Organohalogen Compounds 65, 142-145 (2003)

IDENTIFICATION OF NOVEL AGONISTS AND ANTAGONISTS OF
THE Ah RECEPTOR SIGNAL TRANSDUCTION PATHWAY.
David S Baston1, Scott R. Nagy1, Michael Nantz2, Mark Kurth2, Mark Springsteel2, and Michael
S. Denison1
1

Department of Environmental Toxicology, Meyer Hall, University of California, Davis, CA
2
Department of Chemistry, University of California, Davis, CAIntroduction
The aryl hydrocarbon receptor (AhR) is a ligand dependent transcription factor that is activated bythe high-affinity binding of a variety of halogenated and polycyclic aromatic hydrocarbons (HAHsand PAHs, respectively)1. The prototypical and most potent HAH, 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD), results in a variety of toxic and biochemical effects, the majority of which aremediated by the AhR2. Expression of the toxic and biological effects are a result of liganddependent activation of the AhR which leads to its dimerization with AhR nuclear translocator(ARNT), nuclear accumulation, and consequent induction of gene expression. Recently, we andothers have observed that the AhR can bind and be activated by a structurally diverse range ofchemicals3-5. In an effort to better understand the structural diversity of AhR ligands and theirability to activate the AhR signaling pathway we have used a high-throughput green fluorescentprotein (GFP) based recombinant cell bioassay to screen ~180 novel flavonoids and flavonerelated chemicals for their ability to activate or inhibit AhR signal transduction5,6.
Materials and Methods
Chemicals. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was a gift from Dr. Steven Safe (TexasA&M University) and flavonoids were synthesized as previously defined7.
AhR-Dependent GFP Reporter Gene Expression Analysis. Recombinant mouse hepatoma(H1G1.1c3) cells containing the stably transfected AhR-responsive green fluorescent protein(GFP) reporter plasmid pGreen1.1 was used. In the screening process these cells respond to AhR
agonists with the induction of the GFP reporter gene activity in a time-, dose-, ligand- and AhRdependent manner6. For induction studies, 75,000 cells were plated into individual wells of black
clear-bottomed 96-well microplates and allowed to attach for 24 hours at 37oC. After 24 hours,
the media was replaced with media containing DMSO (1% maximum final concentration), TCDD
or the test chemical followed by incubation for 24 hours 33oC. GFP activity was measured in aTecan microplate fluorometer (Molecular Dynamics) with excitation and emission wavelengths of485 and 515 nm respectively. Samples were run in triplicate and wells containing media were used
as blanks and subtracted as background.

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Organohalogen Compounds 65, 142-145 (2003)

Results and Discussion
The GFP bioassay has been previously established as a relatively inexpensive, quick and effective
screening method for identifying compounds that exhibit dioxin like response of the AhR
signaling pathway6. Agonist activity screens were incubated in 100 µLs of a media solution
containing DMSO and the compound of interest for 24 hours at 1 µM and 10 µM concentrations,
GFP response was measured in relative fluorescence units (RFU) and calculated as a percent of 1
nM TCDD response. Similarly, antagonist activity was determined by co-incubating 1 nM TCDD
with the compound of interest at 1 µM and 10 µM for 24 hours. Normalization between
experimental fluorescence was accomplished by adjusting the fluorometer gain so that thefluorescence level of the GFP induced by 1 nM TCDD produced relative fluorescence units of~9000 RFU. A selection of the 10 µM induction/inhibition GFP response, presented in Figure 1,
by novel compounds is expressed as a percentage of 1 nM TCDD, currently the most potent ligandof the AhR, after normalizing the response to the DMSO/media background. Initial screening ofthese compounds for agonist/antagonist activity has indicated that many of the compounds canbind and activate/inhibit the AhR-dependent GFP gene expression. Response of individualcompounds exhibited varied induction/inhibition responses, indicated by the selectedinduction/inhibition data provided in Figure 1, where compounds exhibit agonist and antagonist
activity, only agonist activity, or only antagonist activity.
From a total of 184 compounds agonist expression of GFP resulted at greater than 50% of 1 nM
TCDD by 46 compounds at a concentration of 10µM, 9 of these compounds displayed activity
equal to or greater than that of TCDD. Antagonistic activity for compounds with less than 50% 1
nM TCDD activity occurred with 58 compounds at a concentration of 10 µM, 7 of thesecompounds completely inhibited TCDD activity. Compounds from Figure 1 displaying bothagonist and antagonist activity are AL7 and BH2. Compounds displaying mostly agonist activity,
similar to β-Naphthoflavone (BNF), are W1, AY9, and BB9, and some compounds displaying
mostly antagonistic activity, similar to α-Naphthoflavone (ANF), are AJ5 and AJ7.
Currently our lab is assessing the EC50 by fitting dose-response data to a logistic curve using
Sigma Plot (SPSS, Chicago, IL), ligand and DNA binding as well as functional activity forcompounds at 1 µM that display agonist activity exhibiting activity greater than 50% andantagonistic activity less than 50% than that of 1nM TCDD.

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Organohalogen Compounds 65, 142-145 (2003)

144

140% Induction% Inhibition
120

100% Induction

8060

40

20-20U3U5V1W1Z7Z8Z9Z10AA1AA2AA3AJ1AJ2AJ3AJ4AJ5AJ6AJ7AJ8AJ9AJ10AL3AL4AL5AL6AL7AT7AU1AY7AY9AY10AZ1AZ2BB8BB9BC5BC6BC7BC8BC9BG7BG8BG9BG10BH1BH2BH3BL5BL6BM10Figure 1. Selected GFP response to novel compounds as agonists and antagonists expressed as a percentage of 1 nM
TCDD

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145

α -Naphthoflavone(Antagonist 1 µM)

β-Naphthoflavone(10-100 nM)
OOOOµ M)

Agonist Activity (>85%@1µM)

Antagonist Activity (>90%@1FHNOOA9OA10O

AJ6A4OONOOOCF 3OO3ONO

AT7Q10N H2OSNH 2OOO

R5OHOH

R9O

R10OBrBrO

V1OIOOOO
OCH 3H 3C OOO

T8O

G10O

O BH 3Figure 2. Selected agonist and antagonist structures of flavone and flavonoid compounds.
References
1. Denison MS, Phelen D, Elferink CJ. 1998. in: Xenobiotics, Receptors and Gene Expression(Denison MS, Helferich WG eds), pp. 3-33, Taylor and Francis, Philadelphia.
2. Safe S (1990) Crit Rev Toxicol 21:51-88.
3. Dension, MS, et al (1998) Bull Environ Contam Toxicol 61,557-568
4. Denison, MS, Nagy SR. Annual Review of Pharm and Toxicol. 43:309-334, 2003.
5. Nagy SR,. Liu G. Lam KS. Denison MS. Biochemistry. 41(3):861-868, 2002 Jan 22.
6. Nagy SR, Sanborn JR, Hammock BD, Denison MS. 2002. Toxicol. Sci. 65:200-10.
7. Galietta LJ et al. Jour. Biol. Chem. 2001, 276(23):19723-8
Acknowledgements
This work was supported by the national Institutes of Environmental Health Sciences (ES07865,ES04699 and ES05707) and the California Agricultural Experiment Station.

Organohalogen Compounds, Volumes 60-65, Dioxin 2003 Boston, MA