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

https://doi.org/10.1016/j.phrs.2003.12.010Get rights and content

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.

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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