Céline Xhrouet

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.

Importance of clean-up for comparison of TEQ-values obtained by CALUX and chemo-analysis

This paper presents Chemically Activated LUciferine gene eXpression (CALUX) TEQ-values obtained for nine plasma samples following two different purification procedures, one of them involving fractionation. CALUX results obtained for the dioxin (DX) and dioxin + PCB (DX + PCB) fractions were then compared to the GC-HRMS TEQ-values calculated for the 17 polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (17 PCDD/F) and 17 PCDD/F + 4 cPCB congeners, respectively. The overestimation of the CALUX (DX fraction) TEQ-values in comparison with the chemo-analyses of the 17 PCDD/F is mainly explained by the presence of other AhR agonists, like brominated compounds. Otherwise, the constancy of the CALUX (DX + PCB fraction) TEQ-value which compares to increasing the GC-HRMS (17 PCDD/F + 4 cPCB) TEQ results raises questions concerning (1) the significance of CALUX results obtained without fractionation as well as (2) the toxicological effect of a cocktail of contaminants on the human health.