| Author/Editor | Žegura, Bojana; Lah, Tamara T; Filipič, Metka | |
| Title | The role of reactive oxygen species in microcystin-LR-induced DNA damage | |
| Type | članek | |
| Source | Toxicology | |
| Vol. and No. | Letnik 200, št. 1 | |
| Publication year | 2004 | |
| Volume | str. 59-68 | |
| Language | eng | |
| Abstract | Microcystins are cyclic heptapeptides produced by different freshwater cyanobacterial speeies such as Microcystis aeruginosa. l hey have been shown to induce DNA damage in vitro and in vivo, however, the meehanisms of their genotoxic activity remain unclear. With the comet assay we demonstrate that, in human hepatoma HepG2 cells, microcystin-LR (MCLR) induced DNA strand breaks which were transiently present and probably produced during the cellular repair of MCLR-induced DNA damage. Digestion of DNA from MCLR-treated HepG2 cells with purified formamidopyrimidine-DNA glycosylase (Fpg), which recognizes specific oxidized purines, displayed a greater extent of DNA strand breaks than non-digested DNA, providing evidence that MCLR induced oxidation ofpurines. The number of DNA strand breaks detected after digestion with Fpg increased with time of exposure of the cells to MCLR, indicating that oxidized purines were not repaired. Using the 2',7'-dichlorofluorescin diacetate (DCFH-DA) fiuoroprobe we showed that MCLR, at non-cytotoxic concentrations, induced a time and dose dependent increase of intracellular reactive oxygen species (ROS) formation in HepG2 cells. The role of ROS in MCLR-induced DNA damage was further confirmed by exposing the ce11s to MCLR in the presence of differentROS scavengers. The formation of DNA strand breaks and oxidized purines was completely prevented by a superoxide dismutase mimic, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL), an iron chelator, deferoxamine (DFO), a precursor of glutathione (GSH) and intracellular ROS scavenger, N-acetyl-L-cysteine (NAC), and partly by hydroxyl radical scavengers dimethylsulphoxide (DMSO) and 1,3-dimetlryl-2-thiourea (DMTU). The results provide evidence that the genotoxicity of MCI,R is mediated by ROS. | |
| Descriptors | REACTIVE OXYGEN SPECIES DNA DAMAGE ENZYME INHIBITORS PEPTIDES, CYCLIC CELLS, CULTURED FREE RADICAL SCAVENGERS CYCLIC N-OXIDES |