Cell-Based Assays for Identification of Aryl Hydrocarbon Receptor (AhR) Activators

Abstract

The Ah receptor (AhR) is a ligand-dependent transcription factor that mediates a wide range of biological and toxicological effects from exposure to structurally diverse synthetic and naturally occurring chemicals. The role of the AhR and its signaling pathway in endogenous physiological functions and its involvement in immune cell development and human diseases has made it a target for development of therapeutic agents. The ability of the AhR to stimulate gene expression in a ligand-specific manner in recombinant mammalian cell lines containing a stably transfected AhR-responsive firefly luciferase or enhanced green fluorescent protein (EGFP) reporter gene permits high throughput chemical screening for AhR activators. The induction of luciferase activity or EGFP fluorescence in these readily available recombinant cell lines occurs in a time-, dose-and AhR-dependent and chemical-specific manner where the magnitude of reporter gene induction is directly proportional to the concentration and potency of the inducing chemical. The AhR agonist activity of positive test chemicals can be confirmed by demonstrating their ability to stimulate expression of CYP1A1, an endogenous AhR-responsive gene, using quantitative real-time PCR. The detailed protocols described here provide step-by-step instructions for detection and characterization of activators of AhR-dependent gene expression that can readily be applied to other appropriate cell lines.

Comparative In Vitro and In Silico Analysis of the Selectivity of Indirubin as a Human Ah Receptor Agonist

“…Recombinant mouse (H1L6.1c3) and human (HG2L6.1c1) hepatoma cells containing a stably integrated AhR-responsive luciferase reporter gene plasmid (pGudLuc6.1) were plated (750,000 cells/well) in 96-well plates and incubated at 37 °C for 24 h prior to chemical treatment. Cells were incubated with DMSO (1% ( v / v )) or the indicated concentration of test chemical in DMSO for 4 h, followed by visual inspection of the cells for toxicity, washing of the cells with phosphate-buffered saline (PBS), addition of 50 µL of passive lysis buffer (Promega) and lysis of cells for 20 min at room temperature with shaking [ 50 ]. Luciferase activity in each well was measured (integrating luminescence over 10 s with a 10 s delay) in an Orion microplate luminometer (Berthold Detection Systems, Bad Wildbad, Germany) following automatic injection of Promega stabilized luciferase reagent.…”

Relationship between the molecular composition, visible light absorption, and health-related properties of smoldering woodsmoke aerosols

“…Screening of WSA extracts for AhR-or ER-active compounds were carried out using recombinant mouse hepatoma (H1L6.1c2) cells containing a stably integrated AhR-responsive luciferase reporter gene plasmid (pGudLuc6.1) and human breast carcinoma (VM7Luc4E2) cells containing a stably integrated ER-responsive luciferase reporter gene plasmid (pGudLuc7ere), as previously described (He et al, 2014;Brennan et al, 2016). Briefly, for AhR analysis, H1L6.1c2 cells in growth medium (alpha minimal essential media (aMEM) containing 10% FBS) were plated (75,000 cells/well) into white, clear-bottomed 96-well tissue culture plates and incubated at 37°C for 24 h prior to the addition of WSA extracts.…”

“…After incubation, cells were visually inspected for signs of toxicity, rinsed twice with phosphate-buffered saline, followed by the addition of Promega cell lysis buffer, and shaking for 20 min at room temperature to allow complete cell lysis. Luciferase activity in each well was measured by using an Orion microplate luminometer as previously described (He et al, 2014).…”

Pityriazepin and other potent AhR ligands isolated from Malassezia furfur yeast

“…Preparation, treatment, incubation and luciferase analysis of these recombinant cell lines was carried out as previously described. 10 Briefly, the recombinant cell lines were grown in 100-mm cell culture plates (Corning Glass Works; Corning, NY) using sterile technique, maintained in alpha-minimum essential media (α-MEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and G418 (1mg/mL) and incubated at >80% humidity and 37°C. For luciferase activity analysis, plates of stable cell clones (approximately 80–100% confluent) were trypsinized and resuspended in 20 mL α-MEM.…”

“…Luciferase activity was measured using an automated microplate luminometer (Anthos Lucy2, Austria) in enhanced flash mode with the automatic injection of 50μL of Promega stabilized luciferase reagent as previously described. 10 …”