Potent inhibition of peroxynitrite-induced DNA strand breakage by ethanol: possible implications for ethanol-mediated cardiovascular protection

Abstract

Epidemiological studies have conclusively demonstrated that moderate consumption of ethanol is causally associated with a significant reduction in cardiovascular events. However, the exact mechanisms underlying the ethanol-mediated cardiovascular protection remain to be elucidated. Because peroxynitrite has been extensively implicated in the pathogenesis of various forms of cardiovascular disorders via its cytotoxic effects, this study was undertaken to investigate if ethanol could inhibit peroxynitrite-induced DNA strand breaks, a critical event leading to peroxynitrite-elicited cytotoxicity. Toward this goal, φX-174 RF I plasmid DNA was used as an in vitro model to determine the protective effects of ethanol on peroxynitrite-induced DNA strand breaks. Incubation of φX-174 plasmid DNA with the peroxynitrite generator, 3-morpholinosydnonimine (SIN-1) led to the formation of both single- and double-stranded DNA breaks in a concentration- and time-dependent fashion. The presence of ethanol at concentrations ranging from 0.01 to 1% (w/v) resulted in a significant inhibition of SIN-1-induced DNA strand breaks. Ethanol also showed inhibitory effects on SIN-1-induced DNA strand breakage in the presence of bicarbonate. The inhibition of SIN-1-induced DNA strand breaks by ethanol exhibited a concentration-dependent manner. Notably, a marked inhibition of SIN-1-elicited DNA strand breaks was observed with 0.01% ethanol. Ethanol at 0.01–1% was unable to affect SIN-1-mediated oxygen consumption, indicating that ethanol did not affect the auto-oxidation of SIN-1 to form peroxynitrite. Furthermore, incubation of the plasmid DNA with authentic peroxynitrite resulted in a significant formation of DNA strand breaks, which could be dramatically inhibited by the presence of 0.02–0.1% ethanol. Taken together, this study demonstrates for the first time that ethanol at physiologically relevant concentrations can potently inhibit peroxynitrite-induced DNA strand breakage. In view of the critical involvement of peroxynitrite in cardiovascular disorders, the results of this study might have implications for the cardiovascular protection associated with moderate consumption of ethanol in humans.