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Identification of biological ligands of uranium in Danio rerio gonads: Impact on their function

​Yvan Eb-Levadoux had defended his thesis on 3rd April 2017 in "Université de Pau et des Pays de l'Adour"

Document type > *Mémoire/HDR/Thesis

Keywords >

Research Unit > IRSN/PRP-ENV/SERIS/L2BT

Authors > EB-LEVADOUX Yvan

Publication Date > 03/04/2017

Summary

​Uranium (U) is naturally present at trace level (microgramme.L-1) in aquatic environment; its concentration can increase up to a few microgramme by liter due to human activity. Several ecotoxological studies have shown uranium toxicity in contamined zebrafishes Danio rerio, e.g. oxidative stress, genotoxicity and reprotoxicity (i.e. lower number of spawn and eggs laid). However, merchanisms of toxicity are not well known.


The objective of this study is to contribute to the understanding of uranium reprotoxicity by elucidating the disrupted molecular mechanisms, i.e. associated to proteins, after contamination. Therefore, investigations have been carried out on ovaries from reproduced (R) and non-reproduced (NR) zebrafishes after waterborne exposure in laboratory conditions at environmentally relevant concentrations.


This project was divided into two parts. Firstly, analytical investigations were carried out to continue the development of non-denaturing methods for U-protein identification by coupling separative techniques (size exclusion chromatography SEC, off gel electrophoresis OGE) with elemental (ICP MS) and molecular (ESI MS) sensitive mass spectrometry detection. Secondly, studies of U reprotoxicity were investigated by studying i) native U-protein complexes (metallomics approach) and ii) differential analysis of protein expression (proteomics approach).


Analytical developments allowed keeping the physiological and non-denaturing extraction buffer for OGE separation step, improving U recovery. In ecotoxicology, the major results showed that ovary is an U accumulating organ and that the reproduction status modifies the accumulation level (R<NR). However, this status is of little influence on its distribution on proteins with 4 fractions (including a major one) determined, all of them coeluting with phosphorus. The identification of U potential targets and of protein expression differences (vtg, GST, GAPDH...) showed that biological processes disrupted after contamination are at two levels: 1/ generic (oxidative stress) and 2/ more specific to gonad (oocyte development and maturation).


As a conclusion these two complementaty approaches showed a direct (complexation) and indirect (modification of protein expression) effects on U, and enabled to hypotesize a lack of oocyte maturation after contamination. This defect could impact the number of spawn and in fine explain the reprotoxicity observed in previous ecotoxicological studies.


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