ABSTRACT
Iron could be a relevant risk factor for carcinogenesis since it catalyses the formation of reactive oxygen species (ROS), which damage DNA. We previously demonstrated genotoxic effects by ferric iron using the human colon cancer cell line HT29. Here we investigated ferric iron in primary non-transformed colon cells and in a preneoplastic colon adenoma cell line (LT97), which both are suitable models to study effects of carcinogens during early stages of cell transformation. Genetic damage was determined using the Comet assay. Comet FISH (fluorescence in situ hybridization) was used to assess specific effects on TP53. Fe-NTA (0-1000 microM, 30 min, 37 degrees C) significantly induced single strand breaks in primary colon cells (500 microM Fe-NTA: Tail intensity [TI] 22.6%+/-5.0% versus RPMI control: TI 10.6%+/-3.9%, p<0.01) and in LT97 cells (1000 microM Fe-NTA: TI 26.8%+/-7.3% versus RPMI control: TI 11.1%+/-3.7%, p<0.01). With the Comet FISH protocol lower concentrations of Fe-NTA significantly increased DNA damage already at 100 and 250 microM Fe-NTA in primary colon and LT97 adenoma cells, respectively. This damage was detected as an enhanced migration of TP53 signals into the comet tail in both cell types, which indicates a high susceptibility of this tumor relevant gene towards Fe-NTA. In conclusion, Fe-NTA acts genotoxic in non-transformed and in preneoplastic human colon cells, in which it also enhances migration of TP53 at relatively low concentrations. Translated to the in vivo situation these results suggest that iron overload putatively contributes to a genotoxic risk during early stages of colorectal carcinogenesis on account of its genotoxic potential in non-tumorigenic human colon cells.
Subject(s)
Colon/drug effects , DNA Damage/drug effects , Ferric Compounds/toxicity , Nitrates/toxicity , Adenoma/genetics , Adenoma/pathology , Cell Line, Tumor , Chromosome Aberrations , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Comet Assay , Dose-Response Relationship, Drug , Humans , In Situ Hybridization, Fluorescence , Reactive Oxygen Species/metabolism , Tumor Suppressor Protein p53/analysisABSTRACT
Iron is a relevant risk factor for colorectal cancer due to its genotoxic properties. Here we hypothesised that iron-overload causes other toxic effects, which contribute to carcinogenesis. For this, we investigated formation of reactive oxygen species (ROS), DNA repair, cell growth and glutathione (GSH) in human colon tumor cells (HT29 clone 19A) treated with ferric nitrilotriacetate (Fe-NTA, 0-2000 microM). Intracellular formation of ROS was analysed with the peroxide-labile fluorescent dye carboxy-dichlorodihydrofluorescine-diacetate. DNA repair, reflected as the persistency of DNA damage induced by selected genotoxins, was determined with the Comet assay. Cell growth and GSH were measured by fluorimetrical analysis. Key findings were that ROS formation increased with time (1000 microM Fe-NTA, p < 0.001). DNA damage was largely repaired after 120 min, but was not affected by 10 microM Fe-NTA. In contrast, 10 microM Fe-NTA significantly increased DNA damage induced by 4-hydroxynonenal. Doses of 25 microM Fe-NTA increased cell growth (p < 0.05), whereas high concentrations (2000 microM) resulted in growth arrest (p < 0.05), that was accompanied by increased GSH levels (p < 0.01). In conclusion, high concentrations of Fe-NTA caused cellular effects, which reflect a stress response, and resulted in formation of ROS. Carcinogenic risks from ferric iron could be derived also from lower concentrations, which enhance tumor cell growth and cause progenotoxic effects.
