Devalckeneer Aude, Jenicot Jérôme, Colet Jean-Marie Use of metabolomics in the exploitation of invertebrate models used in ecotoxicology for biomedical research Devalckeneer Aude, Jenicot Jérôme, Colet Jean-Marie Faculty of Medicine and Pharmacy Human Biology and Toxicology University of Mons (UMONS) – 20, Place du parc 7000 Mons, Belgium Introduction Laboratory studies on mammals are being increasingly the subject of new legislation, it becomes important to implement alternative solutions that respond to the law of the 3Rs (Reduce, Replace, Refine). Omics approaches allowing to reduce the number of organisms as well as a refinement of experiments by using non-invasive methods provide a first solution to the 3Rs constraints. However, mammals species remains largely exploited in biomedical research and in toxicology studies in particular. Invertebrate animal models which are already used in the environmental risk assessment could be transposed to ecotoxicology in the evaluation of health risks. Indeed, many similarities exist between the detoxification systems and the involved pathways AIM The aim of our research is to combine metabonomics analysis and invertebrate animal models in view to reduce, or even discard, the use of vertebrate models in health risk assessment Anodonta cygnea and ecotoxicology Many anthropogenic pollutants now reaching aquatic environments, the use of bivalve molluscs is not uncommon in ecotoxicological studies. As benthic filter feeders, they are the first organisms affected by the pollution of water by polycyclic aromatic hydrocarbons (PAHs), both in dissolved forms (low molecular weight PAHs) and aggregates after adsorbing on the particles in suspension they eat [1]. Following laboratory exposure to phenanthrene, analysis of gill and digestive gland tissues (using CME extraction) by 1H-NMR was carried out allowing an overview of early attacks on metabolism. The measurements were performed on a NMR Avance 500 Bruker spectrometer. Metabolic changes detected in the gill tissues following exposure to phenanthrene Carnitine : Antioxydant Taurine et Bétaine : osmolytes Glucuronides : excretion of toxic substances by conjugation Groupe exposé Contrôle Figure 1 : Comparison of 1 H-NMR spectra (500 MHz) of the aqueous phase of gill tissue with an individual exposed (Toxicology Laboratory – UMONS) Lipid phase Aqueous phase Homogenization CME extraction Centrifugation Figure 2 : 1 H-NMR spectra (500 MHz) of the aqueous phase of gill tissue of control (Toxicology Laboratory – UMONS) Eisenia fetida and cell regeneration Several studies on earthworm have shown the effectiveness of the use of 1H-NMR-based metabolomics in detecting biomarkers and metabolic changes [2,3]. The earthworm is an individual able to regenerate following two successive processes : epimorphic and morphallactic regeneration in 40 days. Based on liver regeneration in rats, the metabolomics study of coelomic fluid of the earthworm is used to evaluate, temporally and in a noninvasive way, all changes after amputation of the 8th segment. Anodonta cygnea is a 20 cm freshwater bivalve of the family of Unionidae meeting the criteria to make a good bioindicator species, namely : a sedentary body, abundant and bioaccumulative. Control Stage 0 Stage 1 Stagee 2 Stage 3 Stage 4 Stage 5 Figure 3: 1 H NMR spectra (500 MHz) of coelomic fluid of worms at different stages after amputation of the 8th segment (Toxicology Laboratory – UMONS) Spermidine Maloate α-cétoglutarate Glycine ? Electrocution 9 V, 20-30 sec in 600 μl of NaCl solution 0,1 % The elevation of spermidine levels is indicative of a higher cell proliferation and makes this candidate biomarker of choice for evaluation of cell regeneration Multivariate analysis (ACP, PLS) Figure 4: Scores plot of the distribution of liquid coelomic samples in control compared to a stage 2 after amputation (Toxicology Laboratory – UMONS) Example from litterature of Helix aspersa model Analysis of pulmonary surfactant by mass spectrometry allowed the detection of protein and lipid biomarkers of pulmonary disorders. The discovery of the presence of surfactant in Helix aspersa has been demonstrated [4]. Therefore, the analysis of these components after exposure to air pollution may play a role in assessing the risk to humans The results obtained in invertebrates indicate the involvement of cellular mechanisms similar to those found in mammals, evidencing their conservation through evolution. The use of invertebrate models could therefore be applied to meet the demand of the 3Rs law by replacing vertebrate organisms. Therefore, the association of invertebrates with metabonomic techniques can be a prelude to the implementation of effects to human health. Invertebrate models for health risk? Références : [1] Casas, S. et Bacher, C., Modelling trace metal (Hg and Pb) bioaccumulation in the Mediterranean mussel, Mytilus galloprovincialis, applied to environmental monitoring. 2006. [2] Brown et al., Evaluation of sample preparation methods for nuclear magnetic resonance metabolic studies with eisenia fetida. Environnemental Toxicology and chemistry. 2008. [3] Yuk et al., Coelomic fluid : a complimentary biological medium to asses sub-lethal endosulfan exposure using 1H NMR based earthworm metabolomics. Ecotoxicology. 2012. [4] Daniels et al., Surfactant in Gas Mantle of the Snail Helix aspersa. Physiological and Biochemical Zoology. 1999.