The calux (chemical‐activated luciferase expression) assay adapted and validated for measuring TCDD equivalents in blood plasma

Abstract

AbstractA 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 3,3′,4,4′‐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 × 10−3 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‐μl 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.