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1.
Food Chem Toxicol ; 145: 111602, 2020 Nov.
Article in English | MEDLINE | ID: mdl-32738369

ABSTRACT

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.


Subject(s)
Estrogen Receptor alpha/agonists , PPAR gamma/agonists , Polycyclic Aromatic Hydrocarbons/pharmacology , Receptors, Aryl Hydrocarbon/agonists , Signal Transduction/drug effects , Animals , Biological Assay , Cell Line, Tumor , Estrogen Receptor alpha/antagonists & inhibitors , Humans , Mice , PPAR gamma/antagonists & inhibitors , Rats , Receptors, Aryl Hydrocarbon/antagonists & inhibitors
2.
Chemosphere ; 221: 99-106, 2019 Apr.
Article in English | MEDLINE | ID: mdl-30634153

ABSTRACT

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.


Subject(s)
Biological Assay/methods , Endocrine Disruptors/analysis , Estrogens/analysis , Food Packaging , Dibutyl Phthalate/analysis , Environmental Exposure/adverse effects , Humans , Phthalic Acids/analysis , Recycling
3.
Chemosphere ; 201: 540-549, 2018 Jun.
Article in English | MEDLINE | ID: mdl-29533803

ABSTRACT

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.


Subject(s)
Biological Assay/methods , Endocrine Disruptors/metabolism , Rivers/chemistry , Animals , Belgium , Biodegradation, Environmental , Dioxins/analysis , Endocrine Disruptors/analysis , Endocrine System/drug effects , Environmental Monitoring/methods , Estradiol Congeners/metabolism , Estrogens/analysis , Estrogens/metabolism , Geologic Sediments/chemistry , Humans , Mammals , Rivers/microbiology , Water Pollutants, Chemical/analysis , Water Pollutants, Chemical/metabolism
4.
Sci Total Environ ; 626: 109-116, 2018 Jun 01.
Article in English | MEDLINE | ID: mdl-29335165

ABSTRACT

BACKGROUND: The Scheldt estuary is historically a highly polluted river system. While several studies have focused on contamination with metals, pesticides, Polycyclic Aromatic Hydrocarbons (PAHs) and marker PolyChlorinated Biphenyls (PCBs), no data are available concerning past contamination by dioxin-like compounds. OBJECTIVES: The objective of this study is to determine spatial and time trends of PolyChlorinated Dibenzo-p-Dioxins and DibenzoFurans (PCDD/Fs) and dioxin-like PCBs (dl-PCBs) in sediment samples and Suspended Particulate Matter (SPM) from the Scheldt River basin and the North Sea Coast. METHODS: Dioxin-like compounds (PCDD/F and dl-PCB fractions) were measured with the CALUX-bioassay. Bioanalytical EQuivalent concentrations (BEQs) and Total Organic Carbon (TOC) content of historical (1982-1984) and recent (2011-2015) sediment and SPM samples from different locations in the coastal area and the estuary, were evaluated. RESULTS: A decrease in dioxin-like compound concentrations was found at all stations over time, especially for the PCDD/Fs. Dl-PCBs were relatively low in all samples. The Scheldt mouth and the Antwerp harbor yielded the highest BEQs and levels were higher in SPM than in sediment due to the higher organic carbon content in this fraction. CONCLUSIONS: Current PCDD/F and dl-PCB levels in the Belgian Coastal Zone and Scheldt estuary are much lower than their levels 30 years ago and pose a relatively low risk to the aquatic system. This is the result of a strong decrease in emissions, however, large local variabilities in sediment concentration levels can still exist because of local variability in sedimentation, erosion rates and in organic carbon content.

5.
Environ Sci Process Impacts ; 19(9): 1142-1149, 2017 Sep 20.
Article in English | MEDLINE | ID: mdl-28612856

ABSTRACT

The impact of unmonitored contaminants, also known as contaminants of emerging concern (CECs), on freshwater streams remains largely uncharacterized. Water samples from 31 streams representing urban, agricultural and undeveloped (i.e., open space) land use in Southern California (USA) were analyzed for in vitro and in vivo bioactivity. The extent and magnitude of bioactivity screened using endocrine-responsive cell bioassays and a fish embryo screening assay were low. In contrast, a wider gradient of responses for the aryl hydrocarbon receptor (AhR) assay was observed, which was negatively correlated with a measure of benthic community structure. Both aromatic and non-aromatic CECs were tentatively identified in these samples, but polycyclic aromatic hydrocarbons (PAHs), known AhR agonists in urban environments, were not present at detectable levels. These results suggest that a combination of in vitro and in vivo show potential as screening techniques for biological condition in situ, but that more advanced, comprehensive analytical methods are needed to identify bioactive contaminants.


Subject(s)
Environmental Monitoring/methods , High-Throughput Screening Assays/methods , Polycyclic Aromatic Hydrocarbons/toxicity , Rivers/chemistry , Water Pollutants, Chemical/toxicity , Animals , California , Embryo, Nonmammalian/drug effects , HEK293 Cells , Humans , Polycyclic Aromatic Hydrocarbons/analysis , Receptors, Aryl Hydrocarbon/metabolism , Receptors, Estrogen/metabolism , Receptors, Glucocorticoid/metabolism , Water Pollutants, Chemical/analysis , Zebrafish
6.
J Steroid Biochem Mol Biol ; 155(Pt B): 182-9, 2016 Jan.
Article in English | MEDLINE | ID: mdl-25595043

ABSTRACT

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'.


Subject(s)
Biological Assay , Dust/analysis , Endocrine Disruptors/pharmacology , Environmental Pollutants/pharmacology , Phthalic Acids/pharmacology , Plasticizers/pharmacology , Cell Line, Tumor , Child, Preschool , Endocrine Disruptors/isolation & purification , Endocrine System/drug effects , Endocrine System/physiology , Environmental Pollutants/isolation & purification , Gas Chromatography-Mass Spectrometry , Gene Expression/drug effects , Genes, Reporter , Humans , Luciferases/genetics , Luciferases/metabolism , Phthalic Acids/isolation & purification , Plasticizers/isolation & purification , Response Elements , Sensitivity and Specificity
7.
Talanta ; 113: 99-105, 2013 Sep 15.
Article in English | MEDLINE | ID: mdl-23708629

ABSTRACT

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.


Subject(s)
Benzofurans/analysis , Dioxins/analysis , Environmental Pollutants/analysis , Milk, Human/chemistry , Polychlorinated Biphenyls/analysis , Adult , Animals , Belgium , Benzofurans/metabolism , Biological Assay , Cell Line, Tumor , Dioxins/metabolism , Environmental Monitoring , Environmental Pollutants/metabolism , Female , Gas Chromatography-Mass Spectrometry/methods , Genes, Reporter , Humans , Luciferases, Firefly/genetics , Mice , Mothers , Polychlorinated Biphenyls/metabolism , Receptors, Aryl Hydrocarbon/metabolism , Response Elements , Rural Population , Young Adult
8.
Chemosphere ; 88(7): 881-7, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22560702

ABSTRACT

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(-2)d(-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 (R(2) 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.


Subject(s)
Benzofurans/analysis , Biological Assay/methods , Chromatography, Gas/methods , Polychlorinated Biphenyls/analysis , Polychlorinated Dibenzodioxins/analogs & derivatives , Dioxins/analysis , Environmental Monitoring/methods , Luciferases/genetics , Luciferases/metabolism , Polychlorinated Dibenzodioxins/analysis
9.
Talanta ; 85(5): 2484-91, 2011 Oct 15.
Article in English | MEDLINE | ID: mdl-21962672

ABSTRACT

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.


Subject(s)
Benzofurans/analysis , Liver Neoplasms, Experimental/chemistry , Polychlorinated Biphenyls/analysis , Polychlorinated Dibenzodioxins/analogs & derivatives , Adolescent , Animals , Belgium , Benzofurans/blood , Cell Line, Tumor , Dibenzofurans, Polychlorinated , Dose-Response Relationship, Drug , Gas Chromatography-Mass Spectrometry , Humans , Limit of Detection , Liver Neoplasms, Experimental/pathology , Mice , Polychlorinated Biphenyls/blood , Polychlorinated Dibenzodioxins/analysis , Polychlorinated Dibenzodioxins/blood , Reproducibility of Results
10.
Talanta ; 85(4): 1966-73, 2011 Sep 30.
Article in English | MEDLINE | ID: mdl-21872045

ABSTRACT

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.


Subject(s)
Biological Assay/methods , Environmental Pollutants/analysis , Environmental Pollutants/pharmacology , Luciferases/genetics , Animals , Biological Assay/standards , Cell Line, Tumor , Dose-Response Relationship, Drug , Gene Expression/drug effects , Mice , Polychlorinated Dibenzodioxins/pharmacology , Reference Standards , Regression Analysis
11.
Chemosphere ; 82(5): 718-24, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21094512

ABSTRACT

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.


Subject(s)
Benzofurans/analysis , Biological Assay/methods , Dioxins/analysis , Environmental Monitoring/methods , Polychlorinated Biphenyls/analysis , Polychlorinated Dibenzodioxins/analogs & derivatives , Atmosphere/chemistry , Belgium , Dibenzofurans, Polychlorinated , Gas Chromatography-Mass Spectrometry , Polychlorinated Dibenzodioxins/analysis
12.
SAR QSAR Environ Res ; 21(1): 187-214, 2010 Jan 01.
Article in English | MEDLINE | ID: mdl-20373220

ABSTRACT

The aryl hydrocarbon receptor is a ligand-activated transcription factor responsive to both natural and synthetic environmental compounds, with the most potent agonist being 2,3,7,8-tetrachlotrodibenzo-p-dioxin. The aim of this work was to develop a categorical COmmon REactivity PAttern (COREPA)-based structure-activity relationship model for predicting aryl hydrocarbon receptor ligands within different binding ranges. The COREPA analysis suggested two different binding mechanisms called dioxin- and biphenyl-like, respectively. The dioxin-like model predicts a mechanism that requires a favourable interaction with a receptor nucleophilic site in the central part of the ligand and with electrophilic sites at both sides of the principal molecular axis, whereas the biphenyl-like model predicted a stacking-type interaction with the aryl hydrocarbon receptor allowing electron charge transfer from the receptor to the ligand. The current model was also adjusted to predict agonistic/antagonistic properties of chemicals. The mechanism of antagonistic properties was related to the possibility that these chemicals have a localized negative charge at the molecule's axis and ultimately bind with the receptor surface through the electron-donating properties of electron-rich groups. The categorization of chemicals as agonists/antagonists was found to correlate with their gene expression. The highest increase in gene expression was elicited by strong agonists, followed by weak agonists producing lower increases in gene expression, whereas all antagonists (and non-aryl hydrocarbon receptor binders) were found to have no effect on gene expression. However, this relationship was found to be quantitative for the chemicals populating the areas with extreme gene expression values only, leaving a wide fuzzy area where the quantitative relationship was unclear. The total concordance of the derived aryl hydrocarbon receptor binding categorical structure-activity relationship model was 82% whereas the Pearson's coefficient was 0.88.


Subject(s)
Gene Expression Regulation/genetics , Models, Chemical , Receptors, Aryl Hydrocarbon/agonists , Receptors, Aryl Hydrocarbon/metabolism , Structure-Activity Relationship , Dioxins/metabolism , Molecular Structure , Protein Binding
13.
Toxicol Lett ; 165(3): 230-41, 2006 Sep 10.
Article in English | MEDLINE | ID: mdl-16750337

ABSTRACT

The TEF system for dioxin-like compounds has included assignment of TEF values for mono-ortho polychlorinated biphenyls (MO-PCBs). Small traces of aryl hydrocarbon receptor (AhR)-active impurities could result in artifactually higher relative potency (REP) values. MO-PCBs -105, -118, -156, and -167 were purified on an active charcoal column to remove AhR agonists that could be present as impurities. Activation or inhibition of AhR-dependent gene expression by purified MO-PCBs was studied in stably transfected cell lines (H1G1.1c3 mouse, H4G1.1c2 rat hepatoma), containing an AhR-responsive (AhR-EGFP) reporter gene. In addition, EROD activity was used as marker for CYP1A1 activity in these cell lines. MO-PCBs -105, -118, -156 induced AhR-EGFP expression in both rodent cell lines, with PCB-156 (10microM) being most effectively; inducing gene expression to approximately 27% of TCDD (mouse cells) and 62.5+/-3.4% (rat cells) of TCDD. This concurred with increased EROD activity in both cell lines to maxima of 20.5+/-1.5% and 68+/-3.2% of TCDD, respectively. No induction was observed for PCB-167. In the H1G1.1c3 mouse cells, PCB-105, -118 and -156 (10microM) significantly reduced TCDD-induced AhR-EGFP expression to 50.9+/-2.9%, 58.3+/-2.2% and 70.8+/-1.3% of TCDD. Reduced EROD activity was also observed, of 39.3+/-2.8%, 67+/-5% and 48.3+/-4% compared to TCDD. PCB-167 did not result in significant reduction. In rat cells, only PCB-156 resulted in significant decrease in TCDD-induced AhR-EGFP expression of 35%, suggesting species differences play a role. Our results suggest that purification of MO-PCBs is an essential step in determining accurate REP values, and could very likely lead to lower TEF values than those presently assigned by the WHO.


Subject(s)
Charcoal/chemistry , Polychlorinated Biphenyls/chemistry , Polychlorinated Biphenyls/pharmacology , Animals , Cell Line, Tumor , Cytochrome P-450 CYP1A1/genetics , Cytochrome P-450 CYP1A1/metabolism , Gene Expression Regulation/drug effects , Green Fluorescent Proteins/metabolism , Mice , Rats , Receptors, Aryl Hydrocarbon/genetics , Receptors, Aryl Hydrocarbon/metabolism
14.
Toxicol Sci ; 92(1): 133-42, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16601081

ABSTRACT

Polybrominated diphenyl ethers (PBDEs) are brominated flame retardants that have been in use as additives in various consumer products. Structural similarities of PBDEs with other polyhalogenated aromatic hydrocarbons that show affinity for the aryl hydrocarbon receptor (AhR), such as some polychlorinated biphenyls, raised concerns about their possible dioxin-like properties. We studied the ability of environmentally relevant PBDEs (BDE-47, -99, -100, -153, -154, and -183) and the "planar" congener BDE-77 to bind and/or activate the AhR in stably transfected rodent hepatoma cell lines with an AhR-responsive enhanced green fluorescent protein (AhR-EGFP) reporter gene (H1G1.1c3 mouse and H4G1.1c2 rat hepatoma). 7-Ethoxyresorufin-O-deethylation (EROD) was used as a marker for CYP1A1 activity. Dose- and bromination-specific inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced responses was measured by their ability to inhibit the induction of AhR-EGFP expression and EROD activity. Individual exposure to these PBDEs did not result in any increase in induction of AhR-EGFP or CYP1A1 activity. The lower brominated PBDEs showed the strongest inhibitory effect on TCDD-induced activities in both cell lines. While the highest brominated PBDE tested, BDE-183, inhibited EROD activity, it did not affect the induction of AhR-EGFP expression. Similar findings were observed after exposing stably transfected human hepatoma (xenobiotic response element [XRE]-HepG2) cells to these PBDEs, resulting in a small but statically significant agonistic effect on XRE-driven luciferase activity. Co-exposure with TCDD resulted again in antagonistic effects, confirming that the inhibitory effect of these PBDEs on TCDD-induced responses was not only due to direct interaction at receptor level but also at DNA-binding level. This antagonism was confirmed for BDE-99 in HepG2 cells transiently transfected with a Gal4-AhR construct and the corresponding Gal4-Luc reporter gene. In addition, a chromatin immunoprecipitation assay further confirmed that BDE-99 could bind to the AhR and activate the AhR nuclear translocation and dioxin responsive element (DRE) binding in the context of the CYP1A1 promoter. However, the transactivation function of the BDE-99-activated AhR seems to be very weak. These combined results suggest that PBDEs do bind but not activate the AhR-AhR nuclear translocator protein-XRE complex.


Subject(s)
Polybrominated Biphenyls/pharmacology , Receptors, Aryl Hydrocarbon/drug effects , Animals , Base Sequence , Cell Line, Tumor , Chromatin Immunoprecipitation , DNA Primers , Ethers , Mice , Rats , Transfection
15.
Arch Environ Contam Toxicol ; 48(2): 201-8, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15696347

ABSTRACT

Numerous environmental contaminants have been identified as endocrine disruptors (EDs)--substances that alter endocrine homeostasis by interfering with the biological action, production, or pharmacokinetics of endogenous hormones. Xenoestrogens are those EDs whose biological activity is similar to endogenous estrogen. This report presents data that identified Surflan, a proprietary herbicide emulsion, and its active ingredient oryzalin as xenoestrogens. In vitro, Surflan and oryzalin activated an estrogen-inducible reporter gene, and oryzalin competitively displaced 17beta-estradiol from the estrogen receptor. In vivo, Surflan and oryzalin induced expression of estrogen-regulated high-molecular-weight choriogenin genes in medaka (Oryzias latipes). These results are consistent with the characteristics of previously identified xenoestrogens and indicate that Surflan and oryzalin have the potential to adversely affect numerous estrogen-regulated biological processes.


Subject(s)
Dinitrobenzenes/pharmacology , Dinitrobenzenes/toxicity , Estradiol/biosynthesis , Estrogens/pharmacology , Estrogens/toxicity , Herbicides/pharmacology , Herbicides/toxicity , Sulfanilamides/pharmacology , Sulfanilamides/toxicity , Water Pollutants, Chemical/pharmacology , Water Pollutants, Chemical/toxicity , Animals , Biological Assay , Blotting, Western , Genes, Reporter , Luciferases, Firefly/analysis , Male , Oryzias/physiology , Receptors, Estrogen/physiology , Xenobiotics/toxicity
16.
Toxicol Sci ; 82(2): 488-96, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15456928

ABSTRACT

Polybrominated diphenylethers (PBDEs) are used as additive flame-retardants in consumer products to reduce the chances of ignition and burning. Levels of certain PBDE congeners have been increasing in fish, wildlife, and human tissues during the last decades. Some PBDEs are lipophilic and persistent, resulting in bioaccumulation in the environment. The structural similarity of PBDEs to other polyhalogenated aromatic hydrocarbons such as PCBs, has raised concerns that PBDEs might act as agonists for the aryl hydrocarbon receptor (AhR). To study the possible AhR-mediated effects of the environmentally relevant PBDEs (BDE47, 77, 99, 100, 153, 154, 183, 209), the induction of cytochrome P450-1A1 (CYP1A1) was studied in human breast carcinoma (MCF-7), human hepatocellular carcinoma (HepG2), and rat hepatoma (H4IIE) cells. 7-Ethoxyresorufin-O-deethylase (EROD) was used as a marker for CYP1A1 activity. Cells were exposed for 72 h to various PBDE concentrations (0.01-10 microM). Positive controls were 2,3,7,8-TCDD (0.001-2.5 nM) and PCB126 (0.01-10 nM). None of these PBDEs was capable of inducing EROD activity; this was confirmed by real time RT-PCR for CYP1A1 mRNA. However, in cells exposed to PBDEs in combination with TCDD, a concentration-dependent decrease in TCDD-induced EROD activity occurred. Co-exposure of BDE153 (10 muM) and a maximally inducing concentration of TCDD (1 nM) reduced EROD activity to 49% of the maximum induction by TCDD alone. All tested PBDEs showed similar effects in each cell line, though quantitative differences were observed. The observed decrease in CYP1A1 activity was not due to PBDE-dependent catalytic inhibition of EROD activity or cytotoxicity, nor were decreased CYP1A1 mRNA levels observed. However, inhibition of luciferase induction in mouse (Hepa) and rat (H4IIE) hepatoma cells containing a stably transfected AhR-responsive luciferase reporter gene, suggests that BDE77 is a weak AhR antagonist or partial agonist.


Subject(s)
Cytochrome P-450 CYP1A1/metabolism , Environmental Pollutants/toxicity , Polybrominated Biphenyls/toxicity , Polychlorinated Dibenzodioxins/toxicity , Animals , Breast Neoplasms/metabolism , Carcinoma, Hepatocellular/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Female , Humans , L-Lactate Dehydrogenase/blood , Luciferases/metabolism , Oxazines , RNA, Messenger/biosynthesis , Rats , Receptors, Aryl Hydrocarbon/agonists , Receptors, Aryl Hydrocarbon/antagonists & inhibitors , Reverse Transcriptase Polymerase Chain Reaction , Tetrazolium Salts , Thiazoles , Xanthenes
17.
J Biochem Mol Toxicol ; 15(4): 187-96, 2001.
Article in English | MEDLINE | ID: mdl-11673847

ABSTRACT

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates many of the biological and toxicological actions of a diverse range of chemicals, including the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin). Although no endogenous physiological ligand for the AhR has yet been described, numerous studies support the existence of such a ligand(s). Here we have examined the ability of prostaglandins and related chemicals to activate the AhR signaling system. Using two AhR-based bioassay systems we report that relatively high concentrations of several prostaglandins (namely, PGB3, PGD3, PGF3alpha, PGG2, PGH1, and PGH2) can not only stimulate AhR transformation and DNA binding in vitro, but also induce AhR-dependent reporter gene expression in mouse hepatoma cells in culture. PGG2 also induced AhR-dependent reporter gene expression to a level three-to four fold greater than that observed with a maximal inducing dose of TCDD. Sucrose gradient ligand binding analysis revealed that PGG2 could competitively displace [3H]TCDD from the AhR. Overall, our results demonstrate that selected prostaglandins are weak agonists for the AhR and they represent a structurally distinct and novel class of activator of the AhR signal transduction pathway.


Subject(s)
Prostaglandins/pharmacology , Receptors, Aryl Hydrocarbon/drug effects , Signal Transduction/drug effects , Animals , Cells, Cultured , Centrifugation, Density Gradient , Chromatography, Gel , Cytosol/metabolism , DNA/biosynthesis , DNA/genetics , Dinoprostone/pharmacology , Dose-Response Relationship, Drug , Guinea Pigs , Liver/drug effects , Liver/metabolism , Luciferases/metabolism , Male , Mice , Polychlorinated Dibenzodioxins/pharmacology , Receptors, Aryl Hydrocarbon/genetics , Signal Transduction/genetics , Sucrose
18.
Arch Biochem Biophys ; 393(1): 154-62, 2001 Sep 01.
Article in English | MEDLINE | ID: mdl-11516172

ABSTRACT

Suspension of human epidermal cells in methylcellulose-containing medium induces CYP1A1 by a mechanism requiring functional Ah receptor (AhR). In present work CYP1A1 mRNA was induced in a variety of cultured rat epithelial cells by suspension, but the induction was transient, with CYP1A1 mRNA reaching maximal levels by 5 h and disappearing by 12 h. Though the methylcellulose itself contained no detectable ligand, (a) suspension activated the AhR, as judged by mobility shift assays, (b) the AhR competitive inhibitor alpha-naphthoflavone inhibited suspension-mediated induction, and (c) induction was dependent upon dioxin responsive transcriptional elements in the CYP1A1 promoter. The rapid disappearance of CYP1A1 mRNA after 5 h of suspension was unaffected by the addition of TCDD but was prevented by the inclusion of the protein synthesis inhibitor cycloheximide. Thus the downregulation appears to be mediated by a novel short-lived protein induced or activated by suspension.


Subject(s)
Cytochrome P-450 CYP1A1/genetics , Animals , Base Sequence , Cells, Cultured , Culture Media , Cycloheximide/pharmacology , DNA Primers/genetics , Gene Expression/drug effects , Genes, Reporter , Keratinocytes/drug effects , Keratinocytes/metabolism , Methylcellulose , Polychlorinated Dibenzodioxins/pharmacology , Promoter Regions, Genetic , Protein Synthesis Inhibitors/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Receptors, Aryl Hydrocarbon/antagonists & inhibitors , Receptors, Aryl Hydrocarbon/metabolism
19.
Chem Biol Interact ; 128(3): 211-29, 2000 Nov 01.
Article in English | MEDLINE | ID: mdl-11064004

ABSTRACT

Green tea possesses significant anticancer activity in numerous experimental animal models, including demonstrated protection against aryl hydrocarbon induced cancers. The aryl hydrocarbon receptor (AhR) mediates the transcriptional activation of CYP1A1 and CYP1A2. In the present study, we investigated the effects of commercially available green tea extracts (GTEs) and individual tea catechins on the function of the AhR and on CYP1A gene expression in human hepatoma HepG2 cells and primary cultures of human hepatocytes. GTEs inhibited the transcription of a human CYP1A1 promoter-driven reporter gene induced by the AhR ligand 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) in a concentration-dependent manner and inhibited the induced accumulation of both CYP1A1 and CYP1A2 mRNAs. GTEs blocked TCDD-induced binding of the AhR to DNA in HepG2 cells and in vitro in isolated hepatic cytosol. To determine if the observed effects were due to a single green tea component, we examined the four major catechins present in GTEs. Only (-)-epigallocatechin gallate (EGCG), the most abundant catechin in green tea, was able to inhibit TCDD-induced binding of the AhR to DNA and subsequent CYP1A transcription, however EGCG alone was less effective than GTEs. We next examined GTEs and catechins for AhR agonist activity. GTEs caused a concentration-dependent increase in CYP1A1-promoter driven reporter gene activity and caused accumulation of CYP1A1 mRNA and protein, but we found that individual catechins were unable to induce the expression of CYP1A1. Our results demonstrate that GTEs as a whole exert mixed agonist/antagonist activity on the AhR, while EGCG functions as a strict AhR antagonist. Therefore, modulation of human CYP1A expression by green tea extracts can not be attributed to the action of a single tea catechin, but rather is due to the effects of a complex mixture. These findings may be useful in future studies concerning green tea as a cancer preventive agent.


Subject(s)
Catechin/analogs & derivatives , Catechin/pharmacology , Cytochrome P-450 CYP1A1/genetics , Gene Expression/drug effects , Plant Extracts/pharmacology , Tea/chemistry , Animals , Carcinoma, Hepatocellular/enzymology , DNA/metabolism , Guinea Pigs , Humans , Liver/enzymology , Liver Neoplasms/enzymology , Male , Mice , Polychlorinated Dibenzodioxins/pharmacology , RNA, Messenger/metabolism , Receptors, Aryl Hydrocarbon/drug effects , Receptors, Aryl Hydrocarbon/physiology , Tumor Cells, Cultured
20.
In Vitr Mol Toxicol ; 13(1): 67-82, 2000.
Article in English | MEDLINE | ID: mdl-10900408

ABSTRACT

The ability of a variety of compounds to disrupt normal endocrine homeostasis, and potentially, the physiological and reproductive capacity of an organism, has gained worldwide attention in recent years. In an attempt to identify such compounds, and to examine the mechanism(s) by which they may exert their actions, we have constructed reporter plasmid vectors that contain the firefly luciferase gene under hormone-inducible control of estrogen-, androgen-, or retinoic acid-responsive DNA enhancer elements. Transient transfection of these vectors into appropriate receptor-containing cell lines revealed their ability to respond to their respective hormones with the induction of luciferase. Here, we describe development and optimization of a recombinant human ovarian carcinoma (BG-1) line, which has been stably transfected with the estrogen responsive luciferase reporter plasmid. The resulting recombinant cell line (BG1Luc4E(2)) responds to 17beta-estradiol at concentrations as low as 1 pM. The utility of BG1Luc4E(2) cells as a bioassay screening system for environmental estrogens was demonstrated by their response to known xenoestrogens, and also by the putative identification of two polychlorinated biphenyls (2,3',4, 4,'-tetrachlorobiphenyl and 2,2',3,5',6-pentachlorobiphenyl) as novel estrogenic chemicals. These cell bioassay systems have applications for rapid screening, identification, and characterization of endocrine disrupting chemicals.


Subject(s)
Biological Assay/methods , Estrogen Receptor Modulators/analysis , Estrogen Receptor Modulators/pharmacology , Estrogens/analysis , Estrogens/pharmacology , Ovary/drug effects , Androgens/pharmacology , Animals , Base Sequence , Dose-Response Relationship, Drug , Enhancer Elements, Genetic/genetics , Estrogen Antagonists/pharmacology , Estrogen Receptor Modulators/toxicity , Estrogens/toxicity , Female , Genes, Reporter/genetics , Humans , Molecular Sequence Data , Ovary/metabolism , Polychlorinated Biphenyls/analysis , Polychlorinated Biphenyls/pharmacology , Polychlorinated Biphenyls/toxicity , Receptors, Estrogen/metabolism , Sensitivity and Specificity , Substrate Specificity , Tamoxifen/pharmacology , Time Factors , Transcriptional Activation/drug effects , Tretinoin/pharmacology , Tumor Cells, Cultured
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