Approches bio-analytiques pour la caractérisation, la surveillance et l’identification de perturbateurs endocriniens dans le milieu aquatique

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Modulation of aromatase activity and mRNA by various selected pesticides in the human choriocarcinoma JEG-3 cell line

International audienceAromatase enzyme plays a central role in steroidogenesis by converting androgens to estrogens and has been proposed as an important molecular target for many environmental endocrine disrupters chemicals. In this study, we have screened 30 selected pesticides with known, unknown or supposed effects on aromatase activity, for their ability to modulate aromatase activity in the human choriocarcinoma JEG-3 cell line after both short (2 h) and long exposure (24 h). All pesticides were tested at concentrations up to 10 microM that did not cause cytotoxicity after 24h of exposure, as verified by the MTT viability assay. Four pesticides inhibited aromatase activity after 2 h of exposure: prochloraz (IC(50)<1 microM), fenbuconazole (IC(50)=1.1 microM), propiconazole (IC(50)=1.5 microM) and fenarimol (IC(50)=3.3 microM). Among them, prochloraz and fenbuconazole also exerted inhibitory effects after 24h. Toxaphen (10 microM) and heptachlor (10 microM) inhibited aromatase activity after 24h exposure only. Nine pesticides induced aromatase activity: aldrin, chlordane, cypermethrin, parathion-methyl, endosulfan, methoxychlor, oxadiazon, metolachlor and atrazine after 24 h of exposure, while tributyltin induced aromatase activity at 1 nM and 3 nM after both 2 h and 24 h of exposure, respectively. To further investigate the mechanisms of aromatase induction we measured CYP19 mRNA expression and showed that methoxychlor, aldrin, chlordane and tributyltin induced the transcription of the cyp19 gene. In addition, none of the aromatase inducers transactivated the retinoic acid receptor (RAR) in JEG-3 stably transfected with a RARE-luciferase plasmid while the RAR agonist TTNPB induced both aromatase and luciferase expression in these cells. Our results, which provide new data for fenbuconazole, as an inhibitor of human aromatase, and for eight pesticides as aromatase inducers, are discussed with regards to the regulation of aromatase expression in the JEG-3 cellular context

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 -zfERβ) 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

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

Effect-directed analysis (EDA)-based strategieshave been increasingly used in order to identify the causativelink between adverse (eco-)toxic effects and chemical con-taminants. In this study, we report the development and use ofan EDA approach to identify endocrine-disrupting chemicals(EDCs) in a multi-contaminated river sediment. The battery ofin vitro reporter cell-based bioassays, measuring estrogenic,(anti)androgenic, dioxin-like, and pregnane X receptor(PXR)-like activities, revealed multi-contamination profiles.To isolate active compounds of a wide polarity range, weestablished a multi-step fractionation procedure combining:(1) a primary fractionation step using normal phase-basedsolid-phase extraction (SPE), validated with a mixture of 12non-polar to polar standard EDCs; (2) a secondary fraction-ation using reversed-phase-based high-performance liquidchromatography (RP-HPLC) calibrated with 33 standardEDCs; and (3) a purification step using a recombinant estro-gen receptor (ER) affinity column. In vitro SPE and HPLCprofiles revealed that ER and PXR activities were mainly dueto polar to mid-polar compounds, while dioxin-like and anti-androgenic activities were in the less polar fractions. Theoverall procedure allowed final isolation and identificationof new environmental PXR (e.g., di-iso-octylphthalate) andER (e.g., 2,4-di-tert-butylphenol and 2,6-di-tert-butyl-α-methoxy-p-cresol) ligands by using gas chromatography cou-pled with mass spectrometry with full-scan mode acquisitionin mid-polar fractions. In vitro biological activity of thesechemicals was further confirmed using commercial standards,with di-iso-octylphthalate identified for the first time as apotent hPXR environmental agonist

Comparison of the in vivo biotransformation of two emerging estrogenic contaminants, BP2 and BPS, in zebrafish embryos and adults

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

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

International audienceMost 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 ERβ2 (ZELHβ2) 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 17β-estradiol-equivalents that was more often detected by MELN cells, while ZELHβ2 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

Study of the day- and night-time SOA formation from the oxidation of furans and PAHs performed in an oxidation flow reactor

International audienceBiomass burning significantly contributes to the concentration levels of ambient primary particulate matter (PM) especially in wintertime due to residential wood burning (Vicente et al., 2018). This source also emits large quantities of volatile and semi-volatile organic compounds leading to the formation, via (photo- ) chemical and condensation processes, of secondary organic aerosols (SOA) accounting for a significant part of fine PM concentrations. However, SOA physicochemical properties and formation yields from the corresponding precursors are still poorly documented in the literature particularly regarding nighttime chemistry (involving nitrate radical) (Bruns et al., 2016). The main aim of this work is to study the formation of biomass burning SOA from typical organic precursors namely PAHs and furans, under both daytime and nighttime chemistry

Study of the day- and night-time SOA formation from the oxidation of furans and PAHs performed in an oxidation flow reactor

International audienceBiomass burning significantly contributes to the concentration levels of ambient primary particulate matter (PM) especially in wintertime due to residential wood burning (Vicente et al., 2018). This source also emits large quantities of volatile and semi-volatile organic compounds leading to the formation, via (photo- ) chemical and condensation processes, of secondary organic aerosols (SOA) accounting for a significant part of fine PM concentrations. However, SOA physicochemical properties and formation yields from the corresponding precursors are still poorly documented in the literature particularly regarding nighttime chemistry (involving nitrate radical) (Bruns et al., 2016). The main aim of this work is to study the formation of biomass burning SOA from typical organic precursors namely PAHs and furans, under both daytime and nighttime chemistry