engleski

Genomic and gene expression responses to genotoxic stress in zebrafish (Danio rerio)

Sublethal exposure to genotoxicants in the environment may influence genome integrity in affected organisms and potentially cause permanent alterations in the genome, thus posing the need to more adequately estimate not only DNA damage but also its longevity. Therefore, in this study we assessed primary DNA damage and existence of potentially persistent DNA alterations in the zebrafish genome using three different models (PAC2 cell line, embryos, adults) upon exposure to model genotoxic agents and the following recovery period. Genotoxicity assessed using Comet assay served as an initial screening for evaluation of general impact on DNA integrity under genotoxic stress. To test for genomic effects upon genotoxic stress quantitative RAPD and AFLP were applied. Furthermore, expression of a suite of genes involved in DNA repair, oxidative stress response and xenobiotic metabolism was monitored in order to get better overview of zebrafish genome responses to genotoxic stressors. Additionally, AFLP method was applied on adult specimens one year after their exposure to genotoxicants at larval stage in order to further evaluate longevity of observed DNA alterations. Comet assay detected DNA damage in all three zebrafish models, which in some cases persisted upon recovery period as well. DNA alterations assessed using qRAPD and AFLP were observed even in cases when Comet assay indicated lack of significant damage proving their sensitivity for detection of various DNA alterations that remain undisclosed by the Comet assay, such as DNA adducts, mutations, structural DNA changes and changes in DNA methylation pattern. Zebrafish larvae proved to be the most sensitive zebrafish model for revealing genotoxic effects, which was evidenced by the high incidence of DNA alterations and the lack of significant DNA repair at the early stages of larval development. Some of the genomic alterations observed in larvae were still detectable in the adulthood, indicating the formation of persistent genomic modifications. These results thus underline zebrafish larvae as particularly sensitive model in such surveys and encourage the simultaneous use of various models and methods for more accurate evaluation of the consequences of the genotoxic insult.

Comet assay; qRAPD; AFLP

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