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M . `r u t 1 , A . `t a m b u k 1 , J . - P . B o u r d i n e a u d 2 , G . I . V . K l o b u
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1 D e p a r t m e n t o f Z o o l o g y , F a c u l t y o f S c i e n c e , U n i v e r s i t y o f Z a g r e b , R o o s e v e l t o v t r g 6 , 1 0 0 0 0 Z a g r e b , C r o a t i a ( m s r ut@biol.pmf.hr; astambuk@biol.pmf.hr; gklobuca@biol.pmf.hr)
2Arcachon Marine Station, CNRS, UMR EPOC 5805, University of Bordeaux, Place du Docteur Peyneau, 33120 Arcachon, France (jean-paul.bourdineaud@u-bordeaux1.fr)
DNA damage detected by methods such as Comet assay is not always a reliable indicator of the influence of genotoxic agents on genome integrity of exposed organisms. Therefore, in order to evaluate DNA damage, its reparability and persistence that may lead to alterations of DNA structure upon the genotoxic stress, in vivo and in vitro zebrafish (Danio rerio) models were exposed to benzo[a]pyrene (B[a]P) and ethyl methanesulfonate (EMS) for 6 days followed by 6 days recovery period. DNA damage in zebrafish embryonic cell line (PAC2) and zebrafish larvae was assessed using Comet assay. Furthermore, quantitative RAPD (random amplified polymorphic DNA) and AFLP (amplified fragment length polymorphism) were applied to measure more permanent DNA alterations. While Comet assay revealed significant increase of DNA damage in zebrafish larvae following both toxicant treatments and in PAC2 cell line only upon EMS treatment, RAPD and AFLP revealed alterations even when Comet assay indicated lack of significant damage. Such alterations were evident as changes in number of RAPD hybridization sites and as loss or appearance of fragments as well as changes in fragment amplification intensity in AFLP. Results of this study indicated that RAPD and AFLP are sensitive methods for detection of DNA alterations and thus e n c o u r a g e t h e i r u s e i n b i o m o n i t o r i n g p r o g r a m m e s a s w e l l a s t h e s i m u l t a n e o u s u s e o f v a r i o u s m e t h o d s a n d m o d e l s f o r r e v e a l i n g t h e c o n s e q u e n c e s o f g e n o t o x i c i n s u l t .
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