Neurochemical Research

, Volume 32, Issue 11, pp 1906–1918 | Cite as

Metabolic and Antioxidant System Alterations in an Astrocytoma Cell Line Challenged with Mitochondrial DNA Deletion

  • Alfred Orina Isaac
  • Vikas V. Dukhande
  • James C. K. LaiEmail author
Original Paper


Oxidative stress can induce mitochondrial dysfunction, mitochondrial DNA (mtDNA) depletion, and neurodegeneration, although the underlying mechanisms are poorly understood. The major mitochondrial antioxidant system that protects cells consists of manganese superoxide dismutase (MnSOD), glutathione peroxidase (GPx) and glutathione (GSH). To investigate the putative adaptive changes in antioxidant enzyme protein expression and targeting to mitochondria as mtDNA depletion occurs, we progressively depleted U87 astrocytoma cells of mtDNA by chronic treatment with ethidium bromide (EB, 50 ng/ml). Cellular MnSOD protein expression was markedly increased in a time-related manner while that of GPx showed time-related decreases. The mtDNA depletion also altered targeting or subcellular distribution of GPx, suggesting the importance of intact mtDNA in mitochondrial genome-nuclear genome signaling/communication. Cellular NADP+-ICDH activity also showed marked, time-related increases while their GSH content decreased. Thus, our findings suggest that interventions to elevate MnSOD, GPx, NADP+-ICDH, and GSH levels may protect brain cells from oxidative stress.


Astrocytoma mtDNA Neurodegeneration Astrocytes Oxidative stress Mitochondrial genome-nuclear genome signaling 



Alzheimer’s disease




Bicinchoninic acid


Bovine serum albumin


Dulbecco’s modified essential medium


Electron transport chain


Ethidium bromide


Glutathione peroxidase




N-2 hydroxyethylpiperazine-N`-2 ethane sulfonic acid


Isocitrate dehydrogenase


Lactate dehydrogenase


Manganese superoxide dismutase


Mitochondrial DNA


Nicotinamide adenine dinucleotide phosphate


Nuclear DNA


Polyvinylidene fluoride


Parkinson’s disease


Reactive oxygen species


Sodium dodecyl sulfate


Tricarboxylic acid


A human astrocytoma cell line



Our studies were supported by a grant from Idaho Biomedical Research Infrastructure Network (NIH NCRR BRINIP20RR016454), and a small project grant from the Mountain States Tumor and Medical Research Institute.


  1. 1.
    Ikebe S, Tanaka M, Ohno K, Sato W, Hattori K, Kondo T, Mizuno Y, Ozawa T (1990) Increase in deleted mitochondrial DNA in the striatum in Parkinson’s disease and senescence. Biochem Biophys Res Commun 170(3):1044–1048PubMedCrossRefGoogle Scholar
  2. 2.
    Copeland WC (2002) Mitochondrial DNA Methods and Protocols. Methods Mol Biol 197:5–58Google Scholar
  3. 3.
    Zeviani M, Moraes CT, DiMauro S, Nakase H, Bonilla E, Schon EA, Rowland LP (1988) Deletions of mitochondrial DNA in Kearns–Sayre syndrome. Neurology 38:1339–1346PubMedGoogle Scholar
  4. 4.
    Nekhaeva E, Bodyyak ND, Kraytsberg Y, McGrath SB, Van Orsouw NJ, Pluzhnikoy A, Wei JY, Vijg J, Khrapko K (2002) Clonally expanded mtDNA mutations are abundant in individual cells of human tissues. Proc Natl Acad Sci USA 99:5521–5526PubMedCrossRefGoogle Scholar
  5. 5.
    Arnaudo E, Dalakas M, Shanske S, Moraes CT, DiMauro S, Schon EA (1991) Depletion of mtDNA in AIDS patients with zidovudine-induced myopathy. Lancet 337:508–510PubMedCrossRefGoogle Scholar
  6. 6.
    Schapira AH, Copper JM, Dexter D, Clark JB, Jenner P, Marsden CD (1990) Mitochondrial complex I deficiency in Parkinson’s disease. J Neurochem 54:823–827PubMedCrossRefGoogle Scholar
  7. 7.
    Jenner P, Dexter DT, Sian J, Shapira AH, Marsden CD (1992) Oxidative stress as a cause of nigral cell death in Parkinson’s disease and incidental Lewy body disease. The royal kings and queen’s parkinson’s disease research group. Ann Neurol 32:S82–S87PubMedCrossRefGoogle Scholar
  8. 8.
    Heales SJ, Bolanos JP (2002) Impairment of brain mitochondrial function by reactive nitrogen species. The role of glutathione in dictating susceptibility. Neurochem Int 40:469–474PubMedCrossRefGoogle Scholar
  9. 9.
    Bolanos JP, Almeida A, Stewart V, Peachen S, Land JM, Clark JB, Heales SJ (1997) Nitric oxide mediated mitochondrial damage in the brain: mechanisms and implications for neurodegenerative diseases. J Neurochem 68(6):2227–2240PubMedCrossRefGoogle Scholar
  10. 10.
    Heales SJ, Lam AA, Duncan AJ, Land JM (2004) Neurodegeneration or Neuroprotection: The pivotal Role of Astrocytes. Neurochem Res 29(3):513–519PubMedCrossRefGoogle Scholar
  11. 11.
    Torreilles F, Salman-Tabcheh S, Guerin M, Torreilles J (1999) Neurodegenerative disorders: the role of peroxynitrite. Brain Res Rev 30:153–163PubMedCrossRefGoogle Scholar
  12. 12.
    Estevez AG, Jordan J (2002) Nitric oxide and superoxide, a deadly cocktail. Ann NY Acad Sci 962:207–211PubMedCrossRefGoogle Scholar
  13. 13.
    Smith MA, Richey Harris PL, Sayre LM, Beckman JS, Perry G (1997) Widespread peroxynitrite-mediated damage in Alzheimers disease. J Neurosci 17:2653–2657PubMedGoogle Scholar
  14. 14.
    Vodovoltz Y, Lucia MS, Flanders KC, Chesler L, Xie OW, Smith TW, Weidner J, Mumford R, Webber R, Nathan C, Roberts AB, Lippa CF, Sporn MB (1996) Inducible nitric oxide synthase in tangle-bearing neurons of patients with Alzheimers disease. J Exp Med 184:1425–1433CrossRefGoogle Scholar
  15. 15.
    Kish SJ, Bergon C, Rajput A, Dozic S, Mastrogiacomo E, Chang LJ, Wilson JM, Distefano LM, Nobrega JN (1992) Brain cytochrome oxidase in Alzheimer’s disease. J Neurochem 59:776–779PubMedCrossRefGoogle Scholar
  16. 16.
    Casley CS, Canevari L, Land JM, Clark JB, Sharpe MA (2002) Beta-amyloid inhibits integrated mitochondrial respiration and key enzyme activities. J Neurochem 80:91–100PubMedCrossRefGoogle Scholar
  17. 17.
    Wallace DC (1992) Mitochondrial diseases in man and mouse. Science 283(5407):1482–1488CrossRefGoogle Scholar
  18. 18.
    Bandy B, Davidson AJ (1990) Mitochondrial mutations may increase oxidative stress: implications for carcinogenesis and aging? Free radicals Biol Med 8(6):523–539CrossRefGoogle Scholar
  19. 19.
    Vogel R, Wiesinger H, Hamprecht B, Dringen R (1999) The regeneration of reduced gluthathione in rat forebrain mitochondria identifies metabolic pathways providing the NADPH required. Neurosci Lett 275:97–100PubMedCrossRefGoogle Scholar
  20. 20.
    Jo SH, Son MK, Koh HJ, Lee SM, Song IH, Kim YO, Lee YS, Jeong KS, Kim WB, Park JW, Song BJ, Huh TL, Huh TL (2001) Control of mitochondrial redox balance and cellular defense against oxidative damage by mitochondrial NADP+-dependent isocitrate dehydrogenase. J Biol Chem 276:16168–16176PubMedCrossRefGoogle Scholar
  21. 21.
    Munich T, Yokota S, Dringen R (2003) Cytosolic and mitochondrial isoforms of NADP+-dependent isocitrate dehydrogenases are expressed in cultured rat neurons, astrocytes, oligodenrocytes and microgial cells. J Neurochem 86:605–614CrossRefGoogle Scholar
  22. 22.
    Halliwell B, Gutteridge JM (1999) Antioxidant defenses. Free radicals in biology and medicine, 3rd edn. Clarendon Press, Oxford, pp 200–216Google Scholar
  23. 23.
    Sun J, Folk D, Bradley T, Tower J (2004) Induced over-expression of mitochondrial MnSOD extends life span of adult Drosophila melanogaster. Genetics 161:661–672Google Scholar
  24. 24.
    Li Y, Huang T, Carlson EJ, Melov S, Ursell PC, Olson JL, Noble LJ, Yoshimura MP, Berger C, Chan PH, Wallace DC, Epstein CJ (1995) Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutase. Nat Genet 11:376–381PubMedCrossRefGoogle Scholar
  25. 25.
    Van Remmen H, Ikeno Y, Hamilton M, Pahlavani M, Wolf N, Thorpe SR, Alderson NL, Baynes JW, Epstein CJ, Huang TT, Nelson J, Strong R, Richardson A (2003) Life-long reduction in MnSOD activity results in increased DNA damage and higher incidence of cancer but does not accelerate aging. Physiol Genomics 16:29–37PubMedCrossRefGoogle Scholar
  26. 26.
    Hinerfeld D, Traini MD, Weinberger RP, Cochran B, Doctrow SR, Harry J, Melov S (2004) Endogenous mitochondrial oxidative stress: neurodegeneration, proteomic analysis, speficic respiratory chain defects, and efficacious antioxidant therapy in superoxide dismutase 2 null mice. J Neurochem 88:657–667PubMedCrossRefGoogle Scholar
  27. 27.
    Melov S, Doctrow SR, Schneider JA, Haberson J, Patel M, Coskun PE, Huffman K, Wallace DC, Malfroy B (2001) Lifespan extension and rescue of spongiform encephalopathy in superoxide dismutase 2 nullizygous mice treated with superoxide dismutase-catalase mimetics. J Neurosci 21:8348–8353PubMedGoogle Scholar
  28. 28.
    Hoehn B, Yenari MA, Sapolsky RM, Steinberg GK (2003) Glutathione peroxidase overexpression inhibits cytochrome C release and proapoptotic mediators to protect neurons from experimental stroke. Stroke 34(10):2489–2494PubMedCrossRefGoogle Scholar
  29. 29.
    King MP, Attardi G (1989) Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation. Science 246:500–503PubMedCrossRefGoogle Scholar
  30. 30.
    Lai JC, Walsh JM, Dennis SC, Clark JB (1977) Synaptic and non-synaptic mitochondria from rat brain: isolation and characterization. J Neurochem 28(3):625–631PubMedCrossRefGoogle Scholar
  31. 31.
    Griffith OW (1980) Determination of Glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal Biochem 106:207–212PubMedCrossRefGoogle Scholar
  32. 32.
    Clark JB, Lai JC (1989) Glycolytic, Tricarboxylic acid cycle and related enzymes in brain. In: Boulton AA, Baker GB, Butterworth RF (eds) Neuromethods, vol 11. Humana press, Clifton NJ, pp 233–281Google Scholar
  33. 33.
    Chan PH (1996) Role of oxidants in ischemic brain damage. Stroke 27:1124–1129PubMedGoogle Scholar
  34. 34.
    Hudson EK, Hogue BA, Souza-Pinto NC, Croteau DL, Anson RM, Bohr VA, Hansford RG (1998) Age-associated change in mitochondrial DNA damage. Free Radic Res 29(6):573–579PubMedCrossRefGoogle Scholar
  35. 35.
    Murakami K, Kondo T, Kawase M, Li Y, Sato S, Chen SF, Chan PH (1998) Mitochondrial susceptibility to oxidative stress exacerbates cerebral infarction that follows permanent focal cerebral ischemia in mutant mice with manganese superoxide dismutase deficiency. J Neurosci 18:205–213PubMedGoogle Scholar
  36. 36.
    Stuart JA, Hashiguchi K, Wilson III DM, Copeland WC, Souza-Pinto NC, Bohr VA (2004) DNA base excision repair activities and pathway function in mitochondrial and cellular lysates from cells lacking mitochondrial DNA. Nucleic Acids Res 32(7):2181–2192PubMedCrossRefGoogle Scholar
  37. 37.
    Wallace DC (2002) Animal models for mitochondrial disease. In: Copeland WC (ed) Mitochondrial DNA: methods & protocols, vol 197. Humana Press, Totowa, NJ, pp 3–54Google Scholar
  38. 38.
    Desjardins P, Frost E, Morais R (1985) Ethidium bromide-induced loss of mitochondrial DNA from primary chicken embryo fibroblasts. Mol Cell Biol 5:1163–1169PubMedGoogle Scholar
  39. 39.
    Wiseman A, Attardi G (1978) Reversible tenfold reduction in mitochondria DNA content of human cells treated with ethidium bromide. Mol Gen Genet 167:51–63PubMedGoogle Scholar
  40. 40.
    Attardi G, Shatz G (1988) Biogenesis of mitochondria. Ann Rev Cell Biol 4:289–333PubMedGoogle Scholar
  41. 41.
    Liao X, Butow RA (1993) RTG1 and RTG2: two yeast genes required for a novel path of communication from mitochondria to the nucleus. Cell 72:61–71PubMedCrossRefGoogle Scholar
  42. 42.
    Parikh VS, Morgan MM, Scott R, Clements LS, Butow RA (1987) The mitochondrial genotype can influence nuclear gene expression in yeast. Science 235:576–580PubMedCrossRefGoogle Scholar
  43. 43.
    Davis AF, Ropp PA, Clayton DA, Copeland WC (1996) Mitochondrial DNA polymerase gamma is expressed and translated in the absence of mitochondrial DNA maintenance and replication. Nucleic Acids Res 24(14):2753–2759PubMedCrossRefGoogle Scholar
  44. 44.
    Votyakova TV, Reynolds IJ (2001) DeltaPsi(m)-Dependent and -independent production of reactive oxygen species by rat brain mitochondria. J Neurochem 79(2):266–277PubMedCrossRefGoogle Scholar
  45. 45.
    Loschen G, Azzi A, Flohe L (1973) Mitochondrial H2O2 formation at site II. Hoppe Seylers Z Physiol Chem 354(7):791–794PubMedGoogle Scholar
  46. 46.
    Sipos I, Tretter L, Adam-Vizi V (2003) Quantitative relationship between inhibition of respiratory complexes and formation of reactive oxygen species in isolated nerve terminals. J Neurochem 84(1):112–118PubMedCrossRefGoogle Scholar
  47. 47.
    Bywood PT, Johnson SM (2003) Mitochondrial complex inhibitors preferentially damage substantia nigra dopamine neurons in rat brain slices. Exp Neurol 179:47–59PubMedCrossRefGoogle Scholar
  48. 48.
    Ikebe S, Tanaka M, Ohno K, Sato W, Hattori K, Kondo T, Mizuno Y, Ozawa T (1990) Increase in deleted mitochondrial DNA in the striatum in Parkinson’s disease and senescence. Biochem Biophys Res Commun 170(3):1044–1048PubMedCrossRefGoogle Scholar
  49. 49.
    Jo SH, Son MK, Koh HJ, Lee SM, Song IH, Kim YO, Lee YS, Jeong KS, Kim WB, Park JW, Song BJ, Huh TL (2001) Control of mitochondrial redox balance and cellular defense against oxidative damage by mitochondrial NADP+-dependent isocitrate dehydrogenase. J Biol Chem 276:16168–16176PubMedCrossRefGoogle Scholar
  50. 50.
    Lee HJ, Yang SE, Park WJ (2003) Inactivation of NADP+-dependent isocitrate. dehydrogenase by peroxynitrite. Implications for cytotoxicity and alcohol-induced liver injury. J Biol Chem 278(51):51360–51371PubMedCrossRefGoogle Scholar
  51. 51.
    Arai M, Imai H, Koumura T, Yoshida T, Emoto K, Umeda M, Chiba N, Nakagawa Y (1999) Mitochondrial phospholipid hydroperoxide glutathione peroxidase plays a major role in preventing oxidative injury to cells. J Biol Chem 274:4924–4933PubMedCrossRefGoogle Scholar
  52. 52.
    MacMillan-Crow LA, Crow JP, Kerby JD, Beckman JS, Thompson JA (1996) Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts. Proc Natl Acad Sci USA 93:11853–11858PubMedCrossRefGoogle Scholar
  53. 53.
    Savvides SN, Scheiwein M, Bohme CC, Arteel GE, Karplus PA, Becker K, Schirmer RH (2002) Crystal structure of the antioxidant enzyme glutathione reductase inactivated by peroxynitrite. J Biol Chem 277:2779–2784PubMedCrossRefGoogle Scholar
  54. 54.
    Dukhande VV, Malthankar-Phatak GH, Hugus JJ, Daniels CK, Lai JC (2006) manganese-induced neurotoxicity is differentially enhanced by glutathione depletion in astrocytoma and neuroblastoma cells. Neurochem Res 31(11):1349–1357PubMedCrossRefGoogle Scholar
  55. 55.
    Pong K, Doctrow SR, Baudry M (2000) Prevention of 1-methyl-4-phenylpyridinium- and 6-hydroxydopamine-induced nitration of tyrosine hydroxylase and neurotoxicity by EUK-134, a superoxide dismutase and catalase mimetic, in cultured dopaminergic neurons. Brain Res 881:182–189PubMedCrossRefGoogle Scholar
  56. 56.
    Isaac AO, Kawikova I, Bothwell AL, Daniels CK, Lai JC (2006) Manganese Treatment modulates the expression of peroxisome proliferator-activated receptors in astrocytoma and neuroblastoma cells. Neurochem Res 31(11):1305–1316PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Alfred Orina Isaac
    • 1
  • Vikas V. Dukhande
    • 1
  • James C. K. Lai
    • 1
    • 2
    Email author
  1. 1.Department of Pharmaceutical SciencesCollege of Pharmacy and Biomedical Research Institute, Idaho State UniversityPocatelloUSA
  2. 2.Mountain States Tumor and Medical Research InstituteBoiseUSA

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