Transgenic marine embryos as sentinels detecting CMR toxicity in the sea. – MarineEmbryoTox

The density of human habitation around the Mediterranean sea is the highest in the world making it an ideal site to monitor man-made pollution of a marine environment, which could also affect humans. Here we set out the case for choosing the ascidian as the ideal animal model to monitor pollution. Amongst all the invertebrates the ascidian is the closest to humans (maximizing the transfer of findings to humans). Each ascidian can generate up to 1 million swimming tadpole larva in about 12 hours following fertilization making this animal useful for rapid large-scale screening experiments. Finally, over the past 2 decades we have developed genomic/molecular/imaging tools and solutions for the ascidian Phallusia mammillata, chosen because its eggs and embryos are completely transparent and thus ideal for microscopy. We are now in a position to fully exploit the Phallusia model to tackle the problem of marine pollution by human populations.
This project brings together biologists and engineers to develop new animal-based toxicological tests using state-of-the art molecular imaging techniques to monitor marine pollution. Several expression screens made by the coordinating team have revealed new proteins enriched on the DNA or in nuclei which are potentially new indicators with improved sensitivity to monitor DNA aberrations (micronuclei and DNA bridges). We aim to improve these indicators further in order to devise a completely new 2-in-1 test to detect both carcinogenicity and endocrine disrupting (EDC) activity. The carcinogenicity and the EDC activity test will use be performed simultaneously on the same embryo. Fluorescent transgenic embryos will be imaged in 3D time-lapse during the 10 hours of development from fertilization to hatching of the swimming tadpole and challenged with the several reference chemicals as defined in OECD test guidelines procedures. Carcinogenicity will be determined by scoring micronuclei and DNA bridges (as in the regulatory test OECD guideline procedure 487) while EDCs activity will be scored by imaging activation of the nuclear receptors affected by EDCs (ERR, TR, AR and PXR) using GFP-based molecular indicators of nuclear receptor activity.
Optimization of image acquisition, data saving and management will be performed to reach an estimate of 100 embryos imaged per microscope per day at high resolution and more than 200 embryos per day at low resolution. The team of Nadine Peyrieras (UPR 3294, team 3) will develop software solutions for image analysis to score automatically DNA aberrations and fluorescent nuclei. The performance of the 2-in-1 test will be assessed using reference chemicals but also through a case study. During the last 2 years of the project, marine samples collected in local basin will be quantified by spectrometric analysis by the team of Christophe Migon (UMR 7093, team 4) after being assess for toxicity using our tests.
The most sensitive indicators will be inserted in the genome of Phallusia mammillata to generate strains of fluorescent ascidians that will be maintained in our EMBRC funded marine culture facilities. Managing this task will allow us to exploit the 2-in-1 test in every part of the world where Phallusia mammillata grows.
Finally the potential impact on human health of EDC exposure will be assessed by modifying the test for NRs activation. This test will show that human NRs are activated during exposure of the ascidian embryo to EDCs to demonstrate that such marine pollution can trigger the xenobiotic response and provoke endocrine disruption in humans.
Beyond the molecular tests described above, the deliverables of this project will be patented by teams 1 and 3 of the consortium. Scientific publications will be completed after patent deposition in order to protect the work achieved during the 48 months of this project.