The aryl hydrocarbon receptor (AhR) is a ligand-dependent, basic helix-loop-helix Per-Arnt-Sim (PAS)-containing transcription factor that can bind and be activated by structurally diverse chemicals, including the toxic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Our previous three-dimensional homology model of the mouse AhR (mAhR) PAS B ligand binding domain allowed identification of the binding site and its experimental validation. We have extended this analysis by conducting comparative structural modeling studies of the ligand binding domains of six additional high-affinity mammalian AhRs. These results, coupled with site-directed mutagenesis and AhR functional analysis, have allowed detection of the "TCDD binding-fingerprint" of conserved residues within the ligand binding cavity necessary for highaffinity TCDD binding and TCDD-dependent AhR transformation DNA binding. The essential role of selected residues was further evaluated using molecular docking simulations of TCDD with both wild-type and mutant mAhRs. Taken together, our results dramatically improve our understanding of the molecular determinants of TCDD binding and provide a basis for future studies directed toward rationalizing the observed species differences in AhR sensitivity to TCDD and understanding the mechanistic basis for the dramatic diversity in AhR ligand structure.The aryl hydrocarbon receptor (AhR) 1 is a ligand-dependent transcription factor and a member of the basic helix-loop-helix Per-Arnt-Sim (bHLH/PAS) family of proteins (1-4). † This research was supported by the National Institutes of Environmental Health Sciences (Grants ES07685 and ES012498) and the California Agricultural Experiment Station.*To whom correspondence should be addressed. Phone: (530) 752-3879. Fax: (530) 752-3394. msdenison@ucdavis.edu. SUPPORTING INFORMATION AVAILABLE Expression levels of in vitro synthesized wild-type and mutant AhRs. 35 S-Labeled wild-type (wt) and mutant AhRs (specific mutation indicated) were synthesized in vitro, denatured, and resolved by SDS-polyacrylamide gel electrophoresis and phosphoimager analysis of the dried gels as described in Materials and Methods (Figure 1). This material is available free of charge via the Internet at http://pubs.acs.org. 1 Abbreviations: AhR, aryl hydrocarbon receptor; bHLH, basic helix-loop-helix; PAS, Per-Arnt-Sim; LBD, ligand binding domain; HAH, halogenated aromatic hydrocarbon; PAH, polycyclic aromatic hydrocarbon; TCDD,2,3,7, hsp90, heat shock protein of 90 kDa; XAP2, X-associated protein 2; ARNT, AhR nuclear translocator; HIF-2α, hypoxia-inducible factor 2α; DRE, dioxin responsive element; mAhR, mouse AhR; rtAhR, rat AhR; haAhR, hamster AhR; rbAhR, rabbit AhR; gpAhR, guinea pig AhR; bAhR, beluga whale AhR; sAhR, seal AhR. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 June 30. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptWhile the highest-affinity AhR ligands include persistent environmental co...

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2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related dioxin-like chemicals are widespread and persistent environmental contaminants that produce diverse toxic and biological effects through their ability to bind to and activate the Ah receptor (AhR) and AhR-dependent gene expression. The chemically activated luciferase expression (CALUX) system is an AhR-responsive recombinant luciferase reporter gene-based cell bioassay that has been used in combination with chemical extraction and cleanup methods for the relatively rapid and inexpensive detection and relative quantitation of dioxin and dioxin-like chemicals in a wide variety of sample matrices. Although the CALUX bioassay has been validated and used extensively for screening purposes, it has some limitations when screening samples with very low levels of dioxin-like chemicals or when there is only a small amount of sample matrix for analysis. Here, we describe the development of third-generation (G3) CALUX plasmids with increased numbers of dioxin-responsive elements, and stable transfection of these new plasmids into mouse hepatoma (Hepa1c1c7) cells has produced novel amplified G3 CALUX cell bioassays that respond to TCDD with a dramatically increased magnitude of luciferase induction and significantly lower minimal detection limit than existing CALUX-type cell lines. The new G3 CALUX cell lines provide a highly responsive and sensitive bioassay system for the detection and relative quantitation of very low levels of dioxin-like chemicals in sample extracts.

In this paper, the protective effect of the bioflavonoid quercetin on behaviors, antioxidases, and neurotransmitters in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine-(MPTP-) induced Parkinson's disease (PD) was investigated. Quercetin treatment (50 mg/kg, 100 mg/kg and 200 mg/kg body weight) was orally administered for 14 consecutive days. The results show that quercetin treatment markedly improves the motor balance and coordination of MPTP-treated mice. Significant increases were observed in the activities of glutathione peroxidase (GPx), superoxide dismutase (SOD), and Na+, K+-ATPase, AchE, the content of dopamine (DA) in the quercetin plus MPTP groups compared to those in the MPTP group. Significant reduction the 4-hydroxy-2-nonenal (4-HNE) immunoreactivity in striatum of brains was observed in the quercetin plus MPTP groups in comparison to the MPTP group. Taken together, we propose that quercetin has shown antiparkinsonian properties in our studies. More work is needed to explore detailed mechanisms of action.

The Ah receptor (AhR)-responsive CALUX (chemically-activated luciferase expression) cell bioassay is commonly used for rapid screening of samples for the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), dioxin-like compounds, and AhR agonists/antagonists. By increasing the number of AhR DNA recognition sites (dioxin responsive elements), we previously generated a novel third generation (G3) recombinant AhR-responsive mouse CALUX cell line (H1L7.5c3) with significantly enhanced sensitivity and response to DLCs compared to existing AhR-CALUX cell bioassays. However, the elevated background luciferase activity of these cells and the absence of comparable G3 cell lines derived from other species have limited their utility for screening purposes. Here, we describe the development and characterization of species-specific G3 recombinant AhR-responsive CALUX cell lines (rat, human, and guinea pig) that exhibit significantly improved sensitivity and dramatically increased TCDD induction response. The low background luciferase activity, low minimal detection limit (0.1 pM TCDD) and enhanced induction response of the rat G3 cell line (H4L7.5c2) over the H1L7.5c3 mouse G3 cells, identifies them as a more optimal cell line for screening purposes. The utility of the new G3 CALUX cell lines were demonstrated by screening sediment extracts and a small chemical compound library for the presence of AhR agonists. The increased sensitivity and response of these new G3 CALUX cell lines will facilitate species-specific analysis of DLCs and AhR agonists in samples with low levels of contamination and/or in small sample volumes.