In vitro Effects of Alloxan/Copper Combinations on Lipid Peroxidation, Protein Oxidation and Antioxidant Enzymes


The in vitro effects of alloxan and the product of its reduction dialuric acid (alone or in combination with copper ions) on lipid peroxidation, carbonyl content, GSH level and antioxidant enzyme activities in rat liver and kidney have been studied. The effects of Cu2+/alloxan and Cu2+/dialuric acid were compared with those of Fe3+/alloxan and Fe3+/dialuric acid. Unlike alloxan, dialuric acid increased liver and kidney lipid peroxidation; similar effects were registered in the presence of Fe3+. In the presence of Cu2+/dialuric acid, the lipid peroxidation was strongly inhibited and vice versa - the liver protein oxidation was increased. Alloxan and dialuric acid, as well as their combinations with Fe3+ had no effect on the total GSH level. Both substances did not affect the Cu2+-induced changes in GSH level, glucose-6-phosphate dehydrogenase and gluthatione reductase activities. In contrast, Cu2+ had no effect on dialuric-acid induced changes in gluthatione peroxidase and superoxide dismutase activities. The present in vitro results, concerning the metal dependence of the effects of alloxan and dialuric acid, are a premise for in vivo study of alloxan effects in metal-loaded animals.


  1. 1.

    Alexandrova, A., Georgieva, A., Kirkova, M. (2006) Alloxan and dialuric acid. Effects on.OH-provoked degradation of deoxyribose in the presence of different metal ions. C.R. Acad. Bulg. Sci. 59, 305–312.

    CAS  Google Scholar 

  2. 2.

    Alexandrova, A., Kessiova, M., Tsvetanova, E., Kirkova, M. (2006) Alloxan. Effects on O2--provoked inhibition of nitro-blue tetrazolium reduction in the presence of different metal ions. C.R. Acad. Bulg. Sci. 59, 201–206.

    CAS  Google Scholar 

  3. 3.

    Alexandrova, A., Kirkova, M., Russanov, E. (1998) In vitro effects of alloxan-vanadium combination on lipid peroxidation and on antioxidant enzyme activity. Gen. Pharmac. 31, 489–493.

    CAS  Article  Google Scholar 

  4. 4.

    Beauchamp, C., Fridovich, I. (1971) Superoxide dismutase: Improved assays and assay applicable to acrylamide gels. Anal. Biochem. 44, 276–287.

    CAS  Article  Google Scholar 

  5. 5.

    Becker, D. J., Reul, B., Ozcelikay, A. T., Buchet, J. P., Henquin, J. C., Brichard, S. M. (1996) Oral selenates improves glucose homeostasis and partly reverses abnormal expression of liver glycolytic and gluconeogenesis enzymes in diabetic rats. Diabetologia 39, 3–11.

    CAS  Article  Google Scholar 

  6. 6.

    Cartier, P., Leroux, J. P., Marchand, J. Cl. (1967) Techniques de dosage des enzymes glycocytiques tissulaires. Ann. Biol. Clin. 25, 109–136.

    CAS  Google Scholar 

  7. 7.

    Fischer, L. J., Harman, A. W. (1982) Oxygen free radicals and diabetogenic action of alloxan. In: Autor, A. P. (ed.) Pathology of Oxygen. Academic Press, New York. p. 261.

    Google Scholar 

  8. 8.

    Grankvist, K., Marklund, S. L., Schlin, J., Taljedal, I. B. (1979) Superoxide dismutase, catalase and scavengers of hydroxyl radical protect against the toxic action of alloxan on pancreatic islet cells in vitro. Biochem. J. 182, 17–25.

    CAS  Article  Google Scholar 

  9. 9.

    Gunzler, W. A., Vergin, H., Muller, I., Flohe, L. (1972) Glutathion peroxidase. VI. Die reaction der glutathion peroxidase mit Verschieden hydroperoxiden. Hoppe-Seyler’s Z. Physiol. Chem. 353, 1001–1004.

    CAS  Article  Google Scholar 

  10. 10.

    Halliwell, B., Gutteridge, J. M. C. (1985) Free Radicals in Biology and Medicine. Calderon Press, Oxford.

    Google Scholar 

  11. 11.

    Halliwell, B., Gutteridge, J. M. C. (1984) Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem. J. 219, 1–14.

    CAS  Article  Google Scholar 

  12. 12.

    Halliwell, B., Gutteridge, J. M. C., Aruoma, O. I. (1987) The deoxyribose method: a simple “test-tube” assay for determination of free constants for reactions of hydroxyl radicals. Anal. Biochem. 165, 215–219.

    CAS  Article  Google Scholar 

  13. 13.

    Heikkila, R. E., Winston, B., Cohen, G., Barden, H. (1976) Alloxan-induced diabetes: evidence for hydrohyl radical as a cytotoxic intermediate. Biochem. Pharmac. 25, 1085–1092.

    CAS  Article  Google Scholar 

  14. 14.

    Houee-Levin, C., Gardes-Albert, M., Ferradini, C., Pucheault, J. (1981) Radiolysis study of the allox-an-dialuric acid couple II: the autooxidation of dialuric acid. Radiat. Res. 88, 20–28.

    CAS  Article  Google Scholar 

  15. 15.

    Hunter, F., Gebinski, J., Hoffstein, P., Weinstein, J., Scott, A. (1963) Swelling and lysis of rat liver mitochondria by ferrous ions. J. Biol. Chem. 238, 828–835.

    CAS  PubMed  Google Scholar 

  16. 16.

    Ishibashi, F., Howard, B. V. (1981) Alloxan and H2O2 action on glucose metabolisis in cultured fibroblasts. Generation of oxygen-containing free radicals as a mechanism of alloxan action. J. Biol. Chem. 256, 12134–12139.

    PubMed  Google Scholar 

  17. 17.

    Kirkova, M., Karakashev, P., Russanov, E. (1998) Hydroxyl radicals production in the vanadium ions/dialuric acid systems. Gen. Pharmac. 31, 247–251.

    CAS  Article  Google Scholar 

  18. 18.

    Letelier, M. E., Lepe, A. M., Faundez, M., Salazar, J., Marin, R., Aracena, P., Speiski, H. (2005) Possible mechanisms underlying copper-induced damage in biological membranes leading to cellular toxicity. Chem. Biol. Interact. 151, 71–82.

    CAS  Article  Google Scholar 

  19. 19.

    Lowry, O. H., Rosenbrough, N. J., Farr, A. L., Randal, R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–278.

    CAS  Google Scholar 

  20. 20.

    Malaisse, W. J. (1982) Alloxan toxicity to the pancreatic β-cell. A new hypothesis. Biochem. Pharmac. 31, 3527–3534.

    CAS  Article  Google Scholar 

  21. 21.

    McNeill, J. H., Delgatty, H. L. M., Battell, M. L. (1991) Insulin-like effects of sodium selenate in streptozotocin induced diabetic rats. Diabetes 40, 1675–1678.

    CAS  Article  Google Scholar 

  22. 22.

    Meyerovitch, J., Farfel, Z., Sack, J., Schechter, Y. (1987) Oral administration of vanadate normalizes blood glucose levels in streptozotocin-treated rats. J. Biol. Chem. 262, 6658–6662.

    CAS  PubMed  Google Scholar 

  23. 23.

    Miller, D. M., Grover, A., Nayini, N., Aust, S. D. (1993) Xanthine oxidase- and iron-dependent lipid peroxidation. Arch. Biochem. Biophys. 301, 1–7.

    CAS  Article  Google Scholar 

  24. 24.

    Munday, R. (1988) Effects of transition metals on the reaction rate and on the generation of “active oxygen” species. Biochem. Pharmac. 37, 409–413.

    CAS  Article  Google Scholar 

  25. 25.

    Ozcelikay, A. T., Becker, D. J., Ongemba, L. N., Pottier, A. M., Henquin, J. L., Buchard, S. M. (1996) Improvement of glucose and lipid metabolism in diabetic rats treated with molybdate. Am. J. Physiol. 270, E344-E352.

  26. 26.

    Pinto, R. E., Bartley, W. (1969) The effect of age and sex on glutathione reductase and glutathione peroxidase activities and on aerobic glutathione oxidation in rat liver homogenates. Biochem. J. 112, 109–115.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Reznick, A. Z., Parker, L. (1994) Oxidative damage to ptoteins: Spectrophotometric method for carbonyl assay. Meth. Enzymol. 233, 357–363.

    CAS  Article  Google Scholar 

  28. 28.

    Rodriguez-Gil, J. E., Fernandez-Novell, J. M., Barbera, A., Guinovart, J. J. (2000) Lithium effects on rat glucose metabolism in vivo. Arch. Biochem. Biophys. 375, 377–384.

    CAS  Article  Google Scholar 

  29. 29.

    Schechter, Y. (1990) Insulin-mimetic effects of vanadate. Possible implications for future treatment of diabetes. Diabetes 39, 1–5.

    Article  Google Scholar 

  30. 30.

    Stadtman, E. R., Oliver, C. N. (1991) Metal-catalyzed oxidation of proteins. J. Biol. Chem. 266, 2005–2008.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Tamura, S., Brown, T. A., Whipple, J. H., Fujita-Yamaguchi, Y., Dubler, R. E., Cheng, K., Larner, J. W. (1984) A novel mechanism for the insulin-like effects of vanadate on glycogen synthase rat adipocytes. J. Biol. Chem. 259, 6650–6658.

    CAS  PubMed  Google Scholar 

  32. 32.

    Tibaldi, J., Benjamin, J., Cabbat, F. S., Heikkila, R. E. (1979) Protection against alloxan-induced diabetes by various urea derivatives: relationship between protective effects and reactivity with the hydroxyl radical. J. Pharmac. Exp. Ther. 211, 415–418.

    CAS  Google Scholar 

  33. 33.

    Tietze, F. (1969) Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: Applications to mammalian blood and other tissues. Anal. Biochem. 27, 502–522.

    CAS  Article  Google Scholar 

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This study was supported by Grant MU-B-1001 from the National Research Fund, Bulgaria.

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Correspondence to Albena Alexandrova.

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Alexandrova, A., Petrov, L., Kessiova, M. et al. In vitro Effects of Alloxan/Copper Combinations on Lipid Peroxidation, Protein Oxidation and Antioxidant Enzymes. BIOLOGIA FUTURA 58, 359–367 (2007).

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  • Alloxan
  • dialuric acid
  • copper
  • lipid peroxidation
  • antioxidant enzymes