TY - JOUR
T1 - Development of a green fluorescent protein-based cell bioassay for the rapid and inexpensive detection and characterization of Ah receptor agonists
AU - Nagy, Scott R.
AU - Sanborn, James R.
AU - Hammock, Bruce D.
AU - Denison, Michael S.
PY - 2002
Y1 - 2002
N2 - The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates the toxic and biological effects of a variety of chemicals. Although halogenated and polycyclic aromatic hydrocarbons (HAHs and PAHs, respectively) represent the highest affinity and most toxic ligands, recent studies have demonstrated that the AhR can be activated by chemicals with structures distinctly different from HAHs/PAHs. In order to identify and characterize novel AhR ligands, we developed a rapid and inexpensive high-throughput screening bioassay based on the ability of AhR agonists to induce an HAH/PAH-responsive, enhanced green fluorescent protein (EGFP) reporter gene in a stably transfected mouse hepatoma (Hepa1c1c7) cell line. EGFP induction in the resulting recombinant cell line, H1G1.1c3, is sensitive (with a minimal 1-pM detection limit for 2,3,7,8-tetrachlorodibenzo-p-dioxin, the most potent AhR ligand), and it responds to HAHs and PAHs in a time-, dose-, and chemical-specific manner. Application of this bioassay was demonstrated by the rapid characterization of the relative inducing potency of a series of previously uncharacterized dioxin surrogates. This bioassay system has numerous advantages over currently available AhR-based bioassays including increased rapidity and ease of use, low reagent cost, and application for high-throughput screening.
AB - The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates the toxic and biological effects of a variety of chemicals. Although halogenated and polycyclic aromatic hydrocarbons (HAHs and PAHs, respectively) represent the highest affinity and most toxic ligands, recent studies have demonstrated that the AhR can be activated by chemicals with structures distinctly different from HAHs/PAHs. In order to identify and characterize novel AhR ligands, we developed a rapid and inexpensive high-throughput screening bioassay based on the ability of AhR agonists to induce an HAH/PAH-responsive, enhanced green fluorescent protein (EGFP) reporter gene in a stably transfected mouse hepatoma (Hepa1c1c7) cell line. EGFP induction in the resulting recombinant cell line, H1G1.1c3, is sensitive (with a minimal 1-pM detection limit for 2,3,7,8-tetrachlorodibenzo-p-dioxin, the most potent AhR ligand), and it responds to HAHs and PAHs in a time-, dose-, and chemical-specific manner. Application of this bioassay was demonstrated by the rapid characterization of the relative inducing potency of a series of previously uncharacterized dioxin surrogates. This bioassay system has numerous advantages over currently available AhR-based bioassays including increased rapidity and ease of use, low reagent cost, and application for high-throughput screening.
KW - Ah receptor
KW - Dioxin
KW - GFP
KW - Green fluorescent protein
KW - HAH
KW - TCDD
UR - http://www.scopus.com/inward/record.url?scp=0036158617&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0036158617&partnerID=8YFLogxK
U2 - 10.1093/toxsci/65.2.200
DO - 10.1093/toxsci/65.2.200
M3 - Article
C2 - 11812924
AN - SCOPUS:0036158617
VL - 65
SP - 200
EP - 210
JO - Toxicological Sciences
JF - Toxicological Sciences
SN - 1096-6080
IS - 2
ER -