First assessment of DNA damage in Mediterranean pelagic species loggerhead turtle and swordfish: Application of comet assay and diffusion assay in frozen total blood

The aim of this study was to apply and validate the comet assay and diffusion assay on frozen total blood to assess the DNA damage in swordfish (Xiphias gladius) and loggerhead turtle (Caretta caretta). Until now, a limited number of studies were published utilizing these assays with marine species in comparison to those using fresh water species. This may be due to logistical problems associated with performing the techniques at sea. Before applying the genotoxicity biomarkers in swordfish and loggerhead turtle in order to develop the frozen conservation methodology, we performed the tests in parallel on fresh and cryopreserved blood of Dicentrarchus labrax. The total blood sample collectedwas mixedwith RPMI 1640 and DMSO and immediately stored in −80 °C. We observed no statistically significant differences in the level of DNA damage between fresh and frozen blood samples. The protocol was then applied to blood samples of the two species investigated. In sea turtles, we applied the technique on fresh whole blood too and the results showed DNA fragmentation values in linewith those of the frozen blood. Results were compared with those of ENA assay, a genotoxicity biomarker validated for the first time in these species. This study strongly suggests that comet and diffusion assays in frozen whole blood can be utilized, in combination with ENA assay, to assess genotoxic damage in top predators or long living species exposed to potential toxicological risk.