ZEBRAFISH GENOME STABILITY AFTER EXPOSURE TO MODEL GENOTOXICANTS
Maja Šruta, Anamaria Štambuka, Jean-Paul Bourdineaudb, Göran I.V. Klobučara
a
Department of Zoology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, 10000 Zagreb,
Croatia
b
Arcachon Marine Station, CNRS, UMR EPOC 5805, University of Bordeaux, Place du Docteur
Peyneau, 33120 Arcachon, France
Sublethal exposure to a variety of genotoxic agents in the environment may directly or indirectly
influence genome integrity of exposed organisms and for that reason various methods for detection of
DNA damage were developed. In this study we have tested and compared three different methods for
detection of DNA damage and alterations using in vivo and in vitro zebrafish models. The exposure of
embryonic cell line (PAC-2) and zebrafish embryos to genotoxic agents (benzo[a]pyrene (B[a]P) and
ethyl methanesulfonate (EMS)) was carried out for 6 days followed by 6 days of recovery period.
Genome stability of tested models was assessed using Comet assay, quantitative RAPD (random
amplified polymorphic DNA) and AFLP (amplified fragment length polymorphism). Comet assay
revealed significant increase of DNA damage in zebrafish embryos following both toxicant treatments
and this damage remained unrepaired after the recovery period. However, in PAC-2 cell line
significant increase of DNA damage was observed only upon EMS treatment. RAPD showed
significant increase in number of hybridization sites in DNA of zebrafish embryos and decrease in DNA
of PAC-2 cell line upon exposure to lower B[a]P concentration, while AFLP revealed changes in
profiles, such as loss or appearance of fragments as well as changes in fragment amplification
intensity, following both toxicants treatment. Our results indicate that RAPD and AFLP are sensitive
methods for detection of DNA alterations since they showed changes in the genome even when
Comet assay indicated lack of significant damage. This could be due to the presence of more
persistent DNA changes such as formation of DNA adducts, mutations or conformational and
structural changes in the DNA, all undetected in the Comet assay. These results thus encourage the
simultaneous use of various methods and models for revealing the consequences of genotoxic insult
and suggest that quantitative RAPD and AFLP could be useful biomarkers in such surveys.