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. 

ER-CALUX Estrogen receptor-mediated chemically activated luciferase gene expression... 

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

Site no. MSP Mutatox DR-CALUX (with clean-up dry sediment) Pore water ER-CALUX on polar fraction sediment YES on 10 X diluted sediment... 

Intra- and interlaboratory calibration of the DR CALUX bioassay for the analysis of dioxins and dioxin-like chemicals in sediments... 

The infralaboratory calibration study was performed by the Institute for Environmental Studies. Sediment was extracted and cleaned up as indicated here. The determination of dioxin and/or dioxin-like content was according to the method indicated under the section DR CALUX analysis. For the intralaboratory study, the following parameters were investigated limit of detection (LOD), limit of quantitation (LOQ), and reproducibility and repeatability of the bioassay. 

Phase 2. In the seeond phase of the study, the partieipants were asked to analyze three extraeted and cleaned sediment samples using the DR CALUX bioassay. Sediments used for extraetion and cleanup were freshwater sediments from the Western Seheldt, The Netherlands. The sediment extracts were prepared by the Royal Institute for Fishery Research (RIVODLO), IJmuiden, The Netherlands, aeeording to the protoeol given here. Dilutions of the supplied sediment extracts were prepared by the partieipants in DMSO and tested for dioxin and/or dioxinlike content. On each 96-well mierotiter plate, a 2,3,7,8-TCDD standard ealibration curve was analyzed. Raw data as well as eonverted data were used for statistieal evaluation. 

Defined standard solutions. The two defined standard solutions were prepared by dissolving either 2,3,7,8-TCDD or 2,3,7,8-TCDD, PCB 126, and PCB 169 in DMSO. The 2,3,7,8TCDD standard solution contained 2,3,7,8-TCDD in DMSO at a concentration of 7.5 nM. Sample 2 contained a mixture of 2,3,7,8 TCDD, PCB 126, and PCB 169 at concentrations of 5.0, 25, and 250 nM, respectively. The total 2,3,7,8-TCDD TEQ content of this mixture was calculated using both World Health Organization (Paris, France) WHO-TEF values and DR CALUX-relative potency (REP) values (Hosoe et al, 2002) and found to be 10 and 7.5 nM 2,3,7,8-TCDD TEQ, respectively. Overall, 27 individual measurements per sample and per participant were available for data analysis. 

On each 96-well microtiter plate, a eomplete 2,3,7,8-TCDD standard concentration range was incubated and analyzed in triplieate. A eurve fit of the 2,3,7,8-TCDD standard range was produced for the calculation of DR CALUX TEQ content in the samples tested. The analyzed relative light units (RLU) from the samples were interpolated on the 2,3,7,8-TCDD standard curve, and the DR CALUX TEQ content was quantified between the limit of quantitation (LOQ) and the concentration of 2,3,7,8-TCDD at whieh 50% of the maximum response is observed (EC50). 

For the determination of the limit of detection (LOD) and LOQ, 10 standard 2,3,7,8-TCDD calibration series were analyzed in triplicate using the DR CALUX bioassay. In Figure 1, a typical example of a standard 2,3,7,8-TCDD calibration curve is given. For each individual calibra-... 

For the determination of the repeatability, two sediments originating from coastal areas along the Duteh eoastline were extracted and cleaned up. One ofthe sediments had a low 2,3,7,8-TCDD TEQ eontent, whereas the second sediment had a relatively high 2,3,7,8-TCDD TEQ eontent (4.8 and 26 pg 2,3,7,8-TCDD TEQ/g sediment, respectively). The 2,3,7,8-TCDD TEQ eontent in both extracts was determined by DR CALUX analysis 10 times on the same day. The reprodueibility was determined by analyzing a 3-pM 2,3,7,8-TCDD standard and a cleaned sediment extraet. Both samples were analyzed on 10 different days and by various persons. The results are summarized in Table 1. 

Both the EC50 values and the 3-pM point of the 2,3,7,8TCDD ealibration curve serve as quality criteria. For each participant, the results for both data points from all 96-well plates analyzed during the presented study were collected and reeorded in Shewhart control charts. The Shewhart control chart is used to identify variations on performanee of the DR CALUX bioassay brought about by unexpected or unassigned causes. The Shewhart eontrol chart shows the mean of the EC50 and 3-pM control point and the upper and lower eontrol limits. In Figure 2, a typical Shewhart control chart is shown. Over the analysis period, none of the participants exceeded the aetion levels (AVG 3 S). 

Phase 1 Defined standard solutions. Partieipants were asked to measure the response of the two standard samples in the DR CALUX bioassay three times in triplicate. The total DR CALUX 2,3,7,8-TCDD TEQ content of both the 2,3,7,8TCDD sample as well as the mixed sample was calculated to be 7.5 uM TEQ. Since the DMSO eontent during exposure was 0.4% and the samples were diluted seven times by all partieipants, the expected DR CALUX TEQ... 

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