The Calux (Chemical-Activated Luciferase Expression) Assay Adapted and Validated for Measuring TCDD Equivalents in Blood Plasma

Abstract

A method was developed to isolate lipophilic compounds efficiently from small aliquots of blood plasma and test these for total dioxin-like toxic potency using recombinant rat (H4IIE) and mouse (Hepa1c1c7) hepatoma cell lines, containing the firefly (Photinus pyralis) luciferase gene under trans-activational control of the aryl hydrocarbon receptor (AhR). For this experiment, blood plasma was used originating from eider ducks (Somateria mollissima) that had been dosed with 3,3Ј,4,4Ј-tetrachlorobiphenyl (PCB-77) or with the technical PCB-mixture Clophen A50. For each sample the CALUX (chemical-activated luciferase expression) response of both the fat-containing organic extract and the fat-free, cleaned extract were compared with data from chemical analyses of these samples. The CALUX responses for the extracts were converted into so-called CALUX TEQs (TCDD equivalents), using a 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) standard curve. The CALUX TEQs in both fatty and cleaned extracts correlated significantly with PCB-77 or PCB-153 levels (depending on the dosage group) determined in blood plasma using gas chromatography-mass spectrometry (GC-MS). For PCB-77 a toxic equivalency factor (TEF) of 1.5 ϫ 10 Ϫ3 was calculated based on these correlations. In addition, PCB-118 and PCB-156 levels in abdominal fat (assessed with GC with electron capture detection) and hepatic ethoxyresorufin O-deethylase activities correlated well with the CALUX TEQs in both fatty and cleaned blood plasma extracts, suggesting the TEQ levels in blood offer a good measure for internal dose. Plasma cholesterol and triglyceride levels were determined as a measure of lipid content, in 10-l aliquots of blood plasma using enzymatic spectrophotometric determination. In conclusion, we have demonstrated that the CALUX assay is a rapid, sensitive assay for assessing the toxic potency of (mixtures of) AhR-active compounds in small aliquots of blood plasma. The limit of detection for the CALUX assay is currently less than 0.1 fmol (32 fg) TEQ, which corresponds with the amount of TEQs present in 0.1 to 1 ml of blood plasma in environmentally exposed species or man.

Application of the Luciferase Recombinant Cell Culture Bioassay System for the Analysis of Polycyclic Aromatic Hydrocarbons

“…Furthermore, the rapidity of our analysis (which minimizes PAH metabolism) reveals that PAHs are more active than previously reported, suggesting that previous cell bioassay analyses may have underestimated the induction potency of PAHs. Other studies by our laboratory and collaborating investigators have shown that this bioassay method can also be used to evaluate Ah-active compounds present in a variety of different biological and nonbiological matrices, such as serum, sediment, and whole-product extracts [29,30,[33][34][35][36]. While such cell bioassay systems do not directly determine the toxicity of such AhR ligands, results can contribute to a more complete assessment of the potential biological and toxic effects of such exposure when combined with results using other methods, such as determination of acute toxicity, mutagenicity, and carcinogenicity.…”

“…Recently, several recombinant cell bioassay systems using luciferase-based reporters have been developed for the detection and relative quantitation of AhR-active chemicals and compounds in a variety of different biological and nonbiological matrices. Although most systems have emphasized analysis of HAH congeners [20][21][22][23], several have evaluated induction resulting from PAH analysis [24][25][26]. The most commonly used bioassays rely on recombinant hepatoma cell lines that contain a stably transfected AhR-responsive luciferase gene.…”

In Utero Exposure to Compounds with Dioxin-like Activity and Birth Outcomes

“…the Dr calUX bioassay has shown a high agreement with congener-specific methods and has been used in many epidemiologic and biomonitoring studies. 14,34,37,43-49 the protocol for sample processing has been presented in further details previously, 44,50 and additional details are provided in the eappendix (http://links.lww.com/eDe/a754). the assay analysis was performed on 791 mother and 269 cord blood samples with enough fat to express the bioassay results per gram fat.…”

Characterization of AhR agonist compounds in roadside snow

“…This bioassay is a welldocumented assay for measuring dioxin-like compounds [8][9][10]. The CALUX (also called DR-LUC) assay makes use of an AhR-reporter gene system where dioxins and dioxin-like compounds bind to and activate AhR-mediated luciferase activity in a genetically modified rat liver cell line.…”