Biological Trace Element Research

, Volume 68, Issue 2, pp 159–174 | Cite as

Cytoprotection against lipid hydroperoxides correlates with increased glutathione peroxidase activities, but not selenium uptake from different selenocompounds

  • Marcel Leist
  • Stefanie Maurer
  • Manfred Schultz
  • Angelika Elsner
  • Dieter Gawlik
  • Regina Brigelius-Flohé
Original Articles


Cells cultivated under standard conditions were highly deficient in tocopherol, selenium, and glutathione peroxidase (GPx) activities. We investigated whether and to what extent the addition of different selenocompounds to growth media would alter biochemical, physiological, and pathophysiological parameters of cultured liver cells. Cellular uptake of selenium, GPx activities, and cytoprotection were measured and compared in human hepatoma cells (HepG2). Selenite and selenocystine were Se donors of high bioavailability (i.e., with these culture supplements, the increased Se uptake, induction of GPx isoenzymes, and protection of treated cells from lipid hydroperoxides were well correlated). In contrast, selenium from selenomethionine was incorporated into cellular proteins but had no effect on GPx activities or cytoprotection. The data show that not all selenium donors provide selenium, which is bioactivated to act as antioxidant. Thus, cellular selenium content, in general, did not correlate with cytoprotective activity of this trace element. However, cellular GPx activities at different times, with different concentrations, and with different Se donors always correlated with protection from lipid hydroperoxides and may, thus, represent a more reliable parameter to define adequate Se supply.

Index Entries

Selenium cell culture media glutathione peroxidase fatty acid hydroperoxide oxidative stress 


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  1. 1.
    K. Schwarz and C. M. Foltz, Selenium as an integral part of factor 3 against dietary necrotic liver degeneration,J. Am. Chem. Soc. 79, 3292–3293 (1957).CrossRefGoogle Scholar
  2. 2.
    S. S. Chernick, J. G. Moe, G. P. Rodnan, and K. Schwarz, A metabolic lesion in dietary necrotic liver degeneration.J. Biol. Chem. 217, 829–843 (1958).Google Scholar
  3. 3.
    F. Ursini and A. Bindoli, The role of selenium peroxidases in the protection against oxidative damage of membranes,Chem. Phys. Lipids 44, 255–276 (1987).PubMedCrossRefGoogle Scholar
  4. 4.
    G. C. Mills, Hemoglobin catabolism I. Glutathione peroxidase, an erythrocyte enzyme which protects hemoglobin from oxidative breakdown,J. Biol. Chem. 229, 189–197 (1957).PubMedGoogle Scholar
  5. 5.
    J. T. Rotruck, A. L. Pope, H. E. Ganther, A. B. Swanson, D. G. Hafeman, and W. G. Hoekstra, Selenium: biochemical role as a component of glutathione peroxidase,Science 179, 558–590 (1973).CrossRefGoogle Scholar
  6. 6.
    L. Flohé, W. A. Günzler, and H. H. Schock, Glutathione peroxidase: a selenoenzyme,FEBS Lett. 32, 132–134 (1973).PubMedCrossRefGoogle Scholar
  7. 7.
    F. Ursini, M. Maiorino, R. Brigelius-Flohé, K. D. Aumann, A. Roveri, D. Schomburg, et al., The diversity of glutathione peroxidases,Methods Enzymol. 252, 38–53 (1995).PubMedGoogle Scholar
  8. 8.
    M. Maiorino, F. F. Chu, F. Ursini, K. J. A. Davies, J. H. Doroshow, and R. S. Esworthy, Phospholipid hydroperoxide glutathione peroxidase is the 18 kDa selenoprotein expressed in human tumor cell lines.J. Biol. Chem. 266, 7728–7732 (1991).PubMedGoogle Scholar
  9. 9.
    F. Ursini, M. Maiorino, M. Valente, L. Ferri, and C. Gregolin, Purification from pig liver of a protein which protects liposomes and biomembranes from peroxidative degradation and exhibits glutathione peroxidase activity on phosphatidylcholine hydroperoxides,Biochim. Biophys. Acta 710, 197–211 (1982).PubMedGoogle Scholar
  10. 10.
    R. Brigelius-Flohé, K. D. Aumann, H. Blöcker, G. Gross, M. Kiess, K.-D. Klöppel, M. Maiorino, A. Roveri, R. Schuckelt, F. Ursini, E. Wingendorf, and L. Flohé, Phospholipid-hydroperoxide glutathione peroxidase. Genomic DNA, cDNA, and deduced amino acid sequence,J. Biol. Chem. 269, 7342–7348 (1994).PubMedGoogle Scholar
  11. 11.
    R. F. Burk and K. E. Hill, Regulation of selenoproteins,Annu. Rev. Nutr. 13, 65–81 (1993).PubMedCrossRefGoogle Scholar
  12. 12.
    D. Behne, C. Weiss-Nowak, M. Kalcklösch, C. Westphal, H. Gessner, and A. Kyriakopoulos, Studies on the distribution and characteristics of new mammalian selenium-containing proteins,Analyst 120, 823–825 (1995).CrossRefGoogle Scholar
  13. 13.
    D. Behne, A. Kyriakopoulos, H. Meinhold, and J. Köhrle, Identification of type I iodothyronine 5′-deiodinase as selenoenzyme,Biochem. Biophys. Res. Commun. 173, 1143–1149 (1990).PubMedCrossRefGoogle Scholar
  14. 14.
    S. C. Vendeland, M. A. Beilstein, C. L. Chen, O. N. Jensen, E. Barofsky, and P. D. Whanger, Purification and properties of selenoprotein-W from rat muscle,J. Biol. Chem. 268, 17103–17107 (1993).PubMedGoogle Scholar
  15. 15.
    R. Read, T. Bellew, J.-G. Yang, K. E. Hill, I. S. Palmer, and R. F. Burk, Selenium and amino acid composition of selenoprotein P, the major selenoprotein in rat serum,J. Biol. Chem. 265, 17,899–17,905 (1990).Google Scholar
  16. 16.
    V. N. Gladyshev, K. T. Jeang, and T. Stadtman, Selenocysteine, identified as the penultimate C-terminal residue in human T-cell thioredoxin reductase, corresponds to TGA in the human placental gene,Proc. Natl. Acad. Sci. USA 93, 6146–6151 (1996).PubMedCrossRefGoogle Scholar
  17. 17.
    F. Weitzel and A. Wendel, Selenoenzymes regulate the activity of leukocyte 5-lipoxygenase via the peroxide tone,J. Biol. Chem. 268, 6288–6292 (1993).PubMedGoogle Scholar
  18. 18.
    P. G. Geiger, F. Lin, and A. W. Girotti, Selenoperoxidase-mediated cytoprotection against the damaging effects of tert-butyl hydroperoxide on leukemia cells,Free Radical Biol. Med. 14, 251–266 (1993).CrossRefGoogle Scholar
  19. 19.
    P. G. Geiger, J. P. Thomas, and A. W. Girotti, Lethal damage to murine L1210 cells by exogenous lipid hydroperoxides: protective role of glutathione-dependent selenoperoxidases,Arch. Biochem. Biophys. 288, 671–680 (1991).PubMedCrossRefGoogle Scholar
  20. 20.
    L. Kiremidjian-Schumacher, M. Roy, H. I. Wishe, M. W. Cohen, and G. Stotzky, Regulation of cellular immune responses by selenium,Biol. Trace Element Res. 33, 23–35 (1992).Google Scholar
  21. 21.
    L. Kiremidjian-Schumacher, M. Roy, H. I. Wishe, M. W. Cohen, and G. Stotzky, Selenium and immune cell functions. I. Effect on lymphocyte proliferation and production of interleukin 1 and interleukin 2,Proc. Soc. Exp. Biol. Med. 193, 136–142 (1990).PubMedGoogle Scholar
  22. 22.
    M. A. Beck, Q. Sei, V. C. Morris, and O. A. Levander, Rapid genomic evolution of a non-virulent Coxsackievirus B3 in selenium-deficient mice results in selection of identical virulent isolates,Nature Med. 1, 433–36 (1995).PubMedCrossRefGoogle Scholar
  23. 23.
    R. F. Burk, Biological activity of selenium,Ann. Rev. Nutr. 3, 53–70 (1983).CrossRefGoogle Scholar
  24. 24.
    R. Reiter and A. Wendel, Selenium and drug metabolism—III. Relation of glutathione-peroxidase and other hepatic enzyme modulations to dietary supplements,Biochem. Pharmacol. 34, 2287–2290 (1985).PubMedCrossRefGoogle Scholar
  25. 25.
    R. D. Baker, S. S. Baker, K. LaRosa, C. Whitney, and P. E. Newburger, Selenium regulation of glutathione peroxidase in human hepatoma cell line Hep3B,Arch. Biochem. Biophys. 304, 53–57 (1993).PubMedCrossRefGoogle Scholar
  26. 26.
    G. Bermano, J. R. Arthur, and J. E. Hesketh, Selective control of the cytosolic glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase gene expression by selenium supply,Biochem. J. 320, 891–895 (1996).PubMedGoogle Scholar
  27. 27.
    M. S. Saedi, C. G. Smith, J. Frampton, I. Chambers, P. R. Harrison, and R. A. Sunde, Effect of selenium status on mRNA levels for glutathione peroxidase in rat liver,Biochem. Biophys. Res. Commun. 153, 855–861 (1998).CrossRefGoogle Scholar
  28. 28.
    N. B. Javitt, Hep G2 cells as a resource for metabolic studies: lipoprotein, cholesterol, and bile acids,FASEB J. 4, 161–168 (1990).PubMedGoogle Scholar
  29. 29.
    M. Leist, F. Gantner, I. Bohlinger, P. G. German, G. Tiegs, and A. Wendel, Murine hepatocyte apoptosis induced in vitro and in vivo by TNF-alpha requires transcriptional arrest,J. Immunol. 153, 1778–1787 (1994).PubMedGoogle Scholar
  30. 30.
    A. T. Natarajan and F. Darroudi, Use of human hepatoma cells for in vitro metabolic activation of chemical mutagens/carcinogens,Mutagenesis 6, 339–403 (1991).CrossRefGoogle Scholar
  31. 31.
    M. Leist, B. Raab, S. Maurer, U. Rösick, and R. Brigelius-Flohé, Conventional cell culture media do not adequately supply cells with antioxidants and thus facilitate peroxide-induce genotoxicity,Free Radical Biol. Med. 21, 297–306 (1996).CrossRefGoogle Scholar
  32. 32.
    V. W. Bowry, K. K. Stanley, and R. Stocker, High density lipoprotein is the major carrier of lipid hydroperoxides in human blood plasma from fasting donors,Proc. Natl. Acad. Sci. USA 89, 10,316–10,320 (1992).CrossRefGoogle Scholar
  33. 33.
    T. Y. Aw, M. W. Williams, and L. Gray, Absorption and lymphatic transport of peroxidized lipids by rat small intestine in vivo: role of mucosal GSH,Am. J. Physiol. 262, G99-G106 (1992).PubMedGoogle Scholar
  34. 34.
    M. O. Funk, R. Isaac, and N. A. Porter, Preparation and purification of lipid hydroperoxides from arachidonic and gamma-linolenic acid,Lipids 11,,113–117 (1976).PubMedCrossRefGoogle Scholar
  35. 35.
    F. J. G. M. van Kuijk, D. W. Thomas, and R. J. D. E. A. Stephens, Gas chromatographymass spectrometry assays for lipid peroxidases,Methods Enzymol. 186, 388–398 (1990).PubMedGoogle Scholar
  36. 36.
    M. Maiorino, C. Gregolin, and F. Ursini, Phospholipid hydroperoxide glutathione peroxidase,Methods Enzymol. 186, 448–457 (1990).PubMedCrossRefGoogle Scholar
  37. 37.
    M. M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding,Anal. Biochem. 72, 248–254 (1976).PubMedCrossRefGoogle Scholar
  38. 38.
    R. Brigelius-Flohé, K. Lötzer, S. Maurer, M. Schultz, and M. Leist, Utilization of selenium from different chemical entities for selenoprotein biosynthesis by mammalian cell lines,Biofactors 5, 125–131 (1996).Google Scholar
  39. 39.
    M. Schultz, M. Leist, M. Petrzika, B. Gassmann, and R. Brigelius-Flohé, Novel urinary metabolite of alpha-tocopherol, 2,5,7,8-tetramethyl-2(2′-carboxyethyl)-6-hydroxychroman, as an indicator of an adequate vitamin E supply,Am. J. Clin. Nutr. 62, 1527S-1534S (1995).PubMedGoogle Scholar
  40. 40.
    D. Behne and H. Jürgensen, Determination of trace elements in human blood serum and in the standard reference material “bovine liver” by instrumental neutron activation analysis,J. Radiolyt. Chem. 42, 447–453 (1978).CrossRefGoogle Scholar
  41. 41.
    F. Tietze, Enzymatic method for quantitative determination of nanogram amounts of total and oxidised glutathione: application to mammalian blood and other tissues,Anal. Biochem. 27, 502–522 (1969).PubMedCrossRefGoogle Scholar
  42. 42.
    T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays,J. Immunol. Methods 65, 55–63 (1983).PubMedCrossRefGoogle Scholar
  43. 43.
    H. U. Bergmeyer,Methods of Enzymatic Analysis, vol. 82, 3rd ed., Verlag Chemie, Weinheim (1984).Google Scholar
  44. 44.
    Y. Thomassen and J. Aaseth, Selenium in man, inOccurrence and Distribution of Selenium, M. Ihnat, ed., CRC, Boca Raton, FL, pp. 169–212 (1989).Google Scholar
  45. 45.
    L. J. Machlin,Handbook of Vitamins, Marcel Dekker, New York, p. 113 (1984).Google Scholar
  46. 46.
    D. Behne, A. Kyriakopoulos, S. Scheid, and H. Gessner, Effect of chemical form and dosage on the incorporation of selenium into tissue proteins in rats,J. Nutr. 121, 806–814 (1991).PubMedGoogle Scholar
  47. 47.
    G. Bellomo, S. Jewell, H. Thor, and S. Orrenius, regulation of intracellular calcium compartmentation: studies with isolated hepatocytes and t-butyl hydroperoxide,Proc. Natl. Acad. Sci. USA 79, 6842–6846 (1982).PubMedCrossRefGoogle Scholar
  48. 48.
    N. Masaki, M. E. Kyle, and J. L. Farber, Tert-butyl hydroperoxide kills cultured hepatocytes by peroxidizing membrane lipids,Arch. Biochem. Biophys. 269, 390–399 (1989).PubMedCrossRefGoogle Scholar
  49. 49.
    T. Kaneko, S.-I. Nakano, and M. Matsuo, Protective effect of vitamin E on linoleic acid hydroperoxide-induced injury to human endothelial cells,Lipids 26, 345–348 (1991).PubMedCrossRefGoogle Scholar
  50. 50.
    I. H. Waschulewski and R. A. Sunde, Effect of dietary methionine on utilization of tissue selenium from dietary selenomethionine for glutathione peroxidase in the rat,J. Nutr. 118, 367–374 (1988).PubMedGoogle Scholar
  51. 51.
    M. A. Beilstein and P. D. Whanger, Glutathione peroxidase activity and chemical forms of selenium in tissues of rats given selenite or selenomethionine,J. Inorg. Biochem. 33, 31–6 (1988).PubMedCrossRefGoogle Scholar
  52. 52.
    J. T. Deagen, J. A. Butler, M. A. Beilstein, and P. D. Whanger, Effects of dietary selenite, selenocystine and selenomethionine on selenocysteine lyase and glutathione peroxidase activities and on selenium levels in rat tissues,J. Nutr. 117, 91–98 (1987).PubMedGoogle Scholar
  53. 53.
    T. C. Stadtman, Biosynthesis and function of selenocysteine-containing enzymes,J. Biol. Chem. 266, 16,257–16,260 (1991).Google Scholar
  54. 54.
    K. P. McConnell and J. L. Hoffman, Methionine-selenomethionine parallels in rat liver polypeptide chain synthesis,FEBS Lett. 24, 60–62 (1972).PubMedCrossRefGoogle Scholar
  55. 55.
    R. A. Sunde, G. E. Gutzke, and W. G. Hoekstra, Effect of dietary methionine on the biopotency of selenite and selenomethionine in the rat,J. Nutr. 110,,1096–1100 (1980).Google Scholar
  56. 56.
    K. Yasumoto and K. Y. M. Iwami, Vitamin B6-dependence of selenomethionine and selenite utilization for glutathione peroxidase in the rat,J. Nutr. 109, 760–766 (1979).PubMedGoogle Scholar
  57. 57.
    M. A. Beilstein and P. D. Whanger, Selenium metabolism and glutathione peroxidase activity in cultured human lymphoblasts. Effects of transsulfuration defects and pyridoxal phosphate,Biol. Trace Element Res. 35, 105–118 (1992).Google Scholar

Copyright information

© Humana Press Inc. 1999

Authors and Affiliations

  • Marcel Leist
    • 1
  • Stefanie Maurer
    • 1
  • Manfred Schultz
    • 1
  • Angelika Elsner
    • 1
  • Dieter Gawlik
    • 2
  • Regina Brigelius-Flohé
    • 1
  1. 1.German Institute of Human NutritionPotsdam-RehbrückeGermany
  2. 2.Hahn-Meitner InstituteBerlinGermany

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