Molecular Neurobiology

, Volume 54, Issue 1, pp 125–136 | Cite as

L-Ascorbate Protects Against Methamphetamine-Induced Neurotoxicity of Cortical Cells via Inhibiting Oxidative Stress, Autophagy, and Apoptosis

  • Ya-Ni Huang
  • Ling-Yu Yang
  • Jing-Ya Wang
  • Chien-Cheng Lai
  • Chien-Tsai Chiu
  • Jia-Yi WangEmail author


Methamphetamine (METH)-induced cell death contributes to the pathogenesis of neurotoxicity; however, the relative roles of oxidative stress, apoptosis, and autophagy remain unclear. L-Ascorbate, also called vitamin (Vit.) C, confers partial protection against METH neurotoxicity via induction of heme oxygenase-1. We further investigated the role of Vit. C in METH-induced oxidative stress, apoptosis, and autophagy in cortical cells. Exposure to lower concentrations (0.1, 0.5, 1 mM) of METH had insignificant effects on ROS production, whereas cells exposed to 5 mM METH exhibited ROS production in a time-dependent manner. We confirmed METH-induced apoptosis (by nuclear morphology revealed by Hoechst 33258 staining and Western blot showing the protein levels of pro-caspase 3 and cleaved caspase 3) and autophagy (by Western blot showing the protein levels of Belin-1 and conversion of microtubule-associated light chain (LC)3-I to LC3-II and autophagosome staining by monodansylcadaverine). The apoptosis as revealed by cleaved caspase-3 expression marked an increase at 18 h after METH exposure while both autophagic markers, Beclin 1 and LC3-II, marked an increase in cells exposed to METH for 6 and 24 h, respectively. Treating cells with Vit. C 30 min before METH exposure time-dependently attenuated the production of ROS. Vitamin C also attenuated METH-induced Beclin 1 and LC3-II expression and METH toxicity. Treatment of cells with Vit. C before METH exposure attenuated the expression of cleaved caspase-3 and reduced the number of METH-induced apoptotic cells. We suggest that the protective effect of Vit. C against METH toxicity might be through attenuation of ROS production, autophagy, and apoptosis.


Ascorbate Vitamin C Methamphetamine Oxidative stress Autophagy Apoptosis 



This work was supported in part by a grant (03C0720004A) to YNH from Taipei Medical University, Taiwan, and grants MOST-103-2321-B-038-002 and MOST-104-2320-B-038-057-MY3 from the Ministry of Sciences and Technology and MOHW104-TDU-B-212-124-001 from the Ministry of Health and Welfare surcharge of tobacco products, Taiwan, to JYW.


  1. 1.
    Cadet JL, Jayanthi S, Deng X (2003) Speed kills: cellular and molecular bases of methamphetamine-induced nerve terminal degeneration and neuronal apoptosis. FASEB J: Off Publ Fed Am Soc Exp Biol 17(13):1775–1788CrossRefGoogle Scholar
  2. 2.
    Riddle EL, Fleckenstein AE, Hanson GR (2006) Mechanisms of methamphetamine-induced dopaminergic neurotoxicity. AAPS J 8(2):E413–E418PubMedPubMedCentralCrossRefGoogle Scholar
  3. 3.
    Davidson C, Gow AJ, Lee TH, Ellinwood EH (2001) Methamphetamine neurotoxicity: necrotic and apoptotic mechanisms and relevance to human abuse and treatment. Brain Res Brain Res Rev 36(1):1–22PubMedCrossRefGoogle Scholar
  4. 4.
    Eisch AJ, Marshall JF (1998) Methamphetamine neurotoxicity: dissociation of striatal dopamine terminal damage from parietal cortical cell body injury. Synapse 30(4):433–445PubMedCrossRefGoogle Scholar
  5. 5.
    Deng X, Wang Y, Chou J, Cadet JL (2001) Methamphetamine causes widespread apoptosis in the mouse brain: evidence from using an improved TUNEL histochemical method. Brain Res Mol Brain Res 93(1):64–69PubMedCrossRefGoogle Scholar
  6. 6.
    Giovanni A, Liang LP, Hastings TG, Zigmond MJ (1995) Estimating hydroxyl radical content in rat brain using systemic and intraventricular salicylate: impact of methamphetamine. J Neurochem 64(4):1819–1825PubMedCrossRefGoogle Scholar
  7. 7.
    Jayanthi S, Ladenheim B, Cadet JL (1998) Methamphetamine-induced changes in antioxidant enzymes and lipid peroxidation in copper/zinc-superoxide dismutase transgenic mice. Ann N Y Acad Sci 844:92–102PubMedCrossRefGoogle Scholar
  8. 8.
    Stumm G, Schlegel J, Schafer T, Wurz C, Mennel HD, Krieg JC, Vedder H (1999) Amphetamines induce apoptosis and regulation of bcl-x splice variants in neocortical neurons. FASEB J: Off Publ Fed Am Soc Exp Biol 13(9):1065–1072Google Scholar
  9. 9.
    Jayanthi S, Deng X, Bordelon M, McCoy MT, Cadet JL (2001) Methamphetamine causes differential regulation of pro-death and anti-death Bcl-2 genes in the mouse neocortex. FASEB J: Off Publ Fed Am Soc Exp Biol 15(10):1745–1752CrossRefGoogle Scholar
  10. 10.
    Nopparat C, Porter JE, Ebadi M, Govitrapong P (2010) The mechanism for the neuroprotective effect of melatonin against methamphetamine-induced autophagy. J Pineal Res 49(4):382–389PubMedCrossRefGoogle Scholar
  11. 11.
    Chandramani Shivalingappa P, Jin H, Anantharam V, Kanthasamy A (2012) N-acetyl cysteine protects against methamphetamine-induced dopaminergic neurodegeneration via modulation of redox status and autophagy in dopaminergic cells. Park Dis 2012:424285Google Scholar
  12. 12.
    Li Y, Hu Z, Chen B, Bu Q, Lu W, Deng Y, Zhu R, Shao X et al (2012) Taurine attenuates methamphetamine-induced autophagy and apoptosis in PC12 cells through mTOR signaling pathway. Toxicol Lett 215(1):1–7PubMedCrossRefGoogle Scholar
  13. 13.
    Scherz-Shouval R, Shvets E, Fass E, Shorer H, Gil L, Elazar Z (2007) Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4. EMBO J 26(7):1749–1760PubMedPubMedCentralCrossRefGoogle Scholar
  14. 14.
    Kourtis N, Tavernarakis N (2009) Autophagy and cell death in model organisms. Cell Death Differ 16(1):21–30PubMedCrossRefGoogle Scholar
  15. 15.
    Shintani T, Klionsky DJ (2004) Autophagy in health and disease: a double-edged sword. Science 306(5698):990–995PubMedPubMedCentralCrossRefGoogle Scholar
  16. 16.
    Mizushima N, Levine B, Cuervo AM, Klionsky DJ (2008) Autophagy fights disease through cellular self-digestion. Nature 451(7182):1069–1075PubMedPubMedCentralCrossRefGoogle Scholar
  17. 17.
    Zeng X, Overmeyer JH, Maltese WA (2006) Functional specificity of the mammalian Beclin-Vps34 PI 3-kinase complex in macroautophagy versus endocytosis and lysosomal enzyme trafficking. J Cell Sci 119(Pt 2):259–270PubMedCrossRefGoogle Scholar
  18. 18.
    Maiuri MC, Le Toumelin G, Criollo A, Rain JC, Gautier F, Juin P, Tasdemir E, Pierron G et al (2007) Functional and physical interaction between Bcl-X(L) and a BH3-like domain in Beclin-1. EMBO J 26(10):2527–2539PubMedPubMedCentralCrossRefGoogle Scholar
  19. 19.
    Ichimura Y, Kirisako T, Takao T, Satomi Y, Shimonishi Y, Ishihara N, Mizushima N, Tanida I et al (2000) A ubiquitin-like system mediates protein lipidation. Nature 408(6811):488–492PubMedCrossRefGoogle Scholar
  20. 20.
    Rice ME (2000) Ascorbate regulation and its neuroprotective role in the brain. Trends Neurosci 23(5):209–216PubMedCrossRefGoogle Scholar
  21. 21.
    Hediger MA (2002) New view at C. Nat Med 8(5):445–446PubMedCrossRefGoogle Scholar
  22. 22.
    Kinuta Y, Kikuchi H, Ishikawa M, Kimura M, Itokawa Y (1989) Lipid peroxidation in focal cerebral ischemia. J Neurosurg 71(3):421–429PubMedCrossRefGoogle Scholar
  23. 23.
    Christen S, Schaper M, Lykkesfeldt J, Siegenthaler C, Bifrare YD, Banic S, Leib SL, Tauber MG (2001) Oxidative stress in brain during experimental bacterial meningitis: differential effects of alpha-phenyl-tert-butyl nitrone and N-acetylcysteine treatment. Free Radic Biol Med 31(6):754–762PubMedCrossRefGoogle Scholar
  24. 24.
    Huang YN, Wang JY, Lee CT, Lin CH, Lai CC (2012) L-ascorbate attenuates methamphetamine neurotoxicity through enhancing the induction of endogenous heme oxygenase-1. Toxicol Appl Pharmacol 265(2):241–252PubMedCrossRefGoogle Scholar
  25. 25.
    Rosenkranz AR, Schmaldienst S, Stuhlmeier KM, Chen W, Knapp W, Zlabinger GJ (1992) A microplate assay for the detection of oxidative products using 2′,7′-dichlorofluorescin-diacetate. J Immunol Methods 156(1):39–45PubMedCrossRefGoogle Scholar
  26. 26.
    Munafo DB, Colombo MI (2001) A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation. J Cell Sci 114(Pt 20):3619–3629PubMedGoogle Scholar
  27. 27.
    Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65(1–2):55–63PubMedCrossRefGoogle Scholar
  28. 28.
    Wang JY, Shum AY, Ho YJ (2003) Oxidative neurotoxicity in rat cerebral cortex neurons: synergistic effects of H2O2 and NO on apoptosis involving activation of p38 mitogen-activated protein kinase and caspase-3. J Neurosci Res 72(4):508–519PubMedCrossRefGoogle Scholar
  29. 29.
    Huang YN, Wu CH, Lin TC, Wang JY (2009) Methamphetamine induces heme. Toxicol Appl Pharmacol 240(3):315–326PubMedCrossRefGoogle Scholar
  30. 30.
    Higgins GC, Devenish RJ, Beart PM, Nagley P (2011) Autophagic activity in cortical neurons under acute oxidative stress directly contributes to cell death. Cell Mol Life Sci: CMLS 68(22):3725–3740PubMedCrossRefGoogle Scholar
  31. 31.
    Fernandez-Gajardo R, Matamala JM, Carrasco R, Gutierrez R, Melo R, Rodrigo R (2014) Novel therapeutic strategies for traumatic brain injury: acute antioxidant reinforcement. CNS Drugs 28(3):229–248PubMedCrossRefGoogle Scholar
  32. 32.
    Barr AM, Panenka WJ, MacEwan GW, Thornton AE, Lang DJ, Honer WG, Lecomte T (2006) The need for speed: an update on methamphetamine addiction. J Psychiatry Neurosci 31(5):301–313PubMedPubMedCentralGoogle Scholar
  33. 33.
    Cadet JL, Krasnova IN, Jayanthi S, Lyles J (2007) Neurotoxicity of substituted amphetamines: molecular and cellular mechanisms. Neurotox Res 11(3–4):183–202PubMedCrossRefGoogle Scholar
  34. 34.
    Beauvais G, Atwell K, Jayanthi S, Ladenheim B, Cadet JL (2011) Involvement of dopamine receptors in binge methamphetamine-induced activation of endoplasmic reticulum and mitochondrial stress pathways. PLoS One 6(12):e28946PubMedPubMedCentralCrossRefGoogle Scholar
  35. 35.
    Acikgoz O, Gonenc S, Kayatekin BM, Pekcetin C, Uysal N, Dayi A, Semin I, Gure A (2000) The effects of single dose of methamphetamine on lipid peroxidation levels in the rat striatum and prefrontal cortex. Eur Neuropsychopharmacol: J Eur Coll Neuropsychopharmacol 10(5):415–418CrossRefGoogle Scholar
  36. 36.
    Moszczynska A, Yamamoto BK (2011) Methamphetamine oxidatively damages parkin and decreases the activity of 26S proteasome in vivo. J Neurochem 116(6):1005–1017PubMedPubMedCentralCrossRefGoogle Scholar
  37. 37.
    Shin EJ, Bach JH, Nguyen TT, Nguyen XK, Jung BD, Oh KW, Kim MJ, Ko SK et al (2011) Gastrodia elata bl attenuates methamphetamine-induced dopaminergic toxicity via inhibiting oxidative burdens. Curr Neuropharmacol 9(1):118–121PubMedPubMedCentralCrossRefGoogle Scholar
  38. 38.
    Gloire G, Legrand-Poels S, Piette J (2006) NF-kappaB activation by reactive oxygen species: fifteen years later. Biochem Pharmacol 72(11):1493–1505PubMedCrossRefGoogle Scholar
  39. 39.
    Circu ML, Aw TY (2010) Reactive oxygen species, cellular redox systems, and apoptosis. Free Radic Biol Med 48(6):749–762PubMedPubMedCentralCrossRefGoogle Scholar
  40. 40.
    Cory S, Adams JM (2002) The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer 2(9):647–656PubMedCrossRefGoogle Scholar
  41. 41.
    Billen LP, Kokoski CL, Lovell JF, Leber B, Andrews DW (2008) Bcl-XL inhibits membrane permeabilization by competing with Bax. PLoS Biol 6(6):e147PubMedPubMedCentralCrossRefGoogle Scholar
  42. 42.
    Smith DJ, Ng H, Kluck RM, Nagley P (2008) The mitochondrial gateway to cell death. IUBMB Life 60(6):383–389PubMedCrossRefGoogle Scholar
  43. 43.
    Choi HJ, Yoo TM, Chung SY, Yang JS, Kim JI, Ha ES, Hwang O (2002) Methamphetamine-induced apoptosis in a CNS-derived catecholaminergic cell line. Mol Cells 13(2):221–227PubMedGoogle Scholar
  44. 44.
    Kanthasamy K, Gordon R, Jin H, Anantharam V, Ali S, Kanthasamy AG, Kanthasamy A (2011) Neuroprotective effect of resveratrol against methamphetamine-induced dopaminergic apoptotic cell death in a cell culture model of neurotoxicity. Curr Neuropharmacol 9(1):49–53PubMedPubMedCentralCrossRefGoogle Scholar
  45. 45.
    Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, Mizushima N, Packer M, Schneider MD et al (2005) Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell 122(6):927–939PubMedCrossRefGoogle Scholar
  46. 46.
    Chen H, Wu J, Zhang J, Fujita Y, Ishima T, Iyo M, Hashimoto K (2012) Protective effects of the antioxidant sulforaphane on behavioral changes and neurotoxicity in mice after the administration of methamphetamine. Psychopharmacology (Berl) 222(1):37–45CrossRefGoogle Scholar
  47. 47.
    Wagner GC, Carelli RM, Jarvis MF (1985) Pretreatment with ascorbic acid attenuates the neurotoxic effects of methamphetamine in rats. Res Commun Chem Pathol Pharmacol 47(2):221–228PubMedGoogle Scholar
  48. 48.
    Shin EJ, Suh SK, Lim YK, Jhoo WK, Hjelle OP, Ottersen OP, Shin CY, Ko KH et al (2005) Ascorbate attenuates trimethyltin-induced oxidative burden and neuronal degeneration in the rat hippocampus by maintaining glutathione homeostasis. Neuroscience 133(3):715–727PubMedCrossRefGoogle Scholar
  49. 49.
    Tronel C, Rochefort GY, Arlicot N, Bodard S, Chalon S, Antier D (2013) Oxidative stress is related to the deleterious effects of heme oxygenase-1 in an in vivo neuroinflammatory rat model. Oxid Med Cell Longev 2013:264935PubMedPubMedCentralCrossRefGoogle Scholar
  50. 50.
    Imuta N, Hori O, Kitao Y, Tabata Y, Yoshimoto T, Matsuyama T, Ogawa S (2007) Hypoxia-mediated induction of heme oxygenase type I and carbon monoxide release from astrocytes protects nearby cerebral neurons from hypoxia-mediated apoptosis. Antioxid Redox Signal 9(5):543–552PubMedCrossRefGoogle Scholar
  51. 51.
    Yao P, Nussler A, Liu L, Hao L, Song F, Schirmeier A, Nussler N (2007) Quercetin protects human hepatocytes from ethanol-derived oxidative stress by inducing heme oxygenase-1 via the MAPK/Nrf2 pathways. J Hepatol 47(2):253–261PubMedCrossRefGoogle Scholar
  52. 52.
    Das S, Fraga CG, Das DK (2006) Cardioprotective effect of resveratrol via HO-1 expression involves p38 map kinase and PI-3-kinase signaling, but does not involve NFkappaB. Free Radic Res 40(10):1066–1075PubMedCrossRefGoogle Scholar
  53. 53.
    Ryter SW, Otterbein LE, Morse D, Choi AM (2002) Heme oxygenase/carbon monoxide signaling pathways: regulation and functional significance. Mol Cell Biochem 234–235(1–2):249–263PubMedCrossRefGoogle Scholar
  54. 54.
    Bishop A, Cashman NR (2003) Induced adaptive resistance to oxidative stress in the CNS: a discussion on possible mechanisms and their therapeutic potential. Curr Drug Metab 4(2):171–184PubMedCrossRefGoogle Scholar
  55. 55.
    McDonagh AF (1990) Is bilirubin good for you? Clin Perinatol 17(2):359–369PubMedGoogle Scholar
  56. 56.
    Brouard S, Otterbein LE, Anrather J, Tobiasch E, Bach FH, Choi AM, Soares MP (2000) Carbon monoxide generated by heme oxygenase 1 suppresses endothelial cell apoptosis. J Exp Med 192(7):1015–1026PubMedPubMedCentralCrossRefGoogle Scholar
  57. 57.
    Moon MK, Choi BM, Oh GS, Pae HO, Kim JD, Oh H, Oh CS, Kim DH et al (2003) Catalposide protects Neuro 2A cells from hydrogen peroxide-induced cytotoxicity via the expression of heme oxygenase-1. Toxicol Lett 145(1):46–54PubMedCrossRefGoogle Scholar
  58. 58.
    Leon A, Le Foll I, Charriault-Marlangue C, Leprince J, Vaudry H, Gabriel C, Duval D (2003) Level of haem oxygenase does not obligatorily reflect the sensitivity of PC12 cells to an oxidative shock induced by glutathione depletion. J Neurochem 84(3):459–470PubMedCrossRefGoogle Scholar
  59. 59.
    Lee DW, Gelein RM, Opanashuk LA (2006) Heme-oxygenase-1 promotes polychlorinated biphenyl mixture aroclor 1254-induced oxidative stress and dopaminergic cell injury. Toxicol Sci: Off J Soc Toxicol 90(1):159–167CrossRefGoogle Scholar
  60. 60.
    Wu PH, Shen YC, Wang YH, Chi CW, Yen JC (2006) Baicalein attenuates methamphetamine-induced loss of dopamine transporter in mouse striatum. Toxicology 226(2–3):238–245PubMedCrossRefGoogle Scholar
  61. 61.
    Klongpanichapak S, Govitrapong P, Sharma SK, Ebadi M (2006) Attenuation of cocaine and methamphetamine neurotoxicity by coenzyme Q10. Neurochem Res 31(3):303–311PubMedCrossRefGoogle Scholar
  62. 62.
    Dong Y, Wang S, Zhang T, Zhao X, Liu X, Cao L, Chi Z (2013) Ascorbic acid ameliorates seizures and brain damage in rats through inhibiting autophagy. Brain Res 1535:115–123PubMedCrossRefGoogle Scholar
  63. 63.
    Mohammed BM, Fisher BJ, Kraskauskas D, Farkas D, Brophy DF, Fowler AA 3rd, Natarajan R (2013) Vitamin C: a novel regulator of neutrophil extracellular trap formation. Nutrients 5(8):3131–3151PubMedPubMedCentralCrossRefGoogle Scholar
  64. 64.
    Jiang K, Wang W, Jin X, Wang Z, Ji Z, Meng G (2015) Silibinin, a natural flavonoid, induces autophagy via ROS-dependent mitochondrial dysfunction and loss of ATP involving BNIP3 in human MCF7 breast cancer cells. Oncol Rep 33(6):2711–2718PubMedPubMedCentralGoogle Scholar
  65. 65.
    Sangani R, Periyasamy-Thandavan S, Pathania R, Ahmad S, Kutiyanawalla A, Kolhe R, Bhattacharyya MH, Chutkan N et al (2015) The crucial role of vitamin C and its transporter (SVCT2) in bone marrow stromal cell autophagy and apoptosis. Stem Cell Res 15(2):312–321PubMedPubMedCentralCrossRefGoogle Scholar
  66. 66.
    Cullen JJ (2010) Ascorbate induces autophagy in pancreatic cancer. Autophagy 6(3):421–422PubMedPubMedCentralCrossRefGoogle Scholar
  67. 67.
    Du J, Martin SM, Levine M, Wagner BA, Buettner GR, Wang SH, Taghiyev AF, Du C et al (2010) Mechanisms of ascorbate-induced cytotoxicity in pancreatic cancer. Clin Cancer Res: Off J Am Assoc Cancer Res 16(2):509–520CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Ya-Ni Huang
    • 1
  • Ling-Yu Yang
    • 2
  • Jing-Ya Wang
    • 2
  • Chien-Cheng Lai
    • 3
  • Chien-Tsai Chiu
    • 4
  • Jia-Yi Wang
    • 2
    • 5
    Email author
  1. 1.Department of NursingHsin Sheng Junior College of Medical Care and ManagementTaoyuanTaiwan
  2. 2.Graduate Institute of Medical Sciences and Department of Physiology, College of MedicineTaipei Medical UniversityTaipeiTaiwan
  3. 3.Division of Orthopedics, Department of SurgeryFar Eastern Memorial HospitalNew Taipei CityTaiwan
  4. 4.Department of NeurosurgeryEn Chu Kong HospitalNew Taipei CityTaiwan
  5. 5.Comprehensive Cancer CenterTaipei Medical UniversityTaipeiTaiwan

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