TEQ-value determinations of animal feed; emphasis on the CALUX bioassay validation

Polyhalogenated aromatic hydrocarbons, such as polychlorinated dibenzo-p-dioxins are a large and diverse group of environmental pollutants. Their tendency to accumulate in the food chain and their toxicity make monitoring necessary. The reference analysis method is laborious and very expensive, therefore cheap and rapid bioassays have been developed. The chemical-activated luciferase bioassay (CALUX) bioassay uses a recombinant cell line, which responds to dioxins and dioxin-like molecules with Ah receptor (AhR)-dependent induction of firefly luciferase in a dose related response. The CALUX was tested for its use in the screening of feed. Aliquots of 20 g of enriched feed were extracted with a toluene:methanol mixture (20:4 v/v) and extracts were defatted on 33% H₂SO₄ silica columns and purified on carbon columns. Only the dioxin and furan fraction was analysed, the PCB fraction was discarded. The precision of the method is acceptable and in compliance with an R.S.D. <30% as suggested for cell-based bioassays in the Commission Directive 2002/70/EC of July 2002. The results evidence good agreement between TEQ-values obtained by either CALUX or GC–HRMS. The method is now routinely in use for a feed screening programme designed by the Federal Agency for the Safety of the Food chain. Approximately, 25 samples are analysed weekly. From the obtained results approximately 10% was confirmed by GC–HRMS. The false positive ratio is 1% and no false negatives were found, making the use of the CALUX technology advantageous.     

Bioanalytical methods applied to endocrine disrupting polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans. A review

The usual methods for determining polychlorinated dibenzo-p-dioxins (PCDD), polychlorinated dibenzofurans (PCDF) and polychlorinated biphenyls (PCB) are generally expensive and time consuming. This fact has favored the development of faster and cheaper techniques, based on immunoassays and bioassays. This paper reviews these bioanalytical methods and their analytical importance at the present moment.     

Importance of REP values when comparing the CALUX bioassay results with chemoanalyses results Example with spiked vegetable oils

Differences between chemical activated luciferase gene expression (CALUX) bioassay and chemoanalyses results are observed.This paper shows that calculations of the TEQ values using REP values instead of WHO TEF values give different results. The REP values do affect the results obtained by the CALUX technique. These differences are more marked for the dioxin like PCB compounds (CALUX TEQ values are lower than WHO TEQ values) than for the dioxin compounds (CALUX TEQ values are higher than WHO TEQ values).The CALUX results were compared with the concentrations of the congeners’ spiked into the oil.     

Recombinant cell bioassay systems for the detection and relative quantitation of halogenated dioxins and related chemicals

Proper epidemiological, risk assessment and exposure analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related halogenated aromatic hydrocarbons (HAHs) requires accurate measurements of these chemicals both in the species of interest and in various exposure matrices (i.e. biological, environmental, food and feed). High-resolution instrumental analysis techniques are established for these chemicals, however, these procedures are very costly and time-consuming and as such, they are impractical for large scale sampling studies (i.e. for epidemiological studies and assessment of areas with widespread contamination). Accordingly, numerous bioanalytical methods have been developed for the detection of these chemicals in extracts from a variety of matrices, the majority of which take advantage of the ability of these chemicals to activate the aromatic hydrocarbon receptor (AhR) and the AhR signal transduction pathway. Here we review the currently available in vitro AhR-based cell bioassay systems with a focus on recent recombinant reporter gene cell lines that have been developed for detection and relative quantitation of TCDD and related HAHs. Comparison of the relative sensitivities of the various cell bioassays and examples of their use in screening and analysis of environmental, biological, and food and feed samples are presented. Currently available experimental results and validation studies demonstrate the utility of these cell bioassay systems to provide a relatively rapid, accurate, and cost effective screening approach for the detection of TCDD and related HAHs in a variety of environmental, biological, food and feed samples. The availability of these cell bioassay systems will not only facilitate the large scale sampling studies needed for accurate assessment of contamination and exposure to these environmental chemicals, but they provide avenues for the identification of novel classes of TCDD-like chemicals.     

Interpretation of CALUX results in view of the EU maximal TEQ level in milk

Analyses of dioxins in food have become increasingly important since the European Commission has enforced maximal toxic equivalent concentration (TEQ) levels in various food and feed products. Screening methodologies are usually used to exempt those samples that are below the maximum permitted limit and that can, therefore, be released to the market. In addition, one needs to select those samples that require confirmation of their dioxin TEQ level. When bioassays are used as screening tools, the interpretation of the obtained results should consider the higher variability and uncertainty associated with them. This paper explores the use of CALUX data as quantitative screening results. The validation of the method for the polychlorinated dibenzo-p-dioxins (PCDD)/F TEQ determination in milk samples is described with emphasis on the decision limit (CC) and the precision of the method. The decision limit amounts to 4.53 pg TEQ/g fat. Repeatability and within-lab reproducibility coefficients of variation are below 30%. The newly introduced parameter CC^* of 1.47 pg TEQ/g fat delimits with CC a range of suspicious results. These data are not significantly different from the maximum limit of 3 pg TEQ/g fat and should be confirmed by a confirmatory analytical method such as HRGC–HRMS.     

The German bakery waste incident; use of a combined approach of screening and confirmation for dioxins in feed and food

During the last six years several incidents have occurred with dioxins in feed, stressing the need for rapid screening methods for these compounds. The most recent incident was the contamination of bakery waste used for animal feed due to the use of waste wood for drying of the material. In addition to Germany, the material was also shipped to the Netherlands. Levels up to 12 ng TEQ/kg have been detected, being about 15 times over the current limit of 0.75 ng TEQ/kg. In the Netherlands a combined strategy of screening with the CALUX-bioassay and the HRGC/HRMS confirmatory method was used to rapidly control the incident. Pigs were contaminated by the incident but only to a very limited extent. Despite the rather low limits for pig meat, the CALUX bioassay showed excellent performance, once again confirming the value of this assay.     

Elucidating the aryl hydrocarbon receptor antagonism from a chemical-structural perspective

ABSTRACT

The aryl hydrocarbon receptor (AhR) plays an important role in several biological processes such as reproduction, immunity and homoeostasis. However, little is known on the chemical-structural and physicochemical features that influence the activity of AhR antagonistic modulators. In the present report, in vitro AhR antagonistic activity evaluations, based on a chemical-activated luciferase gene expression (AhR-CALUX) bioassay, and an extensive literature review were performed with the aim of constructing a structurally diverse database of contaminants and potentially toxic chemicals. Subsequently, QSAR models based on Linear Discriminant Analysis and Logistic Regression, as well as two toxicophoric hypotheses were proposed to model the AhR antagonistic activity of the built dataset. The QSAR models were rigorously validated yielding satisfactory performance for all classification parameters. Likewise, the toxicophoric hypotheses were validated using a diverse set of 350 decoys, demonstrating adequate robustness and predictive power. Chemical interpretations of both the QSAR and toxicophoric models suggested that hydrophobic constraints, the presence of aromatic rings and electron-acceptor moieties are critical for the AhR antagonism. Therefore, it is hoped that the deductions obtained in the present study will contribute to elucidate further on the structural and physicochemical factors influencing the AhR antagonistic activity of chemical compounds.     

Validation of the CALUX bioassay for PCDD/F analyses in human blood plasma and comparison with GC-HRMS

Following the dioxin crisis of 1999, several studies were conducted to assess the impact of this crisis on the dioxin body burden in the Belgian population. The Scientific Institute of Public Health identified a population from whom plasma samples were available and from whom, during the follow up survey, plasma samples were obtained in 2000. In total, 496 samples were collected for GC-HRMS and CALUX analyses to verify statistical assessment conclusions. This study was seen as an opportunity to validate the CALUX bioassay for biological sample analysis and to compare toxic equivalency (TEQ) values obtained by the reference GC-HRMS technique and by the screening method. This article focuses on the validation results of the CALUX bioassay for the analyses of the dioxin fractions of blood plasma. The sample preparation is based on a liquid–liquid extraction, followed by an acid silica in series with an activated carbon clean-up. A good recovery (82%) and reproducibility (coefficient of variation less than 25%) were found for this method. Based on 341 plasma samples, a significant correlation was established between the bioassay and chemical method (R = 0.64). However, a proportional systematic error was observed when the results obtained with the CALUX bioassay were regressed with the results from the GC-HRMS analyses. The limit of quantification (LOQ) used to calculate TEQ values from the GC-HRMS determinations, the use of the relative potency values instead of the toxic equivalent factor and the potential of CALUX bioassay to measure all compounds with affinity for the AhR may partly explain this proportional systematic error. Nevertheless, the present results suggest that the CALUX bioassay could be a promising valid screening method for human blood plasma analyses.     

Enhancing the response of CALUX and CAFLUX cell bioassays for quantitative detection of dioxin-like compounds

Reporter genes produce a protein product in transfected cells that can be easily measured in intact or lysed cells and they have been extensively used in numerous basic and applied research applications.Over the past 10 years,reporter gene assays have been widely accepted and used for analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related dioxin-like compounds in various types of matrices,such as biological,environmental,food and feed samples,given that high-resolution instrumental analysis techniques ...     

CALUX measurements: statistical inferences for the dose-response curve

Chemical Activated LUciferase gene eXpression [CALUX] is a reporter gene mammalian cell bioassay used for detection and semi-quantitative analyses of dioxin-like compounds. CALUX dose-response curves for 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD] are typically smooth and sigmoidal when the dose is portrayed on a logarithmic scale. Non-linear regression models are used to calibrate the CALUX response versus TCDD standards and to convert the sample response into Bioanalytical EQuivalents (BEQs). Several complications may arise in terms of statistical inference, specifically and most important is the uncertainty assessment of the predicted BEQ. This paper presents the use of linear calibration functions based on Box-Cox transformations to overcome the issue of uncertainty assessment. Main issues being addressed are (i) confidence and prediction intervals for the CALUX response, (ii) confidence and prediction intervals for the predicted BEQ-value, and (iii) detection/estimation capabilities for the sigmoid and linearized models. Statistical comparisons between different calculation methods involving inverse prediction, effective concentration ratios (ECR_20-50-80) and slope ratio were achieved with example datasets in order to provide guidance for optimizing BEQ determinations and expand assay performance with the recombinant mouse hepatoma CALUX cell line H1L6.1c3.