Third-Generation Ah Receptor–Responsive Luciferase Reporter Plasmids: Amplification of Dioxin-Responsive Elements Dramatically Increases CALUX Bioassay Sensitivity and Responsiveness

Abstract

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related dioxin-like chemicals are widespread and persistent environmental contaminants that produce diverse toxic and biological effects through their ability to bind to and activate the Ah receptor (AhR) and AhR-dependent gene expression. The chemically activated luciferase expression (CALUX) system is an AhR-responsive recombinant luciferase reporter gene-based cell bioassay that has been used in combination with chemical extraction and cleanup methods for the relatively rapid and inexpensive detection and relative quantitation of dioxin and dioxin-like chemicals in a wide variety of sample matrices. Although the CALUX bioassay has been validated and used extensively for screening purposes, it has some limitations when screening samples with very low levels of dioxin-like chemicals or when there is only a small amount of sample matrix for analysis. Here, we describe the development of third-generation (G3) CALUX plasmids with increased numbers of dioxin-responsive elements, and stable transfection of these new plasmids into mouse hepatoma (Hepa1c1c7) cells has produced novel amplified G3 CALUX cell bioassays that respond to TCDD with a dramatically increased magnitude of luciferase induction and significantly lower minimal detection limit than existing CALUX-type cell lines. The new G3 CALUX cell lines provide a highly responsive and sensitive bioassay system for the detection and relative quantitation of very low levels of dioxin-like chemicals in sample extracts.

Gaseous and particulate emission profiles during controlled rice straw burning

“…The PCDD/Fs were eluted and redisolved with 25 µL of dimethylsulfoxide (DMSO). The target compounds were analyzed using the enhanced recombinant mouse hepatoma CALUX cell line (H1L7.5 cells) which had been stably transfected with an Ah receptor-responsive firely luciferase reporter gene plasmid (pGudLuc7.5) (He et al, 2011). The results were expressed as dioxin toxicity equivalents (TEQ).…”

A sensitive, rapid, and simple DR-EcoScreen bioassay for the determination of PCDD/Fs and dioxin-like PCBs in environmental and food samples

“…A number of studies have reported the utility of these reporter gene assay systems as possible alternatives for the screening of dioxins in fish sold for human consumption (Tsutsumi et al 2003), human blood plasma (Croes et al 2011;Van Wouwe et al 2004), bovine milk (Bovee et al 1998), human milk , fly ash (Till et al 1997), and river, lake, and marine sediments (Jung et al 2012;Takigami et al 2005;Vondracek et al 2001;Xia et al 2014). In 2008, we developed a novel sensitive and rapid reporter gene assay (DR-EcoScreen assay) using a genetically engineered stable cell line, designated DR-EcoScreen cells, which can detect 2,3,7,8-TCDD at lower concentrations than other AhR-mediated reporter gene assays (Takeuchi et al 2008;He et al 2011). In this review, we report the application of a bioassay system using highly sensitive DR-EcoScreen cells to the determination of PCDD/Fs and DL-PCBs in ambient air samples, in fish and shellfish samples, and in flue gas samples from incinerators.…”

“…1c). He et al (2011) provided a comparison of the bioassay characteristics of current CALUX-type recombinant cell lines, including the relative potency of 2,3,7,8-TCDD to induce luciferase (EC 50 ) and the MDL for 2,3,7,8-TCDD in each cell line (Table 1). They reported that the EC 50 value in the DR-EcoScreen cell line (2.8 pM) is much lower than those reported for other cell lines.…”

Improved assays for xenosensor activation based on reverse transfection

“…Furthermore, the assays have been formally validated only in few reports (e.g. Cui et al, 2008;He et al, 2011). In addition, the high basal activity of CAR in transfected cells has often complicated the assay development (Molnár et al, 2013).…”