Novel bioanalytical approaches for the detection and characterization of aryl hydrocarbon receptor (AhR) ligands

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcriptional factor which mediates a diverse array of biological and toxicological effects. Mechanistically, the unliganded AhR exists in the cytosol, and following by ligand binding, the ligand:AhR complex translocates into the nucleus, where it dissociates with other protein components and dimerizes with the Ah receptor nuclear translocator (Arnt) protein. This heterodimer has high affinity for its specific DNA recognition sequences known as dioxin response element (DRE) which stimulates transcriptional activation of downstream genes, such as cytochrome P4501A1 (CYP1A1). The best-characterized and high affinity AhR ligands (agonists and antagonists) include environmental contaminants such as halogenated aromatic hydrocarbons (HAHs), the most potent of which is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polycyclic aromatic hydrocarbons (PAHs). However, many other chemicals having dramatically different structural and physicochemical characteristics can bind to the AhR and activate the AhR-dependent signaling pathway. Development of detection methods for AhR ligands and AhR signaling activators will provide insights into the diverse class of AhR ligands how they activate the AhR signaling pathway. In this study, I developed, characterized, and validated three cell-based bioassay methods to detect AhR ligands and AhR signaling activators. In chapter one, I optimized a human cell bioassay for detection of human-specific AhR ligands, in which a human hepatoma cell line (HepG2) was stably transfected with an AhR-responsive luciferase reporter gene. This bioassay was used to screen 147 commercially available human therapeutic chemicals, and the results compared to data using mouse and rat recombinant cell lines also containing an AhR-responsive luciferase reporter gene. In chapter two, I developed a cellular bioanalytical method to detect ligand-dependent nuclear localization of the AhR. A mouse hepatoma cells (TAO) stably transfected with a YFP-AhR expression vector which allowed detection of the subcellular localization of the AhR in living cells. In chapter 3, I developed an assay method to detect ligand-dependent dimerization of AhR:Arnt using fluorescent resonance energy transfer (FRET). In these three bioassays, the signal was detected in a chemical- dose-, and time-dependent manner. The availability of these cell bioassays provides an avenue to identify and characterize novel AhR agonists.