Informace o publikaci
Comet assay - Studying toxicity and protective effects of natural compounds
| Název česky | Analýza komet - studium toxicity a protektivních účinků přírodních látek |
|---|---|
| Autoři | |
| Rok publikování | 2008 |
| Druh | Konferenční abstrakty |
| Fakulta / Pracoviště MU | |
| Citace | |
| Popis | The comet assay is a microelectrophoretic metod for evaluating DNA damage expressed by strand breaks. The method can be virtually used for all eucaryotic cells. The sample is incubated with studied agens that leads to damage of DNA structures resulting in strand breakage. Cell suspension is mixed with low melting point agarose, poured onto agarose coated microscopic slides. Cells are lysed to remove all cell structures except nuclear DNA. Remaining DNA with protein matrix is called the nucleoid. Lysed cells (nucleoids) are denatured with alkali solution so that supercoiled DNA became relaxed. Undamaged DNA is quite tightly aggregated and it is not very mobile in electric field in contrast to fragmented DNA. Slides are put into an electrophoresis chamber and negative charge of DNA molecules causes migration of relaxed and broken strands towards the anode creating a comet-like picture. Comets can be visualized by fluorescent or silver staining. Evaluation can be done by scoring (class sorting according to amount of damage) or by specially designed PC software. In our experiments we used software Lucia. As a marker of DNA damage we use relative tail intensity, normally expressed as % of DNA in tail. The comet assay method was employed to investigate effects of natural compounds on cultured human leukaemia cells (HL-60). Genotoxic effect of benzophenathridine alkaloid chelerythrine (CHEL) was observed in range of concentrations 1-10 ug/ml of growth medium. CHEL in concentration 1 ug/ml caused mild increase of damaged DNA (mean 45% of DNA in tail) whereas 10 ug/ml led to more than 95% DNA in tail. Mechanisms of the alkaloid effect may involve different signalling pathways. Toxicity of CHEL could be partially caused by induction of oxidative stress. Possible DNA integrity protection by compounds with known antioxidative properties were examined. We tested effects of flavonoid baicaline (BA) and phenolic compound caffeic acid (CA) on HL-60 cells incubated with H2O2 (100 uM) in condition with decreased metabolic activity of the cells (0 C, without nutrient solution). The protective effect of BA (100 uM) was demonstrated (40.6% damaged DNA with antioxidant vs. 50.3 % without BA). Protective effect of CA was found in experiments, where HL-60 cells in the growth medium were preincubated with antioxidant followed by 60 min incubation with CHE (3 ug/ml). DNA damage was 45.9 % with CA vs. 57.4% without it. We may conclude that genotoxic effect of CHE could be partially prevented by antioxidants. |