title: Proteome profiling of mouse embryonic stem cells to define markers for cell differentiation and embryotoxicity.
abstract: In search for alternative methods for developmental toxicity testing, we investigated whether embryonic stem cell (ESC) differentiation and its modulation by toxic exposure could be monitored by proteome profiling. We compared the proteomes of mouse ESC, differentiating ESC (DIF) and differentiating ESC exposed to the embryotoxicant monobutyl phthalate (MBP). Experiments were performed in duplicates for each cell culture and the proteins extracted from the cells were separated by one-dimensional SDS-PAGE. The identified proteins were quantified using a label-free quantitative method based on counting the observed peptides as an index of protein abundance. Fifty-seven proteins were upregulated in DIF relative to ESC, whereas 42 proteins were downregulated. Most of the upregulated proteins could be correlated with cardiomyocyte functionality. In contrast, the downregulated proteins were principally pluripotency markers, chaperones and ribosomal proteins. Higher expression levels of enzymes involved in DNA mismatch repair (MSH6) and in methylation reactions (MAT2A, AHCY) were also detected in the ESC, suggesting that these processes are more active in the ESC. In addition, the detection of gluthatione S-transferase alpha 4 (GSTA4) and Park7 DJ-1 protein (antioxidant) in ESC indicates that these cells have potential detoxification mechanisms. Furthermore, MBP affected the expression of 33 proteins, including MYH6, a cardiomyocyte-specific protein, which was significantly downregulated. MBP also affected the expression levels of chaperones, metabolic enzymes and chromatin modulating proteins, suggesting that MBP alters the differentiation process. Western blot analysis of MYH6 and HSPB1 confirmed the proteomic data. In addition, a favorable correlation was observed between protein expression and published changes in the expression of related genes at the transcriptomics level. Together, the results reveal potential protein markers that may be used as endpoints in an ESC based animal free alternative test for embryotoxicity though further studies are required for confirmation.
authors.affiliation: Laboratory for Health Protection Research, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands. Ahmed.Osman@rivm.nl
authors.forename: Ahmed M
authors.initials: AM
authors.lastname: Osman
authors.forename: Dorien A M
authors.initials: DA
authors.lastname: van Dartel
authors.forename: Edwin
authors.initials: E
authors.lastname: Zwart
authors.forename: Marco
authors.initials: M
authors.lastname: Blokland
authors.forename: Jeroen L A
authors.initials: JL
authors.lastname: Pennings
authors.forename: Aldert H
authors.initials: AH
authors.lastname: Piersma
mesh_terms: D000826:Animal Testing Alternatives; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D024722:Cardiac Myosins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects* / Q000235:genetics; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004622:Embryo, Mammalian / Q000187:drug effects* / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018832:Molecular Chaperones; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D010795:Phthalic Acids / Q000633:toxicity*; D010968:Plasticizers / Q000633:toxicity*; D040901:Proteomics
chemical_list: D015415:Biomarkers; D006360:Heat-Shock Proteins; C486113:Hsbp1 protein, mouse; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D010795:Phthalic Acids; D010968:Plasticizers; D024722:Cardiac Myosins; C028577:monobutyl phthalate