Procena osetljivosti in vitro i in vivo testova u ekogenotoksikologiji i formulisanje smernica za njihovu primenu u istraživanjima na velikim ravničarskim rekama

Abstract

metoda uslovljenim efektima (eng. effect based methods), uključujući i genotoksikološke metode, u programe monitoringa i naučna istraživanja na velikim ravničarskim rekama, omogućilo bi sveobuhvatniju procenu stanja ekosistema i odnosa između samog hemijskog i ekološkog statusa. Osnovni ciljevi disertacije su ispitivanje osetljivosti in vitro i in vivo testova u ekogenotoksikologiji i formulisanje smernica za njihovu primenu u istraživanjima velikih ravničarskih reka. Studija je obuhvatila ukupno 17 lokaliteta u slivovima Velike Morave, Save i Dunava. Za procenu genotoksičnog efekta nativnih i koncentrisanih uzoraka rečne vode, in vitro pristup obuhvatio je SOS/umuC test, alkalni komet test na HepG2 i ZFL ćelijama, kao i analizu ćelijskog ciklusa na ZFL ćelijama. In vivo pristup je za procenu genotoksičnosti obuhvatio alkalni, Fpg modifikovani komet test, kao i mikronukleus test na ćelijama krvi Alburnus alburnus (uklija). Dodatno, ispitivana je osetljivost RAPD analize u detekciji genotoksičnog efekta. U in silico pristupu na osnovu literature analiziran je toksični/genotoksični potencijal hemijskih parametara koji su detektovani u vodi i u uklijama, a integrativnim metodama vršena je kvantifikacija pritiska zagađenja. Biotestovi su pokazali različitu osetljivost u detekciji genotoksičnog efekta. U in vitro istraživanju koncentrisani uzorci rečne vode su se pokazali pogodnim za testiranje toksičnosti/genotoksičnosti, a ZFL ćelije kao dobar model sistem. U in vivo istraživanju A. alburnus se pokazao kao pouzdan bioindikator. Najveća osetljivost u detekciji genotoksičnog efekta u ćelijama krvi dobijena je u alkalnom komet testu. In silico analizom ne može se izvršiti potpuna predikcija genotoksičnog efekta ksenobiotika na organizme.

The implementation of effect based methods, including genotoxicological methods, in monitoring and research programs on large lowland rivers, could provide comprehensive assessment of ecosystem quality and relationship between chemical and ecological status itself. The main objectives of the dissertation are examination the sensitivity of in vitro and in vivo tests in ecogenotoxicology and guidelines formulation for their application in studies of large lowland rivers. The study included a total of 17 sites in the Velika Morava, Sava and Danube River basins. To assess the genotoxic effect of native and concentrated river water samples, the in vitro approach included the SOS/umuC test, alkaline comet test on HepG2 and ZFL cells, and cell cycle analysis on ZFL cells. The in vivo approach for genotoxicity assessment included the alkaline, Fpg modified comet and micronucleus test on Alburnus alburnus (bleak) blood cells. Also, the sensitivity of RAPD analysis in the detection of genotoxic effects was examined. Within in silico approach, based on the literature, the toxic/genotoxic potential of chemical parameters detected in water and bleaks was analyzed while the quantification of pollution pressure was performed by integrative methods. Bioassays showed different sensitivity in detecting the genotoxic effect. Within in vitro study, concentrated river water samples proved to be a suitable for toxicity/genotoxicity testing, and ZFL cells a good model system. Within in vivo research, bleak proved to be a reliable bioindicator. The highest sensitivity in the detection of genotoxic effect in blood cells was obtained in the alkaline comet test. A complete prediction of the genotoxic effect of xenobiotics on organisms cannot be performed by in silico analysis.