Antioxidant protection against oxidant-induced damage in cultured gastric mucosal cells

  • Hideyuki Hiraishi
  • Noriaki Yajima
  • Norihisa Yamaguchi
  • Motoo Ishida
  • Yoshihisa Katoh
  • Takashi Harada
  • Akira Terano
  • Kevin J. Ivey
Basic Approach Recent Advances in Peptic Ulcer Research-Quality of Ulcer Healing


Gastric epithelium is exposed not only to oxidants generated within the lumen, but also to those produced by ischemia/reperfusion. This study examined the mechanism(s) of oxidant-induced injury to cultured rat gastric mucosal cells, and characterized the antioxidant profile of these cells. Hydrogen peroxide (H2O2), generated by glucose oxidase, damaged cells dose-dependently, as assessed by increased leakage of labeled51Cr. Glucose oxidase-induced damage was prevented by exogenous catalase (but not by exogenous Superoxide dismutase). Chelation of cellular iron with desferrioxamine or phenanthroline specifically protected cells against H2O2, whereas binding of extracellular iron with apotransferrin failed to. Disruption of the glutathione redox cycle at three independent sites rendered cells less resistant to H2O2, whereas inhibition of cellular catalase did not result in sensitization of cells to H2O2. In conclusion, (1) oxidant injury induced by extracellular H2O2 is mediated by intracellular iron; (2) extracellular Superoxide is not involved in the damaging process; and (3) the glutathione redox cycle plays a principal role in detoxifying H2O2 as a cellular antioxidant in cultured gastric mucosal cells.


H202 Glucose Oxidase BCNU Desferrioxamine 51Cr Release 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Abbreviations used in this paper




buthionine sulfoximine


diethyl maleate


reduced glutathione


oxidized glutathione


hydrogen peroxide


hydroxyl radical


Superoxide anion


Superoxide dismutase


  1. 1.
    Freeman BA, Crapo JD. Biology of disease. Free radicals and tissue injury. Lab Invest 1982;26: 412–426.Google Scholar
  2. 2.
    Taylor AE, Martin DJ. Oxygen radicals and the microcirculation. Physiologist 1983;26: 152–155.PubMedGoogle Scholar
  3. 3.
    Granger DN, Rutili G, McCord JM. Superoxide radicals in feline intestinal ischemia. Gastroenterology 1981;81: 22–29.PubMedGoogle Scholar
  4. 4.
    Granger DN, Hollwarth ME, Parks DA. Ischemia-reperfusion injury: role of oxygen-derived free radicals. Acta Physiol Scand (Suppl) 1986;548: 47–63.Google Scholar
  5. 5.
    McCord JM. Oxygen-derived free radicals in postischemic tissue injury. N Eng J Med 1986;312: 159–163.Google Scholar
  6. 6.
    Haber F, Weiss J. The catalytic decomposition of hydrogen peroxide by iron salts. Proc R Soc Lond A Math Phys Sci 1934; 147: 332–351.Google Scholar
  7. 7.
    Halliwell B. Superoxide-dependent formation of hydroxyl radicals in the presence of iron salt is a feasible source of hydroxyl radicals in vivo. Biochem J 1982;205: 461–462.PubMedGoogle Scholar
  8. 8.
    Sutton HC, Winterbourn CC. On the participation of higher oxidation states of iron and copper in Fenton reactions. Free Radic Biol Med 1989;6: 53–60.PubMedCrossRefGoogle Scholar
  9. 9.
    Kasai H, Nishimura S. Hydroxylation of deoxyguanosine at the C-8 position by ascorbic acid and other reducing agents. Nucleic Acids Res 1984;12: 2137–2145.PubMedCrossRefGoogle Scholar
  10. 10.
    Van Steveninck J, Van Der Zee J, Dubbelman TMAR. Site-specific and bulk-phase generation of hydroxyl radicals in the presence of cupric ions and thiol compounds. Biochem J 1985; 232: 309–311.PubMedGoogle Scholar
  11. 11.
    Halliwell B, Gutteridge JMC. The importance of free radicals and acute catalytic metal ions in human diseases. Mol Aspects Med 1985;8: 89–193.PubMedCrossRefGoogle Scholar
  12. 12.
    Fridovich I. The biology of oxygen radicals. Science Wash DC 1978;201: 875–880.CrossRefGoogle Scholar
  13. 13.
    McCord JM. Free radicals and inflammation: protection of synovial fluid by Superoxide dismutase. Science Wash DC 1974;185: 529–531.CrossRefGoogle Scholar
  14. 14.
    Nayfield SG, Kent TH, Rodman NF. Gastrointestinal effects of acute ferrous sulfate poisoning in rats. Arch Pathol Lab Med 1976;100: 325–328.PubMedGoogle Scholar
  15. 15.
    Thomas EL, Bates KP, Jefferson MM. Hypothiocyanate ion: detection of the antimicrobial agent in human saliva. J Dent Res 1980;59: 1466–1472.PubMedGoogle Scholar
  16. 16.
    Carlsson J, Iwami Y, Yamada T. Hydrogen peroxide excretion by oral streptococci and effect of lactoperoxidase-thiocyanate-hydrogen peroxide. Infect Immun 1983;40: 70–80.PubMedGoogle Scholar
  17. 17.
    Cross CE, Halliwell B, Allen A. Antioxidant protection: a function of tracheobronchial and gastrointestinal mucus. Lancet 1984;1: 1328–1330.PubMedCrossRefGoogle Scholar
  18. 18.
    Nakayama T, Kodama M, Nagata C. Generation of hydrogen peroxide and Superoxide anion radical from cigarette smoke. Jpn J Cancer Res (Gann) 1984;75: 95–98.Google Scholar
  19. 19.
    Grisham MB, Hernandez LA, Granger DN. Xanthine oxidase and neutrophil infiltration in intestinal ischemia. Am J Physiol 1986; 251: G567-G574.PubMedGoogle Scholar
  20. 20.
    Boyd SC, Sasame HA, Boyd MR. High concentrations of glutathione in glandular stomach: possible implications for carcinogenesis. Science 1979;205: 1010–1012.CrossRefGoogle Scholar
  21. 21.
    Terano A, Ivey KJ, Stachura T, et al. Cell culture of rat gastric fundic mucosa. Gastroenterology 1982;83: 1280–1291.PubMedGoogle Scholar
  22. 22.
    Hiraishi H, Terano A, Ota S, et al. Effect of cimetidine on indomethacin-induced damage in cultured rat gastric mucosal cells: comparison with prostaglandin. J Lab Clin Med 1986;108: 608–615.PubMedGoogle Scholar
  23. 23.
    Terano A, Ota S, Mach T, et al. Prostaglandin protects against taurocholate-induced damage to rat gastric mucosal cell culture. Gastroenterology 1987;92: 669–677.PubMedGoogle Scholar
  24. 24.
    Hiraishi H, Terano A, Ota S, et al. Oxygen metabolite-induced cytotoxicity to cultured rat gastric mucosal cells. Am J Physiol 1987;253: G40-G48.PubMedGoogle Scholar
  25. 25.
    Hiraishi H, Terano A, Ota S, et al. Role for iron in reactive oxygen species-mediated cytotoxicity to cultured rat gastric mucosal cells. Am J Physiol 1991;260: G556-G563.PubMedGoogle Scholar
  26. 26.
    Hiraishi H, Terano A, Ota S, et al. Antioxidant defenses of cultured gastric cells against oxygen metabolites: role of GSH redox cycle and endogenous catalase. Am J Physiol 1991;261: G921-G928.PubMedGoogle Scholar
  27. 27.
    Kosower NS, Kosower EM. The glutathione status of cells. Int Rev Cytol 1978;54: 109–160.PubMedCrossRefGoogle Scholar
  28. 28.
    Ota S, Razandi M, Sekhon S, et al. Salicylate effects on a monolayer culture of gastric mucous cells from adult rats. Gut 1988;29: 1705–1714.PubMedCrossRefGoogle Scholar
  29. 29.
    Hiraishi H, Terano A, Ota S, et al. Regulation of prostaglandin production in cultured gastric mucosal cells. Prostaglandins 1989;38: 65–78.PubMedCrossRefGoogle Scholar
  30. 30.
    Keberle H. The biochemistry of deferoxamine and its relation to iron metabolism. Ann NY Acad Sci 1964;119: 758–768.PubMedCrossRefGoogle Scholar
  31. 31.
    Halliwell B, Gutteridge JMC. Oxygen free radicals and iron in relation to biology and medicine: some problems and concepts. Arch Biochem Biophys 1986;246: 501–514.PubMedCrossRefGoogle Scholar
  32. 32.
    Halliwell B. Protection against tissue damage in vivo by desferrioxamine: what is its mechanism of action? Free Radic Biol Med 1989;7: 645–651.PubMedCrossRefGoogle Scholar
  33. 33.
    Burgess J, Prince RH. Kinetics of reactions of ligand-substituted tris-(2,2′-bipyridyl)iron (II) complexes. J Chem Soc 1965;6061-6066.Google Scholar
  34. 34.
    Nunez M, Cole ES, Glass J. The reticulocyte plasma membrane pathway of iron uptake as determined by the mechanism of α,α′-dipyridyl inhibition. J Biol Chem 1983;258: 1146–1151.PubMedGoogle Scholar
  35. 35.
    Kamath NS, Satamoorthy K, Chitnis MP, et al. 1,10-Phenanthroline potentiates cytotoxicity of hydroxyurea in human chronic myeloid leukemia cells. Oncology 1989;46: 193–197.PubMedCrossRefGoogle Scholar
  36. 36.
    Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol Rev 1979;59: 527–605.PubMedGoogle Scholar
  37. 37.
    Meister A, Anderson ME. Glutathione. Annu Rev Biochem 1983; 52: 711–760.PubMedCrossRefGoogle Scholar
  38. 38.
    Griffith OW, Meister A. Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine). J Biol Chem 1979;254: 7558–7560.PubMedGoogle Scholar
  39. 39.
    Chasseaud LF. The role of glutathione and glutathione-S-transferases in the metabolism of chemical carcinogen and other electrophilic agents. Adv Cancer Res 1979;29: 175–274.PubMedCrossRefGoogle Scholar
  40. 40.
    Fischer H, Ahmad T. Severe generalized glutathione reductase deficiency after antitumor chemotherapy with BCNU [1,3-bis (chloroethyl)-l-nitrosourea]. J Lab Clin Med 1977;89: 1080–1091.Google Scholar
  41. 41.
    Margoliash E, Novogrodsky A. A study of the inhibition of catalase by 3-amino-l,2,4-triazole. Biochem J 1958;68: 468–475.PubMedGoogle Scholar
  42. 42.
    Sobala GM, Schorah CJ, Sanderson M, et al. Ascorbic acid in the human stomach. Gastroenterology 1989;97: 357–363.PubMedGoogle Scholar
  43. 43.
    Ippoliti A, Elashoff J, Valenzuela J, et al. Recurrent ulcer after successful treatment with cimetidine or antacid. Gastroenterology 1983;85: 875–880.PubMedGoogle Scholar
  44. 44.
    Melvin GK, Hansky J, Eaves ER, et al. Influence of cigarette smoking on healing and relapse in duodenal ulcer disease. Gastroenterology 1983;85: 871–874.Google Scholar
  45. 45.
    Salim AS. Scavenging free radicals to prevent stress-induced gastric mucosal injury. Lancet 1989;2: 1390.PubMedCrossRefGoogle Scholar
  46. 46.
    Salim AS. Oxygen-derived free radicals and the prevention of duodenal ulcer relapse: a new approach. Am J Med Sci 1990;300: 1–6.PubMedCrossRefGoogle Scholar
  47. 47.
    Hiraishi H, Terano A, Ivey KJ. Gastric mucosal cell culture for toxicological study. In: Methods in Toxicology, Vol. 1A, In Vitro Biological Systems: New York: Academic Press, 1993;182–192.Google Scholar
  48. 48.
    Takahashi M, Asada K. Superoxide anion permeability of phospholipid membranes and chloroplast thylakoids. Arch Biochem Biophys 1983;226: 558–563.PubMedCrossRefGoogle Scholar
  49. 49.
    Hiraishi H, Terano A, Razandi M, et al. Role of iron and superoxide in mediating hydrogen peroxide injury to cultured rat gastric cells. Gastroenterology (in press).Google Scholar
  50. 50.
    Suttorp N, Toepfer W, Roka L. Antioxidant defense mechanisms of endothelial cells: glutathione redox cycle versus catalase. Am J Physiol 1986;251: C671-C680.PubMedGoogle Scholar
  51. 51.
    Starke PE, Farber JL. Endogenous defenses against the cytotoxicity of hydrogen peroxide in cultured rat hepatocytes. J Biol Chem 1985;260: 86–92.PubMedGoogle Scholar
  52. 52.
    Hiraishi H, Terano A, Ota S, et al. Oxygen radical-induced cytotoxicity to cultured rat hepatocytes: protective effects of glutathione and endogenous catalase (Abstract). Gastroenterology 1987;92: 1740.Google Scholar
  53. 53.
    Grisham MB, Von Ritter C, Smith BF, et al. Interaction between oxygen radicals and gastric mucin. Am J Physiol 1987;253: G93-G96.PubMedGoogle Scholar
  54. 54.
    Hiraishi H, Terano A, Ota S, et al. Role of mucous glycoprotein in protecting cultured rat gastric mucosal cells against toxic oxygen metabolites. J Lab Clin Med (in press).Google Scholar

Copyright information

© The Japanese Society of Gastroenterology 1993

Authors and Affiliations

  • Hideyuki Hiraishi
    • 1
  • Noriaki Yajima
    • 1
  • Norihisa Yamaguchi
    • 1
  • Motoo Ishida
    • 1
  • Yoshihisa Katoh
    • 1
  • Takashi Harada
    • 1
  • Akira Terano
    • 2
  • Kevin J. Ivey
    • 3
  1. 1.Second Department of Internal MedicineDokkyo University School of MedicineTochigiJapan
  2. 2.Second Department of Medicine, Faculty of MedicineUniversity of TokyoTokyoJapan
  3. 3.Department of MedicineUniversity of CaliforniaIrvineUSA

Personalised recommendations