Cytotoxicity and site-specific DNA damage induced by nitroxyl anion (NO-) in the presence of hydrogen peroxide - Implications for various pathophysiological conditions

Nitroxyl anion (NO-), the one-electron reduction product of nitric oxide (N O.), is formed under various physiological conditions. We have used four di fferent assays (DNA strand breakage, 8-oxo-deoxyguanosine formation in calf thymus DNA, malondialdehyde generation from 2'-deoxyribose, and analysis o f site-specific DNA damage using P-32-5'-end-labeled DNA fragments of the h uman p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene) to study t he effects of NO- generated from Angeli's salt on DNA damage. It was found that strong oxidants are generated from NO-, especially in the presence of H2O2 plus Fe(III)-EDTA or Cu(II), NO. released from diethylamine-NONOate ha d no such effect. Distinct effects of hydroxyl radical (HO.) scavengers and patterns of site-specific DNA cleavage caused by Angeli's salt alone or by Angeli's salt, H2O2, plus metal ion suggest that NO- acts as a reductant t o catalyze the formation of the HO. from H2O2, plus Fe(III) and formation o f Cu(I)-peroxide complexes with a reactivity similar to HO. from H2O2 and C u(II), Angeli's salt and H2O2 exerted synergistically cytotoxic effects to MCF-7 cells, determined by lactate dehydrogenase release assay, Thus NO- ma y play an important role in the etiology of various pathophysiological cond itions such as inflammation and neurodegenerative diseases, especially when H2O2 and transition metallic ions are present.