Innovative mass spectrometry approaches in Lipidomics and Proteomics applied in this project will provide a better understanding of the toxicity mechanisms of lipid-lowering molecules on the energy metabolism in G. fossarum.
Aquatic ecosystems are constantly exposed to environmental stressors such as chemical micropollutants from the environment or human activities. These contaminants can alter the internal chemical homeostasis of aquatic organisms, visible at the molecular level. At present, the lipid disturbances induced by drug residues, which can be found in river waters, on aquatic species such as G. fossarum are not known. A major bottleneck is the lack of molecular information at the genome level in some species of environmental relevance. Multi-Omics (Proteomics, Lipidomics) approaches are used to characterize molecular responses to toxic exposures in aquatic organisms during vulnerable life cycle stages. In crustaceans, lipids play essential roles in vulnerable stages such as molting, reproduction and development. <br />The objective of the PLAN-TOX project is to gain the most comprehensive mechanistic understanding as possible of the toxic effects of lipid-lowering drugs in the sentinel organism G. fossarum through innovative Multi-Omics approaches by targeted mass spectrometry and multimodal imaging. This project will provide a better understanding of the molecular plasticity of the energy metabolism and identification of the proteins and lipids involved in exposure-related physiological changes and responses.
To access the molecular characterization of lipids and proteins in G. fossarum, methods employing high-resolution mass spectrometry (Shotgun Lipidomics) and innovative multiplexed approaches in targeted mass spectrometry have been developed and applied in adult organisms (male, female), embryos and in different organs (cephalon, muscle, hepatopancreas). The use of multimodal mass spectrometry imaging (MALDI with ion mobility, ToF-SIMS) enable to localize in situ the spatial distribution of lipids in tissue sections of adult gammarids before and after exposure to pravastatin.
The use of multivariate statistical tools will allow integrated analysis of lipidomics and proteomics data to better understand metabolic disturbances after exposure.
The first significant result is the comprehensive characterization of the lipidome by high-resolution mass spectrometry in the sentinel species G. fossarum. These results have enabled the development of a proof of concept in targeted mass spectrometry for the monitoring of a large lipid and protein multiplex assay (Scout-MRM approach).
This project also permit to characterize the spatial distribution of metabolites and lipids in situ at the organ level in G. fossarum by multimodal imaging (MALDI imaging with ion mobility and ToF-SIMS). These approaches allowed rapid observation of lipid disturbances in female reproductive organs following exposure to lipid-lowering molecules during reproductive cycles.
The analytical approaches employed (multi-modal imaging, shotgun lipidomics, Scout-MRM) were used for the first time in ecotoxicology.
The analytical approaches used (multi-modal imaging, shotgun lipidomics, Scout-MRM) in this project will gather molecular information on other sentinel species in the environment and in different contexts of exposure to contaminants (drugs, cadmium pollution, etc.).
A scientific paper dealing with MALDI imaging with ion mobility applied in G. fossarum samples was published in Journal of Mass Spectrometry (DOI: 10.1002/jms.4531). A proof-of-concept paper on the creation of a large protein multiplex by Scout-MRM is under review in Journal of Proteomics. These articles provide new insights into lipid metabolism and the molecular approaches applied for this sentinel species. The results obtained have also been presented during scientific events (ASMS, SETAC, GDR Aquatic Ecotoxicology, Bruker Webinar).
Aquatic ecosystems are persistently exposed to environmental stressors such as chemical micropollutants from natural environment or anthropogenic activities. These chemical contaminations may result in alterations of the internal biochemical homeostasis of the aquatic organisms, noticeable at the omics scale. The major limitation in the mechanistic knowledge of environmental chemical toxicity effects on aquatic organisms is the absence of molecular information notably at the genome wide scale in environmentally relevant species. The “omics” technologies – Transcriptomics, Proteomics, Lipidomics, Metabolomics to name a few – offer great promises to help to elucidate molecular responses to exposures in aquatic organisms during specific and vulnerable life cycle stages. Lipid metabolism is the major fundamental metabolic pathway producing energy in animals. In crustacean, lipids play a pivotal role in vulnerable stages like molting, reproduction, development. Recently, it has been shown that chemical compounds interfering with lipid metabolism, recognized as obesogens like tributyltin, mislead the distribution and the synthesis of lipids in the non-target and model organism Daphnia magna. Moreover, pharmaceutical drugs used for hypercholesterolemia to lower cholesterol and triglycerides concentrations like pravastatin or bezafibrate have been detected in sewages. Another study showed that simvastatin exposure in the amphipod Gammarus locusta disturbed the reproduction and population growth at the ng/L range. To understand and predict the effects of toxic exposures, it is crucial to identify the affected metabolic networks. Statins are among the most broadly used pharmaceuticals worldwide and is therefore of emerging environmental concerns. This medication inhibits selectively the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) involved in the synthesis of mevalonate, a precursor of sterols including cholesterol. We hypothesize that lipid metabolism may turn out to be the drug-induced toxicity target in aquatic species. However, we are facing a lack of crucial knowledge about the relationships between the abundance of lipid species, the activation/inhibition of signaling and/or biosynthesis pathways and the observed phenotypes. The project PLAN-TOX aim to gain a mechanistic understanding of toxic effects of hypolidiaemia drugs (statins and fibrates) on the sentinel organism G. fossarum.
We are proposing to develop and apply an innovative multi-omics approach in ecotoxicology, including (i) the functional proteome and lipidome mapping in the sentinel organism G. fossarum before and after exposure, (ii) the development of methods for the integrative analysis of multi-omics data. G. fossarum is a widely developed sentinel species in ecotoxicology or environmental monitoring and is one of the ecologically relevant species for which we can evaluate the toxicity on different development stages and reproductive cycles. This project will allow to better understand molecular plasticity of energetic metabolism and to identify proteins and lipids involved in physiological changes/responses related to exposure.
Madame Sophie Ayciriex (Institut des Sciences Analytiques)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
ISA Institut des Sciences Analytiques
Help of the ANR 190,437 euros
Beginning and duration of the scientific project: December 2018 - 36 Months