Human and Zebrafish Nuclear Progesterone Receptors Are Differently Activated by Manifold Progestins.

The environmental risk of natural and synthetic ligands of the nuclear progesterone receptor (nPR) has been pointed out, however there is still a lack of mechanistic information regarding their ability to interact with nuclear PR in aquatic species. To identify possible interspecies differences, we assessed in vitro the ability of manifold progestins to transactivate zebrafish (zf) and human (h) PRs, using two established reporter cell lines, U2OS-zfPR and HELN-hPR, respectively. Reference ligands highlighted some differences between the two receptors. The reference human agonist ligands promegestone and progesterone induced luciferase activity in both cell lines in a concentration-dependent manner, whereas the natural zebrafish progestin 17α,20ß-dihydroxy-4-pregnen-3-one activated zfPR but not hPR. The potent human PR antagonist mifepristone (RU486) blocked PR-induced luciferase in both cell models but with different potencies. In addition, a set of 22 synthetic progestins were screened on the two cell lines. Interestingly, all of the tested compounds activated hPR in the HELN-hPR cell line, whereas the majority of them acted as zfPR antagonists in U2OS-zfPR. Such zfPR-specific response was further confirmed in zebrafish liver cells. This study provides novel information regarding the activity of a large set of progestins on human and zebrafish PR and highlights major interspecies differences in their activity, which may result in differential effects of progestins between fish and humans.

Chronic simultaneous exposure of common carp (Cyprinus carpio) from embryonic to juvenile stage to drospirenone and gestodene at low ng/L level caused intersex.

Synthetic progestins are emerging contaminants of the aquatic environment with endocrine disrupting potential. The main aim of the present study was to investigate the effects of the synthetic progestins gestodene, and drospirenone on sex differentiation in common carp (Cyprinus carpio) by histological analysis. To gain insights into the mechanisms behind the observations from the in vivo experiment on sex differentiation, we analyzed expression of genes involved in hypothalamus-pituitary-gonad (HPG) and hypothalamus-pituitary-thyroid (HPT) axes, histology of hepatopancreas, and in vitro bioassays. Carp were continuously exposed to concentrations of 2 ng/L of single progestins (gestodene or drospirenone) or to their mixture at concentration 2 ng/L of each. The exposure started 24 h after fertilization of eggs and concluded 160 days post-hatching. Our results showed that exposure of common carp to a binary mixture of drospirenone and gestodene caused increased incidence of intersex (32%) when compared to clean water and solvent control groups (both 3%). Intersex most probably was induced by a combination of multiple modes of action of the studied substances, namely anti-gonadotropic activity, interference with androgen receptor, and potentially also with HPT axis or estrogen receptor.

Differential activity of BPA, BPAF and BPC on zebrafish estrogen receptors in vitro and in vivo.

The high volume production compound bisphenol A (BPA) is of environmental concern largely because of its estrogenic activity. Consequently, BPA analogues have been synthesized to be considered as replacement molecules for BPA. These analogues need to be thoroughly evaluated for their estrogenic activity. Here, we combined mechanism zebrafish-based assays to examine estrogenic and anti-estrogenic activities of BPA and two of its analogues, bisphenol AF (BPAF) and bisphenol C (BPC) in vitro and in vivo. In vitro reporter cell lines were used to investigate agonistic and antagonistic effects of the three bisphenols on the three zebrafish estrogen receptors. The transgenic Tg(5 × ERE:GFP) and Cyp19a1b-GFP zebrafish lines were then used to analyze estrogenic and anti-estrogenic responses of the three bisphenols in vivo. BPA, BPAF and BPC were agonists with different potencies for the three zebrafish estrogen receptors in vitro. The potent zfERα-mediated activity of BPA and BPAF in vitro resulted in vivo by activation of GFP expression in zebrafish larvae in the heart (zfERα-dependent) at lower concentrations, and in the liver (zfERß-dependent) at higher concentrations. BPC induced zfERß-mediated luciferase expression in vitro, and the zfERß agonism led to activation of GFP expression in the liver and the brain in vivo. In addition, BPC acted as a full antagonist on zfERα, and completely inhibited estrogen-induced GFP expression in the heart of the zebrafish larvae. To summarize, applying a combination of zebrafish-based in vitro and in vivo methods to evaluate bisphenol analogues for estrogenic activity will facilitate the prioritization of these chemicals for further analysis in higher vertebrates as well as the risk assessment in humans.

Triclosan Lacks Anti-Estrogenic Effects in Zebrafish Cells but Modulates Estrogen Response in Zebrafish Embryos.

Triclosan (TCS), an antimicrobial agent widely found in the aquatic environment, is suspected to act as an endocrine disrupting compound, however mechanistic information is lacking in regards to aquatic species. This study assessed the ability of TCS to interfere with estrogen receptor (ER) transcriptional activity, in zebrafish-specific in vitro and in vivo reporter gene assays. We report that TCS exhibits a lack of either agonistic or antagonistic effects on a panel of ER-expressing zebrafish (ZELH-zfERα and -zfERß) and human (MELN) cell lines. At the organism level, TCS at concentrations of up to 0.3 µM had no effect on ER-regulated brain aromatase gene expression in transgenic cyp19a1b-GFP zebrafish embryos. At a concentration of 1 µM, TCS interfered with the E2 response in an ambivalent manner by potentializing a low E2 response (0.625 nM), but decreasing a high E2 response (10 nM). Altogether, our study suggests that while modulation of ER-regulated genes by TCS may occur in zebrafish, it does so irrespective of a direct binding and activation of zfERs.

Mixture Concentration-Response Modeling Reveals Antagonistic Effects of Estradiol and Genistein in Combination on Brain Aromatase Gene (cyp19a1b) in Zebrafish.

Comprehension of compound interactions in mixtures is of increasing interest to scientists, especially from a perspective of mixture risk assessment. However, most of conducted studies have been dedicated to the effects on gonads, while only few of them were. interested in the effects on the central nervous system which is a known target for estrogenic compounds. In the present study, the effects of estradiol (E2), a natural estrogen, and genistein (GEN), a phyto-estrogen, on the brain ER-regulated cyp19a1b gene in radial glial cells were investigated alone and in mixtures. For that, zebrafish-specific in vitro and in vivo bioassays were used. In U251-MG transactivation assays, E2 and GEN produced antagonistic effects at low mixture concentrations. In the cyp19a1b-GFP transgenic zebrafish, this antagonism was observed at all ratios and all concentrations of mixtures, confirming the in vitro effects. In the present study, we confirm (i) that our in vitro and in vivo biological models are valuable complementary tools to assess the estrogenic potency of chemicals both alone and in mixtures; (ii) the usefulness of the ray design approach combined with the concentration-addition modeling to highlight interactions between mixture components.

In vitro and in vivo estrogenic activity of BPA, BPF and BPS in zebrafish-specific assays.

Bisphenol A (BPA) is a widely used chemical that has been extensively studied as an endocrine-disrupting chemical (EDC). Other bisphenols sharing close structural features with BPA, are increasingly being used as alternatives, increasing the need to assess associated hazards to the endocrine system. In the present study, the estrogenic activity of BPA, bisphenol S (BPS) and bisphenol F (BPF) was assessed by using a combination of zebrafish-specific mechanism-based in vitro and in vivo assays. The three bisphenols were found to efficiently transactivate all zebrafish estrogen receptor (zfER) subtypes in zebrafish hepatic reporter cell lines (ZELH-zfERs). BPA was selective for zfERα while BPS and BPF were slightly more potent on zfERß subtypes. We further documented the estrogenic effect in vivo by quantifying the expression of brain aromatase using a transgenic cyp19a1b-GFP zebrafish embryo assay. All three bisphenols induced GFP in a concentration-dependent manner. BPS only partially induced brain aromatase at the highest tested concentrations (>30µM) while BPA and BPF strongly induced GFP, in an ER-dependent manner, at 1-10µM. Furthermore, we show that BPF strongly induced vitellogenin synthesis in adult male zebrafish. Overall, this study demonstrates the estrogenic activity of BPA, BPF and BPS in different cell- and tissue-contexts and at different stages of development. Differences between in vitro and in vivo responses are discussed in light of selective ER activation and the fate of the compounds in the models. This study confirms the relevance of combining cellular and whole-organism bioassays in a unique model species for the hazard assessment of candidate EDCs.

Comparison of the In Vivo Biotransformation of Two Emerging Estrogenic Contaminants, BP2 and BPS, in Zebrafish Embryos and Adults.

Zebrafish embryo assays are increasingly used in the toxicological assessment of endocrine disruptors. Among other advantages, these models are 3R-compliant and are fit for screening purposes. Biotransformation processes are well-recognized as a critical factor influencing toxic response, but major gaps of knowledge exist regarding the characterization of functional metabolic capacities expressed in zebrafish. Comparative metabolic studies between embryos and adults are even scarcer. Using ³H-labeled chemicals, we examined the fate of two estrogenic emerging contaminants, benzophenone-2 (BP2) and bisphenol S (BPS), in 4-day embryos and adult zebrafish. BPS and BP2 were exclusively metabolized through phase II pathways, with no major qualitative difference between larvae and adults except the occurrence of a BP2-di-glucuronide in adults. Quantitatively, the biotransformation of both molecules was more extensive in adults. For BPS, glucuronidation was the predominant pathway in adults and larvae. For BP2, glucuronidation was the major pathway in larvae, but sulfation predominated in adults, with ca. 40% conversion of parent BP2 and an extensive release of several conjugates into water. Further larvae/adults quantitative differences were demonstrated for both molecules, with higher residue concentrations measured in larvae. The study contributes novel data regarding the metabolism of BPS and BP2 in a fish model and shows that phase II conjugation pathways are already functional in 4-dpf-old zebrafish. Comparative analysis of BP2 and BPS metabolic profiles in zebrafish larvae and adults further supports the use of zebrafish embryo as a relevant model in which toxicity and estrogenic activity can be assessed, while taking into account the absorption and fate of tested substances.

Cell-specific biotransformation of benzophenone-2 and bisphenol-s in zebrafish and human in vitro models used for toxicity and estrogenicity screening.

Several human and fish bioassays have been designed to characterize the toxicity and the estrogenic activity of chemicals. However, their biotransformation capability (bioactivation/detoxification processes) is rarely reported, although this can influence the estrogenic potency of test compounds. The fate of two estrogenic chemicals, the UV filter benzophenone-2 (BP2) and the bisphenol A substitute bisphenol S (BPS) was deciphered in eight human and zebrafish in vitro cell models, encompassing hepatic and mammary cellular contexts. BP2 and BPS were metabolized into a variety of gluco- and sulfo-conjugated metabolites. Similar patterns of BP2 and BPS biotransformation were observed among zebrafish models (primary hepatocytes, ZFL and ZELH-zfER cell lines). Interestingly, metabolic patterns in zebrafish models and in the human hepatic cell line HepaRG shared many similarities, while biotransformation rates in cell lines widely used for estrogenicity testing (MELN and T47D-KBLuc) were quantitatively low and qualitatively different. This study provides new data on the comparative metabolism of BP2 and BPS in human and fish cellular models that will help characterize their metabolic capabilities, and underlines the relevance of using in vitro zebrafish-based bioassays when screening for endocrine disrupting chemicals.

Linking in Vitro Effects and Detected Organic Micropollutants in Surface Water Using Mixture-Toxicity Modeling.

Surface water can contain countless organic micropollutants, and targeted chemical analysis alone may only detect a small fraction of the chemicals present. Consequently, bioanalytical tools can be applied complementary to chemical analysis to detect the effects of complex chemical mixtures. In this study, bioassays indicative of activation of the aryl hydrocarbon receptor (AhR), activation of the pregnane X receptor (PXR), activation of the estrogen receptor (ER), adaptive stress responses to oxidative stress (Nrf2), genotoxicity (p53) and inflammation (NF-κB) and the fish embryo toxicity test were applied along with chemical analysis to water extracts from the Danube River. Mixture-toxicity modeling was applied to determine the contribution of detected chemicals to the biological effect. Effect concentrations for between 0 to 13 detected chemicals could be found in the literature for the different bioassays. Detected chemicals explained less than 0.2% of the biological effect in the PXR activation, adaptive stress response, and fish embryo toxicity assays, while five chemicals explained up to 80% of ER activation, and three chemicals explained up to 71% of AhR activation. This study highlights the importance of fingerprinting the effects of detected chemicals.

Selectivity of natural, synthetic and environmental estrogens for zebrafish estrogen receptors.

Zebrafish, Danio rerio, is increasingly used as an animal model to study the effects of pharmaceuticals and environmental estrogens. As most of these estrogens have only been tested on human estrogen receptors (ERs), it is necessary to measure their effects on zebrafish ERs. In humans there are two distinct nuclear ERs (hERα and hERß), whereas the zebrafish genome encodes three ERs, zfERα and two zfERßs (zfERß1 and zfERß2). In this study, we established HeLa-based reporter cell lines stably expressing each of the three zfERs. We first reported that estrogens more efficiently activate the zfERs at 28°C as compared to 37°C, thus reflecting the physiological temperature of zebrafish in wildlife. We then showed significant differences in the ability of agonist and antagonist estrogens to modulate activation of the three zfER isotypes in comparison to hERs. Environmental compounds (bisphenol A, alkylphenols, mycoestrogens) which are hER panagonists and hERß selective agonists displayed greater potency for zfERα as compared to zfERßs. Among hERα selective synthetic agonists, PPT did not activate zfERα while 16α-LE2 was the most zfERα selective compound. Altogether, these results confirm that all hER ligands control in a similar manner the transcriptional activity of zfERs although significant differences in selectivity were observed among subtypes. The zfER subtype selective ligands that we identified thus represent new valuable tools to dissect the physiological roles of the different zfERs. Finally, our work also points out that care has to be taken in transposing the results obtained using the zebrafish as a model for human physiopathology.

Identification of synthetic steroids in river water downstream from pharmaceutical manufacture discharges based on a bioanalytical approach and passive sampling.

A bioanalytical approach was used to identify chemical contaminants at river sites located downstream from a pharmaceutical factory, where reproductive alterations in wild fish have been previously observed. By using polar organic compound integrative samplers (POCIS) at upstream and downstream sites, biological activity profiles based on in vitro bioassays revealed the occurrence of xenobiotic and steroid-like activities, including very high glucocorticoid, antimineralocorticoid, progestogenic and pregnane X receptor (PXR)-like activities (µg standard-EQ/g of sorbent range), and weak estrogenic activity (ng E2-EQ/g of sorbent range). Chemical analyses detected up to 60 out of 118 targeted steroid and pharmaceutical compounds in the extracts. In vitro profiling of occurring individual chemicals revealed the ability of several ones to act as agonist and/or antagonist of different steroids receptors. Mass balance calculation identified dexamethasone, spironolactone, and 6-alpha-methylprednisolone as major contributors to corticosteroid activities and levonorgestrel as the main contributor to progestogenic activities. Finally, RP-HPLC based fractionation of POCIS extracts and testing activity of fractions confirmed identified compounds and further revealed the presence of other unknown active chemicals. This study is one of the first to report environmental contamination by such chemicals; their possible contribution to in situ effects on fish at the same site is suggested.

Affinity purification using recombinant PXR as a tool to characterize environmental ligands.

Many environmental endocrine disrupting compounds act as ligands for nuclear receptors. The human pregnane X receptor (hPXR), for instance, is activated by a variety of environmental ligands such as steroids, pharmaceutical drugs, pesticides, alkylphenols, polychlorinated biphenyls and polybromo diethylethers. Some of us have previously reported the occurrence of hPXR ligands in environmental samples but failed to identify them. The aim of this study was to test whether a PXR-affinity column, in which recombinant hPXR was immobilized on solid support, could help the purification of these chemicals. Using PXR ligands of different affinity (10 nM < EC50 < 10 µM), we demonstrated that the PXR-affinity preferentially column captured ligands with medium to high affinities (EC50 < 1 µM). Furthermore, by using the PXR-affinity column to analyze an environmental sample containing ERα, AhR, AR, and PXR activities, we show that (i) half of the PXR activity of the sample was due to compounds with medium to high affinity for PXR and (ii) PXR shared ligands with ERα, AR, and AhR. These findings demonstrate that the newly developed PXR-affinity column coupled to reporter cell lines represents a valuable tool for the characterization of the nature of PXR active compounds and should therefore guide and facilitate their further analysis.

Effect-directed analysis of endocrine-disrupting compounds in multi-contaminated sediment: identification of novel ligands of estrogen and pregnane X receptors.

Effect-directed analysis (EDA)-based strategies have been increasingly used in order to identify the causative link between adverse (eco-)toxic effects and chemical contaminants. In this study, we report the development and use of an EDA approach to identify endocrine-disrupting chemicals (EDCs) in a multi-contaminated river sediment. The battery of in vitro reporter cell-based bioassays, measuring estrogenic, (anti)androgenic, dioxin-like, and pregnane X receptor (PXR)-like activities, revealed multi-contamination profiles. To isolate active compounds of a wide polarity range, we established a multi-step fractionation procedure combining: (1) a primary fractionation step using normal phase-based solid-phase extraction (SPE), validated with a mixture of 12 non-polar to polar standard EDCs; (2) a secondary fractionation using reversed-phase-based high-performance liquid chromatography (RP-HPLC) calibrated with 33 standard EDCs; and (3) a purification step using a recombinant estrogen receptor (ER) affinity column. In vitro SPE and HPLC profiles revealed that ER and PXR activities were mainly due to polar to mid-polar compounds, while dioxin-like and anti-androgenic activities were in the less polar fractions. The overall procedure allowed final isolation and identification of new environmental PXR (e.g., di-iso-octylphthalate) and ER (e.g., 2,4-di-tert-butylphenol and 2,6-di-tert-butyl-α-methoxy-p-cresol) ligands by using gas chromatography coupled with mass spectrometry with full-scan mode acquisition in mid-polar fractions. In vitro biological activity of these chemicals was further confirmed using commercial standards, with di-iso-octylphthalate identified for the first time as a potent hPXR environmental agonist.

Interlaboratory prevalidation of a new in vitro transcriptional activation assay for the screening of (anti-)androgenic activity of chemicals using the UALH-hAR cell line.

We report an interlaboratory evaluation of a recently developed androgen receptor (AR) transactivation assay using the UALH-hAR reporter cell line that stably expresses the luciferase gene under the transcriptional control of androgen receptor elements (AREs) with no glucocorticoid receptor (GR) crosstalk. Herein, a two-step prevalidation study involving three laboratories was conducted to assess performance criteria of the method such as transferability as well as robustness, sensitivity, and specificity. The first step consisted in the validation of the transfer of the cell line to participant laboratories through the testing of three reference chemicals: the AR agonist dihydrotestosterone, the AR antagonist hydroxyflutamide and the glucocorticoid dexamethasone. Secondly, a blinded study was conducted by screening a selection of ten chemicals, including four AR agonists, five AR antagonists, and one non-active chemical. All test compounds yielded the same activity profiles in all laboratories. The logEC50 (agonist assay) or logIC50 (antagonist assay) were in the same range, with intra-laboratory coefficients of variation (CVs) of 0.1-3.4% and interlaboratory CVs of 1-4%, indicating very good within- and between-laboratory reproducibility. Our results were consistent with literature and regulatory data (OECD TG458). Overall, this interlaboratory study demonstrated that the UALH-hAR assay is transferable, produces reliable, accurate and specific (anti)androgenic activity of chemicals, and can be considered for further regulatory validation.

In vitro biomonitoring of contamination by estrogenic compounds in coastal environments: comments on the use of M. galloprovincialis.

The use of mussel extracts in in vitro bioassays which express the estrogen receptor could provide valuable information on the bioavailability of endocrine disruptors in coastal environments. The aim of this study was to assess the temporal variability of the estrogenic responses in bioassays in Mytilus galloprovincialis. A 6-month in situ experiment was conducted in order to follow the estrogenic activity on MELN cell line during the reproduction stages of mussels. Estradiol equivalents (EEQ) determined in mussels using the MELN cell lines ranged from 0.79 to 3.72 ng/g dry weight (d.w.) in males, from 0.42 to 2.33 ng/g d.w. in females and from 3.41 to 4.2 d.w. in undifferentiated bivalves. We observed an increase in EEQ values during the spawning stage for males, not for female. The maximal EEQ values were observed at the indifferent stage. We discuss these results in regards to the actual knowledge on mussels' reproductive cycle and to the possible impact of xeno-estrogens. Variations of E2 levels in mussels must be taken into account for further studies on xeno-estrogens monitoring using hER reporter cell-lines bioassays.

Biological effect and chemical monitoring of Watch List substances in European surface waters: Steroidal estrogens and diclofenac - Effect-based methods for monitoring frameworks.

Three steroidal estrogens, 17α-ethinylestradiol (EE2), 17ß-estradiol (E2), estrone (E1), and the non-steroidal anti-inflammatory drug (NSAID), diclofenac have been included in the first Watch List of the Water Framework Directive (WFD, EU Directive 2000/60/EC, EU Implementing Decision 2015/495). This triggered the need for more EU-wide surface water monitoring data on these micropollutants, before they can be considered for inclusion in the list of priority substances regularly monitored in aquatic ecosystems. The revision of the priority substance list of the WFD offers the opportunity to incorporate more holistic bioanalytical approaches, such as effect-based monitoring, alongside single substance chemical monitoring. Effect-based methods (EBMs) are able to measure total biological activities (e.g., estrogenic activity or cyxlooxygenase [COX]-inhibition) of specific group of substances (such as estrogens and NSAIDs) in the aquatic environment at low concentrations (pg/L). This makes them potential tools for a cost-effective and ecotoxicologically comprehensive water quality assessment. In parallel, the use of such methods could build a bridge from chemical status assessments towards ecological status assessments by adressing mixture effects for relevant modes of action. Our study aimed to assess the suitability of implementing EBMs in the WFD, by conducting a large-scale sampling and analysis campaign of more than 70 surface waters across Europe. This resulted in the generation of high-quality chemical and effect-based monitoring data for the selected Watch List substances. Overall, water samples contained low estrogenicity (0.01-1.3 ng E2-Equivalent/L) and a range of COX-inhibition activity similar to previously reported levels (12-1600 ng Diclofenac-Equivalent/L). Comparison between effect-based and conventional analytical chemical methods showed that the chemical analytical approach for steroidal estrogens resulted in more (76%) non-quantifiable data, i.e., concentrations were below detection limits, compared to the EBMs (28%). These results demonstrate the excellent and sensitive screening capability of EBMs.

Characterization of testicular expression of P450 17α-hydroxylase, 17,20-lyase in zebrafish and its perturbation by the pharmaceutical fungicide clotrimazole.

The aim of the present study was to characterize P450 17α-hydroxylase/17,20-lyase (cyp17a1) expression in zebrafish and to assess the effect of the pharmaceutical clotrimazole, a known inhibitor of various cytochrome P450 enzyme activities, on testicular gene and protein expression of this enzyme as well as on the testicular release of 11-ketotestosterone (11-KT), a potent androgen in fish. We first showed that cyp17a1 is predominantly expressed in gonads of zebrafish, notably in male. In vivo, clotrimazole induced a concentration-dependent increase of cyp17a1 gene expression and Cyp17-I protein synthesis in zebrafish testis. Using zebrafish testicular explants, we further showed that clotrimazole did not directly affect cyp17a1 expression but that it did inhibit 11-KT release. These novel data deserve further studies on the effect of azole fungicides on gonadal steroidogenesis.

Estrogenicity of chemical mixtures revealed by a panel of bioassays.

Estrogenic compounds are widely released to surface waters and may cause adverse effects to sensitive aquatic species. Three hormones, estrone, 17ß-estradiol and 17α-ethinylestradiol, are of particular concern as they are bioactive at very low concentrations. Current analytical methods are not all sensitive enough for monitoring these substances in water and do not cover mixture effects. Bioassays could complement chemical analysis since they detect the overall effect of complex mixtures. Here, four chemical mixtures and two hormone mixtures were prepared and tested as reference materials together with two environmental water samples by eight laboratories employing nine in vitro and in vivo bioassays covering different steps involved in the estrogenic response. The reference materials included priority substances under the European Water Framework Directive, hormones and other emerging pollutants. Each substance in the mixture was present at its proposed safety limit concentration (EQS) in the European legislation. The in vitro bioassays detected the estrogenic effect of chemical mixtures even when 17ß-estradiol was not present but differences in responsiveness were observed. LiBERA was the most responsive, followed by LYES. The additive effect of the hormones was captured by ERα-CALUX, MELN, LYES and LiBERA. Particularly, all in vitro bioassays detected the estrogenic effects in environmental water samples (EEQ values in the range of 0.75-304 × EQS), although the concentrations of hormones were below the limit of quantification in analytical measurements. The present study confirms the applicability of reference materials for estrogenic effects' detection through bioassays and indicates possible methodological drawbacks of some of them that may lead to false negative/positive outcomes. The observed difference in responsiveness among bioassays - based on mixture composition - is probably due to biological differences between them, suggesting that panels of bioassays with different characteristics should be applied according to specific environmental pollution conditions.

Evaluation of an hPXR reporter gene assay for the detection of aquatic emerging pollutants: screening of chemicals and application to water samples.

Many environmental endocrine-disrupting compounds act as ligands for nuclear receptors. Among these receptors, the human pregnane X receptor (hPXR) is well described as a xenobiotic sensor to various classes of chemicals, including pharmaceuticals, pesticides, and steroids. To assess the potential use of PXR as a sensor for aquatic emerging pollutants, we employed an in vitro reporter gene assay (HG5LN-hPXR cells) to screen a panel of environmental chemicals and to assess PXR-active chemicals in (waste) water samples. Of the 57 compounds tested, 37 were active in the bioassay and 10 were identified as new PXR agonists: triazin pesticides (promethryn, terbuthryn, terbutylazine), pharmaceuticals (fenofibrate, bezafibrate, clonazepam, medazepam) and non co-planar polychlorobiphenyls (PCBs; PCB101, 138, 180). Furthermore, we detected potent PXR activity in two types of water samples: passive polar organic compounds integrative sampler (POCIS) extracts from a river moderately impacted by agricultural and urban inputs and three effluents from sewage treatment works (STW). Fractionation of POCIS samples showed the highest PXR activity in the less polar fraction, while in the effluents, PXR activity was mainly associated with the dissolved water phase. Chemical analyses quantified several PXR-active substances (i.e., alkylphenols, hormones, pharmaceuticals, pesticides, PCBs, bisphenol A) in POCIS fractions and effluent extracts. However, mass-balance calculations showed that the analyzed compounds explained only 0.03% and 1.4% of biological activity measured in POCIS and STW samples, respectively. In effluents, bisphenol A and 4-tert-octylphenol were identified as main contributors of instrumentally derived PXR activities. Finally, the PXR bioassay provided complementary information as compared to estrogenic, androgenic, and dioxin-like activity measured in these samples. This study shows the usefulness of HG5LN-hPXR cells to detect PXR-active compounds in water samples, and further investigation will be necessary to identify the detected active compounds.

Monitoring organic contaminants in small French coastal lagoons: comparison of levels in mussel, passive sampler and sediment.

In this study, the distribution of organic contaminants was investigated in the particular context of three Mediterranean coastal lagoons, where pollution input was hypothesised to come mainly from sediments resuspension. Mussels and semi-permeable membrane devices (SPMDs) were exposed to the water column for one month and then their content in estrogen-, benzo[a]pyrene- and dioxin-like substances as well as polycyclic aromatic hydrocarbons (PAH), polychlorinated biphenyls and alkylphenols was determined with biological and chemical analyses. PAH concentration was high in sediments (up to 1028 ng g(-1) dry weight), however the aqueous PAH concentrations estimated from SPMD data could be considered below the levels inducing adverse effects according to the environmental quality standards proposed by the Water Framework Directive. Dioxin-like activity was observed in sediments but not in mussels and SPMDs. In the two sewage-impacted lagoons, nonylphenols could be quantified in sediments, SPMDs and mussels. Nonylphenol concentrations in mussels were among the highest found in the literature. However, since nonylphenols contributed only to a small part of the estrogenic activities observed, natural or synthetic steroids originating from wastewater discharges could be also implicated in these responses in sediments.