Description du sujet de thèse

The PhD student will be involved in the design of phthalate analogues and their synthesis (directly or indirectly depending on his/her technical skills). He/she will have to assess the extent to which these compounds mimic the endocrine-disrupting activity of the corresponding reference phthalates (DBP, DEHP) in models linked to aquatic exposure (D. rerio zebrafish larvae and D. rerio cell lines). He will exploit these synthetic compounds to study their influx, organotropism, bioaccumulation and metabolism in these models at low concentrations representative of environmental phthalate levels.

Contexte de travail

Esters of phthalic acid derivatives (i.e. phthalates) are present in a multitude of everyday consumer products (most often PVC-type plastics, but also solvents, paints, glues, detergents and cosmetics), and are among the endocrine-disrupting compounds frequently singled out in both human health and the environment. Characterizing the fate of phthalates within experimental devices and organisms in terms of distribution and metabolism is a prerequisite for any study of the impact of these compounds on human populations and ecosystems. However, quantifying phthalates is complicated by a number of obstacles. Firstly, the methods used are not very sensitive, with quantification limits sometimes close to, or even higher than, environmental concentrations. Moreover, the ubiquitous presence of the most common phthalates in laboratory equipment further complicates the task, with the analytical equipment and fluids used themselves causing contamination of the samples assessed. To overcome these experimental hurdles, we recently initiated an innovative, interdisciplinary strategy combining chemobiology and (eco)toxicology between our 2 partner laboratories (Laboratoire Interdisciplinaire des Environnements Continentaux, UMR7360 and Laboratoire de Chémo-Biologie Synthétique et Thérapeutique, UMR 7199). This approach will provide a simple, rapid and sensitive method for quantifying model phthalates in cellular and in vivo models (zebrafish D. rerio in particular) and environmental matrices, while enabling them to be distinguished from those present ubiquitously. This tool will be used to characterize the location and fate of phthalates (bioaccumulation, metabolism, excretion) at low concentrations, realistically reflecting chronic human and environmental exposure. This chemobiological approach is based on the use of synthetic analogues representative of 2 phthalates commonly detected in anthropized aquatic environments: di-n-butyl phthalate (DBP) and di-(2-ethylhexyl) phthalate (DEHP). Some of the analogues used will be photoaffinity probes.
The aim of this thesis project is to overcome the analytical barriers to the detection and quantification of phthalates by exploiting a new chemobiological approach to quantification using two analogues of di-n-butyl phthalate (DBP) and di-(2-ethylhexyl) phthalate (DEHP). DBP and DEHP are proven endocrine-disrupting compounds, and are the most widely used and abundant phthalates in our everyday environment and anthropized environments, affecting both human and environmental health. Thanks to their unique structural characteristics, these two analogues can be distinguished from any other phthalate likely to be present, whether a priori ubiquitous in the environment and biota, or a posteriori during the use of analytical equipment, which current assay methods do not allow. Moreover, the structural modification, albeit minimal, will ultimately allow us to considerably improve the detection and quantification limits of these compounds, by enabling selective coupling to a fluorogenic chromophore during the final detection/quantification stage after exposure of the organisms.
To validate this new methodology for quantifying phthalates at low concentrations and to assess its applicability first to in vivo laboratory studies before it can be used on biological and/or environmental matrices, we propose to answer the following specific questions:
1) Can DBP and DEHP analogues mimic the endocrine-disrupting effects of phthalates? Is their metabolism representative of that of DBP and DEHP?
2) After exposure of zebrafish embryos to these original phthalates at low concentrations representative of environmental exposure concentrations, can they be easily quantified in comparison with existing methods for phthalates? What is the gain in terms of detection sensitivity? What is the mechanism of phthalate influx at low concentrations, contributing to the absorption phase of bioconcentration? What are the characteristics of possible organotropism, bioaccumulation and metabolism of phthalates under these exposure conditions? Can the information thus obtained be used to understand these same mechanisms in humans?
The aim of this thesis project is to overcome the analytical barriers to the detection and quantification of phthalates by exploiting a new chemobiological approach to quantification using two analogues of di-n-butyl phthalate (DBP) and di-(2-ethylhexyl) phthalate (DEHP). DBP and DEHP are proven endocrine-disrupting compounds, and are the most widely used and abundant phthalates in our everyday environment and anthropized environments, affecting both human and environmental health. Thanks to their unique structural characteristics, these two analogues can be distinguished from any other phthalate likely to be present, whether a priori ubiquitous in the environment and biota, or a posteriori during the use of analytical equipment, which current assay methods do not allow. Moreover, the structural modification, albeit minimal, will ultimately allow us to considerably improve the detection and quantification limits of these compounds, by enabling selective coupling to a fluorogenic chromophore during the final detection/quantification stage after exposure of the organisms.
To validate this new methodology for quantifying phthalates at low concentrations and to assess its applicability first to in vivo laboratory studies before it can be used on biological and/or environmental matrices, we propose to answer the following specific questions:
1) Can DBP and DEHP analogues mimic the endocrine-disrupting effects of phthalates? Is their metabolism representative of that of DBP and DEHP?
2) After exposure of zebrafish embryos to these original phthalates at low concentrations representative of environmental exposure concentrations, can they be easily quantified in comparison with existing methods for phthalates? What is the gain in terms of detection sensitivity? What is the mechanism of phthalate influx at low concentrations, contributing to the absorption phase of bioconcentration? What are the characteristics of possible organotropism, bioaccumulation and metabolism of phthalates under these exposure conditions? Can the information thus obtained be used to understand these same mechanisms in humans?

Contraintes et risques

The candidate should have experimental knowledge and skills in one or, as far as possible, several of the following fields: ecotoxicology, toxicology, biochemistry and molecular biology, organic synthesis, analytical methods (particularly HPLC).
He/she should be comfortable communicating, presenting results and writing reports and scientific articles in English. He/she should be dynamic, capable of initiative, scientifically curious, open to collaboration, able to work in a team and quickly autonomous.

Biological experimentation will be carried out at the Laboratoire Interdisciplinaire des Environnements Continentaux (LIEC, UMR7360, Bridoux site, Metz, Environmental Toxicology team) under the supervision of Eric BATTAGLIA (university professor) and the co-responsibility of Bénédicte SOHM (research engineer). The organic syntheses of the reference phthalate analogues will be carried out at the Laboratoire de Chémo-Biologie Synthétique et Thérapeutique (CBST, UMR 7199, University of Strasbourg) under the supervision of Alexandre SPECHT (CNRS research director). The bi-disciplinary nature of the project, combining organic synthesis, chemical and biological characterization of compounds and biological experiments, will require travel between LIEC and CBST. Travel will be adjusted according to the skills of the successful candidate, but most of the experimentation will take place at the Metz site (biological experiments). Embryo-larval exposures (D. rerio) may require occasional on-call duty at weekends. Travel to congresses is to be expected. The doctoral student will be attached to the SIRENA doctoral school (http://doctorat.univ-lorraine.fr/fr/les-ecoles-doctorales/sirena/presentation), which will require the completion of several training modules leading to the validation of credits.
Information on the two laboratories is available via the links below:
https://liec.univ-lorraine.fr/.
https://cbst.unistra.fr/

Given the nature of the project, there will be close interaction between the PhD student and the heads of LIEC's “Environmental Biology” and “Environmental Analytical Chemistry” divisions. The PhD student will be attached to the “Environmental Toxicology” (TEv) team. Occasional travel will be required between Metz and Nancy to apply analytical methods (uHPLC). The position also involves experiments in connection with analog synthesis at the CBST laboratory in Strasbourg.