CREOLE
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Predictive potential of molecular markers of endocrine disruption in aquatic invertebrates: relationships between transcriptome, proteome and reprotoxicity |
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Research
Context and Issues
The new directive EC 1107/2009 for risk assessment of plant protection products (PPP) has introduced cut-off criteria, among which the endocrine-disrupting potential is used to exclude the substance from the risk assessment procedure. The evaluation of endocrine disruption potential of a chemical is currently based on in vitro tests designed for vertebrates, and thus may not be relevant for invertebrates. In order to apply this cut-off criterion in invertebrates, it is required to develop sub-individual level screening tests to assess endocrine disruption in invertebrates specifically, and link the results of these tests (which reveal the mode of action of the chemical) with the presence or absence of adverse effects at the individual level, as determined in reprotoxicity tests.
Objectives
This project aims at developing a strategic, comprehensive scheme for the screening and assessment of endocrine disruptors (EDs) in invertebrates, based upon the coupling of transcriptomics and proteomics approaches, and further assessment of reproductive performances. This screening strategy is developed in two species, the gastropod mollusc Lymnaea stagnalis and the copepod crustacean Eurythemora affinis, which allows accounting for different types of aquatic environments (freshwater vs brackish water) and different exposure routes (water/substrate interface vs. water column). Molecular and individual responses to mixtures of pesticides and biocides with different putative endocrine-disrupting modes of action (androgenic, anti-androgenic, estrogenic and mimics of arthropod juvenile hormones) and sublethal, reproductive effects in invertebrates are studied.Methodology
Molecular responses and individual performances are monitored in chronic toxicity tests in L. stagnalis and E. affinis. Sexually matures young adults are exposed to single substances and their binary mixtures. Animals are sampled after 4 days of exposure, to assess differences in genes and proteins expression, and measure internal ED concentrations. A screening without a priori will allow detecting genes and proteins that are up- or down- regulated by the substances and their mixtures. Among those genes and proteins, we will identify those which are involved in the hormonal control of growth and reproduction, or directly involved in the functioning of the endocrine systems. Responses of these candidate genes and protein signatures will be used to identify substances and mixtures acting as endocrine disruptors in L. stagnalis and E. affinis. Those results will be compared to the outputs of in vitro screening tests (YES and YAS tests) that are commonly used in risk assessment, but are not specific of L. stagnalis and E. affinis. Early warning responses, as assessed through the transcriptomics and proteomics approaches, will then be related to individual performances of exposed animals and their progeny in order to investigate the links (or the absence thereof) between the induction of changes in the endocrine function and putative reprotoxicity in exposed organisms.
Results
- Laboratory toxicity tests have been conducted for a suite of ED and their binary mixtures i.e. vinclozolin, tributyltin, chlordecone, pyriproxyfen, methoprene, ethynilestradiol. Adverse effects on survival, growth and reproduction of exposed animals have been monitored. Exposed animals were samples and prepared for transcriptomic and proteomic analyses. Exposure concentrations (in water and animals) are currently under being measured. YES and YAS tests have been conducted.
- Unexpectedly, mixtures of EDs had equivalent adverse effect on the reproductive performances of L. stagnalis than the most potent compound, showing no addition, synergy or antagonism of effects from compounds which have similar or opposite modes of action in vertebrates. This shows that classical rules for the interpretation of mixture effects might not apply to L. stagnalis exposed to ED. Results of toxicity tests with E. affinis are being analysed.
- cDNA libraries have been generated successfully from exposed animals and sequenced. The analysis of differential RNA expression between animals exposed to different compounds/mixtures and concentrations will be done in the second party of the project.
- This project will provide stakeholders with a screening strategy enabling them to apply the directive EC 1107/2009. Furthermore, it will bring additional insight towards (i) the use of transcriptomics and proteomics approaches as screening tools for the identification of EDs, and (ii) the relevance of using ED potential as a cut-off criterion for ecological risk assessment of chemicals.
Publications issued from the project
GIUSTI, A. LAGADIC, L. BARSI, A. THOME, J.-P. JOAQUIM-JUSTO, C. DUCROT, V. (2014) .Investigating apical adverse effects of four endocrine active substances in the freshwater gastropod Lymnaea stagnalis
People involved
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BESNARD Anne-Laure, Technician Phone : +33 2 23 48 54 43 Email : anne-laure.besnard(at)inrae.fr |
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COLLINET Marc, Technician Phone : +33 2 23 48 55 29 Email : marc.collinet@inrae.fr |
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COUTELLEC Marie-Agnès, Scientist Phone : +33 2 23 48 52 48 Email : marie-agnes.coutellec@inrae.fr |
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DUCROT Virginie, Scientist Phone : +33 2 23 48 56 25 Email : virginie.ducrot@inrae.fr |
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ECHASSERIAU Yann, Scientist Phone : +33 2 23 48 56 25 Email : yann.echasseriau@inrae.fr |
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LAGADIC Laurent, Scientist Phone : +33 2 23 48 52 37 Email : laurent.lagadic@inrae.fr |
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VASSAUX Danièle, Technician Phone : +33 2 23 48 56 69 Email : daniele.vassaux@inrae.fr |
Partners
Funding and Support
This project is funded and supported ANSES French Agency for Food, Environmental and Occupational Health & Safety under the programme “Environnement-Santé-Travail” (contract CREOLE, number EST-2011/1/190)