title: A method for the assessment of DNA damage in individual, one day old, zebrafish embryo (Danio rerio), without prior cell isolation.
abstract: Zebrafish (Danio rerio) embryos are increasingly used as an experimental model in toxicology for the detection of lethal and sub-lethal effects of diverse chemicals. DNA damage, an early biomarker of long-term effects such as mutagenesis and carcinogenesis, is commonly assessed in vitro and in vivo using the comet assay - single cell gel electrophoresis. Here we describe a new rapid method for the detection of DNA strand breaks in individual, one day old, zebrafish embryos, without the need for prior cell isolation. After the completed spawning, the embryos were exposed to non-toxic concentrations of model genotoxic compounds for 24h. The embryos were then treated with Pronase E, embedded on microscope slides and squashed to release the cells. After alkaline electrophoresis, the nuclei were stained with ethydium bromide and analyzed by fluorescence microscopy. Preparation of slides by the described method resulted in well separated cell nuclei with low background DNA damage. A significant increase in DNA damage was detected after exposure to the model genotoxic compounds, methylmethan sulphonate (MMS) and benzo(a)pyrene (BaP), while no DNA damage was induced by NaCl. Our method proved to be sensitive and suitable for the detection of DNA damage in one day old zebrafish embryos, suggesting it could serve as a useful tool for monitoring the genotoxic potential of chemicals and environmental pollutants.
authors.affiliation: Department for Genetic Toxicology and Cancer Biology, National Institute of Biology, Večna pot 111, SI-1000 Ljubljana, Slovenia. Electronic address: tina.elersek@nib.si.
authors.forename: Tina
authors.initials: T
authors.lastname: Eleršek
authors.forename: Janja
authors.initials: J
authors.lastname: Plazar
authors.forename: Metka
authors.initials: M
authors.lastname: Filipič
mesh_terms: D000818:Animals; D001564:Benzo(a)pyrene / Q000633:toxicity; D002469:Cell Separation; D020552:Comet Assay / Q000379:methods*; D004249:DNA Damage; D004625:Embryo, Nonmammalian; D008741:Methyl Methanesulfonate / Q000633:toxicity; D009153:Mutagens / Q000633:toxicity*; D011402:Pronase / Q000494:pharmacology*; D015027:Zebrafish / Q000196:embryology*
chemical_list: D009153:Mutagens; D001564:Benzo(a)pyrene; D008741:Methyl Methanesulfonate; D011402:Pronase
keywords: Comet assay; Genotoxic monitoring; Zebrafish (Danio rerio) embryo