Title: Determination of dioxins and planar PCBs in fish using the GC/MS and CALUX bioassay: introduction of the screening approach for control purposes

Auteur(s): Hoogenboom R ; Portier L ; Onstenk C ; Polman T ; Hamers A ; Traag W ;  ; 

Year: 2000

Journal: Organohalogen Compounds

Subject(s): bioassay ; CALUX ; dioxin ; dioxins ; PCB ; PCBs ; validation ; Reporter gene ; Reporter gene assay ; reportergene assay ; Ah receptor ; Luciferase ; TCDD ; PAH ; clean-up ; milk ;  ; 

Summary: During the Belgian dioxin crisis in 1999 it has once again been demonstrated that there is a strong need for rapid screening methods for dioxins and related compounds. Together with the Agricultural University m Wageningen and the University of California in Davis, RIKILT has been involved in the development and validation of the so-called CALUX bioassay, a reporter-gene assay for Ah-receptor agonists. The assay is based on the increased production of the enzyme luciferase by hepatoma cells, following binding of e.g. dioxins to the Ah-receptor, transport of the complex to the nucleus and subsequent binding to a xenobiotic responsive element in the DNA. Exposure of the cells to TCDD will result in a dose-related formation of luciferase, which at the end of the exposure time can be measured in an enzyme assay by the production of light. The test has been shown to respond to the different dioxin and PCB-congeners in accordance to their TEF-values, although there is a tendancy for weaker agonists to give a relatively poor response. The response factor for e.g. PCB 105 (TEF of 10-4) was only 2.10-6. Other compounds like certain polyaromatlc hydrocarbons (PAHs), flavones and benzimidazoles may also cause a positive response, due to their capacity to bind to the Ah-receptor. When aiming at the selective detection of dioxins and planar PCBs this can partly be overcome by the use of longer incubation periods (24 h). allowing the cells to metabolise certain agonists and to degrade the luciferase produced during the first part of the incubation period in response to tbe exposure. An additional selection is obtained by the use of a clean-up procedure with acid silica (33% H2SO4) columns. The combined approach, CALUX-cells and acid silica clean-up, has been validated for milk fat, animal blood and citrus pulp. During the validation of the test for milk fat it became clear that suitable controls should be included for correction of results for 1) contaminants from the chemicals used in the clean-up, 2) deviations between WHO-TEFs and response factors in the assay and 3) recovery losses, especially since the test does not allow the use of internal standards. However, during the Brazilian citrus pulp crisis it turned out that any sample exceeding the residue limit still required GC/MS confirmation. Furthermore, quantification of dioxin levels in samples would not acknowledge the fact that the test is in principle a test for Ah-receptor agonists and not exclusively for dioxins or planar PCBs and that other agonists are not included in the TEQ-principle. For these reasons a more simple approach was chosen during last years crisis in which the response of the sample is compared with that of a reference sample included in the test. The level of the reference sample is such that a sample with a lower response is considered as negative, and a sample with a higher response as suspected, thus requiring GC/MS confirmation. The test approach included testing 10% of the negative samples with GC/MS. The test and in particular the procedure was accreditated by the Dutch Sterlab system. The present paper will demonstrate that based on the very low chance on false-negative results, the test is especially suitable for pointing out samples with dioxin levels below the residue limit. Furthermore, regarding the possible action of unknown agonist, the test functions best when operated in combination with te GC/MS reference method, allowing rapid investigation of suspected samples.