Elsevier

Food and Chemical Toxicology

Volume 49, Issue 9, September 2011, Pages 2439-2444
Food and Chemical Toxicology

Myricetin affords protection against peroxynitrite-mediated DNA damage and hydroxyl radical formation

https://doi.org/10.1016/j.fct.2011.06.066Get rights and content

Abstract

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 myricetin is associated with inhibition of peroxynitrite-mediated DNA damage, a critical event leading to peroxynitrite elicited cytotoxicity. We observed that peroxynitrite can cause DNA damage in ϕX-174 plasmid DNA and rat primary astrocytes. The presence of myricetin at physiological concentration was found to significantly inhibit DNA strand breakage induced by both peroxynitrite and its generator 3-morpholinosydnonimine (SIN-1). Moreover, the consumption of oxygen caused by SIN-1 was found to be decreased in the presence of myricetin, indicating that myricetin might affect the auto-oxidation of SIN-1. Furthermore, EPR spectroscopy demonstrated that the formation of DMPO-hydroxyl radical adduct (DMPO-OH) from peroxynitrite, and that myricetin inhibited the adduct signal in a concentration-dependent manner. Taken together, these results demonstrate for the first time that myricetin can inhibit peroxynitrite-mediated DNA damage and hydroxyl radical formation.

Highlights

Peroxynitrite can cause DNA damage in ϕX-174 plasmid DNA and rat primary astrocytes. ► Myricetin significantly inhibit DNA damage induced by peroxynitrite and SIN-1. ► Myricetin can decrease the consumption of oxygen caused by SIN-1. ► Myricetin significantly scavenge hydroxyl radicals from peroxynitrite.

Introduction

Peroxynitrite, a potent oxidizing and nitrating species, is generated from the bi-radical reaction of nitric oxide and superoxide at a diffusion-limited rate (Beckman and Koppenol, 1996). Extensive studies have demonstrated that peroxynitrite formation and subsequent reactions play an important role in the pathogenesis of various forms of neurodegenerative disorders, including Alzheimer’s disease, Parkinsonism, and Huntington’s disease (Nanetti et al., 2005, Zhang et al., 2006). There is now substantial evidence that peroxynitrite can impair most cellular components, including DNA, proteins, and phospholipid membranes (Bartesaghi et al., 2006). The addition of peroxynitrite to biomolecules, cells and tissue leads to oxidation, nitration, and eventually to cytotoxicity, cell death and tissue injury. The mechanisms account for cytotoxicity elicited by peroxynitrite are multiple, among them, DNA strand breakage and the subsequent poly (ADP-ribose) polymerase activation are considered to be critical events (Szabo, 2003). In this context, DNA single-stranded breakage initially trigged by endogenous or exogenous peroxynitrite may lead to cell death (Komjati et al., 2005). Moreover, studies have also demonstrated that some peroxynitrite-scavenging compounds can protect against peroxynitrite-mediated DNA strand breaks (Klotz and Sies, 2003).

Myricetin is a naturally occurring flavonol with hydroxyl substitutions at the 3,5,7,3′,4′ and 5′ positions, and commonly consumed in our diet in fruits, vegetables, tea, berries and red wine (German and Walzem, 2000, Hakkinen et al., 1999, Wang et al., 2011). The chemical structure of multiple hydroxyl may lead to strong antioxidative activity and oxy-radical scavenging effect (Shimmyo et al., 2008, Valdez et al., 2004). Accumulating evidence indicated that myricetin has a variety of biological activities, such as antioxidant, anti-tumor, and anti-inflammatory activities (Morel et al., 1998, Surh, 2003). Recently, Myricetin has been shown to protect against oxidative injury in neurodegenerative disorders at physiological concentration (Laabich et al., 2007, Shimmyo et al., 2008). Although the antioxidant property of myricetin appear to be partially responsible for its neuroprotective effect, the mechanism underlying myricetin-mediated neuroprotection is still unclear. Since peroxynitrite is involved in the pathogenesis of neurodegenerative diseases, and induction of DNA strand breakage is a critical initial lesion leading to cell death induced by peroxynitrite (Komjati et al., 2005, Szabo, 2003), in this study, using ϕX-174 plasmid DNA and rat primary astrocytes as model systems, we investigated the effects of myricetin on peroxynitrite-induced DNA strand breaks. Our results demonstrate for the first time that myricetin can inhibit peroxynitrite-mediated DNA damage and hydroxyl radical generation at physiological concentration.

Section snippets

Reagents

Peroxynitrite was obtained from Calbiochem (San Diego, CA). ϕX-174 RF I DNA and Lambda DNA-HindIII digest were purchased from New England Biolabs (Beverley, MA). SIN-1, myricetin and other chemicals were purchased from Sigma Chemical (St. Louis, MO).

Preparation of SIN-1 and peroxynitrite

SIN-1 was dissolved in phosphate-buffer saline (PBS, pH 5.5), and stored at −80 °C. The concentration of peroxynitrite was measured at 302 nm and calculated with a molar extinction coefficient of 1670 M−1 cm−1 according to the manufacture’s instruction.

SIN-1 triggers DNA strand breakage

Induction of single-strand breaks to the supercoiled double-stranded ϕX-174 RF I DNA leads to generation of open circular DNA, while the formation of a linear form of DNA is indicative of double-strand breaks . At a physiological pH, SIN-1 is believed to undergo auto-oxidation to produce equal nitric oxide and superoxide, which in turn lead to the generation of peroxynitrite (Malan et al., 2003). Since the generation of peroxynitrite from SIN-1 auto-oxidation mimics the in vivo formation of

Conclusions

In summary, this work demonstrates for the first time that myricetin is able to inhibit peroxynitrite-induced DNA damage at physiological concentration (5–10 μM) (Peng and Kuo, 2003). Accumulating evidence suggests that peroxynitrite is critically involved in the pathogenesis of various forms of neuronal diseases (Floyd, 1999, Ischiropoulos and Beckman, 2003), and induction of DNA strand breakage by peroxynitrite is suggested to be a critical event, leading to peroxynitrite-elicited

Conflict of Interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (No. 31000775), the Key Project of Chinese Ministry of Education (No. 210086), Research Fund for the Doctoral Program of Higher Education of China (No. 20103326120006), National Key Technology R & D Program (No.NC2010KB0120), and Research Fund for the Distinguished Young Teachers of Higher Education of Zhejiang Province (1110JF220911).

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