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, Set 2
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, Set 2. ..more
BioActive Compounds: 8
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_SET2 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, Set 2.
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.
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.
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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).
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 uL 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 uL 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).
EC50 values from sigmoidal concentration-response curves were determined using the four-parameter Hill equation (SigmaPlot (Systat)).
PubChem Activity Outcome and Score:
Compounds that induced (increased) reporter activity less than 20% were considered inactive. Compounds that led to a negative response (AhR reporter activity less than 0) were considered inactive, and may be cytotoxic. compounds that induced (increased) reporter activity by 20% or greater were considered active.
The reported PubChem Activity Score has been normalized to 100% observed max % activation.
The PubChem Activity Score range for active compounds is 100-10, and for inactive compounds 6-0.
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 - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)