Assay CALUX

The toxicology of PCBs is complex and not fully understood. Coplanar PCBs interact with the Ah-receptor, with consequent induction of cytochrome P4501A1/2 and Ah-receptor-mediated toxicity. Induction of P4501A1 provides the basis of valuable biomarker assays, including bioassays such as CALUX. Certain PCBs, for example, 3,3, 4,4 -TCB, are converted to monohydroxymetabolites, which act as thyroxine antagonists. PCBs can also cause immunotoxicity (e.g., in seals). 

Stronkhurst, J., Leonards, P., and Murk, A.J. (2002). Using the dioxin receptor Calux in vitro assay to screen marine harhour sediments for a dioxin-hke mode of action. Environmental Toxicology and Chemistry 21, 2552-2561. 

Mol-based estradiol equivalency factors of (xeno-)estrogens in the ER-CALUX assay (adapted from Ref. [28])... 

Houtman, C. J., Booij, R, Jover, E., Pascual del Rio, D., Swart, K., Velzen, M. van, Vreuls, R., Legler, J., Brouwer, A. and Lamoree, M.H. (2006). Estrogenic and dioxin-like compounds in sediment fi om Zierikzee harbour identified with CALUX assay-directed fractionation combined with one and two dimensional chromatography analyses. Chemosphere, 65, 2244-2252. 

Murk, A.J., Denison, M.S., Leonards, P.E.G., Bulder, A.S., Jonas, A.S., Rozemeijer, M.J.C., Koeman, J.H., Brouwer, A. (1997). The CALUX assay adapted and validated for measuring TCDD equivalents in blood plasma. Environ Toxicol Chem 16 1583—1589. 

Table 5.1 Reproducibility of the CALUX assay with milk fat samples. Spiked samples were tested singly in three independent test series (adapted from 12)...

CALUX determined levels were corrected for the blank sample being respectively 6.3, 2.0 and 3.5 pg/g fat for series 1, 2 and 3 respectively. In addition values were corrected for the difference between relative responses in the CALUX assay and i-TEF values. 

An additional screening test for TCDD-like (aryl hydrocarbon receptor, AhR, active) chemicals has been developed (Garrison et al. 1996) and is available commercially (Anonymous 1997). Dubbed the CALUX (for chemically activated luciferase gene expression) system, the assay is based on recombinant cell lines into which researchers have inserted a firefly luciferase gene. When exposed to dioxin-like compounds, the recombinant cells luminesce. The method is sensitive to ppt levels of 2,3,7,8-TCDD equivalents in blood, serum, and milk (Anonymous 1997). Samples testing positive can be subjected to more definitive and specific analytical testing. 

The CALUX assay described in Section 6.1 has been applied to Ah receptor-active compounds (not limited to dioxins) in sediments and pore waters (Murk et al. 1996) and to blood with mixed results. Sensitivities as low as 0.5 fmol of 2,3,7,8-TCDD were reported. Two polychlorinated terphenyl mixtures, the PCB-substituted Ugilec 141, polybrominated diphenyl ethers, and the PCB mixture Clophen 150 were tested in the CALUX assay and had induction potencies that were 10"4 to 10"7 compared to TCDD. Thus, this assay is more selective than earlier, induction-based assays, although clearly not as selective as GC/MS. 

Biosensors differ from bioassays mainly by the fact that in bioassays the transducer is not an integral part of the analytical system and biosensors can extract quantitative analytical information of single compounds in complex mixtures. One example is the determination of concentrations of dioxin-like compounds in the blood and environmental samples using the Calux assay, where within a complex matrix its levels are determined with great accuracy (see, e.g., Murk et al. 1997). Additionally, compounds that are difficult to detect (e.g., surfactants, chlorinated hydrocarbons, sulfophenyl carboxylates, dioxins, pesticide metabolites) can more easily be evaluated using biosensors. 

This section presents relative estrogenic potential determined by E-SCREEN and ER-CALUX, two well recognized in-vitro assays for the determination of estrogenicity (see Table 5.3.3). 

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