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Stoimir Kolarevic defended his PhD thesis within the CytoThreat Project

Our team member Stoimir Kolarevic successfully defended his PhD thesis on the 25th of December 2014, based on the research work he carried out within the CytoThreat Project. Below is the abstract of his thesis. The entire CytoThreat team congratulates him on his accomplishment!



Application of the comet assay on freshwater mussels Unio pictorum (Lianneus, 1758), Unio tumidus (Philipsson, 1788) and Sinanodonta woodiana (Lea, 1834) in ecogenotoxicological assessment of aquatic ecosystems


Mussels have several characteristics, such as wide distribution, filter feeding, a sessile life form and an ability to accumulate pollutants, which makes them favorable organisms for estimating the environmental pollution level and the bioavailability of various types of pollutants and therefore, mussels are valuable for ecogenotoxicological studies. The comet assay or the single cell gel electrophoresis (SCGE) represent sensitive and rapid technique for detection of DNA damage and therefore this method is commonly employed for detection of genotoxic pollution in aquatic ecosystems.

To evaluate the sensitivity of freshwater mussels as bioindicators in ecogenotoxicological studies, the level of DNA damage, assessed by alcaline comet assay, was studied in haemocytes of freshwater mussels of U. pictorum and U. tumidus (autochtonous species) and Sinanodonta woodiana (alochtonous species) in in situ and ex situ experimental conditions.

For in situ studies, active and passive biomonitoring were employed for the assessment of the impact of environmental stress on the level of DNA damage. Water quality assessment on studied sites was based on physical, chemical and microbiological parameters. Passive biomonitoring was performed on specimens of S. woodiana collected from the Velika Morava River, and on specimens of genus Unio collected from the Danube River. Active biomonitoring was performed on the specimens of the same genus on the Sava and Danube rivers in the urban teritory of the Belgrade city.

Ex situ research implied studing of the impact of exposure to cytostatics: 5-fluorouracil, cisplatin, etoposide, vincristine sulphate and imatinib mesylate, in vivo by treatment of the whole animals for 72, and in vitro by treatment of freshly isolated haemocyes for 30 min or by treatment of haemocytes in primary cultures (22 h). Treatment with cadmium chloride (model mutagen) resulted in significant increase of DNA damage in all treatments.

The results of in situ studies indicated that specimens of U. pictorum, U. tumidus and S. woodiana give response to presence of genotoxic pollution which can be measured and quantified by comet assay. The level of DNA damage in haemocytes showed significant correlation with concentration of genotoxic pollutants measured in water and with microbioloical indicators of faecal pollution.

For in vivo treatment, impact of imatinib mesylate was not detected while for 5-fluorouracil, cisplatin, etoposide and vinkristine sulphat the lowest observable effective concentrations (LOECs) were few orders of magnitude of their concentrations in surface waters. The LOECs for 5-fluorouracil and cisplatin were in range of concentrations in which these cytostatics can be found in hospital wastewaters. The results obtained for threatments in vitro indicated that exposure of freshly isolated haemocytes is to short for deetection of the studied effects. Treatment in primary culture of haemocytes can be proposed for detection of genotoxic potential in vitro with few modifications, such as stimulation of haemocytes on proliferation.

Research field: Biology

Specific research field: Genotoxicology, Microbiology

UDC No.: [574.64:594.141][57.088:577.113](043.3)


Key words: ecogenotoxicology, aquatic ecosystems, microbial and chemical pollution, cytostatics, Unio pictorum, U. tumidus, Sinanodonta woodiana, DNA damage risk assessment

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