Skip to main content
SpringerLink
Log in

Ethanol-induced oxidative stress in rat astrocytes: role of HSP70

  • Published:
Cell Biology and Toxicology Aims and scope Submit manuscript

Abstract

Ethanol intake is associated with increase in lipid peroxidation and formation of reactive oxygen species in different cerebral areas, in neurons as well as in astrocytes. The latter's integrity is essential for the normal growth of neurons. In previous studies we observed, in different cerebral areas of both acutely and chronically ethanol-treated rats, correlation between ethanol-induced oxidative stress and the increased expression of HSP70 (70 kDa heat shock proteins), chaperonins having a protective and stabilizing effect on stress–induced cell injury. In this study we examined, in vitro, the role of HSP70 on chronically ethanol-treated rat astrocytes by transfection with an anti-HSP70 antisense oligonucleotide. The results show that treatment with ethanol, from 50 to 100 mmol/L, induces a dose-dependent increase in the production of reactive oxygen species and of HSP70 levels, together with an impairment of the respiratory chain activity and a decrease in cell viability. In addition, our data indicate a drastic reduction of cellular metabolism in HSP70-deprived astrocytes, particularly when these cells were also ethanol-treated. In fact, transfection with HSP70 antisense induced moderate oxidative damage in control astrocytes and, consequently, a drastic decrease in the viability of ethanol-treated cells, with the mitochondrial functionality being particularly affected. Our results confirm that heat shock proteins confer a survival advantage to the astrocytes, preventing oxidative damage and nuclear DNA damage as well, and suggest the development of new drugs exerting a cytoprotective role either in physiological, or pathological conditions.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Andreoli SP, Mallett CP, Bergstein JM. Role of gluathione in protecting endothelial cells against hydrogen peroxide oxidant injury J Lab Clin Med. 1986;108(3):190–8.

    PubMed  CAS  Google Scholar 

  • Aquino DA, Peng D, Lopez C, Farooq, M. The constitutive heat shock protein 70 is required for optimal expression of myelin basic protein during differentiation of oligodendrocytes. Neurochem Res. 1998;23:413–20.

    Article  PubMed  CAS  Google Scholar 

  • Bailey SM, Pietsh EC, Cunningham C. Ethanol stimulates the production of reactive oxygen species at mitochondrial complexes I and III. Free Radic Biol Med. 1999;27:891–900.

    Article  PubMed  CAS  Google Scholar 

  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54.

    Article  PubMed  CAS  Google Scholar 

  • Bukau B and Horwich AL. The HSP70 and HSP60 chaperone machines. Cell. 1998;92:351–66.

    Article  PubMed  CAS  Google Scholar 

  • Buzzard KA, Giaccia AJ, Killender M, Anderson RL. Heat shock protein 72 modulates pathways of stress-induced apoptosis. J Biol Chem. 1998;273:17147–53.

    Article  PubMed  CAS  Google Scholar 

  • Calabrese V, Renis M, Calderone A, Russo A, Barcellona ML and Rizza V. Stress proteins and SH-groups in oxidant-induced cell damage after acute ethanol administration in rat. Free Radic Biol Med. 1996;20:391–7.

    Article  PubMed  CAS  Google Scholar 

  • Calabrese V, Renis M, Calderone A, et al. Stress proteins and SH-groups in oxidant-induced cellular injury after chronic ethanol administration in rat. Free Radic Biol Med. 1998;24:1159–67.

    Article  PubMed  CAS  Google Scholar 

  • Cammer W, Tansey F, Abramovitz M, Ishigaki S, Listowsky I. Differential localization of glutathione-S-transferase Yp and Yb subunits in oligodendrocytes and astrocytes of rat brain. J Neurochem. 1989;52:876–83.

    PubMed  CAS  Google Scholar 

  • Chanvitayapongs S, Draczynska-Lusiak B, Sun AY. Amelioration of oxidative stress by antioxidants and resveratrol in PC12 cells. Neuro Rep. 1997;8:1499–502.

    CAS  Google Scholar 

  • Chen YC, Lin Shian SY, Lin JK. Involvement of heat shock protein 70 and P53 proteins in attenuation of UVC-induced apoptosis by thermal stress in hepatocellular carcinoma cells. Photochem Photobiol. 1999;70:78–86.

    Article  PubMed  CAS  Google Scholar 

  • Copin JC, Leding M, Tholey G. Free radicals scavenging systems of rat astroglias cells in primary culture: effects of anoxia and drug treatment. Neurochem Res. 1992;17:677–82.

    Article  PubMed  CAS  Google Scholar 

  • Diomede L, Colotta B, Piovana F, Re EJ, Modest M, Salmona M. Induction of apoptosis in human leukemic cells by the ether lipid 1-octadecyl-2-methyl-rac-glycero-3-phosphocoline: a possible basis for its selective action. Int J Cancer. 1993;53:124–30.

    PubMed  CAS  Google Scholar 

  • Esteve JM, Mompo J, Garcia De La Asuncion J, et al. Oxidative damage to mitochondrial DNA and glutathione oxidation in apoptosis. Studies “in vivo” and “in vitro”. FASEB J. 1999;13:1055–64.

    PubMed  CAS  Google Scholar 

  • Fonseca VA, Stone A, Munshi M, et al. Oxidative stress in diabetic macrovascular disease: does homocysteine play a role. South Med J. 1997;90:903–6.

    PubMed  CAS  Google Scholar 

  • French SW, Wong K, Jui L, Albano E, Hagbjork AL, Ingelman-Sundberg M. Effect of ethanol on cytochrome P450 2E1 (CYP2E1), lipid peroxidation, and serum protein adduct formation in relation to liver pathology pathogenesis. Exp Mol Pathol. 1993;58:61–75.

    Article  PubMed  CAS  Google Scholar 

  • Gabai VL, Meriin AB, Mosser DD, et al. HSP70 prevents activation of stress kinase. A novel pathway of cellular thermotolerance. J Biol Chem. 1997;272:18033–7.

    Article  PubMed  CAS  Google Scholar 

  • Gassen M, Youdim MB. The potential role of iron chelators in the treatment of Parkinson's disease and related neurological disorders. Pharmacol Toxicol. 1997;80:159–66.

    Article  PubMed  CAS  Google Scholar 

  • Good PF, Werner P, Hsu A, Olanow CW, Perl DP. Evidence of neuronal oxidative damage in Alzheimer's disease. Am J Pathol. 1996;149:21–8.

    PubMed  CAS  Google Scholar 

  • Holownia A. Ethanol-induced cell death in cultured rat astroglia. Neurotoxicol Teratol. 1997;19:141–6.

    Article  PubMed  CAS  Google Scholar 

  • Hunot S, Brugg B, Ricarp D, et al. Nuclear traslocation of NFκB is increased in dopaminergic neurons of patients with Parkinson disease. Proc Natl Acad Sci USA. 1997;94:7531–6.

    Article  PubMed  CAS  Google Scholar 

  • Jaattela M, Wissing D, Kokholm K, Kallunk T, Egebland M. HSP70 exerts its antiapoptotic function downstream of caspase-3-like proteases. EMBO J. 1998;17:6124–34.

    Article  PubMed  CAS  Google Scholar 

  • King TE. Preparation of succinate cytochrome c reductase and the cytochrome b-cl particle and reconstitution of succinate cytochrome c reductase. Methods Enzymol. 1967;10:216–25.

    CAS  Google Scholar 

  • Kowaltowski AJ, Vercesi AE. Mitochondrial damage induced by conditions of oxidative stress. Free Radic Biol Med. 1999;26:463–71.

    Article  PubMed  CAS  Google Scholar 

  • Laemli UK. Cleavage of the structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;277:680–5.

    Article  Google Scholar 

  • Montoliu C, Sancho-Tello H, Azorin I, et al. Ethanol increases cytochrome P-450 2E1 and induces oxidative stress in astrocytes. J Neurochem. 1995;65:2561–70.

    Article  PubMed  CAS  Google Scholar 

  • Morimoto RI. Cells in stress: transcriptional activation of heat shock genes. Science. 1993;259:1409–10.

    PubMed  CAS  Google Scholar 

  • Morimoto RI, Santoro G. Stress-inducible responses and heat shock proteins: new pharmacologic targets for cytoprotection. Nature Biotechnol. 1998;16:833–8.

    Article  CAS  Google Scholar 

  • Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55–63.

    Article  PubMed  CAS  Google Scholar 

  • Murphy TH, Baraban JM. Glutamate toxicity in immature cortical neurons precedes development of glutamate receptor currents. Dev Brain Res. 1990;57:146–50.

    Article  CAS  Google Scholar 

  • Nordmann R, Ribiere C, Rouach H. Implication of free radical mechanisms in ethanol-induced cellular injury. Free Radic Biol Med. 1992;12:219–40.

    Article  PubMed  CAS  Google Scholar 

  • Polla BS, Kantengwa S, Francois D, et al. Mithocondria are selective targets for the protective effects of heat shock against oxidative injury. Proc Natl Acad Sci USA. 1996;93:6458–63.

    Article  PubMed  CAS  Google Scholar 

  • Ragan CI, Wilson MT, Darley-Usmar VM, Lowe PN. Subfraction of mitochondria and isolation of the proteins of oxidative phosphorilation. In: Darley-Usmar VM, Rickwood D, Wilson MT, eds. Mithocondria, a pratical approach. London: IRL Press; 1987:79–112.

    Google Scholar 

  • Renis M, Di Giacomo C, Sorrenti V, et al. Heat shock proteins following rat cerebral ischemic/reperfusion episode: effect of ketamine. Biochem Mol Biol Int. 1994;33:345–54.

    PubMed  CAS  Google Scholar 

  • Renis M, Calabrese V, Russo A, Calderone A, Barcellona ML, Rizza V. Nuclear DNA strand breaks during ethanol-induced oxidative stress in rat brain. FEBS Lett. 1996;390:153–6.

    Article  PubMed  CAS  Google Scholar 

  • Renis M, Cardile V, Russo A, Campisi A, Collovà F. Glutamine synthetase activity and HSP70 levels in cultured rat astrocytes: effect of 1-octadecyl-2-methyl-rac-glycero-3-phosphocoline. Brain Res. 1998;783:143–50.

    Article  PubMed  CAS  Google Scholar 

  • Rossi A, Elia G, Santoro MG. 2-Cyclopenten-1-one, a new inducer of heat shock protein 70 with antiviral activity. J Biol Chem. 1996;271:32192–6.

    Article  PubMed  CAS  Google Scholar 

  • Sagara J, Miura K, Bannai S. Maintenance of neuronal glutathione by glial cells. J Neurochem. 1993;61:1672–6.

    PubMed  CAS  Google Scholar 

  • Sharp FR. Heat-shock protein protection. TINS. 1999;3:97–9.

    Google Scholar 

  • Shawn S, Jayatilleke E, Lieber CS. The effect of chronic alcohol feeding on lipid peroxidation in microsomes: lack of relationship to hydroxyl radical generation. 1984;118(1):233–8.

    Google Scholar 

  • Singh NP, Tice RR, Stephen RE, Schneider EL. A microgel electrophoresis technique for the direct quantification of DNA damage and repair in individual fibroblasts cultured on microscope slides. Mut Res. 1991;252:289–96.

    CAS  Google Scholar 

  • Vanella A, Avola R, Condorelli DF, et al. Antioxidant enzymatic activitics and resistance to oxidative stress in primary and subcultured rat astroglial cells. Int J Dev Neurosci. 1989;7:233–41.

    Article  PubMed  CAS  Google Scholar 

  • Volloch V, Mosser DD, Massie B, Sherman MY. Reduced thermotolerance in aged cells results from a loss of an hsp72-mediated control of JNK signaling pathway. Cell Stress Chaperones. 1998;3:265–71.

    PubMed  CAS  Google Scholar 

  • Wharton DC, Tzagoloff A. Cytochrome oxidase from beef heart mitochondria. Methods Enzymol. 1967;10:245–50.

    Article  CAS  Google Scholar 

  • Yenari MA, Fink SL, Sun GH, et al. Gene therapy with HSP72 is neuroprotective in rat models of stroke and epilepsy. Ann Neurol. 1998;44:584–91.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biological Chemistry, Medical Chemistry and Molecular Biology, Faculty of Pharmacy, University of Catania, Catania, Italy

    A. Russo, M. Palumbo, C. Scifo, M.L. Barcellona & M. Renis

  2. Department of Physiological Sciences, Faculty of Medicine, University of Catania, Catania, Italy

    V. Cardile

Authors
  1. A. Russo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. Palumbo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. C. Scifo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. V. Cardile
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. M.L. Barcellona
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. M. Renis
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Russo, A., Palumbo, M., Scifo, C. et al. Ethanol-induced oxidative stress in rat astrocytes: role of HSP70. Cell Biol Toxicol 17, 153–168 (2001). https://doi.org/10.1023/A:1011936313510

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1011936313510

Search

Navigation