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Zebrafish-based reporter gene assays reveal different estrogenic activities in river waters compared to a conventional human-derived assay
Science of the Total Environment 550 (2016) 934–939
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Zebrafish-based reporter gene assays reveal different estrogenic activities in river waters compared to a conventional human-derived assay Manoj Sonavane a, Nicolas Creusot a, Emmanuelle Maillot-Maréchal a, Alexandre Péry b,c, François Brion a,⁎, Selim Aїt-Aïssa a,⁎ a b c
Institut National de l'Environnement Industriel et des risques (INERIS), Unité Ecotoxicologie in vitro et in vivo, Parc Technologique ALATA, BP2, 60550 Verneuil-en-Halatte, France AgroParisTech, UMR 1402 INRA-AgroParisTech Ecosys, 78850 Thivernal Grignon, France INRA, UMR 1402 INRA-AgroParisTech Ecosys, 78850 Thivernal Grignon, France
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• New zebrafish assays as bioanalytical tools for monitoring surface waters • Different estrogenic patterns are provided by human and zebrafish in vitro assays • In vivo assay revealed brain specific responses at hot-spot sites • Relevance of fish-based integrated assessment for environmental diagnosis
a r t i c l e
i n f o
Article history: Received 6 December 2015 Received in revised form 27 January 2016 Accepted 27 January 2016 Available online 4 February 2016 Editor: D. Barcelo Keywords: Zebrafish assays Estrogen receptor subtypes Cross-species differences Complex mixtures (eco)Toxicological relevance
a b s t r a c t Endocrine disrupting chemicals (EDCs) act on the endocrine system through multiple mechanisms of action, among them interaction with estrogen receptors (ERs) is a well-identified key event in the initiation of adverse outcomes. As the most commonly used estrogen screening assays are either yeast- or human-cell based systems, the question of their (eco)toxicological relevance when assessing risks for aquatic species can be raised. The present study addresses the use of zebrafish (zf) derived reporter gene assays, both in vitro (i.e. zf liver cell lines stably expressing zfERα, zfERβ1 and zfERβ2 subtypes) and in vivo (i.e. transgenic cyp19a1b-GFP zf embryos), to assess estrogenic contaminants in river waters. By investigating 20 French river sites using passive sampling, high frequencies of in vitro zfER-mediated activities in water extracts were measured. Among the different in vitro assays, zfERβ2 assay was the most sensitive and responsive one, enabling the detection of active compounds at all investigated sites. In addition, comparison with a conventional human-based in vitro assay highlighted sites that were able to active zfERs but not human ER, suggesting the occurrence of zf-specific ER ligands. Furthermore, a significant in vivo estrogenic activity was detected at the most active sites in vitro, with a good accordance between estradiol equivalent (E2-EQ) concentrations derived from both in vitro and in vivo assays. Overall, this study shows the relevance and usefulness of such novel zebrafish-based assays as screening tools to monitor estrogenic
⁎ Corresponding authors. E-mail addresses: [email protected] (F. Brion), [email protected] (S. Aїt-Aïssa).
http://dx.doi.org/10.1016/j.scitotenv.2016.01.187 0048-9697/© 2016 Elsevier B.V. All rights reserved.
M. Sonavane et al. / Science of the Total Environment 550 (2016) 934–939
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activities in complex mixtures such as water extracts. It also supports their preferred use compared to human-based assays to assess the potential risks caused by endocrine disruptive chemicals for aquatic species such as fish. © 2016 Elsevier B.V. All rights reserved.
1. Introduction The occurrence of endocrine disrupting chemicals (EDCs) in aquatic ecosystems has raised global concern about their adverse health effects for humans and wildlife (Sumpter, 2005; Hotchkiss et al., 2008). EDCs act on the endocrine system through multiple mechanisms of action, among them interaction with estrogen receptors (ERs) is a well-identified key event in the initiation of adverse outcomes. As a member of the nuclear receptor super family, ER acts as a liganddependant transcription factor that modulates the transcription of target genes involved in essential physiological processes (e.g. development, reproduction). A number of environmental contaminants of different chemical classes and origins are xeno-estrogens (XEs) (reviewed by Kiyama and Wada-Kiyama, 2015), i.e. they have the ability to interfere with ER signaling pathway, modulate its transcriptional activity, and thereby disrupt the normal cellular response to hormones and thus may trigger adverse effects in exposed organisms. While this mechanism is well conserved among species, it can be influenced by several intracellular factors such as the promoter context, the cellular context or the origin species of the receptor. Depending on the cell type or species, one chemical can thus elicit differential estrogenic response in terms of receptor binding affinity, activation and subsequent gene transcription. In this context, the development of species-specific assays has been identified as an important challenge (Hotchkiss et al., 2008). Overall, given the risks posed by these compounds in the environment, increased knowledge about their occurrence, identity and effects in aquatic ecosystems is still needed in order to improve environmental risk assessments. In environmental monitoring, analytical strategies based on only target chemical analyses are insufficient to depict environmental contamination by XEs. It is now well admitted that effect-based tools are needed to take into account the complexity of environmental contamination (Altenburger et al., 2015). Mechanism-based bioassays were shown to be powerful bioanalytical tools to assess contamination of environmental matrices and are being increasingly used to monitor estrogenic activity in complex mixtures (Wernersson et al., 2015). In particular, in vitro assays based on stable reporter gene expression driven by ER provide efficient screening tools as they allow specific, sensitive and quantitative detection of active compounds (Leusch and Snyder, 2015). To date, most of these bioassays are based on the use of human ER alpha (hERα) expressed in either human (Legler et al., 1999) or yeast cells (Leskinen et al., 2005). However, since crossspecies species variations may affect ER transactivation by environmental ligands (Matthews et al., 2000; Molina-Molina et al., 2008, Cosnefroy et al., 2009, Miyagawa et al., 2014), the relevance of human based reporter cell lines to address environmental hazard of XE for aquatic species can be questioned. In this context, efforts have been made to develop screening tools based on model fish species. These include both in vitro assays based on fish ER expressed in either human (Tohyama et al., 2015) or fish cells (Ackermann et al., 2002; Cosnefroy et al., 2009, Cosnefroy et al., 2012) and in vivo assays based on transgenic fish models (Brion et al., 2012). Although in vitro assays are useful and relevant for the integrative quantification of estrogenic chemicals in environmental matrices, they remain simplified biological models that provide only limited information on estrogenic hazard of environmental contaminants. For this purpose, assessing a response at the whole organism level is crucial as it allows addressing these mechanisms while taking into account pharmacokinetics
(e.g. bioavailability, metabolism, transport, excretion) process in a model species. In the present study, recently developed zebrafish-based in vitro and in vivo reporter gene assays were used for the detection of (xeno)estrogens in surface waters. The in vitro assays consist of zebrafish liver cells (ZFL) that were stably transfected by an ERE-driven luciferase gene and the three different zfER subtypes (i.e. zfERα, zfERβ1 and zfERβ2) (Cosnefroy et al., 2012). In vivo estrogenic activity was monitored by using the transgenic cyp19a1b-GFP zebrafish line (Tong et al., 2009, Brion et al., 2012). This fluorescent reporter system allows sensitive detection of environmental estrogens at early developmental stages (0 to 4 days post fertilization) by activation of cyp19a1b, an ERregulated gene coding for brain aromatase (Menuet et al., 2005). This in vivo assay has been shown to sensitively respond to a diversity of ER-active compounds that belong to different chemical classes (Brion et al., 2012). In this study, the application of this panel of in vitro and in vivo bioassays to assess estrogenic activity in river water extracts showed their functionality, sensitivity and complementarity as biomonitoring tools to quantify E2-EQs in complex matrices. Furthermore, the comparison of zebrafish bioassay responses to a human-based in vitro assay was performed to assess the possible occurrence of speciesspecific estrogenic responses in the samples. 2. Materials and methods 2.1. Chemicals, cell culture and bioassay reagents 17β-estradiol (E2), 17α-ethinylestradiol (EE2), 4-tert-octylphenol (4tOP), bisphenol A (BPA), and zearalenone were purchased from Sigma-Aldrich (France). Dichloromethane (DCM), methanol (MeOH), heptane and acetone (HPLC reagent grade) were purchased from VWR (France). Dimethylsulfoxide (DMSO), Leibovitz 15 culture medium (L-15), fetal calf serum (FCS), 4-(2-hydroxy-ethyl)-1piperazineethanesulfonic acid (HEPES), epidermal growth factor (EGF), G418, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) and D-luciferin were purchased from Sigma Aldrich (St-Quentin Fallavier, France); Dulbecco's Modified Eagle Medium High Glucose (DMEM HG) powder, F-12 nutrient mixture (Ham's F12) powder, penicillin and streptomycin were from Gibco (France); insulin, hygromycin B and sodium bicarbonate were from Dominique Dutscher (France). 2.2. Study sites, sampling and extraction procedures Twenty French river sites were investigated to assess the environmental occurrence of estrogen-like compounds in the water phase, using passive sampling based on polar organic chemical integrative samplers (POCIS). POCIS are designed to monitor polar to mid-polar organic bioavailable compounds in the water soluble phase (Alvarez et al., 2004) and have been shown to be suitable for bioassay-based monitoring of bioactive compounds (Tapie et al., 2011, Creusot et al., 2014). These investigated sites were characterized by different anthropogenic pressures (urban, industrial and agricultural) (Table S1) and are currently included in surveillance programs in the frame of the Water Framework Directive (WFD). Sampling was carried out from September to November 2012. At each site, POCIS (obtained from CDTA, EPOC-LPTC, Bordeaux, France) were deployed for a period of three weeks. POCIS preparation and extraction procedures were performed as previously described with
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some minor modifications (Tapie et al., 2011; Creusot et al., 2010; Creusot et al., 2014). After collection, POCIS-sorbent (Oasis HLB phase) was extracted with a solid phase extraction (SPE) system with sequential elution of 10 ml DCM, 10 ml DCM/methanol mixture (50:50 v/v) and 10 ml methanol. These eluates were combined and further evaporated until dryness by using a rotary evaporator (EZ2-Genevac, UK). Residues were redissolved in 1 ml of MeOH/DCM (50:50, v:v) before storage at −20 °C. Identical procedure was applied to unexposed POCIS to obtain procedural blanks. For biotesting, an aliquot of the MeOH/DCM extract was evaporated under N2 and dissolved in DMSO. This concentrated organic extract was finally diluted in culture medium in order to obtain final DMSO concentration of 0.5% (v/v) in the microplate wells. 2.3. In vitro assays In vitro estrogenic activity was assessed using a panel of reporter gene assays established in both zebrafish and human cell lines. The zebrafish in vitro assays were derived from the zebrafish liver cell (ZFL) line that was stably transfected by first an ERE-driven luciferase gene, yielding the ZELH cell line, and then by each of the three zfER subtypes, yielding the ZELH-zfERα, ZELH-zfERβ1 and ZELH-zfERβ2 cell lines (Cosnefroy et al., 2012). Establishment of these cell models and their response to different classes of well-known xeno-estrogens have been previously described (Cosnefroy et al., 2012) and further documented in this paper (Table S2). About the ZELH-zfERβ1 cell line, new stable transfection experiments were carried out to improve the previously established assay. The new ZELH-zfERβ1 clone was shown to be quite stable and well-inducible (i.e. by more than 2-fold) by reference ER ligands (Sonavane, 2015). In addition, the ZELH cell line, which stably expresses the luciferase reporter gene but no functional ER, was used as a control assay to check for possible nonspecific activation or inhibition of luciferase by the extracts. In addition to zebrafish cell models, estrogenic potential of the extracts was also assessed by using the human-derived MELN cell line (Balaguer et al., 1999). MELN cells were kindly provided by Dr. Patrick Balaguer (INSERM Montpellier, France). These cells are derived from the MCF-7 cells, which endogenously express the hERα, but no functional hERβ (P. Balaguer, personal communication). Conditions for routine cell culture and exposure to chemicals and environmental extracts have been detailed previously (Balaguer et al., 1999, Cosnefroy et al., 2012, Creusot et al., 2014). Briefly, cells were seeded in 96-well white opaque culture plates (Greiner CellStar™, Dutscher, France) at 25,000 cells per well for all ZELH-zfER cell lines in phenol red free LDF-DCC medium (containing 50% of L-15, 35% of DMEM HG, 15% of Ham's F12, 15 mM of HEPES, 0.15 g/l of sodium bicarbonate, 0.01 mg/ml of insulin, 50 ng/ml of EGF and 50 U/ml of penicillin and streptomycin antibiotics, 5% v/v stripped FCS), and at 80,000 cells per well for MELN cell line in DMEM white medium. Cells were left to adhere for 24 h. Then, they were exposed in triplicates to serial dilutions of reference ligand (E2), chemical standards or environmental extracts for either 72 h at 28 °C for zebrafish cells or 16 h at 37 °C for the MELN cell line. After exposure, the medium was removed and replaced by 50 μl per well of medium containing 0.3 mM luciferin. The luminescence signal was measured in living cells using a microtiter plate luminometer (Synergy H4, BioTek).
group and exposed for 96 h in 15 ml of acclimated water in glass crystallizers. Serial dilutions of each extract were tested and the final volume of solvent (DMSO) was 0.1% v/v. This solvent concentration did not alter either embryo development or GFP expression. In each experimental series, positive control (EE2) and solvent control were included as separate experimental groups. Exposed embryos were incubated at 28 °C, under static conditions. After the exposure period, each zebrafish larva was photographed using a Zeiss AxioImager.Z1 microscope equipped with an AxioCam Mrm camera (Zeiss GmbH, Gottingen, Germany) to measure GFP expression. Image analysis was performed using the ImageJ software, and fluorescence data was treated exactly as previously described (Brion et al., 2012). 2.5. Bioassay data analysis Environmental samples were tested in at least two independent experiments. In each individual experiment, values are means of triplicates for in vitro assays and a minimum of 15 fish per concentration for the transgenic zebrafish in vivo assay. Dose–response curves (DRCs) were modeled with the Hill equation model by using the freely available RegTox Microsoft Excel™ Macro (http://www.normalesup.org/ ~vindimian/fr_index.html). All of the DRCs of sample extracts were modeled by fixing the slope and maximal effect values to that of E2, hence enabling to calculate effective concentration leading to 20% (EC20) or 50% effect (EC50) relative to E2 (Villeneuve et al., 2000, Kinani et al., 2010). For environmental chemicals, relative estrogenic potencies (REP) were determined as the ratio of EC50 of E2 to that of chemical. Estrogenic activity in environmental samples was expressed as E2-EQs, which were determined as the ratio of the EC20 of the reference compound expressed as g/l to that of the sample expressed as gram equivalents of Oasis HLB sorbent per liter (g EQ HLB/l) for POCIS extracts. 3. Results and discussion 3.1. Estrogenic potency of individual chemicals in zebrafish and human bioassays A set of different in vitro assays was used for comparative assessment of estrogenicity. All in vitro assays were well sensitive to the reference ligand E2 (Fig. 1), with EC50 values ranging from 0.01–0.02 nM for zfERβ1, zfERβ2 and hERα, to 0.2 nM for zfERα (Fig. 1, Table S2). This good sensitivity as well as the higher affinity of zfERβ subtypes than zfERα to E2 is in perfect line with our previous data reported with the
2.4. In vivo assay In vivo estrogenic potential was assessed by using the transgenic cyp19a1b-GFP zebrafish line that has been previously developed (Tong et al., 2009) and characterized with different classes of estrogens and xeno-estrogen compounds (Brion et al., 2012, Table S2). The assay procedure for individual chemical testing has been described in detail by Brion et al. (2012). In the present study, it has been adapted to the testing of organic extracts coming from the aquatic environment. In brief, 20 fertilized transgenic eggs were selected for each experimental
Fig. 1. Concentration-dependant luciferase induction by 17β-estradiol (E2) in the different in vitro reporter gene assays based on zebrafish and human estrogen receptors (ER). Values are presented as percentage of luciferase activity induced by E2 10 nM for zfERα and E2 1 nM for zfERβ1, zfERβ2 and hERα (mean values of triplicates ± standard deviation). The results are representative of at least 10 independent assays.
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same cell models (Cosnefroy et al., 2012), which confirms the stability and the robustness of the established systems. These assays were also further characterized for their response to a panel of XEs from different chemical classes (Figure S1, Table S2). First, a zfER subtype-dependent selective activation is observed in zebrafish in vitro assays, which has been well described and discussed previously (Cosnefroy et al., 2012, Pinto et al., 2014). This is shown by a better affinity of zfERβ subtypes compared to zfERα for natural and synthetic estrogens (E2 and EE2), or a selective activation of zfERα by bisphenol A or genistein. This confirms previous studies using this cell model (Cosnefroy et al., 2012) or human cells expressing zfERs (Le Page et al., 2006; Pinto et al., 2014). Second, it is noteworthy that such selectivity differs in human where natural and synthetic estrogens present better affinity for hERα than for hERβ and phytoestrogens, such as genistein, are selective hERβ modulators (Escande et al., 2006). Overall, marked differences were noticed regarding ERβ selectivity for natural estrogens, which seems to be a common feature in different fish species (Chakraborty et al., 2011, Tohyama et al., 2015). While the intimate roles of ER subtypes are still poorly understood, there exist evidences that show their tissue- and life stage-specific expression (Menuet et al., 2004), hence supporting different physiological functions (Griffin et al., 2013). The differences of affinity of (xeno)-estrogens on ER subtypes may explain their tissue and life-stage dependent effects on ER-signaling pathways in fish (Gorelick et al., 2014). Interestingly, the spatio–temporal analysis of ER mRNAs in zebrafish embryos revealed an early and predominant expression of ERβ subtypes in the hypothalamus (Mouriec et al., 2009). Altogether, this suggests that ERregulated genes in the brain of zebrafish embryos are particularly sensitive to compounds having a good affinity for ERβ subtypes. In this regard, the tested chemicals were also active in the in vivo cyp19a1b-GFP assay, but with some differences in their relative estrogenic potency as compared to in vitro data (Table S2). In several cases, the in vivo assay showed a comparable to better sensitivity to xenoestrogens (e.g. EE2, hexestrol, DES, zearalenone, o,p’-DDT) than in vitro assays. Interestingly, benzophenone-3 (BP3), an anti-UV screen, which showed weak estrogenicity in vivo, was unable to induce any activity in vitro. This could be explained by in vivo biotransformation of BP3 into active metabolites, most likely benzophenone-1. Overall, zebrafish in vitro and in vivo data illustrate the suitability of established assays to detect well-known estrogenic ligands. 3.2. In vitro screening of estrogenic activities in surface waters To assess the usefulness of newly developed in vitro zfER assays to quantify estrogenic activity in environmental matrices, we applied our set of bioassays on POCIS extracts from 20 French river sites, which were known to be subjected to various anthropogenic pressures (Table S1). Overall, estrogenic activities could be detected by all in vitro assays in the water phase at several of the investigated river sites (Table 1). By using the human MELN assay, we previously reported the suitability of using of POCIS sampling to detect estrogenic activity both in surface waters and effluents (Creusot et al., 2013, Tapie et al., 2011, Creusot et al., 2014). Most of known (xeno)-estrogens are polar to mid-polar compounds and are known to be sampled by POCIS (Alvarez et al., 2004, Togola and Budzinski, 2007). In this study, we further demonstrate the relevance of this approach on a larger scale study at WFD surveillance sites. Interestingly, the zebrafish-based assays, and notably the zfERβ2 assay, confirmed the presence of ER ligands in the extracts but also highlighted several other contaminated sites that were not revealed by the human assay. Such important finding was not expected since no species-specific ER activation could be highlighted from the screening of individual chemicals, even though some chemicals showed differences in their relative estrogenic potency between bioassays (Cosnefroy et al., 2012, Table S2). Nevertheless, our data suggest the occurrence of hydrosoluble contaminants that are specifically active on the zebrafish
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Table 1 Estrogenic activities in POCIS extracts from 20 French river sites using zf and human in vitro assays. Results are expressed in ng E2-EQ/g sorbent with confidence interval 95% in bracket and are derived from the dose–response curves presented in Figures S2, S3, S4 and S5. River Human sites hERα
Zebrafish zfERα
zfERβ1
zfERβ2
BED
198.6 (168.0–214.2) 29.6 (24.5–33.2)
131.3 (106.9–181.6) n.a.
10.9 (6.8–14.2) n.a.
LOV REV SOU
11.9 (9.1–12.8) 2.9 (2.7–3.1) 9.1 (7.0–9.8)
n.a. n.a. n.a.
n.a. 1.6 (1.0–4.4) n.a.
MAD
3.8 (2.9–4.2)
8.3 (6.2–11.2)
5.6 (2.4–17.1)
RIS SAR LOI
3.9 (3.0–4.1) 3.7 (3.0–3.9) 2.4 (2.0–2.8)
n.a. n.a. n.a.
51.5 (46.5–61.7) 26.7 (23.6–39.2) 11.5 (9.8–16.0) 2.3 (1.9–3.1) 0.5 (0.4–0.7) 15.8 (13.6–21.2) 12.2 (9.1–16.3) 10.6 (6.6–17.6) 5.0 (2.9–7.4) 15.7 (11.4–23.3) 14.4 (9.2–18.6) 3.5 (2.4–5.3) 99.7 (75.2–150.1) 35.4 (27.1–57.7) 27.4 (20.8–40.9) 10.3 (7.6–15.9) 8.2 (5.9–10.6) 7.8 (6.0–10.6) 5.9 (4.1–10.7) 5.9 (4.0–9.1) 0.4
LOM
AUV
1.9 (1.6–2.3)
25.3 (18.5–45.0)
BRA LUE OLL
1.3 (1.1–1.3) 1.0 (0.8–1.1) n.a.
n.a. n.a. 3.2 (2.2–6.4)
1.0 (0.8–1.5) n.a. 2.6 (1.7–4.2) 63.7 (52.4–99.0) n.a. n.a. n.a.
HER
n.a.
n.a.
8.4 (3.5–19.1)
YER
n.a.
4.6 (3.4–5.4)
n.a.
CHE LOB GAR JOU MAR LOQ
n.a. n.a. n.a. n.a. n.a. 0.1
8.0 (6.0–19.7) n.a. n.a. 5.2 (3.6–7.3) n.a. 2.3
1.8 (1.2–2.5) 0.5 (0.3–0.6) 9.8 (6.5–15.1) n.a. 4.1 (2.7–9.6) 0.1
LOQ: limit of quantification; n.a. non active (bLOQ).
in vitro models. For instance, the OLL site was highly active in zfERβ2 but negative in MELN. This site is known to be contaminated by several substances, including corticoids and progestagenic compounds, while steroid estrogens were not detected (Creusot et al., 2014). At this site, it is likely that such compounds, which can be metabolized into estrogenic metabolites, could have contributed to the observed estrogenic activity in the metabolically competent ZELH cells (Le Fol et al., 2015). Indeed, it is noted that both zebrafish and human in vitro assays used different cell systems (i.e. ZFL cells versus MCF-7 cells, respectively), that could possibly result in different metabolic capacities, which may have also influenced the responses of the assays (Olsen et al., 2005, Le Fol et al., 2015). It is noteworthy that a higher number of POCIS extracts were found to be more active on zfERβ1 and zfERβ2 than on zfERα. This is in line with the better sensitivity of zfERβ subtypes to E2 as compared to zfERα, although the detection of zfER subtypes-specific active compounds cannot be excluded. Furthermore, the importance of zfERβ2 assay was further highlighted by its ability to detect and quantify estrogenic contaminants in all the tested river sites, some at significantly high E2-EQ concentrations (i.e. OLL, HER, YER). The higher responsiveness of zfERβ2 to environmental samples is an important finding as this estrogen receptor subtype is present in fish species but not in humans (Hawkins et al., 2000, Hawkins and Thomas, 2004) and plays a crucial role in mediating estrogen-dependent physiological processes in fish (Nelson and Habibi, 2013, Yost et al., 2014). Overall, our results support the use of zebrafish-based (zfERs) assays as relevant in vitro models to assess estrogenic activity in water samples and raises questions on the relevance of using human-based assay to assess hazard posed by environmental estrogens on aquatic species. 3.3. In vivo estrogenic potency of environmental mixtures To gain further insight on (xeno)-estrogens associated hazard in complex samples, we used the transgenic cyp19a1b-GFP zebrafish
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embryo assay (Tong et al., 2009). After exposure of zf embryos to POCIS extracts, we identified some of the investigated sites that induced the cyp19a1b promoter in a dose-dependent manner (Figure S6). These data were used to calculate in vivo E2-EQs that are reported in Table 2. Our results demonstrate the capacity of the in vivo assay to assess estrogenic potential of environmental mixtures on the whole organism. This reporter system has been shown to be useful to assess estrogenic potency of individual chemicals by establishing dose–response curve and deriving EC50 and relative estrogenic potency (Brion et al., 2012, Table S2). Here, we report the ability of this system to detect active chemicals within complex mixtures and establish E2-EQ. Furthermore, the comparison between in vitro and in vivo estrogenic profiles provided further supports on the relevance of such an integrated assessment of estrogenic activity. Interestingly, the positive sites highlighted and quantified by the in vivo assay were also the most active ones in vitro, notably through the zfERβ2 assay (Table 2). Furthermore, E2-EQ values quantified by the in vivo cyp19a1b-GFP assay at these active sites closely relate to the in vitro estrogenic activity quantified by the zfERβ2 assay. Among these sites, the OLL river site was strongly active in zebrafish in vivo and in vitro assays but not in the human assay (Table 2, Figure S6), which reinforces a possible fish-specific response at this site, as discussed above. Similar observations were also documented using medaka that predicted more accurately in vivo estrogenic activity of wastewater by in vitro assays of the same species as compared to human in vitro assay (Ihara et al., 2015). It is noticeable that POCIS extracts that were weakly active on in vitro assays were not able to elicit any GFP expression in vivo. This could be explained by the differences in the highest test concentration of extracts used for in vivo testing (0.1% v/v) as compared to in vitro assays (0.5% v/ v). Such limitation of extract concentration can however be solved by redefining the sample preparation procedure. Another possible explanation could be due to differential bioavailability and metabolism of compounds between zebrafish embryos and ZFL cells. A higher metabolic capacity of the zebrafish embryo can lead either activation of the parent compound into estrogenic metabolites but also de-activation of some estrogenic compounds into inactive metabolites. It is clear that the identification of the compound(s) responsible for observed effects could help in identifying reasons for observed differences between in vitro and in vivo responses. Altogether, our study suggests a good agreement between in vitro zfERβ2 and in vivo cyp19a1b-GFP to identify hot-spot sites contaminated by endocrine active compounds in fish. In the future, extending this data set to other surface waters will help to increase the statistical value of the correlation and help to define in vitro E2-EQ trigger values that can predict in vivo estrogenic responses in fish. The applied in vivo assay is a highly sensitive assay to detect zebrafish ER ligands in environmental water extracts. It also provides critical toxicological information regarding the impact of environmental contaminants on the developing brain. It revealed that environmental contaminants can target radial glial cells and disrupt brain aromatase that has been associated with altered neurogenesis and behavioral activities in zebrafish (Diotel et al., 2013, Kinch et al., 2015). Impact of environmental estrogens on neurodevelopment and their potential effects on wild fish population has been until now poorly documented but should be further investigated in light of these data. Overall, the toxicological relevance of examined
Table 2 Comparison of in vivo and in vitro estrogenic activities in POCIS extracts from 3 river sites. Results are expressed as ng E2-EQ/g sorbent. In vivo data are derived from the dose–response curves presented in Figure S6. In vitro
In vivo
River sites OLL BED LOM n.a.: not active.
zfERα
zfERβ1
zfERβ2
hERα
cyp19a1b
3 n.a. 131
n.a. n.a. 11
100 27 51
n.a. 30 199
88 44 83
ER-signaling pathway at very early critical developmental stages makes the cyp19a1b-GFP assay a relevant screening tool fitting current bioanalytical challenges (Altenburger et al., 2015). 4. Conclusion This study reports the combined use of in vitro and in vivo fish-based reporter gene assays as bioanalytical tools and its relevance to highlight fish specific estrogenic activities in environmental samples. Among examined zfER assays, zfERβ2 was the most sensitive and responsive one to water extracts, although the presence of other zfER subtype-specific active compounds cannot be excluded. Compared to more commonly used human-based assay, these novel zebrafish reporter gene assays revealed significant differences to detect ER ligands. Specific and sensitive detection of zebrafish estrogen receptor ligands in their native cell (e.g. liver cell in vitro) or tissue (e.g. brain in vivo) contexts, makes them highly relevant as (eco)toxicological models to assess estrogenic effect in aquatic vertebrates. From an environmental risk assessment perspective, the use of such an integrated assessment in fish allows to gain information on the occurrence and concentration range of endocrine active substances (biodetection) as well as the potential estrogenic effect (hazard). Acknowledgments This work was funded by the EU Seventh Framework Programme (FP7-PEOPLE-2011-ITN) as a part of EDA-EMERGE project under the grant agreement number 290100, the French National Agency for Water and Aquatic Environments (ONEMA) as part of the 2012 national prospective campaign on emerging contaminants (“Etude prospective sur les contaminants émergents”) and the French Ministry of Ecology (P190- Axe de Recherche Ecotoxicologie). We wish to warmly thank Sandrine Joachim for her help in improving the English of this manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2016.01.187. References Ackermann, G.E., Brombacher, E., Fent, K., 2002. Development of a fish reporter gene system for the assessment of estrogenic compounds and sewage treatment plant effluents. Environ. Toxicol. Chem. 21, 1864–1875. Altenburger, R., Ait-Aissa, S., Antczak, P., Backhaus, T., Barceló, D., Seiler, T.-B., et al., 2015. Future water quality monitoring—adapting tools to deal with mixtures of pollutants in water resource management. Sci. Total Environ. 512–513, 540–551. Alvarez, D.A., Petty, J.D., Huckins, J.N., Jones-Lepp, T.L., Getting, D.T., Goddard, J.P., et al., 2004. Development of a passive, in situ, integrative sampler for hydrophilic organic contaminants in aquatic environments. Environ. Toxicol. Chem. 23, 1640–1648. Balaguer, P., Francois, F., Comunale, F., Fenet, H., Boussioux, A.M., Pons, M., et al., 1999. Reporter cell lines to study the estrogenic effects of xenoestrogens. Sci. Total Environ. 233, 47–56. Brion, F., Le Page, Y., Piccini, B., Cardoso, O., Tong, S.K., Chung, B.C., et al., 2012. Screening estrogenic activities of chemicals or mixtures in vivo using transgenic (cyp19a1bgfp) zebrafish embryos. PLoS One 7, e36069. Chakraborty, T., Katsu, Y., Zhou, L.Y., Miyagawa, S., Nagahama, Y., Iguchi, T., 2011. Estrogen receptors in medaka (Oryzias latipes) and estrogenic environmental contaminants: an in vitro–in vivo correlation. J. Steroid Biochem. Mol. Biol. 123, 115–121. Cosnefroy, A., Brion, F., Guillet, B., Laville, N., Porcher, J.M., Balaguer, P., et al., 2009. A stable fish reporter cell line to study estrogen receptor transactivation by environmental (xeno)estrogens. Toxicol. in Vitro 23, 1450–1454. Cosnefroy, A., Brion, F., Maillot-Marechal, E., Porcher, J.M., Pakdel, F., Balaguer, P., et al., 2012. Selective activation of zebrafish estrogen receptor subtypes by chemicals by using stable reporter gene assay developed in a zebrafish liver cell line. Toxicol. Sci. 125, 439–449. Creusot, N., Kinani, S., Balaguer, P., Tapie, N., Lemenach, K., Maillot-Marechal, E., Porcher, J.M., Budzinski, H., Aït-Aïssa, S., 2010. Evaluation of an hPXR reporter gene assay for the detection of aquatic emerging pollutants: screening of chemicals and application to water samples. Anal. Bioanal. Chem. 396, 569–583. Creusot, N., Tapie, N., Piccini, B., Balaguer, P., Porcher, J.M., Budzinski, H., Aït-Aïssa, S., 2013. Distribution of steroid- and dioxin-like activities between sediments, POCIS and SPMD in a French river subject to mixed pressures. Environ. Sci. Pollut. Res. 20, 2784–2794.
M. Sonavane et al. / Science of the Total Environment 550 (2016) 934–939 Creusot, N., Aït-Aïssa, S., Tapie, N., Pardon, P., Brion, F., Sanchez, W., et al., 2014. Identification of synthetic steroids in river water downstream from pharmaceutical manufacture discharges based on a bioanalytical approach and passive sampling. Environ. Sci. Technol. 48, 3649–3657. Diotel, N., Vaillant, C., Gabbero, C., Mironov, S., Fostier, A., Gueguen, M.M., et al., 2013. Effects of estradiol in adult neurogenesis and brain repair in zebrafish. Horm. Behav. 63, 193–207. Escande, A., Pillon, A., Servant, N., Cravedi, J.P., Larrea, F., Muhn, P., et al., 2006. Evaluation of ligand selectivity using reporter cell lines stably expressing estrogen receptor alpha or beta. Biochem. Pharmacol. 71, 1459–1469. Gorelick, D.A., Iwanowicz, L.R., Hung, A.L., Blazer, V.S., Halpern, M.E., 2014. Transgenic zebrafish reveal tissue-specific differences in estrogen signaling in response to environmental water samples. Environ. Health Perspect. 122, 356–362. Griffin, L.B., January, K.E., Ho, K.W., Cotter, K.A., Callard, G.V., 2013. Morpholino-mediated knockdown of ERα, ERβa, and ERβb mRNAs in zebrafish (Danio rerio) embryos reveals differential regulation of estrogen-inducible genes. Endocrinology 154, 4158–4169. Hawkins, M.B., Thomas, P., 2004. The unusual binding properties of the third distinct teleost estrogen receptor subtype erbetaa are accompanied by highly conserved amino acid changes in the ligand binding domain. Endocrinology 145, 2968–2977. Hawkins, M.B., Thornton, J.W., Crews, D., Skipper, J.K., Dotte, A., Thomas, P., 2000. Identification of a third distinct estrogen receptor and reclassification of estrogen receptors in teleosts. Proc. Natl. Acad. Sci. U. S. A. 97, 10751–10756. Hotchkiss, A.K., Rider, C.V., Blystone, C.R., Wilson, V.S., Hartig, P.C., Ankley, G.T., et al., 2008. Fifteen years after "wingspread" — environmental endocrine disrupters and human and wildlife health: where we are today and where we need to go. Toxicol. Sci. 105, 235–259. Ihara, M., Kitamura, T., Kumar, V., Park, C.B., Ihara, M.O., Lee, S.J., et al., 2015. Evaluation of estrogenic activity of wastewater: comparison among in vitro eralpha reporter gene assay, in vivo vitellogenin induction, and chemical analysis. Environ. Sci. Technol. 49, 6319–6326. Kinani, S., Bouchonnet, S., Creusot, N., Bourcier, S., Balaguer, P., Porcher, J.M., et al., 2010. Bioanalytical characterisation of multiple endocrine- and dioxin-like activities in sediments from reference and impacted small rivers. Environ. Pollut. 158, 74–83. Kinch, C.D., Ibhazehiebo, K., Jeong, J.H., Habibi, H.R., Kurrasch, D.M., 2015. Low-dose exposure to bisphenol A and replacement bisphenol S induces precocious hypothalamic neurogenesis in embryonic zebrafish. Proc. Natl. Acad. Sci. U. S. A. 112, 1475–1480. Kiyama, R., Wada-Kiyama, Y., 2015. Estrogenic endocrine disruptors: molecular mechanisms of action. Environ. Int. 83, 11–40. Le Fol, V., Aït-Aïssa, S., Cabaton, N., Dolo, L., Grimaldi, M., Balaguer, P., et al., 2015. Cell-specific biotransformation of benzophenone-2 and bisphenol-s in zebrafish and human in vitro models used for toxicity and estrogenicity screening. Environ. Sci. Technol. 49, 3860–3868. Le Page, Y., Scholze, M., Kah, O., Pakdel, F., 2006. Assessment of xenoestrogens using three distinct estrogen receptors and the zebrafish brain aromatase gene in a highly responsive glial cell system. Environ. Health Perspect. 114, 752–758. Legler, J., van den Brink, C.E., Brouwer, A., Murk, A.J., van der Saag, P.T., Vethaak, A.D., et al., 1999. Development of a stably transfected estrogen receptor-mediated luciferase reporter gene assay in the human t47d breast cancer cell line. Toxicol. Sci. 48, 55–66. Leskinen, P., Michelini, E., Picard, D., Karp, M., Virta, M., 2005. Bioluminescent yeast assays for detecting estrogenic and androgenic activity in different matrices. Chemosphere 61, 259–266. Leusch, F.D., Snyder, S., 2015. Bioanalytical tools: half a century of application for potable reuse. Environ. Sci. Water Res. Technol. 1, 606–621.
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Matthews, J., Celius, T., Halgren, R., Zacharewski, T., 2000. Differential estrogen receptor binding of estrogenic substances: a species comparison. J. Steroid Biochem. Mol. Biol. 74, 223–234. Menuet, A., Le Page, Y., Torres, O., Kern, L., Kah, O., Pakdel, F., 2004. Analysis of the estrogen regulation of the zebrafish estrogen receptor (ER) reveals distinct effects of ERalpha, ERbeta1 and ERbeta2. J. Mol. Endocrinol. 32, 975–986. Menuet, A., Pellegrini, E., Brion, F., Gueguen, M.M., Anglade, I., Pakdel, F., et al., 2005. Expression and estrogen-dependent regulation of the zebrafish brain aromatase gene. J. Comp. Neurol. 485, 304–320. Miyagawa, S., Lange, A., Hirakawa, I., Tohyama, S., Ogino, Y., Mizutani, T., et al., 2014. Differing species responsiveness of estrogenic contaminants in fish is conferred by the ligand binding domain of the estrogen receptor. Environ. Sci. Technol. 48, 5254–5263. Molina-Molina, J.M., Escande, A., Pillon, A., Gomez, E., Pakdel, F., Cavailles, V., et al., 2008. Profiling of benzophenone derivatives using fish and human estrogen receptorspecific in vitro bioassays. Toxicol. Appl. Pharmacol. 22, 384–395. Mouriec, K., Lareyre, J.J., Tong, S.K., Le Page, Y., Vaillant, C., Pellegrini, E., et al., 2009. Early regulation of brain aromatase (cyp19a1b) by estrogen receptors during zebrafish development. Dev. Dyn. 238, 2641–2651. Nelson, E.R., Habibi, H.R., 2013. Estrogen receptor function and regulation in fish and other vertebrates. Gen. Comp. Endocrinol. 192, 15–24. Olsen, C.A., Meussen-Elholm, E.T.M., Hongslo, J.K., Stenersen, J., Tollefsen, K.E., 2005. Estrogenic effects of environmental chemicals: an interspecies comparison. Comp. Biochem. Physiol. C 141, 267–274. Pinto, C., Grimaldi, M., Boulahtouf, A., Pakdel, F., Brion, F., Ait-Aissa, S., et al., 2014. Selectivity of natural, synthetic and environmental estrogens for zebrafish estrogen receptors. Toxicol. Appl. Pharmacol. 280, 60–69. Sonavane, M., 2015. Intérêt d'une approche combinant bioessais in vitro et in vivo chez le poisson-zèbre pour l'identification de perturbateurs endocriniens dans les milieux aquatiques. PhD dissertation AgroParisTech, Paris, France. Sumpter, J.P., 2005. Endocrine disrupters in the aquatic environment: an overview. Acta Hydrochim. Hydrobiol. 33, 9–16. Tapie, N., Devier, M.H., Soulier, C., Creusot, N., Le Menach, K., Aït-Aïssa, S., et al., 2011. Passive samplers for chemical substance monitoring and associated toxicity assessment in water. Water Sci. Technol. 63, 2418–2426. Togola, A., Budzinski, H., 2007. Development of polar organic integrative samplers for analysis of pharmaceuticals in aquatic systems. Anal. Chem. 79, 6734–6741. Tohyama, S., Shinichi, M., Anke, L., Yukiko, O., Takeshi, M., Norihisa, T., et al., 2015. Understanding the molecular basis for differences in responses of fish estrogen receptor subtypes to environmental estrogens. Environ. Sci. Technol. 49, 7439–7447. Tong, S.K., Mouriec, K., Kuo, M.W., Pellegrini, E., Gueguen, M.M., Brion, F., et al., 2009. A cyp19a1b-gfp (aromatase b) transgenic zebrafish line that expresses gfp in radial glial cells. Genesis 47, 67–73. Villeneuve, D.L., Blankenship, A.L., Giesy, J.P., 2000. Derivation and application of relative potency estimates based on in vitro bioassay results. Environ. Toxicol. Chem. 19, 2835–2843. Wernersson, A.-S., Carere, M., Maggi, C., Tusil, P., Soldan, P., James, A., Sanchez, W., Dulio, V., Broeg, K., et al., 2015. The European technical report on aquatic effect-based monitoring tools under the water framework directive. Environ. Sci. Eur. 27, 1–11. Yost, E.E., Pow, C.L., Hawkins, M.B., Kullman, S.W., 2014. Bridging the gap from screening assays to estrogenic effects in fish: potential roles of multiple estrogen receptor subtypes. Environ. Sci. Technol. 18, 5211–5219.
Contents lists available at ScienceDirect
Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Zebrafish-based reporter gene assays reveal different estrogenic activities in river waters compared to a conventional human-derived assay Manoj Sonavane a, Nicolas Creusot a, Emmanuelle Maillot-Maréchal a, Alexandre Péry b,c, François Brion a,⁎, Selim Aїt-Aïssa a,⁎ a b c
Institut National de l'Environnement Industriel et des risques (INERIS), Unité Ecotoxicologie in vitro et in vivo, Parc Technologique ALATA, BP2, 60550 Verneuil-en-Halatte, France AgroParisTech, UMR 1402 INRA-AgroParisTech Ecosys, 78850 Thivernal Grignon, France INRA, UMR 1402 INRA-AgroParisTech Ecosys, 78850 Thivernal Grignon, France
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• New zebrafish assays as bioanalytical tools for monitoring surface waters • Different estrogenic patterns are provided by human and zebrafish in vitro assays • In vivo assay revealed brain specific responses at hot-spot sites • Relevance of fish-based integrated assessment for environmental diagnosis
a r t i c l e
i n f o
Article history: Received 6 December 2015 Received in revised form 27 January 2016 Accepted 27 January 2016 Available online 4 February 2016 Editor: D. Barcelo Keywords: Zebrafish assays Estrogen receptor subtypes Cross-species differences Complex mixtures (eco)Toxicological relevance
a b s t r a c t Endocrine disrupting chemicals (EDCs) act on the endocrine system through multiple mechanisms of action, among them interaction with estrogen receptors (ERs) is a well-identified key event in the initiation of adverse outcomes. As the most commonly used estrogen screening assays are either yeast- or human-cell based systems, the question of their (eco)toxicological relevance when assessing risks for aquatic species can be raised. The present study addresses the use of zebrafish (zf) derived reporter gene assays, both in vitro (i.e. zf liver cell lines stably expressing zfERα, zfERβ1 and zfERβ2 subtypes) and in vivo (i.e. transgenic cyp19a1b-GFP zf embryos), to assess estrogenic contaminants in river waters. By investigating 20 French river sites using passive sampling, high frequencies of in vitro zfER-mediated activities in water extracts were measured. Among the different in vitro assays, zfERβ2 assay was the most sensitive and responsive one, enabling the detection of active compounds at all investigated sites. In addition, comparison with a conventional human-based in vitro assay highlighted sites that were able to active zfERs but not human ER, suggesting the occurrence of zf-specific ER ligands. Furthermore, a significant in vivo estrogenic activity was detected at the most active sites in vitro, with a good accordance between estradiol equivalent (E2-EQ) concentrations derived from both in vitro and in vivo assays. Overall, this study shows the relevance and usefulness of such novel zebrafish-based assays as screening tools to monitor estrogenic
⁎ Corresponding authors. E-mail addresses: [email protected] (F. Brion), [email protected] (S. Aїt-Aïssa).
http://dx.doi.org/10.1016/j.scitotenv.2016.01.187 0048-9697/© 2016 Elsevier B.V. All rights reserved.
M. Sonavane et al. / Science of the Total Environment 550 (2016) 934–939
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activities in complex mixtures such as water extracts. It also supports their preferred use compared to human-based assays to assess the potential risks caused by endocrine disruptive chemicals for aquatic species such as fish. © 2016 Elsevier B.V. All rights reserved.
1. Introduction The occurrence of endocrine disrupting chemicals (EDCs) in aquatic ecosystems has raised global concern about their adverse health effects for humans and wildlife (Sumpter, 2005; Hotchkiss et al., 2008). EDCs act on the endocrine system through multiple mechanisms of action, among them interaction with estrogen receptors (ERs) is a well-identified key event in the initiation of adverse outcomes. As a member of the nuclear receptor super family, ER acts as a liganddependant transcription factor that modulates the transcription of target genes involved in essential physiological processes (e.g. development, reproduction). A number of environmental contaminants of different chemical classes and origins are xeno-estrogens (XEs) (reviewed by Kiyama and Wada-Kiyama, 2015), i.e. they have the ability to interfere with ER signaling pathway, modulate its transcriptional activity, and thereby disrupt the normal cellular response to hormones and thus may trigger adverse effects in exposed organisms. While this mechanism is well conserved among species, it can be influenced by several intracellular factors such as the promoter context, the cellular context or the origin species of the receptor. Depending on the cell type or species, one chemical can thus elicit differential estrogenic response in terms of receptor binding affinity, activation and subsequent gene transcription. In this context, the development of species-specific assays has been identified as an important challenge (Hotchkiss et al., 2008). Overall, given the risks posed by these compounds in the environment, increased knowledge about their occurrence, identity and effects in aquatic ecosystems is still needed in order to improve environmental risk assessments. In environmental monitoring, analytical strategies based on only target chemical analyses are insufficient to depict environmental contamination by XEs. It is now well admitted that effect-based tools are needed to take into account the complexity of environmental contamination (Altenburger et al., 2015). Mechanism-based bioassays were shown to be powerful bioanalytical tools to assess contamination of environmental matrices and are being increasingly used to monitor estrogenic activity in complex mixtures (Wernersson et al., 2015). In particular, in vitro assays based on stable reporter gene expression driven by ER provide efficient screening tools as they allow specific, sensitive and quantitative detection of active compounds (Leusch and Snyder, 2015). To date, most of these bioassays are based on the use of human ER alpha (hERα) expressed in either human (Legler et al., 1999) or yeast cells (Leskinen et al., 2005). However, since crossspecies species variations may affect ER transactivation by environmental ligands (Matthews et al., 2000; Molina-Molina et al., 2008, Cosnefroy et al., 2009, Miyagawa et al., 2014), the relevance of human based reporter cell lines to address environmental hazard of XE for aquatic species can be questioned. In this context, efforts have been made to develop screening tools based on model fish species. These include both in vitro assays based on fish ER expressed in either human (Tohyama et al., 2015) or fish cells (Ackermann et al., 2002; Cosnefroy et al., 2009, Cosnefroy et al., 2012) and in vivo assays based on transgenic fish models (Brion et al., 2012). Although in vitro assays are useful and relevant for the integrative quantification of estrogenic chemicals in environmental matrices, they remain simplified biological models that provide only limited information on estrogenic hazard of environmental contaminants. For this purpose, assessing a response at the whole organism level is crucial as it allows addressing these mechanisms while taking into account pharmacokinetics
(e.g. bioavailability, metabolism, transport, excretion) process in a model species. In the present study, recently developed zebrafish-based in vitro and in vivo reporter gene assays were used for the detection of (xeno)estrogens in surface waters. The in vitro assays consist of zebrafish liver cells (ZFL) that were stably transfected by an ERE-driven luciferase gene and the three different zfER subtypes (i.e. zfERα, zfERβ1 and zfERβ2) (Cosnefroy et al., 2012). In vivo estrogenic activity was monitored by using the transgenic cyp19a1b-GFP zebrafish line (Tong et al., 2009, Brion et al., 2012). This fluorescent reporter system allows sensitive detection of environmental estrogens at early developmental stages (0 to 4 days post fertilization) by activation of cyp19a1b, an ERregulated gene coding for brain aromatase (Menuet et al., 2005). This in vivo assay has been shown to sensitively respond to a diversity of ER-active compounds that belong to different chemical classes (Brion et al., 2012). In this study, the application of this panel of in vitro and in vivo bioassays to assess estrogenic activity in river water extracts showed their functionality, sensitivity and complementarity as biomonitoring tools to quantify E2-EQs in complex matrices. Furthermore, the comparison of zebrafish bioassay responses to a human-based in vitro assay was performed to assess the possible occurrence of speciesspecific estrogenic responses in the samples. 2. Materials and methods 2.1. Chemicals, cell culture and bioassay reagents 17β-estradiol (E2), 17α-ethinylestradiol (EE2), 4-tert-octylphenol (4tOP), bisphenol A (BPA), and zearalenone were purchased from Sigma-Aldrich (France). Dichloromethane (DCM), methanol (MeOH), heptane and acetone (HPLC reagent grade) were purchased from VWR (France). Dimethylsulfoxide (DMSO), Leibovitz 15 culture medium (L-15), fetal calf serum (FCS), 4-(2-hydroxy-ethyl)-1piperazineethanesulfonic acid (HEPES), epidermal growth factor (EGF), G418, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) and D-luciferin were purchased from Sigma Aldrich (St-Quentin Fallavier, France); Dulbecco's Modified Eagle Medium High Glucose (DMEM HG) powder, F-12 nutrient mixture (Ham's F12) powder, penicillin and streptomycin were from Gibco (France); insulin, hygromycin B and sodium bicarbonate were from Dominique Dutscher (France). 2.2. Study sites, sampling and extraction procedures Twenty French river sites were investigated to assess the environmental occurrence of estrogen-like compounds in the water phase, using passive sampling based on polar organic chemical integrative samplers (POCIS). POCIS are designed to monitor polar to mid-polar organic bioavailable compounds in the water soluble phase (Alvarez et al., 2004) and have been shown to be suitable for bioassay-based monitoring of bioactive compounds (Tapie et al., 2011, Creusot et al., 2014). These investigated sites were characterized by different anthropogenic pressures (urban, industrial and agricultural) (Table S1) and are currently included in surveillance programs in the frame of the Water Framework Directive (WFD). Sampling was carried out from September to November 2012. At each site, POCIS (obtained from CDTA, EPOC-LPTC, Bordeaux, France) were deployed for a period of three weeks. POCIS preparation and extraction procedures were performed as previously described with
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some minor modifications (Tapie et al., 2011; Creusot et al., 2010; Creusot et al., 2014). After collection, POCIS-sorbent (Oasis HLB phase) was extracted with a solid phase extraction (SPE) system with sequential elution of 10 ml DCM, 10 ml DCM/methanol mixture (50:50 v/v) and 10 ml methanol. These eluates were combined and further evaporated until dryness by using a rotary evaporator (EZ2-Genevac, UK). Residues were redissolved in 1 ml of MeOH/DCM (50:50, v:v) before storage at −20 °C. Identical procedure was applied to unexposed POCIS to obtain procedural blanks. For biotesting, an aliquot of the MeOH/DCM extract was evaporated under N2 and dissolved in DMSO. This concentrated organic extract was finally diluted in culture medium in order to obtain final DMSO concentration of 0.5% (v/v) in the microplate wells. 2.3. In vitro assays In vitro estrogenic activity was assessed using a panel of reporter gene assays established in both zebrafish and human cell lines. The zebrafish in vitro assays were derived from the zebrafish liver cell (ZFL) line that was stably transfected by first an ERE-driven luciferase gene, yielding the ZELH cell line, and then by each of the three zfER subtypes, yielding the ZELH-zfERα, ZELH-zfERβ1 and ZELH-zfERβ2 cell lines (Cosnefroy et al., 2012). Establishment of these cell models and their response to different classes of well-known xeno-estrogens have been previously described (Cosnefroy et al., 2012) and further documented in this paper (Table S2). About the ZELH-zfERβ1 cell line, new stable transfection experiments were carried out to improve the previously established assay. The new ZELH-zfERβ1 clone was shown to be quite stable and well-inducible (i.e. by more than 2-fold) by reference ER ligands (Sonavane, 2015). In addition, the ZELH cell line, which stably expresses the luciferase reporter gene but no functional ER, was used as a control assay to check for possible nonspecific activation or inhibition of luciferase by the extracts. In addition to zebrafish cell models, estrogenic potential of the extracts was also assessed by using the human-derived MELN cell line (Balaguer et al., 1999). MELN cells were kindly provided by Dr. Patrick Balaguer (INSERM Montpellier, France). These cells are derived from the MCF-7 cells, which endogenously express the hERα, but no functional hERβ (P. Balaguer, personal communication). Conditions for routine cell culture and exposure to chemicals and environmental extracts have been detailed previously (Balaguer et al., 1999, Cosnefroy et al., 2012, Creusot et al., 2014). Briefly, cells were seeded in 96-well white opaque culture plates (Greiner CellStar™, Dutscher, France) at 25,000 cells per well for all ZELH-zfER cell lines in phenol red free LDF-DCC medium (containing 50% of L-15, 35% of DMEM HG, 15% of Ham's F12, 15 mM of HEPES, 0.15 g/l of sodium bicarbonate, 0.01 mg/ml of insulin, 50 ng/ml of EGF and 50 U/ml of penicillin and streptomycin antibiotics, 5% v/v stripped FCS), and at 80,000 cells per well for MELN cell line in DMEM white medium. Cells were left to adhere for 24 h. Then, they were exposed in triplicates to serial dilutions of reference ligand (E2), chemical standards or environmental extracts for either 72 h at 28 °C for zebrafish cells or 16 h at 37 °C for the MELN cell line. After exposure, the medium was removed and replaced by 50 μl per well of medium containing 0.3 mM luciferin. The luminescence signal was measured in living cells using a microtiter plate luminometer (Synergy H4, BioTek).
group and exposed for 96 h in 15 ml of acclimated water in glass crystallizers. Serial dilutions of each extract were tested and the final volume of solvent (DMSO) was 0.1% v/v. This solvent concentration did not alter either embryo development or GFP expression. In each experimental series, positive control (EE2) and solvent control were included as separate experimental groups. Exposed embryos were incubated at 28 °C, under static conditions. After the exposure period, each zebrafish larva was photographed using a Zeiss AxioImager.Z1 microscope equipped with an AxioCam Mrm camera (Zeiss GmbH, Gottingen, Germany) to measure GFP expression. Image analysis was performed using the ImageJ software, and fluorescence data was treated exactly as previously described (Brion et al., 2012). 2.5. Bioassay data analysis Environmental samples were tested in at least two independent experiments. In each individual experiment, values are means of triplicates for in vitro assays and a minimum of 15 fish per concentration for the transgenic zebrafish in vivo assay. Dose–response curves (DRCs) were modeled with the Hill equation model by using the freely available RegTox Microsoft Excel™ Macro (http://www.normalesup.org/ ~vindimian/fr_index.html). All of the DRCs of sample extracts were modeled by fixing the slope and maximal effect values to that of E2, hence enabling to calculate effective concentration leading to 20% (EC20) or 50% effect (EC50) relative to E2 (Villeneuve et al., 2000, Kinani et al., 2010). For environmental chemicals, relative estrogenic potencies (REP) were determined as the ratio of EC50 of E2 to that of chemical. Estrogenic activity in environmental samples was expressed as E2-EQs, which were determined as the ratio of the EC20 of the reference compound expressed as g/l to that of the sample expressed as gram equivalents of Oasis HLB sorbent per liter (g EQ HLB/l) for POCIS extracts. 3. Results and discussion 3.1. Estrogenic potency of individual chemicals in zebrafish and human bioassays A set of different in vitro assays was used for comparative assessment of estrogenicity. All in vitro assays were well sensitive to the reference ligand E2 (Fig. 1), with EC50 values ranging from 0.01–0.02 nM for zfERβ1, zfERβ2 and hERα, to 0.2 nM for zfERα (Fig. 1, Table S2). This good sensitivity as well as the higher affinity of zfERβ subtypes than zfERα to E2 is in perfect line with our previous data reported with the
2.4. In vivo assay In vivo estrogenic potential was assessed by using the transgenic cyp19a1b-GFP zebrafish line that has been previously developed (Tong et al., 2009) and characterized with different classes of estrogens and xeno-estrogen compounds (Brion et al., 2012, Table S2). The assay procedure for individual chemical testing has been described in detail by Brion et al. (2012). In the present study, it has been adapted to the testing of organic extracts coming from the aquatic environment. In brief, 20 fertilized transgenic eggs were selected for each experimental
Fig. 1. Concentration-dependant luciferase induction by 17β-estradiol (E2) in the different in vitro reporter gene assays based on zebrafish and human estrogen receptors (ER). Values are presented as percentage of luciferase activity induced by E2 10 nM for zfERα and E2 1 nM for zfERβ1, zfERβ2 and hERα (mean values of triplicates ± standard deviation). The results are representative of at least 10 independent assays.
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same cell models (Cosnefroy et al., 2012), which confirms the stability and the robustness of the established systems. These assays were also further characterized for their response to a panel of XEs from different chemical classes (Figure S1, Table S2). First, a zfER subtype-dependent selective activation is observed in zebrafish in vitro assays, which has been well described and discussed previously (Cosnefroy et al., 2012, Pinto et al., 2014). This is shown by a better affinity of zfERβ subtypes compared to zfERα for natural and synthetic estrogens (E2 and EE2), or a selective activation of zfERα by bisphenol A or genistein. This confirms previous studies using this cell model (Cosnefroy et al., 2012) or human cells expressing zfERs (Le Page et al., 2006; Pinto et al., 2014). Second, it is noteworthy that such selectivity differs in human where natural and synthetic estrogens present better affinity for hERα than for hERβ and phytoestrogens, such as genistein, are selective hERβ modulators (Escande et al., 2006). Overall, marked differences were noticed regarding ERβ selectivity for natural estrogens, which seems to be a common feature in different fish species (Chakraborty et al., 2011, Tohyama et al., 2015). While the intimate roles of ER subtypes are still poorly understood, there exist evidences that show their tissue- and life stage-specific expression (Menuet et al., 2004), hence supporting different physiological functions (Griffin et al., 2013). The differences of affinity of (xeno)-estrogens on ER subtypes may explain their tissue and life-stage dependent effects on ER-signaling pathways in fish (Gorelick et al., 2014). Interestingly, the spatio–temporal analysis of ER mRNAs in zebrafish embryos revealed an early and predominant expression of ERβ subtypes in the hypothalamus (Mouriec et al., 2009). Altogether, this suggests that ERregulated genes in the brain of zebrafish embryos are particularly sensitive to compounds having a good affinity for ERβ subtypes. In this regard, the tested chemicals were also active in the in vivo cyp19a1b-GFP assay, but with some differences in their relative estrogenic potency as compared to in vitro data (Table S2). In several cases, the in vivo assay showed a comparable to better sensitivity to xenoestrogens (e.g. EE2, hexestrol, DES, zearalenone, o,p’-DDT) than in vitro assays. Interestingly, benzophenone-3 (BP3), an anti-UV screen, which showed weak estrogenicity in vivo, was unable to induce any activity in vitro. This could be explained by in vivo biotransformation of BP3 into active metabolites, most likely benzophenone-1. Overall, zebrafish in vitro and in vivo data illustrate the suitability of established assays to detect well-known estrogenic ligands. 3.2. In vitro screening of estrogenic activities in surface waters To assess the usefulness of newly developed in vitro zfER assays to quantify estrogenic activity in environmental matrices, we applied our set of bioassays on POCIS extracts from 20 French river sites, which were known to be subjected to various anthropogenic pressures (Table S1). Overall, estrogenic activities could be detected by all in vitro assays in the water phase at several of the investigated river sites (Table 1). By using the human MELN assay, we previously reported the suitability of using of POCIS sampling to detect estrogenic activity both in surface waters and effluents (Creusot et al., 2013, Tapie et al., 2011, Creusot et al., 2014). Most of known (xeno)-estrogens are polar to mid-polar compounds and are known to be sampled by POCIS (Alvarez et al., 2004, Togola and Budzinski, 2007). In this study, we further demonstrate the relevance of this approach on a larger scale study at WFD surveillance sites. Interestingly, the zebrafish-based assays, and notably the zfERβ2 assay, confirmed the presence of ER ligands in the extracts but also highlighted several other contaminated sites that were not revealed by the human assay. Such important finding was not expected since no species-specific ER activation could be highlighted from the screening of individual chemicals, even though some chemicals showed differences in their relative estrogenic potency between bioassays (Cosnefroy et al., 2012, Table S2). Nevertheless, our data suggest the occurrence of hydrosoluble contaminants that are specifically active on the zebrafish
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Table 1 Estrogenic activities in POCIS extracts from 20 French river sites using zf and human in vitro assays. Results are expressed in ng E2-EQ/g sorbent with confidence interval 95% in bracket and are derived from the dose–response curves presented in Figures S2, S3, S4 and S5. River Human sites hERα
Zebrafish zfERα
zfERβ1
zfERβ2
BED
198.6 (168.0–214.2) 29.6 (24.5–33.2)
131.3 (106.9–181.6) n.a.
10.9 (6.8–14.2) n.a.
LOV REV SOU
11.9 (9.1–12.8) 2.9 (2.7–3.1) 9.1 (7.0–9.8)
n.a. n.a. n.a.
n.a. 1.6 (1.0–4.4) n.a.
MAD
3.8 (2.9–4.2)
8.3 (6.2–11.2)
5.6 (2.4–17.1)
RIS SAR LOI
3.9 (3.0–4.1) 3.7 (3.0–3.9) 2.4 (2.0–2.8)
n.a. n.a. n.a.
51.5 (46.5–61.7) 26.7 (23.6–39.2) 11.5 (9.8–16.0) 2.3 (1.9–3.1) 0.5 (0.4–0.7) 15.8 (13.6–21.2) 12.2 (9.1–16.3) 10.6 (6.6–17.6) 5.0 (2.9–7.4) 15.7 (11.4–23.3) 14.4 (9.2–18.6) 3.5 (2.4–5.3) 99.7 (75.2–150.1) 35.4 (27.1–57.7) 27.4 (20.8–40.9) 10.3 (7.6–15.9) 8.2 (5.9–10.6) 7.8 (6.0–10.6) 5.9 (4.1–10.7) 5.9 (4.0–9.1) 0.4
LOM
AUV
1.9 (1.6–2.3)
25.3 (18.5–45.0)
BRA LUE OLL
1.3 (1.1–1.3) 1.0 (0.8–1.1) n.a.
n.a. n.a. 3.2 (2.2–6.4)
1.0 (0.8–1.5) n.a. 2.6 (1.7–4.2) 63.7 (52.4–99.0) n.a. n.a. n.a.
HER
n.a.
n.a.
8.4 (3.5–19.1)
YER
n.a.
4.6 (3.4–5.4)
n.a.
CHE LOB GAR JOU MAR LOQ
n.a. n.a. n.a. n.a. n.a. 0.1
8.0 (6.0–19.7) n.a. n.a. 5.2 (3.6–7.3) n.a. 2.3
1.8 (1.2–2.5) 0.5 (0.3–0.6) 9.8 (6.5–15.1) n.a. 4.1 (2.7–9.6) 0.1
LOQ: limit of quantification; n.a. non active (bLOQ).
in vitro models. For instance, the OLL site was highly active in zfERβ2 but negative in MELN. This site is known to be contaminated by several substances, including corticoids and progestagenic compounds, while steroid estrogens were not detected (Creusot et al., 2014). At this site, it is likely that such compounds, which can be metabolized into estrogenic metabolites, could have contributed to the observed estrogenic activity in the metabolically competent ZELH cells (Le Fol et al., 2015). Indeed, it is noted that both zebrafish and human in vitro assays used different cell systems (i.e. ZFL cells versus MCF-7 cells, respectively), that could possibly result in different metabolic capacities, which may have also influenced the responses of the assays (Olsen et al., 2005, Le Fol et al., 2015). It is noteworthy that a higher number of POCIS extracts were found to be more active on zfERβ1 and zfERβ2 than on zfERα. This is in line with the better sensitivity of zfERβ subtypes to E2 as compared to zfERα, although the detection of zfER subtypes-specific active compounds cannot be excluded. Furthermore, the importance of zfERβ2 assay was further highlighted by its ability to detect and quantify estrogenic contaminants in all the tested river sites, some at significantly high E2-EQ concentrations (i.e. OLL, HER, YER). The higher responsiveness of zfERβ2 to environmental samples is an important finding as this estrogen receptor subtype is present in fish species but not in humans (Hawkins et al., 2000, Hawkins and Thomas, 2004) and plays a crucial role in mediating estrogen-dependent physiological processes in fish (Nelson and Habibi, 2013, Yost et al., 2014). Overall, our results support the use of zebrafish-based (zfERs) assays as relevant in vitro models to assess estrogenic activity in water samples and raises questions on the relevance of using human-based assay to assess hazard posed by environmental estrogens on aquatic species. 3.3. In vivo estrogenic potency of environmental mixtures To gain further insight on (xeno)-estrogens associated hazard in complex samples, we used the transgenic cyp19a1b-GFP zebrafish
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embryo assay (Tong et al., 2009). After exposure of zf embryos to POCIS extracts, we identified some of the investigated sites that induced the cyp19a1b promoter in a dose-dependent manner (Figure S6). These data were used to calculate in vivo E2-EQs that are reported in Table 2. Our results demonstrate the capacity of the in vivo assay to assess estrogenic potential of environmental mixtures on the whole organism. This reporter system has been shown to be useful to assess estrogenic potency of individual chemicals by establishing dose–response curve and deriving EC50 and relative estrogenic potency (Brion et al., 2012, Table S2). Here, we report the ability of this system to detect active chemicals within complex mixtures and establish E2-EQ. Furthermore, the comparison between in vitro and in vivo estrogenic profiles provided further supports on the relevance of such an integrated assessment of estrogenic activity. Interestingly, the positive sites highlighted and quantified by the in vivo assay were also the most active ones in vitro, notably through the zfERβ2 assay (Table 2). Furthermore, E2-EQ values quantified by the in vivo cyp19a1b-GFP assay at these active sites closely relate to the in vitro estrogenic activity quantified by the zfERβ2 assay. Among these sites, the OLL river site was strongly active in zebrafish in vivo and in vitro assays but not in the human assay (Table 2, Figure S6), which reinforces a possible fish-specific response at this site, as discussed above. Similar observations were also documented using medaka that predicted more accurately in vivo estrogenic activity of wastewater by in vitro assays of the same species as compared to human in vitro assay (Ihara et al., 2015). It is noticeable that POCIS extracts that were weakly active on in vitro assays were not able to elicit any GFP expression in vivo. This could be explained by the differences in the highest test concentration of extracts used for in vivo testing (0.1% v/v) as compared to in vitro assays (0.5% v/ v). Such limitation of extract concentration can however be solved by redefining the sample preparation procedure. Another possible explanation could be due to differential bioavailability and metabolism of compounds between zebrafish embryos and ZFL cells. A higher metabolic capacity of the zebrafish embryo can lead either activation of the parent compound into estrogenic metabolites but also de-activation of some estrogenic compounds into inactive metabolites. It is clear that the identification of the compound(s) responsible for observed effects could help in identifying reasons for observed differences between in vitro and in vivo responses. Altogether, our study suggests a good agreement between in vitro zfERβ2 and in vivo cyp19a1b-GFP to identify hot-spot sites contaminated by endocrine active compounds in fish. In the future, extending this data set to other surface waters will help to increase the statistical value of the correlation and help to define in vitro E2-EQ trigger values that can predict in vivo estrogenic responses in fish. The applied in vivo assay is a highly sensitive assay to detect zebrafish ER ligands in environmental water extracts. It also provides critical toxicological information regarding the impact of environmental contaminants on the developing brain. It revealed that environmental contaminants can target radial glial cells and disrupt brain aromatase that has been associated with altered neurogenesis and behavioral activities in zebrafish (Diotel et al., 2013, Kinch et al., 2015). Impact of environmental estrogens on neurodevelopment and their potential effects on wild fish population has been until now poorly documented but should be further investigated in light of these data. Overall, the toxicological relevance of examined
Table 2 Comparison of in vivo and in vitro estrogenic activities in POCIS extracts from 3 river sites. Results are expressed as ng E2-EQ/g sorbent. In vivo data are derived from the dose–response curves presented in Figure S6. In vitro
In vivo
River sites OLL BED LOM n.a.: not active.
zfERα
zfERβ1
zfERβ2
hERα
cyp19a1b
3 n.a. 131
n.a. n.a. 11
100 27 51
n.a. 30 199
88 44 83
ER-signaling pathway at very early critical developmental stages makes the cyp19a1b-GFP assay a relevant screening tool fitting current bioanalytical challenges (Altenburger et al., 2015). 4. Conclusion This study reports the combined use of in vitro and in vivo fish-based reporter gene assays as bioanalytical tools and its relevance to highlight fish specific estrogenic activities in environmental samples. Among examined zfER assays, zfERβ2 was the most sensitive and responsive one to water extracts, although the presence of other zfER subtype-specific active compounds cannot be excluded. Compared to more commonly used human-based assay, these novel zebrafish reporter gene assays revealed significant differences to detect ER ligands. Specific and sensitive detection of zebrafish estrogen receptor ligands in their native cell (e.g. liver cell in vitro) or tissue (e.g. brain in vivo) contexts, makes them highly relevant as (eco)toxicological models to assess estrogenic effect in aquatic vertebrates. From an environmental risk assessment perspective, the use of such an integrated assessment in fish allows to gain information on the occurrence and concentration range of endocrine active substances (biodetection) as well as the potential estrogenic effect (hazard). Acknowledgments This work was funded by the EU Seventh Framework Programme (FP7-PEOPLE-2011-ITN) as a part of EDA-EMERGE project under the grant agreement number 290100, the French National Agency for Water and Aquatic Environments (ONEMA) as part of the 2012 national prospective campaign on emerging contaminants (“Etude prospective sur les contaminants émergents”) and the French Ministry of Ecology (P190- Axe de Recherche Ecotoxicologie). We wish to warmly thank Sandrine Joachim for her help in improving the English of this manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2016.01.187. References Ackermann, G.E., Brombacher, E., Fent, K., 2002. Development of a fish reporter gene system for the assessment of estrogenic compounds and sewage treatment plant effluents. Environ. Toxicol. Chem. 21, 1864–1875. Altenburger, R., Ait-Aissa, S., Antczak, P., Backhaus, T., Barceló, D., Seiler, T.-B., et al., 2015. Future water quality monitoring—adapting tools to deal with mixtures of pollutants in water resource management. Sci. Total Environ. 512–513, 540–551. Alvarez, D.A., Petty, J.D., Huckins, J.N., Jones-Lepp, T.L., Getting, D.T., Goddard, J.P., et al., 2004. Development of a passive, in situ, integrative sampler for hydrophilic organic contaminants in aquatic environments. Environ. Toxicol. Chem. 23, 1640–1648. Balaguer, P., Francois, F., Comunale, F., Fenet, H., Boussioux, A.M., Pons, M., et al., 1999. Reporter cell lines to study the estrogenic effects of xenoestrogens. Sci. Total Environ. 233, 47–56. Brion, F., Le Page, Y., Piccini, B., Cardoso, O., Tong, S.K., Chung, B.C., et al., 2012. Screening estrogenic activities of chemicals or mixtures in vivo using transgenic (cyp19a1bgfp) zebrafish embryos. PLoS One 7, e36069. Chakraborty, T., Katsu, Y., Zhou, L.Y., Miyagawa, S., Nagahama, Y., Iguchi, T., 2011. Estrogen receptors in medaka (Oryzias latipes) and estrogenic environmental contaminants: an in vitro–in vivo correlation. J. Steroid Biochem. Mol. Biol. 123, 115–121. Cosnefroy, A., Brion, F., Guillet, B., Laville, N., Porcher, J.M., Balaguer, P., et al., 2009. A stable fish reporter cell line to study estrogen receptor transactivation by environmental (xeno)estrogens. Toxicol. in Vitro 23, 1450–1454. Cosnefroy, A., Brion, F., Maillot-Marechal, E., Porcher, J.M., Pakdel, F., Balaguer, P., et al., 2012. Selective activation of zebrafish estrogen receptor subtypes by chemicals by using stable reporter gene assay developed in a zebrafish liver cell line. Toxicol. Sci. 125, 439–449. Creusot, N., Kinani, S., Balaguer, P., Tapie, N., Lemenach, K., Maillot-Marechal, E., Porcher, J.M., Budzinski, H., Aït-Aïssa, S., 2010. Evaluation of an hPXR reporter gene assay for the detection of aquatic emerging pollutants: screening of chemicals and application to water samples. Anal. Bioanal. Chem. 396, 569–583. Creusot, N., Tapie, N., Piccini, B., Balaguer, P., Porcher, J.M., Budzinski, H., Aït-Aïssa, S., 2013. Distribution of steroid- and dioxin-like activities between sediments, POCIS and SPMD in a French river subject to mixed pressures. Environ. Sci. Pollut. Res. 20, 2784–2794.
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