Aurélie Escande

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

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

New stably transfected bioluminescent cells expressing FLAG epitope-tagged estrogen receptors to study their chromatin recruitment

BackgroundBiological actions of estrogens are mediated by the two specific estrogen receptors ERα and ERβ. However, due to the absence of adequate cellular models, their respective transcriptional activities are still poorly understood. For instance, the evaluation of such differing properties on the transcription of responsive genes using ChIP experiments was hindered by the deficiency of cells exhibiting the same genotypic background and properties but expressing only one of the ERs. We describe here the generation of such cells, using an estrogen receptor negative HELN cell line that was derived from HeLa cells stably transfected with an ERE-driven luciferase plasmid. These HELN-Fα and HELN-Fβ cell lines stably express either the alpha or beta (full length) estrogen receptor tagged with the FLAG epitope. The use of antibodies directed against the FLAG epitope allowed a direct comparative evaluation of the respective actions of both ERs using ChIP.ResultsHELN-Fα and HELN-Fβ cell lines were found to express comparable levels of their corresponding tagged receptors with a Kd for estradiol binding of 0.03 and 0.27 nM respectively. The presence of a stably transfected ERE-driven luciferase plasmid in these cells allowed the direct evaluation of the transcriptional activity of both tagged receptors, using natural or synthetic estrogens. FLAG-ERα and FLAG-ERβ were found to exhibit similar transcriptional activity, as indicated by a kinetic evaluation of the transcriptional activation of the luciferase gene during 10 hrs of treatment with estradiol. The validity of these model cells was further confirmed by the predictable transcriptional regulations measured upon treatments with ERα or ERβ specific ligands. The similar immunoprecipitation efficiency of both tagged receptors by an anti-FLAG antibody allowed the assessment of their kinetic recruitment on the synthetic luciferase promoter (containing an estrogen response element) by ChIP assays during 8 hours. A biphasic curve was obtained for both FLAG-ERα and FLAG-ERβ, with a peak occurring either at 2 hr or at 1 hr, respectively, and a second one following 4 hr of E2 stimulation in both cases. In MCF-7 cells, the recruitment of ERα also exhibited a biphasic behaviour; with the second peak however not so important than in the HeLa cell lines.ConclusionIn HELN derived cell lines, no fundamental differences between kinetics were observed during 8 hours for FLAG-ERα and FLAG-ERβ, as well as for polymerase II recruitment. However, the relative importance of recruitment between 1 hr and 4 hr was found to be different in HeLa cell line expressing exogenous tagged ERα and in MCF-7 cell line expressing endogenous ER.

Diclofenac in the marine environment: A review of its occurrence and effects

Interest in the presence and effects of diclofenac (DCF) and other pharmaceutical products (PPs) in the aquatic environment has been growing over the last 20u202fyears. DCF has been included in the First Watch List of the EU Water Framework Directive in order to gather monitoring data in surface waters. Despite PP input in water bodies, few studies have been conducted to determine the extent of DCF occurrence and effects on marine ecosystems, which is usually the final recipient of surface waters. The present article reviews available published data on DCF occurrence in marine water, sediment and organisms, and its effects on marine organisms. The findings highlight the scarcity of available data on the occurrence and effects of DCF in marine ecosystems, and the need for further data acquisition to assess the risks associated with the presence of this compound in the environment.

Extensive Treatment System For Recycling Water For Flushing Fresh Manure And Recovering Nutrients

From preliminary researches on a pilot scale, a complete demonstration plant was built to treat the effluents of a 30 pregnant sow’s piggery. It includes a screen, a vermifilter, a macrophyte lagooning, and a complementary water storage pond; the recycled water is used for flushing, and rainfall is collected to compensate for evapotranspiration. After functioning in 2008 and 2009, it was showed that, during the warm season, the whole plant produced an effluent suitable for flushing, where the concentration decrease was over 70% for the phosphorus and potassium, 95% for the COD and nitrogen, 99.8% for endocrine disruptors (estrogenic activity), and 99.99% for pathogenic micro‐organisms. During the cold season, the dilution by the rain water and the treatment effect of the constructed wetlands lead to similar results. Nevertheless, for this season, suitable floating macrophytes that will cover the lagoons remain to be settled.