Establishment of a cell-free bioassay for detecting dioxin-like compounds

Abstract

Dioxins are byproducts from incomplete combustion processes and belong to a group of mostly toxic chemicals known as persistent organic pollutants, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is considered to be the most toxic species of all dioxin-like compounds. Analytical chemical processes are employed to determine the specific dioxin content in environmental samples. However, cost-ineffectiveness and excess time consumption limit their routine utilization. The aryl hydrocarbon receptor (AhR) is the major TCDD receptor. Upon binding to dioxin, the AhR dissociates from Hsp90 and other cofactors. TCDD-bound AhR subsequently translocates to the nucleus and interacts with the AhR nuclear translocator (Arnt) to induce signal transduction. Here, we describe a highly sensitive and cost-effective alternative assay based on detecting stability of bioluminescence signals. We generated cells that stably co-express Renilla luciferase tagged-AhR (AhR-RL), Ah receptor-interacting protein (AIP), p23 and yellow fluorescent protein-tagged Arnt (Arnt-YFP) (AAPA cells) for detection of dioxin-like compounds. Treatment with 3-methylcholanthrene (3MC), AhR agonist, enhanced the interaction between AhR and Arnt and avoided proteosomal degradation. In addition, treatment with 3MC or TCDD stabilized Renilla luminescence from AhR-RL of AAPA cell-free extracts in a concentration-dependent manner. The TCDD detection limit in this cell-free system was as low as 10(-18 )M. These results highlight the potential of AAPAA cell-free extracts to detect dioxin-like pollutants.

Pharmacological activation of lysophosphatidic acid receptors regulates erythropoiesis

“…Production of lentiviral stocks was described in detail in our previous study 47 . The LPA 2 shRNA template was inserted into the pLKO.1 lentiviral vector = purchased from the National RNAi Core Facility Platform, Academia Sinica.…”

Quinone-mediated induction of cytochrome P450 1A1 in HepG2 cells through increased interaction of aryl hydrocarbon receptor with aryl hydrocarbon receptor nuclear translocator

“…To assess AhR activation by 1,2-NQ, we used a cell free assay developed by Wang et al (2013) to detect dioxin and dioxin-like compounds, also AhR activators. Exposure to 3-methylcholanthrene (1 nM) caused an approximately 1.3-fold activation, compared with control, of the luminescence signal (data not shown), suggesting that the assay could sense low concentrations of AhR activator.…”

“…The bioluminescence-based bioassay was performed as previously described (Wang et al, 2013) with modifications. Briefly, the coding region sequences of AhR (NM_001621), ARNT (NM_001668) and HSP90 (NM_007355) were amplified using cDNA derived from HEK293 cells.…”

Aryl hydrocarbon receptor‐based bioassays for dioxin detection: Thinking outside the box

“…Moreover, cells expressing heat shock protein 90‐YFP were used to monitor the decrease of Renilla luminescence in the presence of dioxin‐like compounds, which is dose‐dependent and reflects the degradation of the AhR‐Renilla fusion by the proteasome. Quite remarkably, this method allowed the detection of 10 −18 m TCDD (Wang, Wu et al, ).…”