Blood mononucleocytes are sensitive to the DNA damaging effects of iron overload— In vitro and ex vivo results with human and rat cells

Abstract

Iron exposure enhances colorectal carcinogeneis, by producing reactive oxygen species, which damage lipids, proteins and DNA. We recently demonstrated that ferric-nitrilotriacetate (Fe-NTA) damages DNA of human colon cells in different stages of malignant transformation. Opposed to this, little is known on systemic effects of iron and it is still difficult to determine the border between essential iron supplementation and iron overload in humans. The aim of this study was to determine whether Fe-NTA causes global and specific DNA damage in peripheral leucocytes. Human leucocytes were treated in vitro with Fe-NTA for 30 min at 37 °C. Male Sprague Dawley rats were fed (6 weeks) with an iron-overload diet (9.9 g Fe/kg DM) and whole blood was collected. DNA damage was measured in human and rat blood cells using the alkaline version of the Comet Assay with repair specific enzymes. In human cells the distribution of TP53 in the comet images was detected using fluorescence in situ hybridization (Comet FISH) to measure DNA damage in the region of the TP53 gene. Fe-NTA (10–500 μM) was clearly genotoxic in human leucocytes in vitro, and also in leucocytes of rats fed the iron overload diet. The induced damage in human leucocytes was approximately two-fold that observed previously in human colon cells. Oxidized bases were induced by iron in rat leucocytes in vivo, while they were not induced in human leucocytes in vitro. Fe-NTA enhanced the migration of TP53 signals into the comet tail of human leucocytes, indicating a high susceptibility of this tumour-relevant gene towards DNA damage induced by iron overload. In conclusion, iron markedly induced DNA damage in human and rat leucocytes, which shows that these white blood cells are sufficiently sensitive to assess exposure to iron. The measurement of DNA damage in human leucocytes could be used as a sensitive biomarker to study iron overload in vivo in humans and thus to determine whether supplementation results in genotoxic risk.