Development of an Untargeted Metabolomics Approach Based on LC-(ESI)-HRMS, Applied to Three-Spined Sticklebacks Exposed to Diclofenac
Résumé
Introduction The aquatic systems are the ultimate repository of a large part of the compounds due to human activities. In response to these chemical stresses which aquatic environment may suffer, most studies have focused on the use of biomarkers as early responses induced by these pressures. In recent years, several studies showed that metabolomics can be used to compare and determine the causal link between exposure and effects for predicting toxic effects of contaminants in aquatic organisms. Especially, untargeted approaches based on high resolution mass spectrometry has gained more and more popularity due to its benefit of analysing the whole metabolome in a global view. This approach is a promising tool to provide biomarkers of effect and information on exposure ways in organisms in view of aquatic biomonitoring and environmental risk assessment. The aims of this study were : - To develop an exhaustive untargeted approach able to cover a huge range of metabolites within three-spined sticklebacks (Gasterosteus aculeatus) - To identify impacted metabolites after a sub-chronic exposure of this species to diclofenac. Materials and methods A solid liquid extraction followed by liquid chromatography (RSLC U3000 from Thermo Scientific®) coupled to QtoF (Maxis Plus from Bruker Daltonics®) were used. The data were processed using Metaboscape 4.0 (Bruker Daltonics®). The extraction process was performed on 25 or 50 mg of whole fish, liver, gills or brain with 2.5 mL of MeOH/H2O/heptane. Two complementary analytical methods were then combined : a reverse phase separation on HSS T3 C18 column for positive ionization mode, and an HILIC separation on Nucleodur column for negative ionization mode. This development was done to identify finer and then more relevant biomarkers by a comparative strategy between control and exposed individuals to diclofenac. Results and discussion Two LC-HRMS methods have been developed based on the responses of analytical standards representative of different metabolite groups (amino acids, sugars...). The extraction protocol has been optimized in all matrices, taking into account the number of features (m/z, tr) observed in LC-HRMS. The study shows the influence of the nature of the solvents during the extraction stage as well as the choice of chromatographic conditions. These methods were applied to adult fish exposed to diclofenac, a non-steroidal anti-inflammatory drug ubiquitous in the environment. Individuals were exposed for 21 days at 1 and 100 µg/L. The results of the untargeted metabolomic approach will be presented during the speech. Conclusions This study provides a complete reliable analytical method for the characterization of metabolites in three-spined sticklebacks. Thus, it enables to identify biomarkers through a comparative strategy, for biomonitoring of water bodies quality.