Embryonic exposures to chemicals acting on brain aromatase lead to different locomotor effects in zebrafish larvae.

Pathways underlying neurodevelopmental effects of endocrine disruptors (EDs) remain poorly known. Expression of brain aromatase (aroB), responsible for estrogen production in the brain of teleosts, is regulated by estrogenic EDs and could play a role in their behavioral effects. We exposed zebrafish eleutheroembryos (0-120 h post-fertilization) to various concentrations of 16 estrogenic chemicals (incl. bisphenols and contraceptives), and of 2 aroB inhibitors. Behavior was monitored using a photomotor response test procedure. Both aroB inhibitors (clotrimazole and prochloraz) and a total of 6 estrogenic EDs induced significant behavioral alterations, including DM-BPA, BPC and BPS-MPE, three bisphenol substitutes which behavioral effects were, to our knowledge, previously unknown. However, no consensus was reported on the effects among tested substances. It appears that behavioral changes could not be linked to groups of substances defined by their specificity or potency to modulate aroB expression, or by their structure. Altogether, behavioral effects of estrogenic EDs in 120 h post-fertilization larvae appear unrelated to aroB but are nonetheless not to be neglected in the context of environmental safety.

ReadEDTest: A tool to assess the readiness of in vitro test methods under development for identifying endocrine disruptors.

Growing evidence shows that endocrine disruptors (EDs), known to affect the reproductive system, may also disturb other hormone-regulated functions leading to cancers, neurodevelopmental defects, metabolic and immune diseases. To reduce exposure to EDs and limit their health effects, development of screening and mechanism-based assays to identify EDs is encouraged. Nevertheless, the crucial validation step of test methods by regulatory bodies is a time- and resource-consuming process. One of the main raisons of this long duration process is that method developers, mainly researchers, are not fully aware of the regulatory needs to validate a test. We propose an online self-assessment questionnaire (SAQ) called ReadEDTest easy to be used by all researchers. The aim of ReadEDTest is to speed up the validation process by assessing readiness criteria of in vitro and fish embryo ED test methods under development. The SAQ is divided into 7 sections and 13 sub-sections containing essential information requested by the validating bodies. The readiness of the tests can be assessed by specific score limits for each sub-section. Results are displayed via a graphical representation to help identification of the sub-sections having sufficient or insufficient information. The relevance of the proposed innovative tool was supported using two test methods already validated by the OECD and four under development test methods.

Hypericum lanceolatum Lam. Medicinal Plant: Potential Toxicity and Therapeutic Effects Based on a Zebrafish Model.

Hypericum lanceolatum Lam. (H. lanceolatum) is a traditional medicinal plant from Reunion Island used for its pleiotropic effects mainly related to its antioxidant activity. The present work aimed to 1) determine the potential toxicity of the plant aqueous extract in vivo and 2) investigate its putative biological properties using several zebrafish models of oxidative stress, regeneration, estrogenicity, neurogenesis and metabolic disorders. First, we characterized the polyphenolic composition by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and identified chlorogenic acid isomers, quercetin and kaempferol derivatives as the major compounds. We then evaluated for the first time the toxicity of an aqueous extract of H. lanceolatum and determined a maximum non-toxic concentration (MNTC) in zebrafish eleutheroembryos from 0 to 96 hpf following OECD (Organization for Economic Cooperation and Development) guidelines. This MNTC test was also determined on hatched eleutheroembryos after 2 days of treatment (from 3 to 5 dpf). In our study, the anti-estrogenic effects of H. lanceolatum are supported by the data from the EASZY assay. In a tail amputation model, we showed that H. lanceolatum at its MNTC displays antioxidant properties, favors immune cell recruitment and tissue regeneration. Our results also highlighted its beneficial effects in metabolic disorders. Indeed, H. lanceolatum efficiently reduces lipid accumulation and body mass index in overfed larva- and adult-models, respectively. In addition, we show that H. lanceolatum did not improve fasting blood glucose levels in a hyperglycemic zebrafish model but surprisingly inhibited neurogenesis impairment observed in diabetic conditions. In conclusion, our study highlights the antioxidant, pro-regenerative, anti-lipid accumulation and pro-neurogenic effects of H. lanceolatum in vivo and supports the use of this traditional medicinal plant as a potential alternative in the prevention and/or treatment of metabolic disorders.

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.

Estrogenic activity of surface waters using zebrafish- and human-based in vitro assays: The Danube as a case-study.

Most in vitro reporter gene assays used to assess estrogenic contamination are based on human estrogen receptor α (hERα) activation. However, fish bioassays can have distinct response to estrogenic chemicals and mixtures, questioning the relevance of human-based bioassays for assessing risk to this species. In this study, zebrafish liver cells stably expressing zebrafish ERß2 (ZELHß2) and human breast cancer cells expressing hERα (MELN) were used to quantify the estrogenic activity of 25 surface water samples of the Danube River, for which chemicals have been previously quantified. Most samples had a low estrogenic activity below 0.1 ng/L 17ß-estradiol-equivalents that was more often detected by MELN cells, while ZELHß2 response tend to be lower than predicted based on the chemicals identified. Nevertheless, both bioassays quantified well a higher estrogenic activity at two sites, which was confirmed in vivo using a transgenic zebrafish assay. The results are discussed considering the effect-based trigger values proposed for water quality monitoring.

Neurodevelopmental effects of natural and synthetic ligands of estrogen and progesterone receptors in zebrafish eleutheroembryos.

Natural and synthetic estrogens and progestins are widely used in human and veterinary medicine and are detected in waste and surface waters. Our previous studies have clearly shown that a number of these substances targets the brain to induce the estrogen-regulated brain aromatase expression but the consequences on brain development remain virtually unexplored. The aim of the present study was therefore to investigate the effect of estradiol (E2), progesterone (P4) and norethindrone (NOR), a 19-nortestosterone progestin, on zebrafish larval neurogenesis. We first demonstrated using real-time quantitative PCR that nuclear estrogen and progesterone receptor brain expression is impacted by E2, P4 and NOR. We brought evidence that brain proliferative and apoptotic activities were differentially affected depending on the steroidal hormone studied, the concentration of steroids and the region investigated. Our findings demonstrate for the first time that steroid compounds released in aquatic environment have the capacity to disrupt key cellular events involved in brain development in zebrafish embryos further questioning the short- and long-term consequences of this disruption on the physiology and behavior of organisms.

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.

Monitoring estrogenic activities of waste and surface waters using a novel in vivo zebrafish embryonic (EASZY) assay: Comparison with in vitro cell-based assays and determination of effect-based trigger values.

This study reports the use of the recently developed EASZY assay that uses transgenic cyp19a1b-GFP zebrafish (Danio rerio) embryos to assess in vivo estrogenic activity of 33 surface (SW) and waste water (WW) samples collected across Europe that were previously well-characterized for estrogen hormones and in vitro estrogenic activity. We showed that 18 out of the 33 SW and WW samples induced estrogenic responses in the EASZY assay leading to a significant and concentration-dependent up-regulation of the ER-regulated cyp19a1b gene expression in the developing brain. The in vivo 17ß-estradiol-equivalents (EEQs) were highly correlated with, both, the chemical analytical risk quotient (RQ) based on steroidal estrogen concentrations and EEQs reported from five different in vitro reporter gene assays. Regression analyses between the vitro and in vivo effect concentrations allowed us to determine an optimal cut-off value for each in vitro assay, above which in vivo responses were observed. These in vitro assay-specific effect-based trigger values (EBTs), ranging from 0.28 to 0.58 ng EEQ/L define the sensitivity and specificity of the individual in vitro assays for predicting a risk associated with substances acting through the same mode of action in water samples. Altogether, this study demonstrates the toxicological relevance of in vitro-based assessment of estrogenic activity and recommends the use of such in vitro/in vivo comparative approach to refine and validate EBTs for mechanism-based bioassays.

Combined effects of environmental xeno-estrogens within multi-component mixtures: Comparison of in vitro human- and zebrafish-based estrogenicity bioassays.

Some recent studies showed that in vitro bioassays based on fish or human estrogen receptor (ER) activation may have distinct responses to environmental samples, highlighting the need to better understand bioassay-specific ER response to environmental mixtures. For this purpose, we investigated a 12-compound mixture in two mixture ratios (M1 and M2) on zebrafish (zf) liver cells stably expressing zfERα (ZELHα cells) or zfERß2 (ZELHß2 cells) and on human ER-reporter gene (MELN) cells. The mixture included the well-known ER ligands bisphenol A (BPA) and genistein (GEN), and other compounds representatives of a freshwater background contamination. In this context, the study aimed at assessing the robustness of concentration addition (CA) model and the potential confounding influence of other chemicals by testing subgroups of ER activators, ER inhibitors or ER activators and inhibitors combined. Individual chemical testing showed a higher prevalence of ER inhibitors in zebrafish than human cells (e.g. propiconazole), and some chemicals inhibited zfER but activated hER response (e.g. benzo(a)pyrene, triphenylphosphate). The estrogenic activity of M1 and M2 was well predicted by CA in MELN cells, whereas it was significantly lower than predicted in ZELHß2 cells, contrasting with the additive effects observed for BPA and GEN binary mixtures. When testing the subgroups of ER activators and inhibitors combined, the deviation from additivity in ZELHß2 cells was caused by zebrafish-specific inhibiting chemicals. This study provides novel information on the ability of environmental pollutants to interfere with zfER signalling and shows that non-estrogenic chemicals can influence the response to a mixture of xeno-estrogens in a bioassay-specific manner.

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.

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.

An integrative approach combining passive sampling, bioassays, and effect-directed analysis to assess the impact of wastewater effluent.

Wastewater treatment plant (WWTP) effluents are major sources of endocrine-disrupting chemicals (EDCs) and other chemicals of toxicological concern for the aquatic environment. In the present study, we used an integrated strategy combining passive sampling (Chemcatcher®), developmental toxicity, and mechanism-based in vitro and in vivo bioassays to monitor the impacts of a WWTP on a river. In vitro screening revealed the WWTP effluent as a source of estrogen, glucocorticoid, and aryl hydrocarbon (AhR) receptor-mediated activities impacting the downstream river site where significant activities were also measured, albeit to a lesser extent than in the effluent. Effect-directed analysis of the effluent successfully identified the presence of potent estrogens (estrone, 17α-ethinylestradiol, and 17ß-estradiol) and glucocorticoids (clobetasol propionate and fluticasone propionate) as the major contributors to the observed in vitro activities, even though other unidentified active chemicals were likely present. The impact of the WWTP was also assessed using zebrafish embryo assays, highlighting its ability to induce estrogenic response through up-regulation of the aromatase promoter-dependent reporter gene in the transgenic (cyp19a1b-green fluorescent protein [GFP]) zebrafish assay and to generate teratogenic effects at nonlethal concentrations in the zebrafish embryo toxicity test. The present study argues for the use of such an integrated approach, combining passive sampling, bioassays, and effect-directed analysis, to comprehensively identify endocrine active compounds and associated hazards of WTTP effluents. Environ Toxicol Chem 2018;37:2079-2088. © 2018 SETAC.

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.

Integrating chemical analysis and bioanalysis to evaluate the contribution of wastewater effluent on the micropollutant burden in small streams.

Surface waters can contain a range of micropollutants from point sources, such as wastewater effluent, and diffuse sources, such as agriculture. Characterizing the source of micropollutants is important for reducing their burden and thus mitigating adverse effects on aquatic ecosystems. In this study, chemical analysis and bioanalysis were applied to assess the micropollutant burden during low flow conditions upstream and downstream of three wastewater treatment plants (WWTPs) discharging into small streams in the Swiss Plateau. The upstream sites had no input of wastewater effluent, allowing a direct comparison of the observed effects with and without the contribution of wastewater. Four hundred and five chemicals were analyzed, while the applied bioassays included activation of the aryl hydrocarbon receptor, activation of the androgen receptor, activation of the estrogen receptor, photosystem II inhibition, acetylcholinesterase inhibition and adaptive stress responses for oxidative stress, genotoxicity and inflammation, as well as assays indicative of estrogenic activity and developmental toxicity in zebrafish embryos. Chemical analysis and bioanalysis showed higher chemical concentrations and effects for the effluent samples, with the lowest chemical concentrations and effects in most assays for the upstream sites. Mixture toxicity modeling was applied to assess the contribution of detected chemicals to the observed effect. For most bioassays, very little of the observed effects could be explained by the detected chemicals, with the exception of photosystem II inhibition, where herbicides explained the majority of the effect. This emphasizes the importance of combining bioanalysis with chemical analysis to provide a more complete picture of the micropollutant burden. While the wastewater effluents had a significant contribution to micropollutant burden downstream, both chemical analysis and bioanalysis showed a relevant contribution of diffuse sources from upstream during low flow conditions, suggesting that upgrading WWTPs will not completely reduce the micropollutant burden, but further source control measures will be required.

Several synthetic progestins disrupt the glial cell specific-brain aromatase expression in developing zebra fish.

The effects of some progestins on fish reproduction have been recently reported revealing the hazard of this class of steroidal pharmaceuticals. However, their effects at the central nervous system level have been poorly studied until now. Notwithstanding, progesterone, although still widely considered primarily a sex hormone, is an important agent affecting many central nervous system functions. Herein, we investigated the effects of a large set of synthetic ligands of the nuclear progesterone receptor on the glial-specific expression of the zebrafish brain aromatase (cyp19a1b) using zebrafish mechanism-based assays. Progesterone and 24 progestins were first screened on transgenic cyp19a1b-GFP zebrafish embryos. We showed that progesterone, dydrogesterone, drospirenone and all the progesterone-derived progestins had no effect on GFP expression. Conversely, all progestins derived from 19-nortesterone induced GFP in a concentration-dependent manner with EC50 ranging from the low nM range to hundreds nM. The 19-nortestosterone derived progestins levonorgestrel (LNG) and norethindrone (NET) were further tested in a radial glial cell context using U251-MG cells co-transfected with zebrafish ER subtypes (zfERα, zfERß1 or zfERß2) and cyp19a1b promoter linked to luciferase. Progesterone had no effect on luciferase activity while NET and LNG induced luciferase activity that was blocked by ICI 182,780. Zebrafish-ERs competition assays showed that NET and LNG were unable to bind to ERs, suggesting that the effects of these compounds on cyp19a1b require metabolic activation prior to elicit estrogenic activity. Overall, we demonstrate that 19-nortestosterone derived progestins elicit estrogenic activity by inducing cyp19a1b expression in radial glial cells. Given the crucial role of radial glial cells and neuro-estrogens in early development of brain, the consequences of exposure of fish to these compounds require further investigation.

Zebrafish-based reporter gene assays reveal different estrogenic activities in river waters compared to a conventional human-derived assay.

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, zfERß1 and zfERß2 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, zfERß2 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 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.

Inhibitory effect of cadmium on estrogen signaling in zebrafish brain and protection by zinc.

The present study was conducted to assess the effects of Cd exposure on estrogen signaling in the zebrafish brain, as well as the potential protective role of Zn against Cd-induced toxicity. For this purpose, the effects on transcriptional activation of the estrogen receptors (ERs), aromatase B (Aro-B) protein expression and molecular expression of related genes were examined in vivo using wild-type and transgenic zebrafish embryos. For in vitro studies, an ER-negative glial cell line (U251MG) transfected with different zebrafish ER subtypes (ERα, ERß1 and ERß2) was also used. Embryos were exposed either to estradiol (E2 ), Cd, E2 +Cd or E2 +Cd+Zn for 72 h and cells were exposed to the same treatments for 30 h. Our results show that E2 treatment promoted the transcriptional activation of ERs and increased Aro-B expression, at both the protein and mRNA levels. Although exposure to Cd, does not affect the studied parameters when administered alone, it significantly abolished the E2 -stimulated transcriptional response of the reporter gene for the three ER subtypes in U251-MG cells, and clearly inhibited the E2 induction of Aro-B in radial glial cells of zebrafish embryos. These inhibitory effects were accompanied by a significant downregulation of the expression of esr1, esr2a, esr2b and cyp19a1b genes compared to the E2 -treated group used as a positive control. Zn administration during simultaneous exposure to E2 and Cd strongly stimulated zebrafish ERs transactivation and increased Aro-B protein expression, whereas mRNA levels of the three ERs as well as the cyp19a1b remained unchanged in comparison with Cd-treated embryos. In conclusion, our results clearly demonstrate that Cd acts as a potent anti-estrogen in vivo and in vitro, and that Cd-induced E2 antagonism can be reversed, at the protein level, by Zn supplement. Copyright © 2016 John Wiley & Sons, Ltd.

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.

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.

Effect-directed analysis for estrogenic compounds in a fluvial sediment sample using transgenic cyp19a1b-GFP zebrafish embryos.

Xenoestrogens may persist in the environment by binding to sediments or suspended particulate matter serving as long-term reservoir and source of exposure, particularly for organisms living in or in contact with sediments. In this study, we present for the first time an effect-directed analysis (EDA) for identifying estrogenic compounds in a sediment sample using embryos of a transgenic reporter fish strain. In the tg(cyp19a1b-GFP) transgenic zebrafish strain, the expression of GFP (green fluorescent protein) in the brain is driven by an oestrogen responsive element in the promoter of the cyp19a1b (aromatase) gene. The selected sediment sample of the Czech river Bilina had already been analysed in a previous EDA using the yeast oestrogen screening assay and had revealed fractions containing estrogenic compounds. When normal phase HPLC (high performance liquid chromatography) fractionation was used for the separation of the sediment sample, the biotest with transgenic fish embryos revealed two estrogenic fractions. Chemical analysis of candidate compounds in these sediment fractions suggested alkylphenols and estrone as candidate compounds responsible for the observed estrogenic effect. Alkylphenol concentrations could partially explain the estrogenicity of the fractions. However, xenoestrogens below the analytical detection limit or non-targeted estrogenic compounds have probably also contributed to the sample's estrogenic potency. The results indicated the suitability of the tg(cyp19a1b-GFP) fish embryo for an integrated chemical-biological analysis of estrogenic effects.