Neurochemical Research

, Volume 35, Issue 2, pp 336–342 | Cite as

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

  • Wei Chen
  • Zhenquan Jia
  • Hong Zhu
  • Kequan Zhou
  • Yunbo Li
  • Hara P. Misra
Original Paper


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 φX-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 μM 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.


Ethyl pyruvate Peroxynitrite DNA strand breaks Neuroprotection 





DMPO-hydroxyl adduct


DMPO-superoxide spin adduct


Electron paramagnetic resonance


Ethyl pyruvate


Superoxide dismutase


Reactive oxygen species


Reactive nitrogen species





This work was supported by grants from the NIH grant R01HL071190, and Harvey Peters Research Center Foundation.


  1. 1.
    Pong K (2003) Oxidative stress in neurodegenerative diseases: therapeutic implications for superoxide dismutase mimetics. Expert Opin Biol Ther 3:127–139CrossRefPubMedGoogle Scholar
  2. 2.
    Jenner P (2003) Oxidative stress in Parkinson’s disease. Ann Neurol 53(Suppl 3):S26–S36 discussion S36-8CrossRefPubMedGoogle Scholar
  3. 3.
    Klein JA, Ackerman SL (2003) Oxidative stress, cell cycle, and neurodegeneration. J Clin Invest 111:785–793PubMedGoogle Scholar
  4. 4.
    Malkus KA, Tsika E, Ischiropoulos H (2009) Oxidative modifications, mitochondrial dysfunction, and impaired protein degradation in Parkinson’s disease: how neurons are lost in the Bermuda triangle. Mol Neurodegener 4:24CrossRefPubMedGoogle Scholar
  5. 5.
    Ischiropoulos H, Beckman JS (2003) Oxidative stress and nitration in neurodegeneration: cause, effect, or association? J Clin Invest 111:163–169PubMedGoogle Scholar
  6. 6.
    Mancuso C, Scapagini G, Curro D, Giuffrida Stella AM, De Marco C, Butterfield DA, Calabrese V (2007) Mitochondrial dysfunction, free radical generation and cellular stress response in neurodegenerative disorders. Front Biosci 12:1107–1123CrossRefPubMedGoogle Scholar
  7. 7.
    Beckman JS, Koppenol WH (1996) Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol 271:C1424–C1437PubMedGoogle Scholar
  8. 8.
    Estevez AG, Radi R, Barbeito L, Shin JT, Thompson JA, Beckman JS (1995) Peroxynitrite-induced cytotoxicity in PC12 cells: evidence for an apoptotic mechanism differentially modulated by neurotrophic factors. J Neurochem 65:1543–1550PubMedCrossRefGoogle Scholar
  9. 9.
    Yamamoto T, Maruyama W, Kato Y, Yi H, Shamoto-Nagai M, Tanaka M, Sato Y, Naoi M (2002) Selective nitration of mitochondrial complex I by peroxynitrite: involvement in mitochondria dysfunction and cell death of dopaminergic SH-SY5Y cells. J Neural Transm 109:1–13CrossRefPubMedGoogle Scholar
  10. 10.
    Kaji T, Kaieda I, Hisatsune T, Kaminogawa S (2002) 3-Morpholinosydnonimine hydrochloride induces p53-dependent apoptosis in murine primary neural cells: a critical role for p21(ras)-MAPK-p19(ARF) pathway. Nitric Oxide 6:125–134CrossRefPubMedGoogle Scholar
  11. 11.
    Szabo C (2003) Multiple pathways of peroxynitrite cytotoxicity. Toxicol Lett 140–141:105–112CrossRefPubMedGoogle Scholar
  12. 12.
    Komjati K, Besson VC, Szabo C (2005) Poly (adp-ribose) polymerase inhibitors as potential therapeutic agents in stroke and neurotrauma. Curr Drug Targets CNS Neurol Disord 4:179–194CrossRefPubMedGoogle Scholar
  13. 13.
    Cai L, Klein JB, Kang YJ (2000) Metallothionein inhibits peroxynitrite-induced DNA and lipoprotein damage. J Biol Chem 275:38957–38960CrossRefPubMedGoogle Scholar
  14. 14.
    Klotz LO, Sies H (2003) Defenses against peroxynitrite: selenocompounds and flavonoids. Toxicol Lett 140–141:125–132CrossRefPubMedGoogle Scholar
  15. 15.
    Dobsak P, Courderot-Masuyer C, Zeller M, Vergely C, Laubriet A, Assem M, Eicher JC, Teyssier JR, Wolf JE, Rochette L (1999) Antioxidative properties of pyruvate and protection of the ischemic rat heart during cardioplegia. J Cardiovasc Pharmacol 34:651–659CrossRefPubMedGoogle Scholar
  16. 16.
    Vonkorff RW (1964) Pyruvate-C14, Purity and Stability. Anal Biochem 8:171–178CrossRefPubMedGoogle Scholar
  17. 17.
    Sims CA, Wattanasirichaigoon S, Menconi MJ, Ajami AM, Fink MP (2001) Ringer’s ethyl pyruvate solution ameliorates ischemia/reperfusion-induced intestinal mucosal injury in rats. Crit Care Med 29:1513–1518CrossRefPubMedGoogle Scholar
  18. 18.
    Kim JB, Yu YM, Kim SW, Lee JK (2005) Anti-inflammatory mechanism is involved in ethyl pyruvate-mediated efficacious neuroprotection in the postischemic brain. Brain Res 1060:188–192CrossRefPubMedGoogle Scholar
  19. 19.
    Liu J, Segal M, Yoo S, Yang GY, Kelly M, James TL, Litt L (2009) Antioxidant effect of ethyl pyruvate in respiring neonatal cerebrocortical slices after H2O2 stress. Neurochem Int 54:106–110CrossRefPubMedGoogle Scholar
  20. 20.
    Yu YM, Kim JB, Lee KW, Kim SY, Han PL, Lee JK (2005) Inhibition of the cerebral ischemic injury by ethyl pyruvate with a wide therapeutic window. Stroke 36:2238–2243CrossRefPubMedGoogle Scholar
  21. 21.
    Tokumaru O, Kuroki C, Yoshimura N, Sakamoto T, Takei H, Ogata K, Kitano T, Nisimaru N, Yokoi I (2009) Neuroprotective effects of ethyl pyruvate on brain energy metabolism after ischemia–reperfusion injury: a 31P-nuclear magnetic resonance study. Neurochem Res 34:775–785CrossRefPubMedGoogle Scholar
  22. 22.
    Beckman JA, Goldfine AB, Gordon MB, Garrett LA, Keaney JF Jr, Creager MA (2003) Oral antioxidant therapy improves endothelial function in Type 1 but not Type 2 Diabetes mellitus. Am J Physiol Heart Circ Physiol 285:H2392–H2398PubMedGoogle Scholar
  23. 23.
    Hsu CS, Li Y (2002) Aspirin potently inhibits oxidative DNA strand breaks: implications for cancer chemoprevention. Biochem Biophys Res Commun 293:705–709CrossRefPubMedGoogle Scholar
  24. 24.
    Jia Z, Zhu H, Vitto MJ, Misra BR, Li Y, Misra HP (2009) Alpha-lipoic acid potently inhibits peroxynitrite-mediated DNA strand breakage and hydroxyl radical formation: implications for the neuroprotective effects of alpha-lipoic acid. Mol Cell Biochem 323:131–138CrossRefPubMedGoogle Scholar
  25. 25.
    Jia Z, Zhu H, Misra BR, Mahaney JE, Li Y, Misra HP (2008) EPR studies on the superoxide-scavenging capacity of the nutraceutical resveratrol. Mol Cell Biochem 313:187–194CrossRefPubMedGoogle Scholar
  26. 26.
    Pieper GM, Felix CC, Kalyanaraman B, Turk M, Roza AM (1995) Detection by ESR of DMPO hydroxyl adduct formation from islets of langerhans. Free Radic Biol Med 19:219–225CrossRefPubMedGoogle Scholar
  27. 27.
    Li Y, Trush MA (1993) DNA damage resulting from the oxidation of hydroquinone by copper: role for a Cu(II)/Cu(I) redox cycle and reactive oxygen generation. Carcinogenesis 14:1303–1311CrossRefPubMedGoogle Scholar
  28. 28.
    Ruan RS (2002) Possible roles of nitric oxide in the physiology and pathophysiology of the mammalian cochlea. Ann N Y Acad Sci 962:260–274CrossRefPubMedGoogle Scholar
  29. 29.
    Malan D, Levi RC, Alloatti G, Marcantoni A, Bedendi I, Gallo MP (2003) Cyclic AMP and cyclic GMP independent stimulation of ventricular calcium current by peroxynitrite donors in guinea pig myocytes. J Cell Physiol 197:284–296CrossRefPubMedGoogle Scholar
  30. 30.
    Kim SY, Lee JH, Yang ES, Kil IS, Park JW (2003) Human sensitive to apoptosis gene protein inhibits peroxynitrite-induced DNA damage. Biochem Biophys Res Commun 301:671–674CrossRefPubMedGoogle Scholar
  31. 31.
    Cao Z, Li Y (2004) Potent inhibition of peroxynitrite-induced DNA strand breakage by ethanol: possible implications for ethanol-mediated cardiovascular protection. Pharmacol Res 50:13–19CrossRefPubMedGoogle Scholar
  32. 32.
    Roussyn I, Briviba K, Masumoto H, Sies H (1996) Selenium-containing compounds protect DNA from single-strand breaks caused by peroxynitrite. Arch Biochem Biophys 330:216–218CrossRefPubMedGoogle Scholar
  33. 33.
    Szabo C, Ischiropoulos H, Radi R (2007) Peroxynitrite: biochemistry, pathophysiology and development of therapeutics. Nat Rev Drug Discov 6:662–680CrossRefPubMedGoogle Scholar
  34. 34.
    Frejaville C, Karoui H, Tuccio B, Le Moigne F, Culcasi M, Pietri S, Lauricella R, Tordo P (1995) 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide: a new efficient phosphorylated nitrone for the in vitro and in vivo spin trapping of oxygen-centered radicals. J Med Chem 38:258–265CrossRefPubMedGoogle Scholar
  35. 35.
    Buettner GR (1987) Spin trapping: ESR parameters of spin adducts. Free Radic Biol Med 3:259–303CrossRefPubMedGoogle Scholar
  36. 36.
    Lemercier JN, Squadrito GL, Pryor WA (1995) Spin trap studies on the decomposition of peroxynitrite. Arch Biochem Biophys 321:31–39CrossRefPubMedGoogle Scholar
  37. 37.
    Zang LY, Misra HP (1992) EPR kinetic studies of superoxide radicals generated during the autoxidation of 1-methyl-4-phenyl-2, 3-dihydropyridinium, a bioactivated intermediate of parkinsonian-inducing neurotoxin 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine. J Biol Chem 267:23601–23608PubMedGoogle Scholar
  38. 38.
    Tsou TC, Lai HJ, Yang JL (1999) Effects of mannitol or catalase on the generation of reactive oxygen species leading to DNA damage by Chromium(VI) reduction with ascorbate. Chem Res Toxicol 12:1002–1009CrossRefPubMedGoogle Scholar
  39. 39.
    Fink MP (2008) Ethyl pyruvate. Curr Opin Anaesthesiol 21:160–167CrossRefPubMedGoogle Scholar
  40. 40.
    Ainscow EK, Brand MD (1999) Top–down control analysis of ATP turnover, glycolysis and oxidative phosphorylation in rat hepatocytes. Eur J Biochem 263:671–685CrossRefPubMedGoogle Scholar
  41. 41.
    Floyd RA (1999) Neuroinflammatory processes are important in neurodegenerative diseases: an hypothesis to explain the increased formation of reactive oxygen and nitrogen species as major factors involved in neurodegenerative disease development. Free Radic Biol Med 26:1346–1355CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Division of Biomedical Sciences, Edward Via Virginia College of Osteopathic MedicineVirginia Tech Corporate Research CenterBlacksburgUSA
  2. 2.College of Food Science and Biological EngineeringZhejiang Gongshang UniversityHangzhouChina
  3. 3.Department of Food Science and TechnologyVirginia Polytechnic Institute and State UniversityBlacksburgUSA

Personalised recommendations