Title: Development of improved DR-CALUX bioassay for sensitive measurement of aryl hydrocarbon receptor activating compounds

Auteur(s): Sonneveld E ; van den Brink CE ; Zeinstra L ; Jansen JHJ ; van der Saag PT ; Brouwer A ; Van der Burg B ;  ; 

Year: 2002

Journal: Organohalogen Compounds

Subject(s): bioassay ; DR CALUX ; polyhalogenated aromatic hydrocarbons ; dioxins ; dioxin ; Ah receptor ; CYP1A1 ; Luciferase ; dioxin-like compounds ; gene expression ; analysis ; CALUX ; TCDD ;  ; 

Summary: Polyhalogenated aromatic hydrocarbons (PHAHs) including dioxins induce a wide variety of effects in mammals, birds and fish such as immunotoxicity, carcinogenicity and metabolic changes. The mechanism of action of PHAHs has been extensively studied over the past decades. These compounds bind to an intracellular receptor, known as the aryl hydrocarbon (Ah) receptor. Binding to the Ah receptor is followed by transportation of the PHAH-Ah receptor complex into the nucleus ofthe cell and subsequent binding to specific sequences in the DNA called dioxin responsive elements (DREs). Binding of the PHAH-Ah receptor to the DRE triggers the expression of DRE associated genes. By using this knowledge, a genetically modified cell-line (rat-hepatoma H4IIE) was designed containing part of the mouse CYP1A1 promoter including DREs coupled to part of the MMTV promoter and the firefly luciferase gene as a reporter for the presence of PHAHsl (pGudLuc1.1: Figure lA). This cell-line contains the complete machinery that is involved in the mode of action of dioxins and dioxin-like compounds. The thus created stable cell-line expresses the firefly luciferase gene in addition to genes that are normally expressed in the parental cells upon exposure to dioxins or dioxin-like compounds. As a consequence, these cells emit light which can very easily be quantified. The amount of light-production is related to the amount of dioxins or dioxin-like compounds in the exposure mixture. This well-established assay is called the DR-CALUX ® (Dioxin Responsive-Chemical Activated Luciferase gene eXpression) assay and has an advantage over traditional analysis of dioxins by HRGC-MS in terms of sensitivity, speed, sample clean-up/work-up, cost reduction, and biological relevance. Here we report on the development of an improved DR-CALUX ® H4IIE cell line, containing a multimerized DRE in front of a minimal promoter coupled to the firefly luciferase gene, thus lacking the CYPIAI and MMTV promoter regions. This has the advantage that regulation through promoter elements other than DREs is avoided and optimal selectivity for Ah-receptor interacting molecules is obtained. Combined with an optimized CALUX culture medium, a much-improved bioassay has been developed with a higher fold induction combined with a lower detection limit for TCDD.