Development of a Recombinant Yeast Assay to Detect Ah-Receptor Ligands

Abstract

Endocrine systems of humans and animals are disturbed by dioxin-like compounds, which are ligands of the aryl hydrocarbon receptor (AhR). It is important to determine the accumulation of dioxin-like compounds in the environment for maintenance of human health. In this study, we developed a new method for screening ligands of the AhR using a yeast hybrid system. Reporter genes constructed by the insertion of dioxin response elements were integrated into HIS and lacZ yeast genomes. Then yeast was transformed with GAL4-activated domain-fused AhR and aryl hydrocarbon receptor nuclear translocator expression constructs. At 10⁻⁴ M of β-naphthoflavone, which is an AhR ligand, the absorbance of optical density at 600 nm (OD 600) and β-galactosidase activity was significantly increased. β-galactosidase activity was increased when the concentration of 3-methylcholanthrene (MC) was increased. ATP concentration increased as concentration of MC increased up to 10⁻¹⁰ M but decreased at higher concentrations. The concentration of ATP in the cell suspensions increased linearly with OD 600, used as an index of cell density (r² = 0.8366, F = 209.9, p < 0.0001, n = 44). The established yeast assay could possibly be used in the future to detect dioxin-like compounds in environmental samples.

Keywords :

• Dioxin-like Compounds
• Aryl Hydrocarbon Receptor
• Screening