Chemical-activated Luciferase Gene Expression Bioassay Research Articles

Brominated Dioxins in Egg, Broiler, and Feed Additives: Significance of Bioassay-Directed Screening for Identification of Emerging Risks in Food.

Food authorities aim to safeguard our food. This requires sensitive analyses to guarantee detection of both banned and regulated substances at low concentrations. At the same time, broad screening methods are needed to identify new emerging risks. For this purpose, effect-based bioassays combined with mass spectrometric analyses offer an advantage. During the regular monitoring of dioxins in agricultural products, a discrepancy was observed between the results of the DR CALUX (Dioxin-Responsive Chemical Activated Luciferase gene Expression) bioassay and the confirmatory gas chromatographic high resolution mass spectrometric (GC-HRMS) analysis in egg and broiler fat samples. The response in the bioassay was high, suggesting a clear exceedance of the maximum limits of dioxins in these samples, yet regulated dioxins or dl-PCBs were not detected by GC/HRMS analysis. Ultimately, a broad screening analysis using GC-HRMS resulted in the identification of 2,3,7,8-tetrabromo-dibenzofuran (2,3,7,8-TBDF) in both egg and broiler fat. To investigate the potential source of this brominated furan contaminant, different samples were analyzed: bedding material, poultry feed, feed additives (choline chloride and l-lysine), and seaweed. The poultry feed and feed additives all contained 2,3,7,8-TBDF. Using a feed-to-food transfer model, it became clear that the poultry feed was probably the source of 2,3,7,8-TBDF in broilers and eggs through a feed additive like L-lysine or choline chloride. This study underlines the importance of using a combination of effect-based screening assays with sensitive analytical methods to detect potential new and emerging risks.

Bioanalytical screening of low levels of dioxins and dioxin-like PCBs in pig meat (pork) for checking compliance with EU maximum and action levels using highly sensitive \u201cthird generation\u201d recombinant H4L7.5c2 rat hepatoma cells

BackgroundLow maximum and action levels set by the European Union for polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (DL-PCBs) in pig meat (pork) have led to a demand for reliable and cost-effective bioanalytical screening methods implemented upstream of gas chromatography/high-resolution mass spectrometry confirmatory technology, that can detect low levels of contamination in EU-regulated foods with quick turn-around times.ResultsBased on the Chemically Activated LUciferase gene eXpression (CALUX) bioassay, extraction and clean-up steps were optimized for recovery and reproducibility within working ranges significantly lower than in current bioassays. A highly sensitive “3rd generation” recombinant rat hepatoma cell line (H4L7.5c2) containing 20 dioxin responsive elements was exposed to pork sample extracts, and their PCDD/Fs and DL-PCBs levels were evaluated by measuring luciferase activity. The method was validated according to the provisions of Commission Regulation (EU) 2017/644 of 5 April 2017 with spiking experiments performed selectively for PCDD/Fs and DL-PCBs and individual calibration for PCDD/Fs, DL-PCBs and the calculated sum of PCDD/Fs and DL-PCBs. The resulting performance parameters met all legal specifications as confirmed by re-calibration using authentic samples. Cut-off concentrations for assessing compliance with low maximum levels and action levels set for PCDD/Fs and DL-PCBs within a range of 0.50–1.25 pg WHO-TEQ/g fat were derived, ensuring low rates of false-compliant results (ß-error < 1%) and keeping the rate of false-noncompliant results well under control (α-error < 12%).ConclusionsWe present a fast and efficient bioanalytical routine method validated according to the European Union’s legal requirements on the basis of authentic samples, allowing the analyst to reliably identify pork samples and any other EU-regulated foods of animal origin suspected to be noncompliant with a high level of performance and turn-around times of 52 h. This was facilitated in particular by a quick and efficient extraction step followed by selective clean-up, use of a highly sensitive “3rd generation” H4L7.5c2 recombinant rat hepatoma cell CALUX bioassay, and optimized assay performance with improved calibrator precision and reduced lack-of-fit errors. New restrictions are proposed for the calibrator bias and the unspecific background contribution to reportable results. The procedure can utilize comparably small sample amounts and allows an annual throughput of 840–1000 samples per lab technician. The described bioanalytical method contributes to the European Commission's objective of generating accurate and reproducible analytical results according to Commission Regulation (EU) 2017/644 across the European Union.

Expression of aryl hydrocarbon receptor, inflammatory cytokines, and incidence of rheumatoid arthritis in Vietnamese dioxin-exposed people

Many Vietnamese citizens have been and continue to be inadvertently exposed to dioxins and dioxin-like compounds deposited in the country during the Vietnam War. Dioxins may be involved in the pathogenesis of inflammatory diseases in part via by affecting expression of aryl hydrocarbon receptor (Ahr) and inflammatory cytokines in animal models. As the role of the Ahr in dioxin-exposed people is not well defined, a study was conducted to examine gene expression levels of Ahr, inflammatory cytokines, and the incidence of diseases in dioxin-exposed citizens who had/still resided near a heavily dioxin-contaminated area in Vietnam. Whole blood from citizens at/around Da Nang airbase and control individuals living in unsprayed areas was collected. Serum levels of dioxins were analyzed by using a dioxins-responsive chemical-activated luciferase gene expression bioassay. Gene expression of Ahr, interleukin (IL)-1β, TNFα, IL-6, and IL-22 in whole blood was examined by quantitative real-time PCR. The results showed levels of dioxins and expression of Ahr, IL-1β, TNFα, and IL-6 were up-regulated while IL-22 expression was down-regulated in dioxin-exposed people. Various disease incidences in the study subjects was also examined. Interestingly, the incidence of rheumatoid arthritis (RA) in these individuals was increased compared to the estimated prevalence of this disease in the general Vietnamese population. Analyses also showed that expression levels of Ahr correlated to those of IL-6 and IL-22 in the dioxin-exposed people. Taken together, dioxins might be involved in an up-regulated expression of Ahr that might possibly relate to changes in level of inflammatory cytokines and, ultimately, in the incidence of select diseases in residents of Vietnam who had/continue to live near a dioxins-contaminated site.

How much do PCB toxic equivalents account for PHAH toxicity in predatory birds?

Various diffuse polyhalogenated aromatic hydrocarbons (PHAHs) exert common toxicity through the aryl hydrocarbon receptor (AhR). Apex predators spatially and temporally integrate diffuse contamination and simultaneous exposure can cause additive toxicity. We investigated the extent to which PCBs, still amongst the most prevalent PHAHs accumulated by predators, accounted for total PHAH toxicity in raptors and fish eating birds from Britain. We analysed egg or liver extracts from six species and compared chemically determined ΣPCB-TEQs concentrations with total AhR-mediated toxicity determined using the chemical-activated luciferase gene expression bioassay (CALUX-TEQ). Dioxin-like PCB profiles in eggs and livers were dominated by congeners 118, 105 and 167. ΣPCB-TEQ and CALUX-TEQ concentrations were positively associated but not in a 1:1 relationship. ΣPCB-TEQ were broadly similar to CALUX-TEQ concentrations in eggs and livers with CALUX-TEQ concentrations >50–80 and 160–320 pg g−1 lipid respectively, but were lower than CALUX-TEQ concentrations in less contaminated samples.

Determination of PCDD/Fs, PBDD/Fs and dioxin-like PCBs in human milk from mothers residing in the rural areas in Flanders, using the CALUX bioassay and GC-HRMS

Since the CALUX (Chemically Activated LUciferase gene eXpression) bioassay is a fast and inexpensive tool for the determination of dioxin-like compounds in a large number of samples and requires only small sample volumes, the use of this technique in human biomonitoring programs provides a good alternative to GC-HRMS. In this study, a new CALUX method for the separate analysis of PCDD/Fs and dioxin-like PCBs (dl-PCBs) in small amounts of human milk samples with the new sensitive H1L7.5c1 cell line was used to analyze 84 human milk samples, collected from mothers residing in the Flemish rural communities. The geometric mean CALUX-Bioanalytical Equivalent (CALUX-BEQ) values, reported for the 84 mothers from the study area were 10.4 (95% CI: 9.4–11.4) pg CALUX-BEQ per gram lipid or 0.41 (95% CI: 0.37–0.45) pg CALUX-BEQ per gram milk for the PCDD/Fs and 1.73 (1.57–1.91) pg CALUX-BEQ per gram lipid or 0.07 (95% CI: 0.06–0.08) pg CALUX-BEQ per gram milk for the dioxin-like PCBs. Multiple regression analysis showed significant associations between PCDD/Fs and weight change after pregnancy, smoking and consumption of local eggs. One pooled human milk sample was analyzed with both CALUX and GC-HRMS. The ratio of CALUX and GC-HRMS results for this sample were respectively 1.60, 0.58 and 1.23 for the PCDD/Fs, the dl-PCBs and the sum of both fractions, when using the 2005-TEF values. Additionally, also low levels of certain brominated dioxins and furans were detected in the pooled sample with GC-HRMS.

Analysis of PCDD/Fs and dioxin-like PCBs in atmospheric deposition samples from the Flemish measurement network: Correlation between the CALUX bioassay and GC–HRMS

Since the CALUX (Chemically Activated LUciferase gene eXpression) bioassay is a fast, sensitive and inexpensive tool for the analysis of a high number of samples, the use of this technique in routine analysis of atmospheric deposition samples may be a valuable alternative for GC–HRMS. In this study, a validated CALUX method was used for the analysis of PCDD/Fs and dioxin-like PCBs in more than 90 atmospheric deposition samples for different locations in Flanders. The samples were taken in residential and agricultural areas, where a threshold limit of 21pgWHO-TEQm−2d−1 for the sum of PCDD/Fs and dioxin-like PCBs was set, and in industrial zones and natural reserves, where no official threshold limit is available. The results from the Flemish measurement program showed correlation between CALUX and GC–HRMS for all the samples, originating from the different areas (R2 of 0.81, 0.53 and 0.64 for dl-PCBs, PCDD/Fs and sum of both fractions, respectively). Median CALUX/GC–HRMS ratios of 2.0, 0.9 and 1.3 were reported for the PCDD/Fs, dioxin-like PCBs and the sum of both fractions, respectively. The results show that the CALUX bioassay is a valuable alternative tool for the classic GC–HRMS analysis of atmospheric deposition samples in the Flemish measurement network.

Quantification of PCDD/Fs and dioxin-like PCBs in small amounts of human serum using the sensitive H1L7.5c1 mouse hepatoma cell line: Optimization and analysis of human serum samples from adolescents of the Flemish Environment and Health Study (FLEHS II)

Since the CALUX (Chemically Activated LUciferase gene eXpression) bioassay is a fast and inexpensive tool for the throughput analysis of dioxin-like compounds in a large number of samples and requires only small sample volumes, the use of this technique in human biomonitoring programs provides a good alternative to GC–HRMS. In this study, a method for the separate analysis of PCDD/Fs and dioxin-like PCBs (dl-PCBs) in human serum with the new sensitive H1L7.5c1 mouse hepatoma cell line was optimized.Sample dilution factors of 5 and 2.4 were selected for routine analysis of respectively the PCDD/Fs and dl-PCBs. The validation studies showed that repeatability and within-lab reproducibility for the quality control (QC) standard were within the in-house criteria. A long-term within-lab reproducibility of 25% for the PCDD/F fraction and 41% for the dl-PCB fraction for the analysis of pooled serum samples, expressed as pgBEQ/gfat, was determined. CALUX recoveries of the spiked procedural blanks were within the acceptable in-house limits of 80–120% for both fractions and the LOQ was 30.3pgBEQ/gfat for the PCDD/Fs and 14.5pgBEQ/gfat for the dl-PCBs. The GC–HRMS recovery of a C13-spiked pooled serum sample was between 60 and 90% for all PCDD/F congeners and between 67 and 82% for the non-ortho PCBs. An adequate separation between both fractions was found. The CALUX/GC–HRMS ratio for a pooled serum sample was respectively 2.0 and 1.4 for the PCDD/Fs and the dl-PCBs, indicating the presence of additional AhR active compounds. As expected, a correlation was found between human serum samples analyzed with both the new H1L7.5c1 cell line and the more established H1L6.1c3 cell line. The geometric mean CALUX-BEQ values, reported for the adolescents of the second Flemish Environment and Health Study (FLEHS II) recruited in 2009–2010, were 108 (95% CI: 101–114)pgCALUX-BEQ/gfat for the PCDD/Fs and 32.1 (30.1–34.2)pgCALUX-BEQ/gfat for the dioxin-like PCBs.

Analysis of PCDD/Fs and dioxin-like PCBs in atmospheric deposition samples from the Flemish measurement network: Optimization and validation of a new CALUX bioassay method

Since the CALUX (Chemically Activated LUciferase gene eXpression) bioassay is a fast, sensitive and inexpensive tool for the analysis of a high number of samples, validation of new methods is urgently needed. In this study, a new method for the analysis of PCDD/Fs and dioxin-like PCBs in atmospheric deposition samples with the CALUX bioassay was developed, optimized and validated. The method consists of 4 steps: filtration, extraction, clean up and bioassay analysis. To avoid the use of large amounts of toxic solvents, new techniques were used for filtration and extraction: a C18 filter was used instead of a liquid/liquid extraction and an Accelerated Solvent Extractor (ASE) was used instead of the traditional soxhlet extraction. After pre-oxidation of the sample extract, clean up was done using a multi-layer silica gel column coupled to a carbon column. The PCDD/F and PCB fractions were finally analyzed with the H1L7.5c1 and/or the H1L6.1c3 mouse hepatoma cell lines. The limit of quantification was 1.4 pg CALUX-BEQ m −2 d −1 for the PCBs and 5.6 pg CALUX-BEQ m −2 d −1 for the PCDD/Fs, when using the new sensitive H1L7.5c1 cell line. The GC–HRMS recovery for all PCDD/F congeners was between 55% and 112%, with a mean recovery of 90%. CALUX recoveries of spiked procedural blanks were between the accepted ranges of 80–120%. Repeatability and reproducibility were satisfactory and no interferences from metals were detected. The first results from the Flemish measurement program showed good correlation between CALUX and GC–HRMS.

CALUX Bioassay of Dioxin-Like Compounds in Sediments from the Haihe River, China

Chemically Activated LUciferase gene eXpression (CALUX) bioassay is a rapid, effective, and cheap method to analyze dioxin-like compounds (DLCs). Using this method, total levels of DLCs, including PCDD/Fs (polychlorinated dibenzo-p-dioxin and dibenzofurans) fraction and DL-PCBs (dioxin-like polychlorinated biphenyls) fraction, in extracts of sediments from the Haihe River, China, were determined. It was found that the levels of PCDD/Fs in the sediments from the Haihe River were tens of times to hundreds of times higher than those of DL-PCBs. The DLC levels in sediments in March 2005 approximated those in November 2006. The CALUX toxic equivalents (TEQs) of DLCs were independent of the total organic carbon (TOC) of sediments. Significant correlation was observed between the PCDD/Fs TEQs and DL-PCBs TEQs. The two fractions of some samples were also analyzed by HRGC/HRMS (high-resolution gas chromatography/high-resolution mass spectrometry) method. Significant statistical correlations were observed between the CALUX TEQs and HRGC/HRMS TEQs. The difference between TEF (toxic equivalent factor) and REP (relative potency) can explain most of the discrepancy between CALUX and HRGC/HRMS analysis. A high level of DLCs was found at the sites near the influx into Bohai Bay. The dioxin pollution might be associated with industrial activities of pentachlorophenol (PCP) in the adjacent area. More attention should be paid to the increase of DLC levels and further investigation should be done for this area.

Mass loading and partitioning of dioxins in irrigation runoff from Japanese paddy fields: Combination usage of the CALUX assay with HRGC/HRMS

Lack of understanding of dioxins mass loading into the aquatic environment motivated the quantitative investigation of dioxins runoff from paddy fields during one entire irrigation period in the Minakuchi region, Japan. Combination use of the chemically activated luciferase gene expression (CALUX) bioassay together with high resolution gas chromatography and high resolution mass spectrometry (HRGC/HRMS) enabled efficient investigation of dioxins contamination. The result shows that the congener profile in irrigation runoff is quite similar to those in paddy soil samples and that 1,3,6,8-/1,3,7,9-TeCDD and OCDD derived from pesticides (i.e., pentachlorophenol (PCP) and chloronitrophen (CNP)) are predominant congeners in irrigation runoff. Although it is not surprising that dioxins concentration was strongly dependent on the suspended solids (SS) and the particulate organic carbon (POC) concentration, the dioxins toxic equivalency (TEQ) concentration was extremely high in irrigation runoff (max: 16,380 pg/L, corresponding to 12 pg WHO-TEQ/L) due to runoff of highly contaminated paddy soils. The results imply that dioxins concentration in a river must be monitored considering soil contamination level, land use, and soil runoff events. Using experimental data and a theoretical model, the mass loading of dioxins from the paddy fields by irrigation runoff was estimated to be 1.50 × 10 −2% of total amount of dioxins accumulated in the paddy fields. Given the results of other researches, it is implied the following: 1) large portion of paddy soils released into the river appear to be settled on the riverbed due to small water flux, and, then, washed out and transported by rainfall runoff after irrigation period, 2) rainfall runoff itself also wash out paddy soils directly from paddy fields. Combination use of the CALUX bioassay with HRGC/HRMS is demonstrated as an alternative strategy to assess dioxins contamination in the environment.

Validation of the CALUX bioassay as a screening and semi-quantitative method for PCDD/F levels in cow's milk

The objectives of this study were first to set up and validate the quality criteria of the Xenobiotic Detection Systems—chemical activated luciferase gene expression (CALUX) bioassay for the analysis of cow's milk samples spiked with polychlorinated dibenzo- p-dioxins and polychlorinated dibenzofurans (PCDD/Fs). The application of this bioassay was then tested by analyzing 28 commercially available pasteurized milk samples and comparing the data with the reference method for PCDD/F analysis. The CALUX criteria from the U.S. EPA and the European Union (EU) were calculated from 16 replaced spiked milk (SM) samples and 8 performance evaluation (PE) samples to validate the CALUX bioassay system. The CALUX bioassay criteria included control chart for quality control (QC) standards, recovery efficiency, and data comparability. The control chart for QC standards were both within the μ ± 2 σ range. The recovery efficiencies ranged from 60.4% to 106% with an average of 79.9% (relative standard deviation (RSD): 20.7%). The mean of data comparability (i.e., relative percent difference, RPD) between CALUX bioassay and high-resolution gas chromatography and high-resolution mass spectrometry (HRGC/HRMS) was 19.0% for SM samples. For the PE samples, Pearson's correlation coefficient between CALUX and the HRGC/HRMS method was 0.953. The high correlation shows that the CALUX system is suitable as a screening method and a semi-quantitative method to analyze the PCDD/F concentration in milk samples. Next, the validated CALUX bioassay was applied to measure 28 commercially available pasteurized milk samples. These milk samples were also analyzed with the HRGC/HRMS method to compare the analysis data from two different methods. There is no false negative sample when applying the bioassay to pasteurized milk and PE samples as a screening method and a semi-quantitative method. The present study indicates that CALUX is a powerful bioassay method for screening a large number of milk samples.

Agreement between breast milk dioxin levels by CALUX bioassay and chemical analysis in a population survey in Hong Kong

Chemically-activated luciferase gene expression (CALUX) bioassay and gas chromatography/mass spectrometry (GC/MS) are used to determine dioxin levels in food and humans. Valid measures of the agreement between the two methods would improve interpretation of bioassay results. Paired breast milk samples from 250 mothers, as 11 pooled samples, were analysed by GC/MS for total WHO-TEQ (7 polychlorinated dibenzo- para-dioxins, 10 polychlorinated dibenzofurans and 12 dioxin-like polychlorinated biphenyls) and as individual samples by CALUX. Mean difference between total WHO-TEQ (weighted by TEF system derived in 1997) and mean CALUX-TEQ in each pool was 1.6 pg/g fat (95% CI: 0.7, 2.4), indicating a statistically significant overestimation of CALUX-TEQ compared to WHO-TEQ, probably due to the presence of Ah-receptor agonists. CALUX estimated toxicity of 13 pg/g fat was greater than the WHO-TEQ by 0.9, 3.1 and 0.3 pg/g fat for mothers from Hong Kong, mainland China and overseas territories, respectively. When the 2005 TEF system was applied, a reduction of 14–26% in the WHO-TEQ and a larger but less disperse discrepancy between WHO-TEQ and CALUX-TEQ (3.9 pg/g fat, 95% CI: 3.5, 4.4) were observed. Our study suggested that the mothers’ place of residence explained the discrepancy between CALUX-TEQ and WHO-TEQ and should be considered in inter-country comparisons for CALUX-TEQ. For regulatory purposes bioassays for detecting quantitative dioxin contents in any setting must be combined with adequate extraction, clean-up and validation with WHO-TEQs. The larger difference between the two measurements after using the new TEF system warrants further investigation.

Time-course changes of mixture effects on AhR binding-dependent luciferase activity in a crude extract from a compost sample

Crude extracts of environmental samples contain stable and labile aryl hydrocarbon receptor (AhR) ligands, and show huge activities in the cell-based bioassay, and these activities are higher than the chemically calculated induction equivalent values. It is thought that not only unidentified AhR ligands but also mixture effect among compounds might contribute to these activities. In the previous work, we have indicated that hydrophobic compounds in household sewage sludge (HSS) compost may interact synergistically with 2,3,7,8-TCDD in the CALUX (DR-CALUX ®: Dioxin-Responsive Chemical-Activated Luciferase gene eXpression) assay [Suzuki, G., Takigami, H., Kushi, Y., Sakai, S., 2004. Evaluation of mixture effects in a crude extract of compost using the CALUX bioassay and HPLC fractionation. Environ. Int. 30, 1055–1066]. In this study, we focused on co-existing stable compounds such as halogenated aromatic hydrocarbons (HAHs) and labile compounds such as polyaromatic hydrocarbons (PAHs) in the crude extract and investigated the time-course changes of mixture effects among compounds in environmental samples using the CALUX assay and normal-phase high-performance liquid chromatography (NP-HPLC) fractionation. We confirmed that CYP1A-inducing PAHs and HAHs could be separated by NP-HPLC on a nitrophenylpropylsilica (NITRO) column. To determine whether the activities of AhR ligands in environmental samples (including the HSS compost) could be assessed by the additivity theory, we compared the CALUX activity of the crude extract with the arithmetical sum of the activities of all the fractions separated by NP-HPLC. We confirmed a potentiation of CALUX activity at 12-, 24- and 48-h exposure durations. In contrast, CALUX activity increased additively at 6- and 72-h exposure durations. CALUX activity was potentiated when the CALUX activity of the HPLC fractions showed a remarkable reduction resulting in a change of activity profiles. In contrast, additivity was observed at a 72-h exposure duration when the CALUX activity of the HPLC fractions showed neither remarkable reduction nor a change in profile. Our results suggest that differences in the metabolic decomposition of compounds affected mixture effects on CALUX activity in a crude extract from HSS compost.

Chemically Activated Luciferase Gene Expression (CALUX) Cell Bioassay Analysis for the Estimation of Dioxin-Like Activity: Critical Parameters of the CALUX Procedure that Impact Assay Results

The chemically activated luciferase gene expression (CALUX) in vitro cell bioassay is a bioanalytical tool that is increasingly being used by research and commercial laboratories for the screening and relative quantification of dioxins and dioxin-like compounds in sample extracts. Since CALUX analyses provide a biological response to all aryl hydrocarbon receptor active compounds present in a given sample extract containing a complex mixture of chemicals, interpretation of results is significantly more complexthan of chemical analyses. Operators in the laboratory can adjust many parameters when performing CALUX analyses, and the applied procedure strongly affects the result and, hence, the interpretation of the results. This paper examines critical methodological parameters and aspects of the CALUX bioassay that can affect the quality and accuracy of the analyses. Moreover, the study aims to identify the ways that alteration of these parameters influences CALUX measurements. A greater understanding of these characteristics will lead to increased accuracy, precision, and reproducibility of the widely used CALUX bioassay within and between research laboratories.

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.

Comparison of Chemical-Activated Luciferase Gene Expression Bioassay and Gas Chromatography for PCB Determination in Human Serum and Follicular Fluid

We assessed exposure to dioxin-like compounds using chemical and bioassay analysis in different matrices in a female population. A total of 106 serum and 9 follicular fluid samples were collected from infertile women attending Centers for Reproductive Medicine in Belgium from 1996 to 1998. Major polychlorinated biphenyl (PCB) congeners were quantified by chemical analysis using gas chromatography with electron-capture detection, and the chemical-activated luciferase gene expression (CALUX) bioassay was used to determine the total dioxin-like toxic equivalence (TEQ) of mixtures of polyhalogenated aromatic hydrocarbons present in body fluids, such as serum and follicular fluid. To the best of our knowledge, this is the first investigation to determine TEQ values by the CALUX bioassay in follicular fluid. The TEQ levels in both matrices are well correlated (r = 0.83, p = 0.02). As the chemical and bioassay analysis executed in this study do not cover the same span of polyhalogenated aromatic hydrocarbons, we did not expect totally correlated results. Moreover, the sample workup and quantification of the analytes differed completely. Nonetheless, the TEQ values in human extracts correlated well with the sum of four major PCB congeners chemically determined in both serum and follicular fluid. These results indicate that the CALUX bioassay may serve as a simple, relatively inexpensive prescreening tool for exposure assessment in epidemiologic surveys.

Validation and use of the CALUX‐bioassay for the determination of dioxins and PCBs in bovine milk

There is a strong need for the development of relatively cheap and rapid bioassays for the determination of dioxins and related compounds in food. A newly developed CALUX (Chemical‐Activated LUciferase gene expression) bioassay was tested for its possible use to determine low levels of dioxins in bovine milk. Data show that this mammalian cell‐based test is very sensitive for 2,3,7,8‐substituted dioxins and related PCBs, thereby reflecting the relative potencies of these compounds in comparison to TCDD (TEF‐values). The limit of detection was about 50 fg of TCDD. Furthermore, the response obtained with a mixture of dioxins was additive, in accordance with the TEF‐principle. Milk fat was isolated by centrifugation followed by clean‐up of the fat with n‐pentane, removal of the fat on a 33% H2SO4 silica column, and determination of Ah receptor agonist activity with the CALUX‐bioassay. An equivalent of 67 mg fat was tested per experimental unit, resulting in a limit of quantification around 1 pg i‐TEQ/g fat. To investigate the performance of the method, butter fat was cleaned and spiked with a mixture of 17 different 2,3,7,8‐substituted PCDD and PCDF congeners at 1, 3, 6, 9, 12 and 15 pg TEQ/g fat, as confirmed by GC/MS. In this concentration range, the method showed a recovery of TEQs around 67% (58–87%). The reproducibility, determined in three independent series showed a CV varying between 4% and 54%, with the exception of the sample spiked at 1 pg i‐TEQ (CV 97%). The repeatability determined with the sample spiked at 6pg i‐TEQ/g showed a CV of 10%. Testing of 22 bovine milk samples, taken at different sites in The Netherlands, in the CALUX‐assay showed combined dioxin and dioxin‐like PCB levels equivalent to 1.6 pg TCDD/g fat (range 0.2–4.6). GC/MS analysis of these samples revealed an average level of 1.7 pg i‐TEQ/g fat, varying between 0.5 and 4.7 pg i‐TEQ/g fat. All five samples showing a GC/MS determined dioxin content of more than 2 pg i‐TEQ/g fat gave a response in the CALUX‐assay corresponding to more than 2 pg TCDD/g fat. These data clearly show that the CALUX‐bioassay is a promising method for the rapid and low cost screening of dioxins in bovine milk.