The CALUX Bioassay

The chemically activated luciferase gene expression (CALUX) in vitro cell bioassay is a bioanalytical tool increasingly used by research and commercial laboratories for screening of dioxins and dioxin-like compounds in sample extracts. The assay is based on aromatic hydrocarbon receptor (AhR)-mediated firefly luciferase expression in genetically modified rat or mouse hepatoma cell lines that express firefly luciferase upon exposure to dioxins or dioxin-like compounds. Since CALUX analyses provide a biological response to all Ah-receptor active compounds present in a sample extract, interpretation of results is much more complex than that of chemical analyses, as an array of parameters affects the results. The Bioassay Research Laboratory at EU-RL for Dioxins and PCBs examines critical methodological parameters and aspects of the CALUX bioassay that can affect quality and accuracy of the analyses.

CALUX technology comprises various fields of work and attention: Cell culturing and maintenance, sample fat extraction, followed by selective clean-up techniques, exposure of cultured cells to cleaned-up sample extracts containing dioxins and/or dioxin-like compounds, measurement of luciferase activity and statistical evaluation and interpretation of results.

Performance Evaluation of the CALUX Technology

Biologically based techniques hold the promise of screening large numbers of samples and greatly decreasing the time and cost of analyses. These techniques are based on the measurement of cellular response produced by enzyme activation. As one of the tasks assigned by the European Commission, the performance of bioassay technology available on the European market is evaluated with regard to their suitability for routine use by European official laboratories, namely, the DR-CALUX [BioDetection Systems (BDS), Netherlands], involving genetically modified H4IIe rat hepatoma cells, and the XDS-CALUX [Xenobiotic Detection Systems, USA] technologies, the latter originating from the research laboratories of Prof. M.S.Denison, University of California Davis (USA), involving genetically modified H1L6.1c3 mouse hepatoma cells. Included in the scope of EU-RL’s activities were also H1L6.1c3 mouse hepatoma cells directly obtained from Prof. M.S.Denison, for research purposes.

Experimental studies are performed involving optimization of numerous parameters for cell growth, handling, seeding, exposure and measurement of luciferase activity. For a thorough insight into parameters influencing bioanalytical results, and to retrieve information on correspondence between bioanalytical and GC/HRMS results, matrix-matched calibration experiments are performed involving those types of samples for which maximum levels and action levels are set by the European Commission.

A statistical-mathematical approach was developed and implemented allowing to derive besides other important performance characteristics “bioassay cut-off values” for decision over sample compliance with the respective legal limits for each sample matrix of interest.

Comprehensive QC-System maintained

A comprehensive quality control system is maintained to monitor accuracy and reproducibility of bioanalytical results. Information on cell performance, affinity of dioxin-like compounds to the Ah-receptor, gene expression efficiency, inter-assay and intra-assay-variability of results and the working range of the standard curve can, besides other parameters, be derived from these data. Procedure blanks and reference samples for recovery control are analyzed with each series of experimental samples and used for background subtraction and recovery correction, respectively.

Performance Characteristics from Calibration Experiments

The process of analytical method validation should demonstrate that the method is fit for its intended purpose. Parameters evaluated are sample-related LODs and LOQs, trueness, precision, selectivity/specificity, linearity, working range and ruggedness. For this purpose, matrix-matched calibration experiments involving GC/HRMS pre-analyzed samples or spiked samples are carried out. From those experiments, sample-related, robust BEQ concentrations (bioassay cut-off values) are calculated, above which samples must be declared suspected to be non-compliant and submitted to GC/HRMS analysis. A calibration line is constructed and 95%-prediction intervals are calculated from the signal dispersion around the calibration line. Decision limits are calculated from the prediction bands.

Proposal for Amendment of Criteria for Bioanalytical Methods

Criteria for bioanalytical methods as laid down in Annexes I and II to Commission Regulation (EC) No 1883/2006 (food) and in Annex V (B) to Commission Regulation (EC) No 152/2009 (feed), respectively, are re-evaluated since September 2008 by a core working group chaired by an EU-RL representative. Results are included in a draft document submitted to the European Commission in early 2010.