Neurochemical Research

, Volume 37, Issue 10, pp 2150–2160 | Cite as

Differential Toxicity of 6-Hydroxydopamine in SH-SY5Y Human Neuroblastoma Cells and Rat Brain Mitochondria: Protective Role of Catalase and Superoxide Dismutase

  • Javier Iglesias-González
  • Sofía Sánchez-Iglesias
  • Estefanía Méndez-Álvarez
  • Sarah Rose
  • Atsuko Hikima
  • Peter Jenner
  • Ramón Soto-Otero
Original Paper


Oxidative stress and mitochondrial dysfunction are two pathophysiological factors often associated with the neurodegenerative process involved in Parkinson’s disease (PD). Although, 6-hydroxydopamine (6-OHDA) is able to cause dopaminergic neurodegeneration in experimental models of PD by an oxidative stress-mediated process, the underlying molecular mechanism remains unclear. It has been established that some antioxidant enzymes such as catalase (CAT) and superoxide dismutase (SOD) are often altered in PD, which suggests a potential role of these enzymes in the onset and/or development of this multifactorial syndrome. In this study we have used high-resolution respirometry to evaluate the effect of 6-OHDA on mitochondrial respiration of isolated rat brain mitochondria and the lactate dehydrogenase cytotoxicity assay to assess the percentage of cell death induced by 6-OHDA in human neuroblastoma cell line SH-SY5Y. Our results show that 6-OHDA affects mitochondrial respiration by causing a reduction in both respiratory control ratio (IC50 = 200 ± 15 nM) and state 3 respiration (IC50 = 192 ± 17 nM), with no significant effects on state 4o. An inhibition in the activity of both complex I and V was also observed. 6-OHDA also caused cellular death in human neuroblastoma SH-SY5Y cells (IC50 = 100 ± 9 μM). Both SOD and CAT have been shown to protect against the toxic effects caused by 6-OHDA on mitochondrial respiration. However, whereas SOD protects against 6-OHDA-induced cellular death, CAT enhances its cytotoxicity. The here reported data suggest that both superoxide anion and hydroperoxyl radical could account for 6-OHDA toxicity. Furthermore, factors reducing the rate of 6-OHDA autoxidation to its p-quinone appear to enhance its cytotoxicity.


6-Hydroxydopamine Superoxide dismutase Catalase Mitochondria SH-SY5Y cells High-resolution respirometry 





Parkinson’s disease


Reactive oxygen species


Superoxide dismutase




Glutathione peroxidase


Respiratory control ratio


Lactate dehydrogenase


Eagle’s minimal essential medium


p-Quinone of 6-OHDA


Semiquinone radical of 6-OHDA


Superoxide radical


Hydrogen peroxide


Hydroxyl radical


Hydroperoxyl radical



This study was supported by grants SAF2007-66114 (to R. S.-O.) from the Ministerio de Ciencia e Innovación (Madrid, Spain) with the contribution of the European Regional Development Fund and 09CSA005298PR (to E. M.-A.) from the Xunta de Galicia (Santiago de Compostela, Spain). J. I.-G. was supported by a scholarship from the Fundación Obra Social La Caixa (Barcelona, Spain).


  1. 1.
    Blum D, Torch S, Nissou MF, Benabid AL, Verna JM (2000) Extracellular toxicity of 6-hydroxydopamine on PC12 cells. Neurosci Lett 283:193–196. doi: 10.1016/S0304-3940(00)00948-4 PubMedCrossRefGoogle Scholar
  2. 2.
    Bové J, Prou D, Perier C, Przedborski S (2005) Toxin-induced models of Parkinson’s disease. NeuroRx 2:484–494. doi: 10.1602/neurorx.2.3.484 PubMedCrossRefGoogle Scholar
  3. 3.
    Sauer H, Oertel WH (1994) Progressive degeneration of nigrostriatal dopamine neurons following intrastriatal terminal lesions with 6-hydroxydopamine: a combined retrograde tracing and immunocytochemical study in the rat. Neuroscience 59:401–415. doi: 10.1016/0306-4522(94)90605-X PubMedCrossRefGoogle Scholar
  4. 4.
    Rodríguez M, Barroso-Chinea P, Abdala P, Obeso J, González-Hernández T (2001) Dopamine cell degeneration induced by intraventricular administration of 6-hydroxydopamine in the rat: similarities with cell loss in Parkinson’s disease. Exp Neurol 169:163–181. doi: 10.1006/exnr.2000.7624 PubMedCrossRefGoogle Scholar
  5. 5.
    Soto-Otero R, Méndez-Álvarez E, Hermida-Ameijeiras A, López-Real AM, Labandeira-García JL (2002) Effects of (-)-nicotine and (-)-cotinine on 6-hydroxydopamine-induced oxidative stress and neurotoxicity: relevance for Parkinson’s disease. Biochem Pharmacol 64:125–135. doi: 10.1016/S0006-2952(02)01070-5 PubMedCrossRefGoogle Scholar
  6. 6.
    Biswas SC, Ryu E, Park C, Malagelada C, Greene LA (2005) PUMA and p53 play required roles in death evoked in a cellular model of Parkinson disease. Neurochem Res 30:839–845. doi: 10.1007/s11064-005-6877-5 PubMedCrossRefGoogle Scholar
  7. 7.
    Kulich SM, Horbinsky C, Patel M, Chu CT (2007) 6-Hydroxydopamine induces mitochondrial ERK activation. Free Radic Biol Med 43:372–383. doi: 10.1016/j.freeradbiomed.2007.04.028 PubMedCrossRefGoogle Scholar
  8. 8.
    Gomez-Lazaro M, Galindo MF, Concannon CG, Segura MF, Fernandez-Gomez FJ, Llecha N, Comella JX, Prehn JHM, Jordan J (2008) 6-Hydroxydopamine activates the mitochondrial apoptosis pathway through p38 MAPK-mediated, p53-independent activation of Bax and PUMA. J Neurochem 104:1599–1612. doi: 10.1111/j.1471-4159.2007.05115.x PubMedCrossRefGoogle Scholar
  9. 9.
    Marti MJ, James CJ, Oo TF, Kelly WJ, Burke RE (1997) Early developmental destruction of terminals in the striatal target induces apoptosis in dopamine neurons of the substantia nigra. J Neurosci 17:2030–2039PubMedGoogle Scholar
  10. 10.
    Jenner P (2003) Oxidative stress in Parkinson’s disease. Ann Neurol 53:S26–S36. doi: 10.1002/ana.10483 PubMedCrossRefGoogle Scholar
  11. 11.
    Graham DG, Tiffany SM, Bell WR, Gutknecht WF (1978) Autoxidation versus covalent binding of quinones as the mechanism of toxicity of dopamine, 6-hydroxydopamine, and related compounds toward C1300 neuroblastoma cells in vitro. Mol Pharmacol 14:644–653PubMedGoogle Scholar
  12. 12.
    Gee P, Davison AJ (1989) Intermediates in the aerobic autoxidation of 6-hydroxydopamine: relative importance under different reaction conditions. Free Radic Biol Med 6:271–284. doi: 10.1016/0891-5849(89)90054-3 PubMedCrossRefGoogle Scholar
  13. 13.
    Halliwell B, Gutteridge JM, Cross CE (1992) Free radicals, antioxidants, and human disease: where are we now? J Lab Clin Med 119:598–620PubMedGoogle Scholar
  14. 14.
    Soto-Otero R, Méndez-Álvarez E, Hermida-Ameijeiras A, Muñoz-Patiño AM, Labandeira-Garcia JL (2000) Autoxidation and neurotoxicity of 6-hydroxydopamine in the presence of some antioxidants: potential implication in relation to the pathogenesis of Parkinson’s disease. J Neurochem 74:1605–1612. doi: 10.1046/j.1471-4159.2000.0741605.x PubMedCrossRefGoogle Scholar
  15. 15.
    Méndez-Álvarez E, Soto-Otero R, Hermida-Ameijeiras A, López-Martín ME, Labandeira-García JL (2001) Effect of iron and manganese on hydroxyl radical production by 6-hydroxydopamine: mediation of antioxidants. Free Radic Biol Med 31:986–998. doi: 10.1016/S0891-5849(01)00679-7 PubMedCrossRefGoogle Scholar
  16. 16.
    Blum D, Torch S, Lambeng N, Nissou MF, Benabid A-L, Sadoul R, Verna JM (2001) Molecular pathways involved in the neurotoxicity of 6-OHDA, dopamine and MPTP: contribution to the apoptotic theory in Parkinson’s disease. Progress Neurobiol 65:135–172. doi: 10.1016/S0301-0082(01)00003-X CrossRefGoogle Scholar
  17. 17.
    Simola N, Morelli M, Carta AR (2007) The 6-hydroxydopamine model of Parkinson’s disease. Neurotox Res 11:151–167PubMedCrossRefGoogle Scholar
  18. 18.
    Vercesi AE, Kowaltowski AJ, Grijalba MT, Meinicke AR, Castilho RF (1997) The role of reactive oxygen species in mitochondrial permeability transition. Biosci Rep 17:43–52. doi: 10.1023/A:1027335217774 PubMedCrossRefGoogle Scholar
  19. 19.
    Lenaz G, Bovina C, D’Aurelio M, Fato R, Formiggini G, Genova ML, Giuliano G, Merlo Pich M, Paolucci U, Parenti Castelli G, Ventura B (2002) Role of mitochondria in oxidative stress and aging. Ann NY Acad Sci 959:199–213PubMedCrossRefGoogle Scholar
  20. 20.
    Boveris A, Cadenas E, Stoppani AO (1976) Role of ubiquinone in the mitochondrial generation of hydrogen peroxide. Biochem J 156:435–444PubMedGoogle Scholar
  21. 21.
    Cadenas E, Boveris A, Ragan CI, Stopani AO (1977) Production of superoxide radicals and hydrogen peroxide by NADH-ubiquinone reductase and ubiquinol-cytochrome c reductase from beef-heart mitochondria. Arch Biochem Biophys 180:248–257. doi: 10.1016/0003-9861(77)90035-2 PubMedCrossRefGoogle Scholar
  22. 22.
    Turrens JF, Boveris A (1980) Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria. Biochem J 191:421–427PubMedGoogle Scholar
  23. 23.
    Dauer W, Przedborski S (2003) Parkinson’s disease: mechanisms and models. Neuron 39:889–909. doi: 10.1016/S0896-6273(03)00568-3 PubMedCrossRefGoogle Scholar
  24. 24.
    Vila M, Przedborski S (2003) Targeting programmed cell death in neurodegenerative diseases. Nat Rev Neurosci 4:365–375. doi: 10.1038/nrn1100 PubMedCrossRefGoogle Scholar
  25. 25.
    Liss B, Haeckel O, Wildmann J, Miki T, Seino S, Roeper J (2005) K-ATP channels promote the differential degeneration of dopaminergic midbrain neurons. Nat Neurosci 8:1742–1751. doi: 10.1038/nn1570 PubMedCrossRefGoogle Scholar
  26. 26.
    Parker WD Jr, Swerdlow RH (1998) Mitochondrial dysfunction in idiopathic Parkinson disease. Am J Hum Genet 62:758–762. doi: 10.1086/301812 PubMedCrossRefGoogle Scholar
  27. 27.
    Schapira AHV, Cooper JM, Dexter D, Clark JB, Jenner P, Marsden CD (1990) Mitochondrial complex I deficiency in Parkinson’s disease. J Neurochem 54:823–827. doi: 10.1111/j.1471-4159.1990.tb02325.x PubMedCrossRefGoogle Scholar
  28. 28.
    Mazzio EA, Reams RR, Slimaqn KFA (2004) The role of oxidative stress, impaired glycolysis and mitochondrial respiratory redox failure in the cytotoxic effects of 6-hydroxydopamine in vitro. Brain Res 1004:29–44. doi: 10.1016/j.brainres.2003.12.034 PubMedCrossRefGoogle Scholar
  29. 29.
    Glinka Y, Tipton K, Youdim M (1996) Nature of inhibition of mitochondrial respiratory complex I by 6-hydroxydopamine. J Neurochem 66:2004–2010PubMedCrossRefGoogle Scholar
  30. 30.
    Glinka Y, Tipton KF, Youdim MBH (1998) Mechanism of inhibition of mitochondrial respiratory complex I by 6-hydroxydopamine and its prevention by desferroxamine. Eur J Pharmacol 351:121–129. doi: 10.1016/S0014-2999(98)00279-9 PubMedCrossRefGoogle Scholar
  31. 31.
    Tiffany-Castiglioni E, Saneto RP, Proctor PH, Perez-Polo R (1982) Participation of active oxygen species in 6-hydroxydopamine toxicity to a human neuroblastoma cell line. Biochem Pharmacol 31:181–188. doi: 10.1016/0006-2952(82)90208-8 PubMedCrossRefGoogle Scholar
  32. 32.
    Simantov R, Blinder E, Ratovitski T, Tauber M, Gabbay M, Porat S (1996) Dopamine-induced apoptosis in human neuronal cells: inhibition by nucleic acids antisense to the dopamine transporter. Neuroscience 74:39–50. doi: 10.1016/0306-4522(96)00102-9 PubMedCrossRefGoogle Scholar
  33. 33.
    Storch A, Kaftan A, Burkhardt K, Schwarz J (2000) 6-Hydroxydopamine toxicity towards human SH-SY5Y dopaminergic neuroblastoma cells: independent of mitochondrial energy metabolism. J Neural Transm 107:281–293. doi: 10.1007/s007020050023 PubMedCrossRefGoogle Scholar
  34. 34.
    Asanuma M, Hirata H, Cadet JL (1998) Attenuation of 6-hydroxydopamine-induced dopaminergic nigrostriatal lesions in superoxide dismutase transgenic mice. Neuroscience 85:907–917. doi: 10.1016/S0306-4522(97)00665-9 PubMedCrossRefGoogle Scholar
  35. 35.
    Kabuto H, Yokoi I, Iwata-Ichikawa E, Ogawa N (1999) EPC-K1, A hydroxyl radical scavenger, prevents 6-hydroxydopamine-induced dopamine depletion in the mouse striatum by up-regulation of catalase activity. Neurochem Res 24:1543–1548. doi: 10.1023/A:1021152115752 PubMedCrossRefGoogle Scholar
  36. 36.
    Barkats M, Millecamps S, Bilang-Bleuel A, Mallet J (2002) Neuronal transfer of the human Cu/Zn superoxide dismutase gene increases the resistance of dopaminergic neurons to 6-hydroxydopamine. J Neurochem 82:101–109. doi: 10.1046/j.1471-4159.2002.00952.x PubMedCrossRefGoogle Scholar
  37. 37.
    Hanrott K, Gudmunsen L, O’Neill MJ, Wonnacott S (2006) 6-Hydroxydopamine-induced apoptosis is mediated via extracellular auto-oxidation and caspase 3-dependent activation of protein kinase C delta. J Biol Chem 281:5373–5382. doi: 10.1074/jbc.M511560200 PubMedCrossRefGoogle Scholar
  38. 38.
    Choi W-S, Yoon S-Y, Oh TH, Choi E-J, O’Malley KL, Oh YJ (1999) Two distinct mechanisms are involved in 6-hydroxydopamine- and MPP+-induced dopaminergic neuronal cell death: role of caspases, ROS, and JNK. J Neurosci Res 57:86–94. doi: 10.1002/(SICI)1097-4547(19990701)57:1<86::AID-JNR9>3.0.CO;2-E PubMedCrossRefGoogle Scholar
  39. 39.
    Saito Y, Nishio K, Ogawa Y, Kinumi T, Yoshida Y, Masuo Y, Niki E (2007) Molecular mechanisms of 6-hydroxydopamine-induced cytotoxicity in PC12 cells: involvement of hydrogen peroxide-dependent and -independent action. Free Radic Biol Med 42:675–685. doi: 10.1016/j.freeradbiomed.2006.12.004 PubMedCrossRefGoogle Scholar
  40. 40.
    Izumi Y, Sawada H, Sakka N, Yamamoto N, Kume T, Katsuki H, Shimohama S, Akaike A (2005) p-Quinone mediates 6-hydroxydopamne-induced dopaminergic neuronal death and ferrous iron accelerates the conversion of p-quinone into melanin extracellularly. J Neurosci Res 79:849–860. doi: 10.1002/jnr.20382 PubMedCrossRefGoogle Scholar
  41. 41.
    Rosenthal RE, Hamud F, Fiskum G, Varghese PJ, Sharpe S (1987) Cerebral schemia and perfusion: prevention of brain mitochondrial injury by lidofalzine. J Cereb Blood Flow Metab 7:752–758PubMedCrossRefGoogle Scholar
  42. 42.
    Markwell MAK, Haas SM, Bieber LL, Tolbert NE (1978) A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 87:206–210. doi: 10.1016/0003-2697(78)90586-9 PubMedCrossRefGoogle Scholar
  43. 43.
    Chance B, Williams GR (1956) Respiratory enzymes in oxidative phosphorylation. VI. The effects of adenosine diphosphate on azide-treated mitochondria. J Biol Chem 221:477–489PubMedGoogle Scholar
  44. 44.
    Zhang S, Fu J, Zhou Z (2004) In vitro effect of manganese chloride exposure on reactive oxygen species generation and respiratory chain complexes activities of mitochondria isolated from rat brain. Toxicol Vitro 18:71–77. doi: 10.1016/j.tiv.2003.09.002 CrossRefGoogle Scholar
  45. 45.
    Brzozowski MJ, Alcantara SL, Iravani MM, Rose S, Jenner P (2011) The effect of nNOS inhibitors on toxin-induced cell death in dopaminergic cell lines depends on the extent of enzyme expression. Brain Res 1404:21–30. doi: 10.1016/j.brainres.2011.05.063 PubMedCrossRefGoogle Scholar
  46. 46.
    Heikkila RE, Cohen G (1973) 6-Hydroxydopamine: evidence for superoxide radical as an oxidative intermediate. Science 181:456–457. doi: 10.1126/science.181.4098.456 PubMedCrossRefGoogle Scholar
  47. 47.
    Ossola B, Kääräinen TM, Raasmaja A, Männistö PT (2008) Time-dependent protective and harmful effects of quercetin on 6-OHDA-induced toxicity in neuronal SH-SY5Y cells. Toxicology 250:1–8. doi: 10.1016/j.tox.2008.04.001 PubMedCrossRefGoogle Scholar
  48. 48.
    Halliwell B (2006) Oxidative stress and neurodegeneration: where are we now? J Neurochem 97:1634–1658. doi: 10.1111/j.1471-4159.2006.03907.x PubMedCrossRefGoogle Scholar
  49. 49.
    Bové J, Perier C (2012) Neurotoxin-based models of Parkinson’s disease. Neuroscience 211:51–76. doi: 10.1016/j.neuroscience.2011.10.057 PubMedCrossRefGoogle Scholar
  50. 50.
    Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph, and high-resolution respirometry to assess mitochondrial function. In: Dyens J, Will Y (eds) Drug-induced mitochondrial dysfunction. Wiley, Hoboken, pp 327–352Google Scholar
  51. 51.
    Berman SB, Hastings TG (1999) Dopamine oxidation alters mitochondrial respiration and induces permeability transition in brain mitochondria: implications for Parkinson’s disease. J Neurochem 73:1127–1137. doi: 10.1046/j.1471-4159.1999.0731127.x PubMedCrossRefGoogle Scholar
  52. 52.
    Masini A, Ceccarelli-Stanzani D, Muscatello U (1983) The effect of oligomycin on rat liver mitochondria respiring in state 4. FEBS Lett 160:137–140. doi: 10.1016/0014-5793(83)80953-3 PubMedCrossRefGoogle Scholar
  53. 53.
    Pryor WA (1986) Oxy-radicals and related species: their formation, lifetimes, and reactions. Ann Rev Physiol 48:657–667. doi: 10.1146/annurev.physiol.48.1.657 CrossRefGoogle Scholar
  54. 54.
    Enochs WS, Sarna T, Zecca L, Riley PA, Swartz HM (1994) The roles of neuromelanin, binding of metal ions, and oxidative cytotoxicity in the pathogenesis of Parkinson’s disease: a hypothesis. J Neural Transm 7:83–100. doi: 10.1007/BF02260963 CrossRefGoogle Scholar
  55. 55.
    Linert W, Herlinger E, Jameson RF, Kienzl E, Jellinger K, Youdim MBH (1996) Dopamine, 6-hydroxydopamine, iron, and dioxygen-their mutual interactions and possible implication in the development of Parkinson’s disease. Biochim Biophys Acta 1316:160–168. doi: 10.1016/0925-4439(96)00020-8 PubMedCrossRefGoogle Scholar
  56. 56.
    Tatsuta T, Langer T (2008) Quality control of mitochondria: protection against neurodegeneration and ageing. EMBO J 27:306–314. doi: 10.1038/sj.emboj.7601972 PubMedCrossRefGoogle Scholar
  57. 57.
    Aikens J, Dix JA (1991) Perhydroxyl radical (HOO·) initiated lipid peroxidation. The role of fatty acid hydroperoxides. J Biol Chem 266:15091–15098PubMedGoogle Scholar
  58. 58.
    Gebicki S, Gebicki JM (1993) Formation of peroxides in amino acids and proteins exposed to oxygen free radicals. Biochem J 289:743–749PubMedGoogle Scholar
  59. 59.
    Fu S, Gebicki S, Jessup W, Gebicki JM, Dean RT (1995) Biological fate of amino acid, peptide and protein hydroperoxides. Biochem J 311:821–827PubMedGoogle Scholar
  60. 60.
    Hermida-Ameijeiras A, Méndez-Álvarez E, Sánchez-Iglesias S, Sanmartín-Suárez C, Soto-Otero R (2004) Autoxidation and MAO-mediated metabolism of dopamine as a potential cause of oxidative stress: role of ferrous and ferric ions. Neurochem Int 45:103–116. doi: 10.1016/j.neuint.2003.11.018 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Javier Iglesias-González
    • 1
  • Sofía Sánchez-Iglesias
    • 1
  • Estefanía Méndez-Álvarez
    • 1
  • Sarah Rose
    • 2
  • Atsuko Hikima
    • 2
  • Peter Jenner
    • 2
  • Ramón Soto-Otero
    • 1
  1. 1.Group of Neurochemistry for Parkinson’s Disease, Department of Biochemistry and Molecular Biology, Faculty of MedicineUniversity of Santiago de CompostelaSantiago de CompostelaSpain
  2. 2.Neurodegenerative Diseases Research Group, Institute of Pharmaceutical SciencesKing’s College LondonLondonUK

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