The influence of cylindrospermopsin on oxidative DNA damage and apoptosis induction in HepG2 cells

Abstract

Cylindrospermopsin (CYN) a potent cyanobacterial cytotoxin and protein synthesis inhibitor is increasingly being found in surface freshwaters worldwide. Due to its genotoxic activity and potential carcinogenicity it was recognized as a potential threat to humans. However, the mechanisms of CYN genotoxicity are not well understood. We explored whether CYN at non-cytotoxic exposure conditions causes DNA damage through induction of oxidative stress and whether it induces apoptosis in HepG2 cells. With the DCFH-DA probe a significant increase in the intracellular formation of reactive oxygen species (ROS) was observed, which steadily increased with incubation. Induction of oxidative DNA damage was determined with the modified comet assay with formamidopyrimidine glycosylase (Fpg) digestion. No DNA damage was observed after 4h exposure to CYN. After 12 and 24h exposure, CYN (at 0.25 and 0.5μgmL−1) induced significant increase of DNA strand breaks, but not oxidative DNA damage, suggesting minor role of oxidative stress in CYN mediated genotoxicity. CYN also significantly increased the mitochondrial membrane potential (MMP), determined with the JC-1 probe, while no induction of caspase 3 and 7 activity and no increase in the number of apoptotic cells, measured with Annexin V/PI staining, could be determined. These results show that at non-cytotoxic concentrations CYN induced DNA damage was not the consequence of oxidative stress and that CYN did not induce apoptosis, which may add to the hazard of this toxin, as cells with damaged DNA are not removed from the population, enhancing the risk of mutations and consequently carcinogenesis.