title: Fish peripheral blood mononuclear cells preparation for future monitoring applications.
abstract: Fish species possess many specific characteristics that support their use in ecotoxicology. Widely used in clinical research, peripheral blood mononuclear cells (PBMCs) can reasonably be exploited as relevant target cells in the assessment of environmental chemical toxicity. The current article focuses on the methods necessary to isolate, characterize, and culture fish PBMCs. These procedures were successfully applied on an endangered species, the European eel (Anguilla anguilla L.), and on an economically important and worldwide exported species, the Asian catfish (Pangasianodon hypophthalmus S.). Proteomic approaches can be useful to screen xenobiotic exposure at the protein expression level, giving the opportunity to develop early warning signals thanks to molecular signatures of toxicity. To date, a major limitation of proteomic analyses is that most protein expression profiles often reveal the same predominant and frequently differentially expressed families of proteins regardless of the experimental stressing conditions. The current study describes a methodology to get a postnuclear fraction of high quality isolated from fish PBMCs in order to perform subsequent subproteomic analyses. Applied on samples from eel, the subproteomic analysis (two-dimensional differential in-gel electrophoresis) allowed the identification by liquid chromatography-tandem mass spectrometry and searches in the full NCBInr (National Center for Biotechnology Information nonredundant) database of 66 proteins representing 36 different proteins validated through Peptide and Protein Prophet of Scaffold software.
authors.affiliation: Research Unit in Environmental and Evolutionary Biology (URBE-NARILIS), University of Namur (FUNDP), B-5000 Namur, Belgium.
authors.forename: Marie-Aline
authors.initials: MA
authors.lastname: Pierrard
authors.forename: Kathleen
authors.initials: K
authors.lastname: Roland
authors.forename: Patrick
authors.initials: P
authors.lastname: Kestemont
authors.forename: Marc
authors.initials: M
authors.lastname: Dieu
authors.forename: Martine
authors.initials: M
authors.lastname: Raes
authors.forename: Frédéric
authors.initials: F
authors.lastname: Silvestre
mesh_terms: D000818:Animals; D002397:Catfishes; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D030562:Databases, Protein; D004524:Eels; D015180:Electrophoresis, Gel, Two-Dimensional; D004784:Environmental Monitoring; D005434:Flow Cytometry; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism; D040901:Proteomics; D012984:Software; D053719:Tandem Mass Spectrometry; D015262:Xenobiotics / Q000633:toxicity
chemical_list: D020543:Proteome; D015262:Xenobiotics