Organohalogen Compounds 65, 90-93 (2003)

ACTIVATION OF THE Ah RECEPTOR AND Ah RECEPTOR SIGNAL
TRANSDUCTION PATHWAY BY TRYPTOPHAN-DERIVED AGONISTS
Michael S. Denison1, Dal-Ho Han1, Sharon Heath-Pagliuso1, John Scovill2, Laurent Meijer3,Leandros Skaltounis4, Elizabeth M.J. Gillam5 and Scott R. Nagy1
1

Department of Environmental Toxicology, Meyer Hall, University of California, Davis, CA, USA
2
Department of Chemistry, University of Pennsylvania, Philadelphia, PA, USA
3
C.R.N.S, Cell Biology Group & UPS-2682, Station Bioloique, Roscoff cedex, Bretagne, France
4
Department of Pharmacognosy, University of Athens, 15771 Athens, Greece
5Department of Physiology and Pharmacology, University of Queensland, Brisbane, Australia.

Introduction
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related halogenated aromatichydrocarbons (HAHs), such as polychlorinated dibenzo-p-dioxins (PCDDs), biphenyls (PCBs)
and dibenzofurans (PCDFs), exert the majority of their effects via their bind to and activation ofthe Ah receptor (AhR) and AhR-dependent gene expression.1 Recently, it has become abundantly
clear that the AhR can be bound and activated by structurally diverse range of chemicals andnumerous studies have described and characterized a variety of naturally-occurring dietarychemicals that can directly bind to and activate and/or inhibit the AhR signaling pathway.2,3Given that the greatest source of exposure of animals and humans to AhR ligands (both syntheticand natural) comes from the diet, an understanding of the structural diversity of AhR ligands andtheir biological and toxicological activity and potency is needed. Numerous naturally-occurring
indoles and indole-containing chemicals have been identified that can activate the AhR signaling
pathway (Figure 1). The majority of these AhR ligands are formed from tryptophan (TRP) as aresult of various biological and physiochemical processes. Potent AhR activators have beenobserved to be formed from the photooxidation of TRP and the acidic condensation of dietary
indole-3-carbinol, while other naturally-occurring TRP metabolic and biosynthetic products (i.e.
tryptamine, indole acetic acid, indirubin and indigo) were shown to bind to and activate the AhR
and AhR-dependent gene expression.2,3 Given the lack of information regarding the structural
diversity of AhR ligands as well as the lack of information with respect to endogenous
physiological ligands for the AhR, further analysis of this rather large group of naturally occurring
indole ligands is warranted. Accordingly, we have used several AhR-based bioassay systems to
examine the ability of a variety of natural and synthetic indoles and indole-containing chemicals toactivate the AhR and AhR-dependent gene expression.
Materials and Methods
The ability of various chemicals to stimulate AhR-dependent transformation and DNA binding
was determined by gel retardation analysis using guinea pigs cytosol.4 Determination of theability of various chemicals to stimulate AhR-dependent gene expression was carried out using arecombinant mouse hepatoma cell line that contains an AhR-responsive green fluorescent protein(GFP) reporter plasmid pGreen1.1 (H1G1.1c3 cells). These cells respond to AhR agonists with theinduction of GFP in a time-, dose-, ligand-dependent manner and GFP fluorescence activity ismeasured in intact cells as described.5

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Results and Discussion
Numerous indoles and indole-containing chemicals have been shown to be AhR agonists (figure1).2 We have examined a series of derivatives of several of these indole-containing ligands inorder to better understand the structural diversity of AhR ligands and to determine whether key
structural characteristics exist in this class naturally-occurring ligands. Tryptanthrins are a class ofnaturally occurring compounds found in plants as well as being a microbial metabolite that has
some therapeutic uses. Given their high degree of structural similarity to one of the most potent
indole AhR agonists, indolo[3,2-b]carbazole, and previous studies demonstrating their activity at
the AhR,6 we first examined a series of trytpanthrins.7 These results revealed several compounds(2, 4, 5, 11) that were relatively potent activators of AhR-dependent gene expression (with EC50sof between 14-30 nM compared to TCDD at 0.01 nM). Subsequent studies demonstrated theability of these specific compounds to stimulate AhR DNA binding in gel retardation analysis andto induce CYP1A1 expression as determined by rtPCR (data not shown). Our results also
demonstrate that these compounds were significantly more potent than the classical PAH inducers
beta-naphthoflavone and 3-methylcholanthrene. Additional analysis was focused on the ability ofa series of 10 substituted indoles to activate AhR-dependent gene expression.8 The majority ofthese chemicals were relatively weak activators, with only 2-methylindole and oxazolodiindole
inducing reporter gene expression in the low to mid µM concentration range. Previous studies
have also reported that indigo and indirubin were potent activators of the AhR in an AhR-Arnt
containing yeast cell bioassay system and that they were equipotent or 50-fold more potent thanTCDD, respectively.9 Our results, however, demonstrate that while these compounds arerelatively active (figure 3), they are 50,000 to 100,000-fold less potent than TCDD. Thissignificant difference from previously published results likely derives from the metabolic labilityof these chemicals in mammalian cells combined with the limitated solubility of TCDD in
aqueous yeast growth media. Subsequent analysis of 26 substituted synthetic indirubins resulted
in the identification of five compounds that were extremely potent activators of the AhRdependent gene expression (with EC50s between 1-8 nM, compared to TCDD at 0.015 nM).10
These results demonstrate that the substituted indirubins represent some of the most potent
nonHAH AhR activators that have been identified to date. Current studies are examining theaffinity for AhR ligand binding domain and the relative ability to stimulate AhR transformationand DNA binding. While the biological/toxicological significance of these results remain to beelucidated, these results do suggest that indole-containing compounds likely represent one majorgroup of potent naturally-occurring activators of the AhR to which we are exposed.
References
1. Safe S (1990) Crit Rev Toxicol 21,51-88.
2. Denison MS, Nagy SR (2002) Ann Rev Pharmacol Toxicol 43,309-334.
3. Denison MS, Heath-Pagliuso S (1998) Bull Environ Contam Toxicol 61,557-568.
4. Phelan D, Winter GM, Rogers WJ, Lam JC et al. (1998) Arch Biochem Biophys 357,155-163.
5. Nagy S., Sanborn JR, Hammock BD, Denison, M.S. (2002) Toxicol Sci 65, 200-210.
6. Schrenk D, Riegniger D, Till M et al. (1997) Biochem Pharmacol 54, 165-171.
7. Scovill J, Blank E, Konnick M et al. (2002) Antimicrob Agent Chemother 46,882-883.
8. Gillam E., Notley LM, Cai H, DeVoss JJ, Guengerich FP (2000) Biochem 39,13817-13824.
9. Adachi J, Mori Y, Matsui S, Takigama H et al. (2001) J Biol Chem 276,31475-31478.
10. Leclerk S, Garnier M, Hoessel R, Marko D et al. (2001) J Biol Chem 276,251-260.
Acknowledgements
This work was supported by the National Institutes of Environmental Health Sciences (ES07865,ES04699 and ES05707) and the California Agricultural Experiment Station.

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Organohalogen Compounds 65, 90-93 (2003)

Figure 1. Tryptophan-derived chemicals which have been shown to bind to and/or activate
the AhR and AhR-dependent gene expression. Reprinted from reference 2.

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Organohalogen Compounds 65, 90-93 (2003)

Figure 2. Determination of the relative potency of series of tryptanthrins toinduce AhR-dependent GFP reporter gene expression in H1G1.1c3 cells.

Figure 3. Dose-dependent induction of AhR-dependent GFP reporter gene
expression in H1G1.1c3 cells by TCDD, indirubin, indigo and several
substituted indoles.

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