Characterization of DNA--protein cross-links induced by oxanine: cellular damage derived from nitric oxide and nitrous acid

Reactive nitrogen species are implicated in inflammatory diseases and cancers. Oxanine (Oxa) is a DNA lesion derived from the guanine base with nitric oxide, nitrous acid, or N-nitrosoindoles. It was shown by gel electrophoresis that oxanine mediated the formation of DNA-protein cross-links (DPCs) with DNA-binding proteins and in the cell extract. Although 2'-deoxyoxanosine was shown to react with amines including the N-terminal amino group of glycine, the structures of DNA-protein cross-links induced by oxanine have not been characterized. In this study, we find that the thiol group of the amino acid side chain is reactive toward oxanine, forming a thioester. Two reaction products of oxanine, namely, the thioester and the amide adducts, with the endogenous tripeptide glutathione (GSH) as a model protein were characterized on the basis of their UV, NMR (1H- and 13C-), and mass spectra. Interestingly, the disulfide GSSG also reacts with oxanine, forming the thioester adduct. The thioester and the amide adducts are generated when GSH and GSSG react with oxanine-containing calf thymus DNA, and they might be possible forms of cellular DPCs. Because the repair mechanism of DPCs is not extensively investigated, the characterization of oxanine-derived DPC structures should shed light on their detection in vivo and on their biological consequences.