FORMATION OF DNA-PROTEIN CROSS-LINKS IN CULTURED-MAMMALIAN-CELLS UPONTREATMENT WITH IRON IONS

Formation of DNA-protein crosslinks (DPCs) in mammalian cells upon tre atment with iron or copper ions was investigated. Cultured murine hybr idoma cells were treated with Fe(II) or Cu(II) ions by addition to the culture medium at various concentrations. Subsequently, chromatin sam ples were isolated from treated and control cells. Analyses of chromat in samples by gas chromatography/mass spectrometry after hydrolysis an d derivatization revealed a significant increase over the background a mount of ihydro-2,4-dioxopyrimidin-5-yl)-methyl]-L-tyrosine (Thy-Tyr c rosslink) in cells treated with Fe(II) ions in the concentration range of 0.01 to 1 mM. In contrast, Cu(II) ions at the same concentrations did not produce this DPC in cells. No DNA base damage was observed in cells treated with Cu(II) ions, either. Preincubation of cells with as corbic acid or coincubation with dimethyl sulfoxide did not significan tly alleviate the Fe(II) ion-mediated formation of DPCs. In addition, a modified fluorometric analysis of DNA unwinding assay was used to de tect DPCs formed in cells. Fe(II) ions caused significant formation of DPCs, but Cu(II) ions did not. The nature of the Fe(II)-mediated DPCs suggests the involvement of the hydroxyl radical in their formation. The Thy-Tyr crosslink may contribute to pathological processes associa ted with free radical reactions.