Effects of Nitroxide Stable Radicals on Juglone Cytotoxicity

Nitroxides stable radicals are unreactive toward most diamagnetic molecules, but readily undergo one-electron redox reactions with paramagnetic species such as free radicals and transition metals, thus serving as cell-permeable antioxidants. The cytotoxicity of juglone (5-hydroxy-1, 4-naphthoquinone), like that of other naphthoquinones, requires bioreduction to yield the semiquinone which in turn reduces oxygen to O⨪2. Therefore, nitroxides are expected to mitigate cytotoxicity of quinone-based xenobiotics, such as naphthoquinones. In the present study, in vitro scission of isolated DNA was induced upon juglone reduction by glutathione and Fe(II) ions, however, not by xanthine oxidase or cytochrome c reductase. The DNA scission was inhibited by nitroxides, catalase and chelating agents, though not by superoxide dismutase. Juglone was more toxic toward bacterial cells under hypoxia than under air. Nitroxides ≤ 2 mM protected bacterial cells from juglone-induced toxicity under both aerobic and hypoxic conditions. The cytoprotective effect of lipophilic nitroxide was greater than that of hydrophilic ones. Catalase and metal chelating agents decreased juglone-induced cell killing, whereas H2O2 increased it. The mechanisms underlying the nitroxides protective effect involve (a) the reoxidation of reduced transition metal ions, (b) the selective radical-radical reaction with juglone semiquinone, and possibly (c) under aerobic condition catalytic removal of extra- and intracellular O⨪2. The present results suggest also that the cell membrane rather than DNA is the main target of juglone toxicity.