1 nM) is included as the positive control. Vortex all treatments for several seconds. Treatment volumes should account for triplicate wells.

Dump media from plated 96-well plate(s) into appropriate biological waste container, taking care not to contaminate the cells during this procedure but to remove as much media as possible.

Carefully fill the appropriate wells of a 96-well microtiter plate with 100 uL chemical suspension prepared in the above step and incubate at 37 C for 24 hours. All chemicals were examined in at least triplicate incubations.

Protocol for lysing HG2L7.5c1 cells and measurement of luciferase activity:

1. Microscopically examine the health of cells in every treated well in the 96-well plate.

2. Dump media from 96-well plate(s) into appropriate biological waste container.

3. Wash wells twice with 100 microtiters PBS per well. Gently dump liquid into waste container.

4. Check cells health and confluency under the microscope after the PBS rinses to ensure that cells were not lost during washing. Remove any remaining PBS.

5. Add 50 microtiters of room temperature Promega lysis buffer (1X) to each well (1X lysis buffer is prepared by adding 30 mL 5X lysis buffer to 120 mL MilliQ water; store in glass bottle).

6. Shake the plate at a moderate speed for at least 20 minutes to ensure cell lysis.

7. Prepare the luminometer. Add 1 bottle of room temperature luciferase buffer to 1 bottle substrate (buffer and substrate from Luciferase Assay System). Apply white backing tape to plate containing lysed cells. Read luminescence of treated wells after automatic injection of Promega stabilized luciferase reagent.

8. Activity is normalized to that obtained with a maximal inducing concentration of TCDD (10 nM).

PubChem Activity Outcome and Score:

Compounds that induced a change in reporter activity less than 20% were considered inactive. Compounds wthat induced a change in reporter activity equal to or greater than 20% were considered active.

The reported PubChem Activity Score has been normalized to 100% absolute value of observed % max response.

The PubChem Activity Score range for active compounds is 100-17. There are no inactive compounds.
Comment
This assay was run by the assay provider. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well luminescence. 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
BAO: version: 1.4b1090

BAO: bioassay specification: assay stage: confirmatory

BAO: bioassay specification: assay biosafety level: bsl1

BAO: assay format: cell-based 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 gene expression

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

BAO: assay design: enzyme reporter: enzyme activity: enzyme activation

BAO: detection technology: luminescence: chemiluminescence

Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Maximum Response (10μM**)The maximum % of activity of the AHR-dependent-luciferase reporter in the presence of 10 micromolar compound. Values shown are normalized to the activity of the report in the presence of 10 nM TCDDFloat%
2Standard DeviationStandard deviation derived from the normalized percent activity of the triplicate data for each compoundFloat

** Test Concentration.
Additional Information
Grant Number: 1-X01-DA026558-01

Data Table (Concise)
Classification
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