Ethyl Pyruvate Inhibits Peroxynitrite-induced DNA Damage and Hydroxyl Radical Generation: Implications for Neuroprotection

Abstract

Ethyl pyruvate (EP) has recently been reported to afford protection against neurodegenerative disorders. However, the mechanism underlying EP-mediated neuroprotection remains to be elucidated. Because peroxynitrite has been extensively implicated in the pathogenesis of various forms of neurodegenerative disorders via its cytotoxic effects, this study was undertaken to investigate whether the neuroprotective effect of EP is associated with inhibition of peroxynitrite-induced DNA strand breaks, a critical event leading to peroxynitrite elicited cytotoxicity. Incubation of phiX-174 plasmid DNA with 3-morpholinosydnonimine (SIN-1), a peroxynitrite generator, led to the formation of both single- and double-stranded DNA breaks in a concentration- and time- dependent manner. The presence of EP (0.5-10 mM) was found to significantly inhibit SIN-1-induced DNA strand breaks in a concentration-dependent fashion. The consumption of oxygen induced by 250 microM SIN-1 was found to be decreased in the presence of EP (0.5-10 mM), indicating that EP might affect the auto-oxidation of SIN-1. It was observed that incubation of the plasmid DNA with authentic peroxynitrite caused significant DNA strand breaks, which could also be dramatically inhibited by EP (0.5-10 mM). EPR spectroscopy in combination with spin-trapping technique using 5,5-dimethylpyrroline-N- oxide (DMPO) as a spin trap demonstrated the formation of DMPO-hydroxyl radical adducts (DMPO-OH) from authentic peroxynitrite, and that EP at 0.5-10 mM inhibited the adduct signal in a concentration-dependent manner. Taken together, these results demonstrate for the first time that EP can inhibit peroxynitrite-mediated DNA damage and hydroxyl radical generation.