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

Abstract

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.

Host and environmental determinants of polychlorinated aromatic hydrocarbons in serum of adolescents.

“…Previous studies, in which dioxin congeners were determined separately by using gas chromatography with high-resolution mass spectrometry, and in which the TEQ value was calculated by multiplying concentrations of individual dioxin congeners by their respective toxic equivalent factors (TEFs), should be interpreted cautiously, because TEFs for individual congeners are theoretical values based on various toxicologic end points obtained in different studies (16). Moreover, TEQs are usually assumed to be additive and not synergistic or antagonistic (17). The CALUX assay used in the present study is mechanistically based and thus measures all compounds in the serum that act via binding to the AhR.…”

“…We performed the extraction and cleanup procedures as described in detail elsewhere (16)(17)(18). Briefly, the method involved nhexane extraction of the sample and removal of acid-labile matrix components, fat, and nonstable PCAHs by passage through a silica column containing concentrated H 2 SO 4 (33%, w/v).…”

Analysis of dioxins in contaminated soils with the CALUX and CAFLUX bioassays, an immunoassay, and gas chromatography/high‐resolution mass spectrometry

“…However, deviations between the TEF values for individual congeners and their relative effective potency to induce a bioassay response, and contributions from AhR ligands for which no TEFs are available, could in principle lead to discrepancies between bioassay and GC/HRMS results. Nevertheless, these bioassays have been successfully used in several applications, including assessments of dioxin-like potency in sediments and pore water, blood plasma, sewage sludge, food control, and analyses of polycyclic aromatic hydrocarbons (PAHs) [13][14][15][16][17]. Intra-and interlaboratory comparison studies have confirmed their usefulness in dioxin analysis of soils and sediments [18][19][20].…”

Dioxin-like components in human breast milk collected from Hong Kong and Guangzhou

“…The highest induction of EROD activities and thus the Ah receptor binding affinities were by 2,3,7,8-TCDD, followed by B[a]P, TCDT, and b-NF, with EC 50 values of about 1.6, 480, 570, and 25,000 pg/mL, respectively. The detection limit in this assay system was 40 fg TCDD/well, which was comparable to the ChemicalActivated Luciferase Expression (CALUX) assay, with a detection limit of 32 fg/well (Murk et al, 1997). There was no EROD induction by p,p 0 -DDT, p,p 0 -DDE, and DMSO (data not shown).…”