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.

High throughput in vitro and in vivo screening of inland waters of Southern California.

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.

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'.

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.

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.

Determination of in vitro relative potency (REP) values for mono-ortho polychlorinated biphenyls after purification with active charcoal.

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.

Interactions of polybrominated diphenyl ethers with the aryl hydrocarbon receptor pathway.

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.

Identification of the herbicide Surflan and its active ingredient oryzalin, a dinitrosulfonamide, as xenoestrogens.

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.

Effects of polybrominated diphenyl ethers on basal and TCDD-induced ethoxyresorufin activity and cytochrome P450-1A1 expression in MCF-7, HepG2, and H4IIE cells.

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.

Activation of the Ah receptor signaling pathway by prostaglandins.

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.

Comparative studies on the effects of green tea extracts and individual tea catechins on human CYP1A gene expression.

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.

Recombinant cell bioassays for endocrine disruptors: development of a stably transfected human ovarian cell line for the detection of estrogenic and anti-estrogenic chemicals.

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.

Ah receptor-based chemical screening bioassays: application and limitations for the detection of Ah receptor agonists.

The aromatic hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates many of the biologic and toxicologic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related chemicals. Here we utilized two AhR-dependent bioassay systems as screening tools to identify novel AhR agonists and to detect the presence of AhR agonists in sample extracts. These assays measure the ability of a chemical to activate AhR DNA binding in vitro (GRAB bioassay) or AhR-dependent (luciferase) gene expression in cultured cells (CALUX bioassay). Known AhR agonists (halogenated and nonhalogenated aromatic hydrocarbons) were positive in both assays, whereas the AhR antagonist alpha-naphthoflavone exhibited agonist activity only in the GRAB assay. In vitro GRAB analysis has identified several imidazoline receptor ligands and beta-carbolines as AhR agonists and also revealed the presence of AhR agonist activity in crude DMSO extracts of commercial newspapers. In contrast to their positive activity in the GRAB assay, the majority of these chemicals/extracts were only weakly active or inactive in the cell-based CALUX assay. Our results not only reveal that the ability of a chemical to activate the AhR in vitro does not necessarily correlate with its ability to induce gene expression in intact cells, but the high level of false positives obtained with the GRAB assay clearly demonstrates its inability to accurately identify AhR agonists or agonist activity. Screening of unknown chemicals, chemical classes, and samples for AhR agonist activity will require the use of intact cell bioassays.

Development and modification of a recombinant cell bioassay to directly detect halogenated and polycyclic aromatic hydrocarbons in serum.

Polycyclic and halogenated aromatic hydrocarbons (PAHs/HAHs) are a diverse group of widespread and persistent environmental contaminants that can cause a variety of detrimental effects in vertebrates. As most available methods to detect these contaminants are expensive, labor and time intensive, and require large amounts of tissue for extraction and analysis, several rapid mechanistically based bioassay systems have been developed to detect these chemicals. Here we describe application and optimization of a recently developed recombinant mouse cell bioassay system that responds to both PAHs and HAHs with the rapid induction of firefly luciferase for the detection of these chemicals in whole serum samples. This chemically activated luciferase expression (CALUX) bioassay has been modified to allow rapid (4-h) and direct analysis of small volumes (25-50 microl) of whole serum in a 96-well microtiter plate format without the need for solvent extraction. This bioassay can detect as little as 10 parts per trillion of the most potent HAH, 2,3,7,8-TCDD, and is also sensitive to other HAHs and PAHs. The use of simple procedures corrects for interplate and intraplate variability and the Ah receptor dependence of the induction response is accounted for by use of the antagonist 4-amino-3-methoxyflavone.

Effect of in utero and postnatal exposure to environmental tobacco smoke on the developmental expression of pulmonary cytochrome P450 monooxygenases.

Pulmonary cytochrome P450 monooxygenases metabolize xenobiotic chemicals, including those found in environmental tobacco smoke (ETS). Exposure to ETS beginning at birth has been shown to induce the P450 CYP1A1 by seven days of life. The effects of perinatal exposure to ETS of the rat lung on the expression of CYP1A1, 1B1, 2B1, and NADPH cytochrome P450 reductase were measured using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Timed pregnant dams and their pups were exposed to aged and diluted sidestream cigarette smoke (ADSS) as a surrogate for ETS for four hours/ day from gestational day 5 through postnatal day 21. For all genes analyzed, mRNA could be detected in the fetal lung beginning at gestational day 17 but were not altered by ADSS. In contrast, intraperitoneal injection of dams with beta-naphthoflavone significantly elevated both CYP1A1 and 1B1 at gestational day 21, indicating that these genes are inducible. Continued exposure to ADSS significantly induced CYP1A1 but not other P450 genes as early as one day after birth.. We conclude that (1) ADSS induces pulmonary CYP1A1 in the first day of life; (2) fetal cytochrome P450 genes are not induced by maternal exposure to ADSS; and (3) in the fetal lung, CYP1A1 and 1B1 can be induced by beta-naphthoflavone.

Effects of histone deacetylase inhibitors on the Ah receptor gene promoter.

The aromatic hydrocarbon receptor (AhR) is a ligand-dependent basic helix-loop-helix-PAS-containing transcription factor which is activated by chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin. Constitutive expression of the AhR gene occurs in a tissue- and developmentally specific manner and appears to be altered by chemicals which affect histone deacetylase (HDAC) activity in cells in culture. Here we have directly characterized the effects of two HDAC inhibitors, n-butyrate and trichostatin A, on the promoter activity of the murine AhR gene. HDAC inhibitors increased the constitutive activity of the AhR gene promoter in a luciferase reporter construct by five- to sevenfold in a dose- and time-dependent manner in several cell lines and was correlated with an increase in endogenous AhR activity in an AhR-deficient cell line. Deletion analysis of the upstream region of the AhR gene localized the HDAC inhibitor effect to a 167-bp region encompassing -77 to +90 of the AhR gene promoter. Cotransfection of an AhR promoter-luciferase reporter plasmid with a vector expressing the E1A(12s) oncoprotein, a negative regulator of p300, a protein with histone acetylase activity, decreased AhR promoter activity fivefold. Overall, our results support a role for histone acetylation in the transcriptional activity of the AhR gene promoter.