Assessing the receptor-mediated activity of PAHs using AhR-, ERα- and PPARγ- CALUX bioassays.

Polycyclic aromatic hydrocarbons (PAH) are a complex group of organic compounds, consisting of at least three fused aromatic rings, which are formed during combustion of organic matter. While some PAHs have been reported to have carcinogenic and/or mutagenic properties, another possible negative health impact is their endocrine disrupting potential. Therefore, the aim of this study was to determine both the agonistic and antagonistic endocrine activity of 9 environmentally relevant PAHs using three different CALUX bioassays: The AhR-CALUX, The ERα-CALUX and PPARγ-CALUX. For the PPARγ-CALUX anthracene, fluoranthene, pyrene and fluorene showed weak agonistic activity, whilst benzo(a)pyrene (B(a)P) was the only one exhibiting weak antagonistic activity. For the AhR-CALUX, chrysene was the only PAH that showed relatively strong agonist activity (except for B(a)P which was used as a standard). Pyrene, anthracene and fluoranthene showed weak AhR agonist activity. In the ERα-CALUX bioassay, fluoranthene had agonistic activity whilst B(a)P exhibited both agonistic and antagonistic activity (lowering E2 activity by 30%). Phenanthrene and anthracene had weak ERα agonist activities. These results indicate that certain PAHs have multiple modes of action and can activate/inhibit multiple receptor signaling pathways known to play critical roles in mediating endocrine disruption.

Assessment of estrogenic compounds in paperboard for dry food packaging with the ERE-CALUX bioassay.

Paperboard used as packaging, a non-inert material, can transfer chemicals into food. Over the years, endocrine disrupting compounds (EDCs), such as NonylPhenols (NPs), BisPhenol A (BPA) and phthalates have been shown to migrate from packaging materials into food. Due to chronic exposure and mixture effects of these EDCs, they could cause health effects even at very low doses. Many EDCs are still unknown and many more are still unregulated. The ERE-CALUX bioassay was used as a bioanalytical tool to investigate estrogenic activities of paperboard food packaging and its characteristics, including recycling rate and printing ink. A "worst case" scenario with full extraction is compared to a dry food migration experiment. By measuring an overall estrogenic activity, known and unknown estrogenic chemicals and mixture effects are taken into account and the data are compared to molecule specific analysis. Estrogenic activities ranged from 682 ±â€¯66 pg E2 eq./dm2 to 3250 ±â€¯400 pg E2 eq./dm2 for "worst case" extraction and from 347 ±â€¯30 pg E2 eq./dm2 to 1350 ±â€¯70 pg E2 eq./dm2 for migration experiments. A two-factor ANOVA revealed a relationship between estrogenic activity and the recycling rate of the paperboard, but no significant difference with printing ink was observed for these paperboard samples. Bis(2-ethylhexyl)phthalate (DEHP), dibutyl phthalate (DBP), butyl benzyl phthalate (BBP) and 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH) were determined in all extraction and migrations experiment samples. A Spearman rank correlation analysis showed a relationship between the estrogenic activity and the total phthalates as well as with each compound individually.

Endocrine activity in an urban river system and the biodegradation of estrogen-like endocrine disrupting chemicals through a bio-analytical approach using DRE- and ERE-CALUX bioassays.

The Zenne River, crossing the Brussels region (Belgium) is an extremely urbanized river impacted by both domestic and industrial effluents. The objective of this study was to monitor the occurrence and activity of Endocrine Active Substances (EAS) in river water and sediments in the framework of the Environmental Quality Standards Directive (2008/105/EC and 2013/39/EU). Activities were determined using Estrogen and Dioxin Responsive Elements (ERE and DRE) Chemical Activated Luciferase Gene Expression (CALUX) bioassays. A potential contamination source of estrogen active compounds was identified in the river at an industrial area downstream from Brussels with a peak value of 938 pg E2 eq./L water (above the EQS of 0.4 ng/L) and 195 pg E2 eq./g sediment. Estrogens are more abundantly present in the sediments than in the dissolved phase. Principal Component Analysis (PCA) showed high correlations between Suspended Particulate Matter (SPM), Particulate (POC) and Dissolved Organic Carbon (DOC) and estrogenic EAS. The dioxin fractions comply with previous data and all were above the United States Environmental Protection Agency (US EPA) low-level risk, with one (42 pg TCDD eq./g sediment) exceeding the high-level risk value for mammals. The self-purifying ability of the Zenne River regarding estrogens was examined with an in vitro biodegradation experiment using the bacterial community naturally present in the river. Hill coefficient and EC50 values (Effective Concentration at 50%) revealed a process of biodegradation in particulate and dissolved phase. The estrogenic activity was decreased by 80%, demonstrating the ability of self-purification of estrogenic compounds in the Zenne River.

Characterisation and implementation of the ERE-CALUX bioassay on indoor dust samples of kindergartens to assess estrogenic potencies.

Estrogen-like endocrine disrupting chemicals (EEDCs) can be found abundantly in the environment. Due to their low-dose effects and the large amount of unknown EEDCs, it is difficult to assess and manage possible human health risks. For young children, who are particularly vulnerable to endocrine disruption due to their development rate, indoor dust is one of the main routes of exposure. In this study, an estrogen responsive elements chemically activated luciferase gene expression (ERE-CALUX) bioassay was characterized and implemented for the analysis of 12 dust samples from kindergartens in Flanders and Brussels (Belgium). The human ovarian carcinoma BG 1CALUX cell line showed reproducible results and a low limit of detection (LOD). The effective concentration at 50% of the maximum response (EC50) yielded 497 fg/well, while the LOD was 16 fg/well. For all dust samples, full dose-response curves and their corresponding EC50 values could be calculated. All samples yielded bio-analytical equivalent concentrations (BEQs) that were significantly higher than the procedural blank level and ranged from 426 to 8710 pg E2 equivalents/g dust. A clear relationship was observed between a semi-quantitative interior score and the ERE-CALUX response of the samples. In addition, the concentration of phthalates, a major group of EEDCs used as plasticizers in plastics, was determined in the samples by GC-MS. Diisoheptyl phthalate (DiHP) and di(2-ethylhexyl) phthalate (DEHP) were present in every dust sample. A good correlation was found between ERE-CALUX activities and phthalate concentrations, when all phthalates except diisononyl phthalate (DiNP) and diisodecyl phthalate (DiDP), which do not bind to the estrogen receptor, were taken into account. This shows that the ERE-CALUX can provide relevant results concerning exposure to EEDCs from indoor dust. This article is part of a Special Issue entitled 'Endocrine disruptors & steroids'.

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 human biomonitoring program 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 pg BEQ/g fat, 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 30.3 pg BEQ/g fat for the PCDD/Fs and 14.5 pg BEQ/g fat 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) pg CALUX-BEQ/g fat for the PCDD/Fs and 32.1 (30.1-34.2) pg CALUX-BEQ/g fat for the dioxin-like PCBs.

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.

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 1.4pg CALUX-BEQm(-2)d(-1) for the PCBs and 5.6pgCALUX-BEQm(-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.

High resolution profiles of thallium, manganese and iron assessed by DET and DGT techniques in riverine sediment pore waters.

High resolution profiles of Mn, Tl and Fe concentrations have been assessed in the pore waters of river Leie sediments at Warneton and Menen (at the border of Belgium and France) by DET (Diffusive Equilibrium in Thin Films) and DGT (Diffusive Gradients in Thin Films) techniques. The oxidized, solid Mn (IV), Tl (III) and Fe (III) compounds were reduced in the suboxic (+255 to -20 mV versus Standard Hydrogen Electrode (SHE)) riverine sediments and since these reduced species are much more soluble also they are released into the pore waters. The highest DET (total dissolved) concentrations of Fe (76 mg l(-1)), Mn (2 mg l(-1)) were observed at the station of Menen, while Tl maxima differed only slightly between the 3 surveys (21 to 27 microg l(-1)). The average ratios of Fe/Mn/Tl in the pore waters at the 3 sampling stations are fairly constant for both the DET and DGT samplings. However, the results indicate that compared to Fe and Tl a greater proportion of the Mn measured by DET is accumulated by DGT, reflecting the ready supply of Mn from solid phase to solution.

The role of oxidative stress on the effect of 1,4,7,10,13,16-hexathiacyclooctadecane on copper and zinc toxicity in HepG2 cells.

Experiments have shown that 1,4,7,10,13,16-hexathiacyclooctadecane (L3) increased the Cu2+ toxicity on HepG2 cells, whereas the combination Zn(2+)/L3 was less toxic relative to the metal control. In all cases, glutathione (GSH) levels were decreased and vitamins C and E supplementation partially counteracted the increased toxicity in the Cu(2+)/L3-treated cells. The previously observed effects of this hexathiamacrocyclic ligand (L3) on the Cu2+ and Zn2+ toxicity were further investigated by first depleting the intracellular GSH levels by means of L-buthionine S,R-sulphoximine. Combined treatment with Cu(2+)/L3 resulted in complete cell death, whereas for Zn(2+)/L3 no severe effects were observed. Direct measurement of reactive oxygen species (ROS) revealed that Cu2+ induced a high degree of oxidative stress on the cells. This was not the case for Zn2+. The results proved a previously proposed mechanism in which GSH is used to conjugate the metal-ligand complex, but as a result of this, GSH is no longer available for inactivation of ROS. Also, both the intracellular copper and zinc content were determined for each experiment by means of inductively coupled plasma-atomic emission spectroscopy. According to these data, zinc is depleted in Cu(2+)/L3-treated cells, which could have consequences on superoxide dismutase and as a result of this on the amount of oxidative stress.

Thiram and dimethyldithiocarbamic acid interconversion in Saccharomyces cerevisiae: a possible metabolic pathway under the control of the glutathione redox cycle.

A rapid decrease of intracellular glutathione (GSH) was observed when exponentially growing cells of Saccharomyces cerevisiae were treated with sublethal concentrations of either dimethyldithiocarbamic acid or thiram [bis(dimethylthiocarbamoyl) disulfide]. The underlying mechanism of this effect possibly involves the intracellular oxidation of dimethyldithiocarbamate anions to thiram, which in turn oxidizes GSH. Overall, a linear relationship was found between thiram concentrations up to 21 microM and production of oxidized GSH (GSSG). Cytochrome c can serve as the final electron acceptor for dimethyldithiocarbamate reoxidation, and it was demonstrated in vitro that NADPH handles the final electron transfer from GSSG to the fungicide by glutathione reductase. These cycling reactions induce transient alterations in the intracellular redox state of several electron carriers and interfere with the respiration of the yeast. Thiram and dimethyldithiocarbamic acid also inactivate yeast glutathione reductase when the fungicide is present within the cells as the disulfide. Hence, whenever the GSH regeneration rate falls below its oxidation rate, the GSH:GSSG molar ratio drops from 45 to 1. Inhibition of glutathione reductase may be responsible for the saturation kinetics observed in rates of thiram elimination and uptake by the yeast. The data suggest also a leading role for the GSH redox cycle in the control of thiram and dimethyldithiocarbamic acid fungitoxicity. Possible pathways for the handling of thiram and dimethyldithiocarbamic acid by yeast are considered with respect to the physiological status, the GSH content, and the activity of glutathione reductase of the cells.

Glutathione as an endogenous sulphur source in the yeast Saccharomyces cerevisiae.

Glutathione-deficient mutants (gshA) of the yeast Saccharomyces cerevisiae, impaired in the first step of glutathione (GSH) biosynthesis were studied with respect to the regulation of enzymes involved in GSH catabolism and cysteine biosynthesis. Striking differences were observed in the content of the sulphur amino acids when gshA mutants were compared to wild-type strains growing on the same minimal medium. Furthermore, all mutants examined showed a derepression of gamma-glutamyltranspeptidase (gamm-GT), the enzyme initiating GSH degradation. However, gamma-cystathionase and cysteine synthase were unaffected by the GSH deficiency as long as the nutrient sulphate source was not exhausted. The results suggest that the mutants are probably not impaired in the sulphate assimilation pathway, but that the gamma-glutamyl cycle could play a leading role in the regulation of the sulphur fluxes. Studies of enzyme regulation showed that the derepression of gamma-GT observed in the gshA strains was most probably due to an alteration of the thiol status. The effectors governing the biosynthesis of cysteine synthase and gamma-cystathionase seemed different from those playing a role in gamma-GT regulation and it was only under conditions of total sulphate deprivation that all these enzymes were derepressed. As a consequence the endogenous pool of GSH was used in the synthesis of cysteine. GSH might, therefore, fulfil the role of a storage compound.