Potent inhibition of peroxynitrite-induced DNA strand breakage by ethanol: possible implications for ethanol-mediated cardiovascular protection
Introduction
Cardiovascular diseases remain the number one killer of the human population worldwide. Substantial evidence suggests that peroxynitrite generated from the bi-radical reaction of nitric oxide and superoxide is crucially involved in the pathogenesis of various forms of cardiovascular disorders, including atherosclerosis, myocardial ischemia-reperfusion injury, and cardiomyopathy [1], [2], [3], [4], [5], [6], [7]. Multiple mechanisms have been proposed to account for the deleterious effects of peroxynitrite on cardiovascular tissues/cells. Among them induction of DNA strand breaks and the subsequent activation of poly(ADP-ribose) polymerase have been demonstrated to be critical events leading to peroxynitrite-elicited cytotoxicity [8], [9]. In this context, the ability of peroxynitrite to induce DNA strand breakage in both in vivo and in vitro systems has been repeatedly reported in the literature [8], [9], [10], [11], [12], [13]. Moreover, studies have also demonstrated that peroxynitrite-scavenging compounds are able to protect against peroxynitrite-induced DNA strand breaks as well as cytotoxicity in target cells [11], [12], [13], [14].
A number of epidemiological studies have conclusively demonstrated that moderate consumption of ethanol (1–2 drinks per day or 15–30 ml ethanol per day) is causally associated with a marked reduction in the cardiovascular events [15], [16], [17], [18]. Several mechanisms have been proposed to explain the cardiovascular protective effects of ethanol, including increasing production of high density lipoprotein, decreasing platelet aggregability, decreasing plasma concentration of C-reactive protein, and augmentation of the generation of the vascular protective nitric oxide by endothelial cells [16], [19], [20]. The above effects of ethanol have been documented in human studies and/or animal experiments [16], [19], [20]. However, it remains unknown whether the ethanol-mediated cardiovascular protection also occurs through other mechanisms. Since peroxynitrite is crucially involved in the pathogenesis of cardiovascular diseases [1], [2], [3], [4], [5], [6], [7], in this study using φX-174 plasmid DNA as an in vitro system, we have investigated the effects of ethanol on peroxynitrite-induced DNA strand breaks. Our results demonstrate for the first time that ethanol at physiologically relevant concentrations potently inhibits peroxynitrite-induced DNA strand breakage in a concentration-dependent fashion.
Section snippets
Materials
φX-174 RF I plasmid DNA was from New England Biolabs (Beverley, MA). Authentic peroxynitrite was from Calbiochem (San Diego, CA). 3-Morpholinosydnonimine (SIN-1) and other chemicals were from Sigma Chemical (St. Louis, MO).
Preparation of SIN-1 and peroxynitrite
SIN-1 was dissolved in phosphate-buffer saline, pH 5.5, and stored at −80 °C. The concentration of authentic peroxynitrite was determined spectrophotometrically at 302 nm (extinction coefficient=1670 M−1 cm−1). The peroxynitrite was aliquot and stored at −80 °C under nitrogen.
Assay for DNA strand breaks
DNA
Induction of DNA strand breaks by SIN-1
Induction of single-strand breaks to the supercoiled double-stranded φX-174 RF I plasmid DNA leads to formation of open circular DNA, while the formation of a linear form of DNA is indicative of double-strand breaks [24]. Although being stable in acidic environment, SIN-1 can undergo auto-oxidation at a physiological pH to produce equal molar nitric oxide and superoxide, leading to the formation of peroxynitrite [12], [13], [25], [26]. Because the generation of peroxynitrite from SIN-1
Acknowledgements
This work was supported in part by the National Institute of Health Grant CA91895 (Y.L.), the St. John’s University Faculty Research Fund, and the Doctoral Fellowship from St. John’s University (Z.C.).
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