THE CALUX (CHEMICAL-ACTIVATED LUCIFERASE EXPRESSION) ASSAY ADAPTED AND VALIDATED FOR MEASURING TCDD EQUIVALENTS IN BLOOD-PLASMA

A method was developed to isolate lipophilic compounds efficiently fro m small aliquots of blood plasma and test these for total dioxin-like toxic potency using recombinant rat (H4IIE) and mouse (Hepa1c1c7) hepa toma 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 el der ducks (Somateria mollissima) that had been dosed with 3,3',4,4'-te trachlorobiphenyl (PCB-77) or with the technical PCB-mixture Clophen A 50. For each sample the CALUX (chemical-activated luciferase expressio n) response of both the fat-containing organic extract and the fat-fre e, cleaned extract were compared with data from chemical analyses of t hese samples. The CALUX responses for the extracts were converted into so-called CALUX TEQs (TCDD equivalents), using a 2,3,7,8-tetrachlorod ibenzo-p-dioxin (TCDD) standard curve. The CALUX TEQs in both fatty an d cleaned extracts correlated significantly with PCB-77 or PCB-153 lev els (depending on the dosage group) determined in blood plasma using g as chromatography-mass spectrometry (CC-MS). For PCB-77 a toxic equiva lency factor (TEF) of 1.5 x 10(-3) was calculated based on these corre lations. In addition, PCB-118 and PCB-156 levels in abdominal fat (ass essed with GC with electron capture detection) and hepatic ethoxyresor ufin O-deethylase activities correlated well with the CALUX TEQs in bo th fatty and cleaned blood plasma extracts, suggesting the TEQ levels in blood offer a good measure for internal dose. Plasma cholesterol an d triglyceride levels were determined as a measure of lipid content, i n 10-mu l aliquots of blood plasma using enzymatic spectrophotometric determination. In conclusion, we have demonstrated that the CALUX assa y is a rapid, sensitive assay for assessing the toxic potency of (mixt ures of) AhR-active compounds in small aliquots of blood plasma. The l imit 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 mi of blood plasma in environmentally exposed species or man.