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CH223191 Is a Ligand-Selective Antagonist of the Ah (Dioxin) Receptor

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CH223191 Is a Ligand-Selective Antagonist of the Ah (Dioxin) Receptor TOXICOLOGICAL SCIENCES 117(2), 393–403 (2010) doi:10.1093/toxsci/kfq217 Advance Access publication July 15, 2010 CH223191 Is a Ligand-Selective Antagonist of the Ah (Dioxin) Receptor Bin Zhao,*,† Danica E. DeGroot,* Ai Hayashi,* Guochun He,* and Michael S. Denison*,1 *Department of Environmental Toxicology, University of California, Davis, California 95616; and †State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China 1To whom correspondence should be addressed at Department of Environmental Toxicology, Meyer Hall, One Shields Avenue, University of California, Davis, CA 95616-8588. Fax: (530) 752-3394. E-mail: [email protected]. Downloaded from Received April 22, 2010; accepted July 8, 2010 beta-naphthoflavone (BNF) (Denison et al., 1998; Poland and The aryl hydrocarbon (dioxin) receptor (AhR) is a ligand- Knutson, 1982; Safe, 1990). However, a relatively large number http://toxsci.oxfordjournals.org/ dependent transcription factor that produces a wide range of of natural, endogenous, and synthetic AhR agonists have also biological and toxic effects in many species and tissues. Whereas been identified in recent years whose structures and physico- the best-characterized high-affinity ligands include structurally related halogenated aromatic hydrocarbons (HAHs) and poly- chemical characteristics are dramatically different from the cyclic aromatic hydrocarbons (PAHs), the AhR is promiscuous prototypical HAH and PAH AhR ligands (Denison and Heath- and can also be activated by structurally diverse exogenous and Pagliuso, 1998; Denison and Nagy, 2003; Denison et al., 1998; endogenous chemicals. However, little is known about how these Nguyen and Bradfield, 2008), which suggests that the AhR has diverse ligands actually bind to and activate the AhR. Utilizing an extremely promiscuous ligand-binding pocket. AhR ligand binding, DNA binding, and reporter gene expression Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, assays, we have identified a novel ligand-selective antagonist dioxin), the protypical and most potent AhR ligand/agonist, at University of California, Davis - Library on November 5, 2013 (CH223191) that preferentially inhibits the ability of some classes and related dioxin-like halogenated aromatic hydrocarbons of AhR agonists (2,3,7,8-tetrachlorodibenzo-p-dioxin and related (dl-HAHs), results in a wide variety of toxic and biological HAHs), but not others (PAHs, flavonoids, or indirubin), to bind to responses, many of which have been shown to require AhR- and/or activate the AhR and AhR signal transduction. HAH- specific antagonism of AhR-dependent reporter gene expression dependent gene expression (Bradshaw and Bell, 2009; Furness by CH223191 was observed with mouse, rat, human, and guinea and Whelan, 2009; Poland and Knutson, 1982; Safe, 1990). pig cell lines. Ligand- and species-selective antagonism was also Although the spectrum of biochemical and toxicological observed with the AhR antagonists 3#-methoxy-4#-nitroflavone responses mediated by the AhR are species and tissue specific, and 6,2#,4#,-trimethoxyflavone. Our results suggest that the ligand-specific differences have also been observed, with differences in the binding by various ligands to the AhR contribute TCDD and other dl-HAHs, but not PAHs or other non-HAH to the observed structural diversity of AhR ligands and could AhR agonists, producing a characteristic spectrum of AhR- contribute in ligand-specific variation in AhR functionality and dependent toxic effects (Bradshaw and Bell, 2009; Denison the toxic and biological effects of various classes of AhR agonists. et al., 1998; Poland and Knutson, 1982; Safe, 1990). Although Key Words: 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD; Ah the greater toxic potency of HAHs appears to be related to their receptor; beta-naphthoflavone; CH223191. resistance to metabolism and persistent AhR activation (Bradshaw and Bell, 2009; Denison et al., 1998; Poland and Glover, 1980; Poland and Knutson, 1982; Safe, 1990), ligand- The aryl hydrocarbon (dioxin) receptor (AhR) is a ligand- dependent differences in the structure and function of the AhR dependent basic helix-loop-helix-Per-Arnt-Sim (PAS)–containing protein and/or AhR protein complex may also play a role transcription factor that responds to exogenous and endogenous (Goue´dard et al., 2004; Matikainen et al., 2001). chemicals with the induction/repression of expression of Although the extreme structural diversity of AhR ligands is a diverse battery of genes in a wide range of species and tissues analogous to the ligand promiscuity reported for some (reviewed in Beischlag et al., 2008; Bradshaw and Bell, 2009; members of the steroid, thyroid, and retinoic acid receptor Kewley et al., 2004; Ma, 2001). The best-characterized high- superfamily (Ghosh et al., 2003; Ngan et al., 2009; Noy, affinity ligands for the AhR include a variety of toxic halogenated 2007), a mechanistic understanding of AhR ligand diversity is aromatic hydrocarbons (HAHs), such as the polychlorinated lacking due to the absence of a three-dimensional (3D) dibenzo-p-dioxins, dibenzofurans, and biphenyls, and numerous structure of the AhR ligand–binding domain (LBD). However, polycyclic aromatic hydrocarbons (PAHs) and PAH-like several studies examining the ability of low- and high-affinity chemicals, such as benzo(a)pyrene, 3-methylcholanthrene, and ligands to bind to AhRs containing mutations within the Ó The Author 2010. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: [email protected] 394 ZHAO ET AL. ligand-binding pocket have provided some evidence for et al., 1996; Han et al., 2004). Cells were plated into white, clear-bottomed 96- differential binding of structurally diverse ligands within the well culture plates (75,000 cells per well), incubated with DMSO (1% final AhR LBD (Backlund and Ingelman-Sundberg, 2004; Goryo concentration) or the indicated concentration of TCDD, BNF, or other agonist in the absence or presence of CH223191 for the indicated time at 37°C and et al., 2007; Whelan et al.,2010). These results suggested to us luciferase activity determined (Han et al., 2004). that if structurally diverse ligands can bind to different amino DNA- and ligand-binding analysis. Male Hartley guinea pig hepatic acids within the AhR ligand–binding pocket, then it should be cytosol (8 mg protein/ml) was incubated with 20nM TCDD, 1lM BNF, or possible to identify an antagonist that would preferentially carrier solvent (DMSO) in the absence or presence of the indicated affect one ligand, or a class of ligands, over another. concentration of CH223191 for 2 h at 20°C, and binding of an in vitro Accordingly, we examined the ability of a variety of chemicals transformed AhR complex to a 32P-labeled oligonucleotide containing the to differently inhibit the ability of the AhR to be activated by dioxin-responsive element (DRE) oligonucleotide was determined using gel retardation analysis as described in detail (Denison et al., 2002). The amount of the prototypical agonists TCDD (an HAH) and BNF (a PAH- ligand-induced AhR:Arnt:[32P]-DRE complex was determined by phosphor- like chemical). Here we report that the compound CH223191, imager analysis and expressed relative to the amount of complex produced by previously shown to be a pure AhR antagonist that competes maximal activating concentrations of TCDD or BNF. For ligand binding, with TCDD for binding to the AhR (Kim et al., 2006), is cytosol (2 mg protein/ml) was incubated with 10nM [3H]TCDD in the absence 3 actually an HAH-selective AhR antagonist, producing little or or presence of 1lM TCDF or 10lM of CH223191 and [ H]TCDD-specific binding determined by sucrose density centrifugation (Denison et al., 2002). no antagonism of AhR activation by maximally inducing Nuclear localization analysis. Ligand-dependent AhR nuclear localiza- concentrations of PAHs or other non-HAH AhR agonists. Not r only is CH223191 the first reported ligand-selective antagonist tion was determined using mouse hepatoma (TAOc1BP c1) cells that have been stably transfected with an N-terminal yellow fluorescent protein (YFP)-AhR of the AhR but also our results provide strong supporting chimera (yAhR) expression plasmid. These cells (referred to as yAHAYc6 evidence for the differential interaction of HAHs and non-HAH cells) respond to AhR agonists with nuclear accumulation of yAhR that occurs agonists within the AhR LBD and provide novel insights into in a chemical-, dose-, and time-dependent manner (Hayashi and Denison, in the mechanisms contributing to the observed structural pro- preparation). One day before microscopy, yAHAYc6 cells were plated onto miscuity of AhR ligands. 4-well Lab-Tek chambered #1.0 borosilicate cover glass (Nunc) in 600 llof media. Prior to chemical treatment, cells were rinsed with PBS and treated with the desired chemicals in Opti-MEM reduced serum media (Invitrogen). After a 1-h incubation at 37°C, cells were visualized using an Olympus IX71 MATERIALS AND METHODS fluorescence microscope with a YFP filter (excitation: HQ500/20, emission: HQ520lp, beam splitter: Q515lp; Chroma Technology Corp.). Live cell images 3 Chemicals. TCDD, [ H]TCDD, and 3#-methoxy-4#-nitroflavone (MNF) were captured and processed using a Hamamatsu Orca camera and Slidebook were generously provided by Dr Steven Safe (Texas A&M University) and 4.2 imaging software (Intelligent Imaging Innovations).
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