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Detection of DNA damage in yolk-sac larvae of the Japanese Medaka, Oryzias latipes, by the comet assay

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Abstract

This study was set up to determine the suitability of the early life stage (ELS) alkaline comet assay for the detection of DNA strand breaks induced by genotoxicants in whole organism. This assay was performed on cells of medaka 2 days posthatch (dph). An efficient procedure for cell dissociation using enzymatic and mechanical digestion was developed. This protocol ensures 80% viability of cells and low DNA damage background. Cells from 2 dph medaka larvae were exposed in vitro to model genotoxicants, hydrogen peroxide, cadmium, and fluoranthene, followed by comet assay analysis. Results show a significant increase in the percentage of DNA damage of dissociated cells by all the tested compounds when compared to controls. The assay was also performed in vivo on medaka larvae (2 dph) exposed for 24 h to waterborne cadmium or fluoranthene. Significant induction of DNA damage levels were observed following larvae exposure to cadmium and fluoranthene at concentrations of 0.1 and 50 μM, respectively. This study demonstrates that cells of embryo life stage medaka respond to known DNA damaging agents and that the ELS comet assay may be a useful biomarker to detect DNA strand breakage in whole body of pluricellular organism induced by a range of agents. This technique may provide a sensitive, nonspecific endpoint of genotoxicity as part of ELS toxicity test.

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Acknowledgments

This study was supported by the GIP Seine-Aval, Haute-Normandie, and Aquitaine regions.

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Authors and Affiliations

  1. Institute of Molecular Sciences (ISM), University Bordeaux 1, UMR 5255 CNRS, Group LPTC, 33405, Talence Cedex, France

    Bénédicte Morin, Julien Filatreau, Ludovic Vicquelin, Iris Barjhoux, Sylvain Guinel & Jérôme Cachot

  2. Laboratory of Ecotoxicology (LEMA), University of Le Havre, UPRES EA 3222, IFRMP 23, 76058, Le Havre Cedex, France

    Ludovic Vicquelin & Joelle Leray-Forget

Authors
  1. Bénédicte Morin
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  2. Julien Filatreau
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Correspondence to Bénédicte Morin.

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Morin, B., Filatreau, J., Vicquelin, L. et al. Detection of DNA damage in yolk-sac larvae of the Japanese Medaka, Oryzias latipes, by the comet assay. Anal Bioanal Chem 399, 2235–2242 (2011). https://doi.org/10.1007/s00216-010-4602-y

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  • DOI: https://doi.org/10.1007/s00216-010-4602-y

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