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, 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 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

Bioanalytical Characterization and Source Identification of a High Glucocorticoid Contamination in a River Under Urban and Industrial Pressures

International audienceSynthetic glucocorticoids (GCs) are a group of steroids that are massively used as anti-inflammatory drugs to treat a broad spectrum of deseases such as asthma or arthritis. They may enter surface waters via urban, hospital and/or pharmaceutical industry effluents and were already detected in surface waters at concentrations ranging from < 1 to tens of ng/L. In organisms, GCs are designed to act through the glucocorticoid receptor (GR) and some studies have reported adverse effects on aquatic vertebrates, such as fish. Overall, there is still a need for knowledge on the sources and driver compounds for GR activity in different contexts. In the frame of a French national survey of EDCs in surface waters using in vitro bioassays, we previously identified a hotspot river site with high GR activity. To further identify the source(s) and the nature of this contamination, an investigative monitoring based on bioassay (MDA-Kb2) and chemical analyses was implemented in the current study. A contamination mapping was established on the basis of bioassay data, along a 40 km streatch upstream and downstream of the contaminated site allowing identification of the discharge of the contamination in the river and of its highly probable industrial origin in the sewerage network, although the contribution of waste waters from a hospital could not be excluded. By using a suspect screening approach based on HR-MS, fluticasone propionate as well as few other GCs were identified in the samples. Mass balance analysis based on quantitave chemical analysis confirmed fluticasone propionate main role in the measured GR activity. The high mobility and persistence of this contamination points out possible high environmental concern in terms of its fate and hazard for aquatic organisms

Bioanalytical Characterization and Source Identification of a High Glucocorticoid Contamination in a River Under Urban and Industrial Pressures

International audienceSynthetic glucocorticoids (GCs) are a group of steroids that are massively used as anti-inflammatory drugs to treat a broad spectrum of deseases such as asthma or arthritis. They may enter surface waters via urban, hospital and/or pharmaceutical industry effluents and were already detected in surface waters at concentrations ranging from < 1 to tens of ng/L. In organisms, GCs are designed to act through the glucocorticoid receptor (GR) and some studies have reported adverse effects on aquatic vertebrates, such as fish. Overall, there is still a need for knowledge on the sources and driver compounds for GR activity in different contexts. In the frame of a French national survey of EDCs in surface waters using in vitro bioassays, we previously identified a hotspot river site with high GR activity. To further identify the source(s) and the nature of this contamination, an investigative monitoring based on bioassay (MDA-Kb2) and chemical analyses was implemented in the current study. A contamination mapping was established on the basis of bioassay data, along a 40 km streatch upstream and downstream of the contaminated site allowing identification of the discharge of the contamination in the river and of its highly probable industrial origin in the sewerage network, although the contribution of waste waters from a hospital could not be excluded. By using a suspect screening approach based on HR-MS, fluticasone propionate as well as few other GCs were identified in the samples. Mass balance analysis based on quantitave chemical analysis confirmed fluticasone propionate main role in the measured GR activity. The high mobility and persistence of this contamination points out possible high environmental concern in terms of its fate and hazard for aquatic organisms

Using mass spectrometry to highlight structures of degradation compounds obtained by photolysis of chloroacetamides: case of acetochlor.

International audienceThe photooxidation of acetochlor (a pesticide belonging to the acetamides group) using a polychromatic UV irradiation in ultrapure water was studied. This study reports the efficiency of mass spectrometry for the characterization of photodegradation products of acetochlor. Decompositions of protonated ions MH+are proposed in electrospray (ESI) mode for LC-MS, while electron ionization (EI) and chemical ionization modes (CI) are used for GC-MS. The knowledge of fragmentation and the use of a combination of experiments (MS/MS, high resolution) allow the characterization of photoproducts. Structural elucidation is assisted by the use of photolysed deuterated compounds. Fifteen major degradation products have been characterized, five by LC-QTOF, six photoproducts by GC-ITMS, and four are observed by both techniques. In vitro bioassays based on the quantification of receptor-mediated activity demonstrated that acetochlor photolysis engenders a moderate but significant estrogenic activity. Moreover, a quantitative structure-activity relationship (QSAR) approach was used to assess the potential toxicity effect of acetochlor and its by-products. The predictions were analyzed showing a variety of toxicity profiles of acetochlor photoproducts depending on the toxicological investigated endpoint

Repeated exposure to nanosecond high power pulsed microwaves increases cancer incidence in rat

International audienceHigh-power microwaves are used to inhibit electronics of threatening military or civilian vehicles. This work aims to assess health hazards of high-power microwaves and helps to define hazard threshold levels of modulated radiofrequency exposures such as those emitted by the first generations of mobile phones. Rats were exposed to the highest possible field levels, under single acute or repetitive exposures for eight weeks. Intense microwave electric fields at 1 MV m-1 of nanoseconds duration were applied from two sources at different carrier frequencies of 10 and 3.7 GHz. The repetition rate was 100 pps, and the duration of train pulses lasted from 10 s to twice 8 min. The effects on the central nervous system were evaluated, by labelling brain inflammation marker GFAP and by performing different behavioural tests: rotarod, T-maze, beam-walking, open-field, and avoidance test. Long-time survival was measured in animals repeatedly exposed, and anatomopathological analysis was performed on animals sacrificed at two years of life or earlier in case of precocious death. Control groups were sham exposed. Few effects were observed on behaviour. With acute exposure, an avoidance reflex was shown at very high thermal level (22 W kg-1); GFAP was increased some days after exposure. Most importantly, with repeated exposures, survival time was 4-months shorter in the exposed group, with eleven animals exhibiting a large sub-cutaneous tumour, compared to two in the sham group. A residual X-ray exposure was also present in the beam (0.8 Gy), which is probably not a bias for the observed result. High power microwaves below thermal level in average, can increase cancer prevalence and decrease survival time in rats, without clear effects on behaviour. The parameters of this effect need to be further explored, and a more precise dosimetry to be performed

Selective activation of zebrafish estrogen receptor subtypes by chemicals by using stable reporter gene assay developed in a zebrafish liver cell line.

International audienceThe number of environmental chemical contaminants suspected to act as endocrine disruptor compounds by interacting with estrogen receptor (ER) signaling pathway has been continuously increasing. To study such interaction, the use of stable reporter gene assays is relevant, but species-specific in vitro screening assays are still lacking to address hazard assessment of estrogenic chemicals in aquatic vertebrates. Here, we describe the development of stable reporter gene assays based on stable expression of subtypes of zebrafish ER (zfERα, zfERβ1, and zfERβ2) coupled to estrogen response element-driven luciferase in a zebrafish liver (ZFL) cell line. The three established cell models, named ZELH-zfERα, ZELH-zfERβ1, and ZELH-zfERβ2, expressed stable and significant basal luciferase signal, which was induced by 17β-estradiol (E2) in a sensitive and dose-response manner at EC(50)s of 0.2, 0.03, and 0.05 nM, respectively. In addition, E2 significantly altered cell proliferation in ZELH-zfERα and ZELH-zfERβ2 cells, but not in parental ZFL and ZELH-zfERβ1 cells, suggesting a functionality of these two receptors to modulate endogenous gene expression in the transfected clones. The screening of various xenoestrogens from different classes in the three models resulted in different luciferase response patterns. Natural and synthetic estrogens and 1,1,1-trichloro-2-(2 chlorophenyl)-2-(4-chlorophenyl)ethane were active at lower concentrations in ZELH-zfERβ1 and ZELH-zfERβ2 than in ZELH-zfERα cells, whereas genistein and zearalenone metabolites as well as three benzophenone derivatives preferentially activated zfERα. Altogether, the newly established models provide specific and convenient in vitro tool for comparative assessment of zfERs selective activation by chemicals within ZFL cell context

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

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