Oxidative stress by menadione affects cellular copper and iron homeostasis

Menadione produces DNA strand breaks (DNA sb) in cultured Chinese hamster fibroblasts which are, to a great extent, mediated by OH radical. A reasonable hypothesis is that H₂O₂, a product of menadione metabolism, reacts with nuclear iron and produces OH radicalin situ. Consistent with that, 1,10-phenanthroline (PHEN) prevents menadione-induced DNA sb at low (<200 M) concentrations of the chelator. However, at higher PHEN concentrations, the effect is reversed and an enhancement of DNA sb is observed. The PHEN-induced enhancement of DNA sb becomes more evident at high (>60 M) menadione concentrations and is strongly prevented by neocuproine (NEO), an efficient copper chelator. However, NEO offers only a slight protection against DNA sb caused by menadione alone. The results are consistent with the following events: (i) the products of menadione metabolism causes copper ion release from some cellular compartment; (ii) in the presence of PHEN, a Cu(PHEN)₂ complex is formed; (iii) the Cu(PHEN)₂ complex is known to be very clastogenic, inducing DNA damage in a reducing environment.Evidence is also presented that menadione metabolism causes an increase in intracellular chelatable iron: in the presence of a constant 2,2-dipyridyl concentration, the DNA sb produced by increasing concentrations of menadione become progressively less susceptible to inhibition by the chelator.Therefore the DNA damage originated from menadione metabolism seems to be caused by two conjugated and synergistic events, viz., the production of reactive oxygen species and the release of copper and iron from a cellular storage site into a free form pool, capable of catalyzing DNA damaging reactions.Abbreviations sb strand breaks - PHEN 1,10-phenanthroline - PBS phosphate-buffered saline (8.1 mM Na₂HPO₄, 1.47 mM KH₂PO₄, 1.68 mM KCl, 137 mM NaCl, pH 7.0) - HP Hydrogen Peroxide - Md Menadione - NEO Neocuproine