Organohalogen Compounds 65, 126-129 (2003)

IDENTIFICATION AND CHARACTERIZATION OF NOVEL FLAVONEAGONISTS OF THE Ah RECEPTOR
Dal-Ho Han1, Bin Zhao1, David S. Baston1, M Springsteel2, M Kurth2, MH Nantz2 andMichael S. Denison1
1

Department of Environmental Toxicology, 2Department of Chemistry,
University of California, Davis, CA, USA

Introduction
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates manyof the biological and toxicological effects of a structurally diverse array of hydrophobic naturaland synthetic chemicals1. Exposure to AhR agonists results in variety of effects, the most studiedof which is induction of gene expression. Following ligand binding, the cytosolic ligand:AhR
complex dissociates from its associated protein subunits, accumulates within the nucleus, anddimerizes with Ah receptor nuclear translocator (ARNT) protein, with the resulting conversion ofthe heteromeric complex into its high-affinity DNA binding form. The binding of theligand:AhR:ARNT complex to its specific recognition site, the dioxin responsive element (DRE),stimulates gene transcription through an adjacent promoter. The AhR signal transduction pathway
is present in a diverse range of species, tissues, and cell types2. To date, the majority of genes
which respond to AhR ligands have been shown to utilize the AhR-DRE-dependent mechanism ofaction. Flavones represent one group of natural chemicals that have been shown to bind to and act
as AhR agonists and antagonists3, 4. Here we describe the analysis of 37 novel flavones andbenzoflavone(BNF) derivatives for their ability to bind and activate AhR using gel retardation
analysis, GFP reporter assays, and RT-PCR.
Materials and Methods
The Enhanced Green Fluorescent Protein (EGFP)-Based Cell Bioassay. Mouse hepatoma(Hepa1c1c7) cells that have been stably transfected with a reporter plasmid containing theenhanced green fluorescent protein reporter gene (H1G1.1c3 cells) were maintained in alphaminimal essential medium (MEM) supplemented with 10% fetal bovine serum,
penicillin/streptomycin (50 units/ml each), and 968 mg/L G418. These cells respond to TCDD andother AhR agonists with the induction of GFP in a time-, dose-, chemical-, and AhR-dependent
manner. GFP activity was expressed as a percentage of that obtained with 1 nM TCDD after
correction for the background fluorescence in the DMSO samples5, 6.
RNA isolation and RT-PCR of AhR. Mouse hepatoma (Hepa1c1c7) cells were grown to 90%
confluence and treated with DMSO or 1 mM chemicals for 4 hr. Total RNA was isolated withTRIzol Reagent (GibcoBRL) and stored at –80 until further use. RT-PCR for CYP1A1 and thecontrol HPRT was carried out as described in detail. The forward and reverse primers for mouse
CYP1A1 mRNA produced a product of 280 bp between exon 5 and exon 7 of the mouse CYP1A1gene7. mCYP1A1 FP: 5’-GCCTTCATTCTGGAGACCTTCC-3’and mCYP1A1 RP: 5’CAATGGTCTCTCCGATGC-3’. Mouse hypoxanthine phosphoribosyl transferase (mHPRT) wasused as an internal control and amplification of mHPRT produced a product of 177 bp8. Primer

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Organohalogen Compounds 65, 126-129 (2003)

sequences are as follows: mHPRT FP: 5’-GTAATGATCAGTCAACGGGGGAC-3’ and mHPRT
RP: 5’-CCAGCAAGCTTGCAACCTTAACCA-3’
Preparation of Nuclear Protein. Nuclear extracts of mouse hepatoma (Hepa1c1c7) cells which
had been incubated with DMSO (1 µL/mL), BNF (1 µM), flavones (1 µM), or TCDD (1 nM) for 1
hr at 37 were prepared as previously described9. Nuclear extracts were stored frozen at -80
until use and protein concentrations determined using the Bradford assay10.
Synthetic Oligonucleotides and Gel Retardation Analysis. A complementary pair of synthetic
oligonucleotides containing the sequence 5’-GATCTGGCTCTTCTCACGCAACTCCG-3’ and5’-GATCCGGAGTTGCGTGAGAAGAGCCA-3’ (corresponding to the AhR binding site of
DRE3 and designated as the DRE oligonucleotide11) was synthesized, purified, annealed, andradiolabeled with [γ32P]ATP as described11. Gel retardation analysis of nuclear extracts (14 µgprotein) from cell lines was carried out as previously described9. The final salt concentration of thebinding reaction was 160 mM KCl and 1.7 µg p[dI·dC] and protein-DNA complexes werevisualized by autoradiography. The amount of [32P]-labeled DRE present in the induced proteinDNA complex was quantified using a Molecular Dynamics phosphoImaging system.
Results and Discussion
EGFP Reporter Assay. Thirty seven flavones were analyzed using the high throughput EGFP
reporter assay, of these 8 compounds were identified that significantly induced GFP expression(Table 1). EC50 values for EGFP induction by these compounds were between 10 nM and 8 µM,
approximately 2 x 103 to 3 x 106-fold lower than that of TCDD (20 pM). Among them, R6 was themost potent on inducer of GFP expression, with an EC50 of 10 nM. These results indicate thatthese compounds are relatively potent AhR inducers in H1G1.1c3 cells.
Effect of Selected Flavones on Expression of CYP1A1 mRNA Levels. To establish that thetransformed AhR generated by these compounds actively induced CYP1A1 mRNA in vivo, RTPCR was carried out (Table 1). Total RNA was harvested following a 4 hr treatment of mouse
hepatoma (hepa1c1c7) cells with the test compounds or DMSO and subsequently analyzed byPCR with CYP1A1 primers and HPRT primers. Our results showed that all of compounds, withexception of R7 and A7, induced expression of CYP1A1 mRNA levels between 12 and 81% ofthat induced by 1 nM TCDD. Among them, compound R6 was the most induction of CYP1A1mRNA to a level 81% of that induced by TCDD. These results indicate that selected flavones areable to induce the CYP1A1 mRNA at the level of transcription.
Effect of Selected Flavones on AhR transformation and DNA binding in Hepa1c1c7 cells. Gel
retardation analysis was performed to confirm that the inducing activity of these compounds is dueto their ability to activate AhR (Table 1). Incubation of nuclear extracts with compounds R6, R9,R10, O3, and BNF stimulated formation of a protein-DNA complex that represents DNA-bound
AhR. These results are consistent with their ability to activate the AhR signal transduction
pathway.
Combinational Effects of Flavones on TCDD-induced EGFP Activity. To demonstrate thecombinational effects of these compounds, H1G1.1c3 cells were treated with 1 µM of theindicated flavone in the absent or presence of 1 nM TCDD at 33 for 24 hr. Our results showed

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Organohalogen Compounds 65, 126-129 (2003)

that various flavones induced GFP expression to between 20 and 90% that of 1 nM TCDD (Table2). Interestingly, in the case of cotreatment, all of the compounds decreased in TCDD-induced
GFP expression, with the exception of compounds O3 and BNF, indicating that these compounds
were relatively weak inducers of GFP expression. These results also indicate that several of theseflavones may act as partial AhR antagonists.
Conclusions
We have been able to identify several novel flavones for their ability to activate/inhibit the AhR
signal transduction pathway using a combination of ligand- and DNA-binding and reporter geneassay. In addition, cotreatment with flavones and TCDD revealed that several of these flavoneswere partial AhR antagonists. Overall, these results have demonstrated that the structuralcharacteristics of several of these flavones are distinctly different from known flavonoids agonistsof the AhR continuing the ongoing debates as to the structural determinants necessary for AhR
agonist and antagonist activity.
Acknowledgments.
This work was supported by the National Institute of Environmental Health Sciences (ES07685,ES04699 and ES05707) and the California Agricultural Experiment Station.
References
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229,231-237.
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5. Nagy SR, Sanborn JR, Hammock BD, Denison MS (2002) Toxicol 65,200-210.
6. Nagy SR, Liu G, Lam KS, Denison MS (2002) Biochemistry 41,861-868.
7. Wei YD, Rannug U, Rannug A (1999) Chem Biol Interact 118(2),127-140.
8. Reiners Jr, J J, Jones CL, Hong N, Myrand SP (1998) Mol Carcinogenesis 146(21),135-146.
9. Denison MS, Rogers JM, Rusing SR, Jones CL, Tetangco SC, Heath-Pagliuso S (2002)
Current Protocols in Toxicol 4.8.1-4.8.45, Wiley, New York.
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Organohalogen Compounds 65, 126-129 (2003)

Table 1. EC50 and Relative Potencies of Selected Flavones in EGFP reporter gene assay, gelretardation, and RT-PCR.
Relative DRE binding
Relative CYP1A1
Chemicals EC50 (nM)a Relative Potencyb(%)cmRNA (%)dTCDD.0.02
1100 ± 29
100 ± 12R610
2.0 x 10-311 ± 2
81 ± 15R1030
6.7 x 10-4
66 ± 635 ± 5O390
1.8 x 10-475 ± 12
49 ± 15BNF
90
1.8 x 10-4
127 ± 1460 ± 4R9200..
1.0 x 10-4
31 ± 749 ± 6A10800..
2.5 x 10-4
ND
44 ± 7A91000…
2.0 x 10-5ND
24 ± 2R81000…
2.0 x 10-52±1
20 ± 5A88000…
2.5 x 10-6ND
12 ± 5e
R7ND
ND
ND
5±2A7NDe
ND
ND
2±2Note. Relative potencies were determined from dose-response relationship studies measuring
induction of EGFP at 24 hr.
aConcentration at which 50% of the maximum EGFP induction.
bValues are expressed relative to the EC for EGFP induction by TCDD.
50c
PhosphorImager quantitation of the amount of inducible AhR:DRE complex formed andexpressed as a percent of TCDD.
dValues are expressed relative fold induction of CYP1A1 mRNA by TCDD.
e
ND: Not Detectable.
Table 2. Combinational Effects of Flavones on TCDD-induced EGFP Activity.
Flavones
Flavone only
Flavone + TCDD
R653 ± 3073 ± 6
R726 ± 1063 ± 6R847 ± 5058 ± 7R982 ± 7098 ± 5R1084 ± 6097 ± 4O391 ± 4
107 ± 4
BNF96 ± 4114 ± 3A7NVa075 ± 1A820 ± 2074 ± 3A945 ± 5062 ± 2A1061 ± 7093 ± 3
H1G1.1c3 cells were incubated with 1 µM of the indicated flavone or 1 nM TCDD for 24 hr at
33 , and the EGFP activity was measured as described under Materials and Methods.Values areexpressed as the mean ± SD of triplicate determinations relative to that induced by 1 nM TCDD(100%). aNV: Values were less than control.

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