Application of induction factor in comparison of the results obtained in comet assay under different conditions of electrophoresis

Komet-test je osjetljiva, brza i ekonomična metoda koja detektira oštećenja DNA u pojedinačnim stanicama. Zbog svoje jednostavne provedbe, često je korišten pri utvrđivanju i procjeni genotoksičnog djelovanja. Različiti uvjeti i načini provedbe komet-testa, otežavaju uspoređivanje dobivenih podataka i rezultata. Stoga je namjera ovog rada istražiti primjenu normiranja, koja bi omogućila usporedbu rezultata dobivenih komet-testom izvođenog u različitim uvjetima elektroforeze. U ovom radu korištene su dvije stanične linije – PLHC-1 i ZFL te dva modelna genotoksikanta - etil-metanosulfonat (EMS) i vodikov peroksid (H2O2). Komet-test je za sve navedene varijable bio izvođen u četiri različita uvjeta elektroforeze, ovisno o jačini (25-35 V) i trajanju (15-20 min). Promatran je genotoksični učinak, koji je prikazan kao postotak DNA koja je tijekom elektroforeze migrirala u rep (% tDNA) za obje stanične linije tretirane s EMS-om i vodikovim peroksidom. Indukcijski faktor (IF) je prikazan kao razlika u % tDNA između kontrole i tretiranih grupa. Comet assay is a sensitive, quick and inexpensive method that detects DNA damage in single cells. Because of this, it is often used in the assessment of genotoxic effects. Different conditions and ways of implementation of Comet assay have made it difficult to compare given data and results. Thus, the aim of this research is to investigate the standardization that could enable comparison of results given by Comet assay performed in different electrophoretic conditions. In this study, two different cell lines were used – PLHC-1 fish hepatoma cell line and ZFL cell line, as well as two genotoxic agens - ethyl methanesulfonate (EMS) and hydrogen peroxide (H2O2). Comet assay was performed for all given variables and in 4 different conditions of electrophoresis, depending on intensity (25-35 V) and duration (15-20 min). Observed genotoxic effect was demonstrated as percentage of DNA that migrated in the tail during the electrophoresis (% tDNA) for both cell lines treated with EMS and H2O2. Induction factor (IF) was represented as difference in % tDNA between the control and treated groups.