Cell-based assays for identification of aryl hydrocarbon receptor (AhR) activators

Guochun He, Jing Zhao, Jennifer C. Brennan, Alessandra A. Affatato, Bin Zhao, Robert H. Rice, Michael S. Denison

Research output: Chapter in Book/Report/Conference proceedingChapter

5 Citations (Scopus)

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.

Original languageEnglish (US)
Title of host publicationMethods in Pharmacology and Toxicology
Pages221-235
Number of pages15
DOIs
StatePublished - 2014

Publication series

NameMethods in Pharmacology and Toxicology
ISSN (Print)15572153
ISSN (Electronic)19406053

Fingerprint

Aryl Hydrocarbon Receptors
Aptitude
Reporter Genes
Cell Line
Ligands
Firefly Luciferases
Gene Expression
Cytochrome P-450 CYP1A1
Human Development
Luciferases
Toxicology
Real-Time Polymerase Chain Reaction
Transcription Factors
Fluorescence

Keywords

  • Ah receptor
  • CAFLUX
  • CALUX
  • Green fluorescent protein
  • Luciferase
  • Quantitative real time PCR

ASJC Scopus subject areas

  • Pharmacology, Toxicology and Pharmaceutics(all)
  • Molecular Medicine
  • Pharmacology (medical)

Cite this

He, G., Zhao, J., Brennan, J. C., Affatato, A. A., Zhao, B., Rice, R. H., & Denison, M. S. (2014). Cell-based assays for identification of aryl hydrocarbon receptor (AhR) activators. In Methods in Pharmacology and Toxicology (pp. 221-235). (Methods in Pharmacology and Toxicology). https://doi.org/10.1007/978-1-62703-742-6-13

Cell-based assays for identification of aryl hydrocarbon receptor (AhR) activators. / He, Guochun; Zhao, Jing; Brennan, Jennifer C.; Affatato, Alessandra A.; Zhao, Bin; Rice, Robert H.; Denison, Michael S.

Methods in Pharmacology and Toxicology. 2014. p. 221-235 (Methods in Pharmacology and Toxicology).

Research output: Chapter in Book/Report/Conference proceedingChapter

He, G, Zhao, J, Brennan, JC, Affatato, AA, Zhao, B, Rice, RH & Denison, MS 2014, Cell-based assays for identification of aryl hydrocarbon receptor (AhR) activators. in Methods in Pharmacology and Toxicology. Methods in Pharmacology and Toxicology, pp. 221-235. https://doi.org/10.1007/978-1-62703-742-6-13
He G, Zhao J, Brennan JC, Affatato AA, Zhao B, Rice RH et al. Cell-based assays for identification of aryl hydrocarbon receptor (AhR) activators. In Methods in Pharmacology and Toxicology. 2014. p. 221-235. (Methods in Pharmacology and Toxicology). https://doi.org/10.1007/978-1-62703-742-6-13
He, Guochun ; Zhao, Jing ; Brennan, Jennifer C. ; Affatato, Alessandra A. ; Zhao, Bin ; Rice, Robert H. ; Denison, Michael S. / Cell-based assays for identification of aryl hydrocarbon receptor (AhR) activators. Methods in Pharmacology and Toxicology. 2014. pp. 221-235 (Methods in Pharmacology and Toxicology).
@inbook{5f634257cbe54d799d402e5fda62f343,
title = "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.",
keywords = "Ah receptor, CAFLUX, CALUX, Green fluorescent protein, Luciferase, Quantitative real time PCR",
author = "Guochun He and Jing Zhao and Brennan, {Jennifer C.} and Affatato, {Alessandra A.} and Bin Zhao and Rice, {Robert H.} and Denison, {Michael S.}",
year = "2014",
doi = "10.1007/978-1-62703-742-6-13",
language = "English (US)",
isbn = "9781627037419",
series = "Methods in Pharmacology and Toxicology",
pages = "221--235",
booktitle = "Methods in Pharmacology and Toxicology",

}

TY - CHAP

T1 - Cell-based assays for identification of aryl hydrocarbon receptor (AhR) activators

AU - He, Guochun

AU - Zhao, Jing

AU - Brennan, Jennifer C.

AU - Affatato, Alessandra A.

AU - Zhao, Bin

AU - Rice, Robert H.

AU - Denison, Michael S.

PY - 2014

Y1 - 2014

N2 - 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.

AB - 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.

KW - Ah receptor

KW - CAFLUX

KW - CALUX

KW - Green fluorescent protein

KW - Luciferase

KW - Quantitative real time PCR

UR - http://www.scopus.com/inward/record.url?scp=84891942236&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84891942236&partnerID=8YFLogxK

U2 - 10.1007/978-1-62703-742-6-13

DO - 10.1007/978-1-62703-742-6-13

M3 - Chapter

SN - 9781627037419

T3 - Methods in Pharmacology and Toxicology

SP - 221

EP - 235

BT - Methods in Pharmacology and Toxicology

ER -