Comparison of two reference systems for biomarker data analysis in a freshwater biomonitoring context

International audienceThe usefulness of fish biomarkers for freshwater biomonitoring is now well recognized, but they still pose several questions to ecotoxicology researchers. The present study, designed to assess the effects of a small city located in an agricultural river basin watershed on sticklebacks living in an adjacent river, underlines the importance of reference selection. Two reference systems were used to analyse responses of a set of biomarkers, including biotransformation enzymes, oxidative stress parameters, neurotoxicity and endocrine disruption end-points, measured in wild sticklebacks electrofished in a contaminated stream. The results showed that the investigated urban pressure disturbed CYP3A activity but also induced hepatic lipoperoxidation and circulating vitellogenine but this result is strongly influenced by the selected reference system. This work therefore demonstrates the need for further research to identify a robust reference system for stickleback biomarker analysis

Comparative toxicity of 24 manufactured nanoparticles in human alveolar epithelial and macrophage cell lines

<p>Abstract</p> <p>Background</p> <p>A critical issue with nanomaterials is the clear understanding of their potential toxicity. We evaluated the toxic effect of 24 nanoparticles of similar equivalent spherical diameter and various elemental compositions on 2 human pulmonary cell lines: A549 and THP-1. A secondary aim was to elaborate a generic experimental set-up that would allow the rapid screening of cytotoxic effect of nanoparticles. We therefore compared 2 cytotoxicity assays (MTT and Neutral Red) and analyzed 2 time points (3 and 24 hours) for each cell type and nanoparticle. When possible, TC50 (Toxic Concentration 50 i.e. nanoparticle concentration inducing 50% cell mortality) was calculated.</p> <p>Results</p> <p>The use of MTT assay on THP-1 cells exposed for 24 hours appears to be the most sensitive experimental design to assess the cytotoxic effect of one nanoparticle. With this experimental set-up, Copper- and Zinc-based nanoparticles appear to be the most toxic. Titania, Alumina, Ceria and Zirconia-based nanoparticles show moderate toxicity, and no toxicity was observed for Tungsten Carbide. No correlation between cytotoxicity and equivalent spherical diameter or specific surface area was found.</p> <p>Conclusion</p> <p>Our study clearly highlights the difference of sensitivity between cell types and cytotoxicity assays that has to be carefully taken into account when assessing nanoparticles toxicity.</p

Adverse effects in wild fish living downstream from pharmaceutical manufacture discharges

International audienceA set of biochemical and histological responses was measured in wild gudgeon collected upstream and downstream of urban and pharmaceutical manufacture effluents. These individual end-points were associated to fish assemblage characterisation. Responses of biotransformation enzymes, neurotoxicity and endocrine disruption biomarkers revealed contamination of investigated stream by a mixture of pollutants. Fish from sampled sites downstream of the industrial effluent exhibited also strong signs of endocrine disruption including vitellogenin induction, intersex and male-biased sex-ratio. These individual effects were associated to a decrease of density and a lack of sensitive fish species. This evidence supports the hypothesis that pharmaceutical compounds discharged in stream are involved in recorded endocrine disruption effects and fish population disturbances and threaten disappearance of resident fish species. Overall, this study gives argument for the utilisation of an effect-based monitoring approach to assess impacts of pharmaceutical manufacture discharges on wild fish populations

Evaluation de la toxicité et de l'activité oestrogénique de substituts du bisphénol A chez le poisson zèbre

International audienc

Evaluation de la toxicité et de l'activité oestrogénique de substituts du bisphénol A chez le poisson zèbre

International audienc

Use of zebrafish cell lines to assess estrogenic compounds in environmental samples

Aquatic endocrine disrupting chemicals (EDCs) encompass a great diversity of chemicals that affect exposed organisms through different molecular mechanisms, including their ability to bind to estrogen receptors (ERs) and to modulate the transcription of target genes. Till date, most of in vitro assays used to assess xeno-estrogens have been established using human ER in either yeast or mammalian cell lines. However, cross-species differences due to cellular context (metabolic capacities, transcription) or different receptor sequences may lead to significant differences between-assay in estrogenic potency of chemicals and eventually influence the response of complex environmental mixtures. We recently established an in vitro luciferase assay based on stable expression of zebrafish ER subtypes (zfERa, zfERß1, zfERß2) in the ZFL zebrafish liver cell line (Cosnefroy et al 2012). Luciferase induction by 17ß-estradiol (E2) in zfERa and zfERß2 cell lines results in EC 50 s of 0.2 and 0.03 nM, respectively. In the present study, we have implemented this assay to screen and characterize the individual effect of different chemical contaminants, including bisphenols, zearalenone, alkylphenols, parabens and phthalates. Comparison with the response in the human cell based MELN assay revealed differences in both sensitivity (i.e. EC50) and receptor binding potency (i.e. full versus partial agonists). In addition, the ability of the zebrafish in vitro assays to detect active compounds in complex mixtures will be tested by screening organic extracts of river sediment, effluents and surface waters. These results will be compared with those obtained with the MELN assay which will provide new information on fish specific effects of environmental xenoestrogens present in aquatic ecosystems

Combined use of passive sampling and in vitro bioassays for the detection of emerging pollutants in surface water

River systems are contaminated by various chemicals, a number of which are described as endocrine disrupting compounds (EDCs) that can be monitored by using in vitro receptor-based bioassays. While estrogen (ER) and aryl hydrocarbon receptors (AhR) activities have been well documented, much fewer information are available on other xenobiotic-sensing or steroid receptors [e.g. pregnane X, (PXR) androgen (AR), glucocorticoid (GR), progestagen (PR) receptors]. In this study, we address the use of multi-receptor approach (ER, AhR, PXR, AR, GR, MR and PR) based on different reporter cell lines combined with passive water sampling with polar organic compounds chemical sampler (POCIS) for the detection of polar emerging compounds in surface waters. This approach has been applied to a river site that is subjected to pharmaceutical industry effluent and where development abnormalities in fish were noted. At three sampling stations located upstream (A) and downstream (B and C) from the pharmaceutical industry, EDC activities were monitored in sediments and POCIS. In sediments, weak to moderate estrogenic and dioxin-like activities were found at the three stations. First analyzed POCIS showed weak PAH-like activity as well as significant PXR and ER activities at the three stations. Interestingly, strong GR activities were recorded at site B and C (Dexa-EQ = 61-69 µg/g) but none at site A. In addition, anti-MR, anti-PR and AR activity were also found downstream from the effluent hence suggesting a release of polar steroid compounds in the water phase. Samples from the other campaigns are still under analyses to assess seasonal variability of the contamination. Chemicals analyses are under investigation to identify contaminants responsible for measured activities

Endocrine disrupting activities profiling of monitored French river sites based on in vitro bio-analytical tools

Regarding thousands of chemicals occurring in aquatic systems at trace levels and as mixtures, current regulatory tools -in the frame of the water framework directive (WFD)- give an incomplete scope of environmental complexity. In this context, development and evaluation of new strategies allowing holistic contamination characterization and identification of environmental pollutants contributing to deleterious effect (e.g. endocrine disruption) on exposed organisms is relevant. To tackle this challenge, the use of in vitro bio-analytical tools is a promising approach since they allow specific, sensitive and quantitative detection of all compounds presenting similar mechanism of action (e.g. binding to nuclear receptors) within environmental mixtures. Hence, monitoring active pollutants by using a panel of complementary bioassays should increase the detection capacity of contaminants and allow a better hazard assessment through biological activity-based profiling. In the frame of a large study on surface waters monitoring in France in 2012 (Botta et al. accompanying communication), we applied a battery of receptor-based in vitro assays (estrogen (ER), androgen (AR), glucorticoid (GR),pregnane (PXR), and aryl hydrocarbon (AhR) receptors) for establishing endocrine disrupting profiles of 20 French rivers sites subject to various anthropogenic pressures (i.e. urban, agricultural, industrial, mixed, reference). This was performed on both sediment and surface water (both grab and POCIS) extracts. Estrogenic (1-10 ng-E2eq/g d.w range), PXR-like (1-10 µg-SReq/g d.w range) and dioxin-like (1-100 ngTCDD-eq/g d.w range) activities were detected in both sediment and surface water while anti-androgenic activity (10-100 µg-DHTeq/g d.w range) was only detected in sediment and glucorticoidic activity (100 µg-DEXeq/g d.wrange) was only detected in one surface water.Overall, the levels of detected activity differed according to sites and to compartments. Altogether, the use of in vitro bio-analytical tools combined to multi-compartment approach offers a cost-effective integrated quality assessment of the chemical status and permits to classify and identify hotspot polluted sites.They constitute a relevant complementary approach to current regulatory chemicals analyses for routine monitoring of chemical quality status