Toxicology 200 (2004) 59–68 The role of reactive oxygen species in microcystin-LR-induced DNA damage Bojana Žegura, Tamara T. Lah, Metka Filipiˇ c ∗ National Institute of Biology, Veˇ cna pot 111, 1000 Ljubljana, Slovenia Received 25 February 2004; received in revised form 25 February 2004; accepted 12 March 2004 Abstract Microcystins are cyclic heptapeptides produced by different freshwater cyanobacterial species such as Microcystis aeruginosa. They have been shown to induce DNA damage in vitro and in vivo, however, the mechanisms 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 of purines. 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) fluoroprobe 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 cells to MCLR in the presence of different ROS 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-dimethyl-2-thiourea (DMTU). The results provide evidence that the genotoxicity of MCLR is mediated by ROS. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Comet assay; DNA damage; Formamidopyrimidine-DNA glycosylase; Microcystin; Reactive oxygen species; Free radical scavengers 1. Introduction Microcystins are the most commonly found hep- atotoxins produced by cyanobacteria and over 60 ∗ Corresponding author. Tel.: +386-1-42-33-388; fax: +386-1-25-73-847. E-mail address: metka.filipic@nib.si (M. Filipiˇ c). microcystin structural variants have been described (reviewed in Codd et al., 1999). The general structure of microcystins is cyclo(-d-Ala-X-d-MeAsp-Z-Adda- d-Glu-Mdha-), where X and Z are variable l-amino acids, d-MeAsp is d-erythro-ß-methylaspartic acid, Mdha is N-methyldehydroalanine and Adda is an un- usual C 20 amino acid 3-amino-9-methoxy-10-phenyl- 2,6,8-trimethyl-deca-4(E)6(E)-dienoic acid (Adda). 0300-483X/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2004.03.006