title: Validation and use of the CALUX-bioassay for the determination of dioxins and PCBs in bovine milk.
abstract: There is a strong need for the development of relatively cheap and rapid bioassays for the determination of dioxins and related compounds in food. A newly developed CALUX (Chemical-Activated LUciferase gene eXpression) bioassay was tested for its possible use to determine low levels of dioxins in bovine milk. Data show that this mammalian cell-based test is very sensitive for 2,3,7,8-substituted dioxins and related PCBs, thereby reflecting the relative potencies of these compounds in comparison to TCDD (TEF-values). The limit of detection was about 50 fg of TCDD. Furthermore, the response obtained with a mixture of dioxins was additive, in accordance with the TEF-principle. Milk fat was isolated by centrifugation followed by clean-up of the fat with n-pentane, removal of the fat on a 33% H2SO4 silica column, and determination of Ah receptor agonist activity with the CALUX-bioassay. An equivalent of 67 mg fat was tested per experimental unit, resulting in a limit of quantification around 1 pg i-TEQ/g fat. To investigate the performance of the method, butter fat was cleaned and spiked with a mixture of 17 different 2,3,7,8-substituted PCDD and PCDF congeners at 1, 3, 6, 9, 12 and 15 pg TEQ/g fat, as confirmed by GC/MS. In this concentration range, the method showed a recovery of TEQs around 67% (58-87%). The reproducibility, determined in three independent series showed a CV varying between 4% and 54%, with the exception of the sample spiked at 1 pg i-TEQ (CV 97%). The repeatability determined with the sample spiked at 6 pg i-TEQ/g showed a CV of 10%. Testing of 22 bovine milk samples, taken at different sites in The Netherlands, in the CALUX-assay showed combined dioxin and dioxin-like PCB levels equivalent to 1.6 pg TCDD/g fat (range 0.2-4.6). GC/MS analysis of these samples revealed an average level of 1.7 pg i-TEQ/g fat, varying between 0.5 and 4.7 pg i-TEQ/g fat. All five samples showing a GC/MS determined dioxin content of more than 2 pg i-TEQ/g fat gave a response in the CALUX-assay corresponding to more than 2 pg TCDD/g fat. These data clearly show that the CALUX-bioassay is a promising method for the rapid and low cost screening of dioxins in bovine milk.
authors.affiliation: State Institute for Quality Control of Agricultural Products (RIKILT-DLO), Wageningen, The Netherlands.
authors.forename: T F
authors.initials: TF
authors.lastname: Bovee
authors.forename: L A
authors.initials: LA
authors.lastname: Hoogenboom
authors.forename: A R
authors.initials: AR
authors.lastname: Hamers
authors.forename: W A
authors.initials: WA
authors.lastname: Traag
authors.forename: T
authors.initials: T
authors.lastname: Zuidema
authors.forename: J M
authors.initials: JM
authors.lastname: Aarts
authors.forename: A
authors.initials: A
authors.lastname: Brouwer
authors.forename: H A
authors.initials: HA
authors.lastname: Kuiper
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002417:Cattle; D004147:Dioxins / Q000032:analysis*; D004305:Dose-Response Relationship, Drug; D008401:Gas Chromatography-Mass Spectrometry; D008156:Luciferases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D008892:Milk / Q000737:chemistry*; D011078:Polychlorinated Biphenyls / Q000032:analysis*; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D051381:Rats; D015203:Reproducibility of Results; D014407:Tumor Cells, Cultured
chemical_list: D004147:Dioxins; D000072317:Polychlorinated Dibenzodioxins; D011078:Polychlorinated Biphenyls; D008156:Luciferases