title: Hazard assessment of methylmercury toxicity to neuronal induction in embryogenesis using human embryonic stem cells.
abstract: Pluripotent human embryonic stem cell (hESC) lines can to some extent mimic in vitro the development of the embryo, providing the scientific rationale for the use of these cells to establish tests for toxicity to embryogenesis. Such humanised in vitro tests have potential to improve human hazard prediction by avoiding interspecies differences. We explored the potential of a hESC-based assay for detection of toxicity to neuronal induction in embryonic development. Neuronal precursor differentiation was performed according to a previous publication, while we established a new protocol for maturation of the precursors into neuron-like cells. Appearance of neuronal derivatives was demonstrated by real-time PCR, showing up-regulation of several neuronal marker genes, and immunohistochemistry, demonstrating the appearance of neurofilament medium polypeptide, beta-tubulin III and microtubule-associated protein 2 positive cells. In order to assess whether the hESC model could detect chemically induced developmental toxicity, we exposed the differentiating cells to methylmercury (MeHg) causing structural developmental abnormalities in the brain. Two separate exposure intervals were used to determine the effects of MeHg on neuronal precursor formation and their further maturation, respectively. The formation of precursors was sensitive to MeHg in non-cytotoxic concentrations, as the expression of several neuronal mRNA markers changed. In contrast, non-cytotoxic MeHg concentrations did not effect the mRNA marker expression in matured cells, indicating that neuronal precursor formation is more sensitive to MeHg than later stages of neuronal differentiation. Overall, our experiments demonstrate that the hESC assay can provide alerts for the adverse effects of MeHg on neuronal induction.
authors.affiliation: European Centre for the Validation of Alternative Methods (ECVAM) T.P. 580, IHCP, JRC, European Commission, via Fermi 2749, 21027 Ispra (VA), Italy. stummann@hotmail.com
authors.forename: Tina C
authors.initials: TC
authors.lastname: Stummann
authors.forename: Lars
authors.initials: L
authors.lastname: Hareng
authors.forename: Susanne
authors.initials: S
authors.lastname: Bremer
mesh_terms: D015415:Biomarkers; D002454:Cell Differentiation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003627:Data Interpretation, Statistical; D047108:Embryonic Development / Q000187:drug effects*; D053595:Embryonic Stem Cells / Q000187:drug effects*; D006801:Humans; D007150:Immunohistochemistry; D020262:Mercury Poisoning, Nervous System / Q000378:metabolism / Q000473:pathology*; D008767:Methylmercury Compounds / Q000633:toxicity*; D009474:Neurons / Q000187:drug effects*; D039904:Pluripotent Stem Cells / Q000187:drug effects*; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018570:Risk Assessment
chemical_list: D015415:Biomarkers; D008767:Methylmercury Compounds; D012313:RNA