Biological Trace Element Research

, Volume 122, Issue 2, pp 127–136 | Cite as

Combination of Selenium and Three Naturally Occurring Antioxidants Administration Protects d-Galactosamine-Induced Liver Injury in Rats

  • Tunc Catal
  • Sehnaz Bolkent


d-Galactosamine (d-GaIN) is a highly selective hepatotoxin that causes liver injury similar to human viral hepatitis via depletion of uridine nucleotides, which subsequently diminishes synthesis of RNA and proteins. The aim of this study was to investigate the role of selenium, ascorbic acid, beta-carotene, and alpha-tocopherol on d-GaIN-induced liver injury of rats by morphological and immunohistochemical means. In this study, Sprague–Dawley female rats were divided into four groups. Group I consists of rats injected physiologic saline solution intraperitoneally. Group II consists of rats given selenium (0.2 mg/kg per day), ascorbic acid (100 mg/kg per day), beta-carotene (15 mg/kg per day), and alpha-tocopherol (100 mg/kg per day) for 3 days via gavage method. Group III consists of the single dose of d-GaIN (500 mg/kg)-injected animals. Group IV are the d-GaIN-injected animals given the same antioxidant combination. In situ terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate nick-end labeling (TUNEL) assay was applied to determine apoptosis for paraffin sections of the liver samples. Moreover, caspase-3 and proliferating cell nuclear antigen antibody were applied for paraffin sections. In the group given d-GaIN, apoptotic cells with TUNEL assays and caspase-3 activity, which are liver injury markers induced by d-GaIN, the hepatocyte proliferation with cell proliferation assay increased. However, selenium and other three antioxidants combination clearly suppressed an increase in apoptotic cells with TUNEL assay and caspase-3 activity. In addition, it suppressed d-GaIN-induced cell proliferation in the liver. As a result, these results indicate that selenium and three naturally occurring antioxidants shows a protective effect against liver injury induced by d-GaIN. These results suggest that supplementation with the combination of selenium, ascorbic acid, beta-carotene, and alpha-tocopherol may help prevent the development of liver injury.


d-Galactosamine Liver injury Apoptosis Selenium Antioxidant Proliferation 





phosphate buffer saline


proliferating cell nuclear antigen


reactive oxygen species


terminal deoxynucleotidyl transferase


tumor necrosis factor


in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling



This study was supported by the Research Fund of Istanbul University (project no. T-290/18062003).


  1. 1.
    Stachlewitz RF, Seabra V, Bradford B, Bradham CA, Rusyn I, Germolec D, Thurman RG (1999) Glycine and uridine prevent d-galactosamine hepatotoxicity in the rat: role of Kupffer cells. Hepatology 29:737–745PubMedCrossRefGoogle Scholar
  2. 2.
    Gujral JS, Farhood A, Jaeschke H (2003) Oncotic necrosis and caspase-dependent apoptosis during galactosamine-induced liver injury in rats. Toxicol Appl Pharmacol 190:37–46PubMedCrossRefGoogle Scholar
  3. 3.
    Aiub CAF, Bortolini R, Azambuja AA, Filho JCA, Nunes FB, de Olievira JR (2003) Alterations in the indexes of apoptosis and necrosis induced by galactosamine in the liver of Wistar rats treated with fructose-1,6-bisphosphate. Hepatol Res 25:83–91CrossRefGoogle Scholar
  4. 4.
    Ferencikova R, Cervinkova Z, Drahota Z (2003) Hepatotoxic effect of d-galactosamine and protective role of lipid emulsion. Physiol Res 52:73–78PubMedGoogle Scholar
  5. 5.
    Bradham CA, Plumpe J, Manns MP, Brenner DA, Trautwein C (1998) Mechanisms of hepatic toxicity. I. TNF-induced liver injury. Am J Physiol 275:387–392Google Scholar
  6. 6.
    Sun F, Hamagawa E, Tsutsui C, Sagaguchi N, Kakuta Y, Tokumaru S, Kojo S (2003) Evaluation of oxidative stress during apoptosis and necrosis caused by d-galactosamine in rat liver. Biochem Pharmacol 65:101–107PubMedCrossRefGoogle Scholar
  7. 7.
    Gate L, Paul J, Ba GN, Tew KD, Tapiero H (1999) Oxidative stress induced in pathologies: the role of antioxidants. Biomed Pharmacother 53:169–80PubMedCrossRefGoogle Scholar
  8. 8.
    Fang YZ, Yang S, Wu G (2002) Free radicals, antioxidants, and nutrition. Nutrition 18:872–879PubMedCrossRefGoogle Scholar
  9. 9.
    Evans P, Halliwell B (2001) Micronutrients: oxidant/antioxidant status. Br J Nutr 85(2):67–74CrossRefGoogle Scholar
  10. 10.
    Liebler DC (1993) The role of metabolism in the antioxidant function of vitamin E. Crit Rev Toxicol 23:147–169PubMedCrossRefGoogle Scholar
  11. 11.
    Jaeschke H, Farhood A, Cai SX, Tseng BY, Bajt ML (2000) Protection against TNF-induced liver parenchymal cell apoptosis during endotoxemia by a novel caspase inhibitor in mice. Toxicol Appl Pharmacol 169:77–83PubMedCrossRefGoogle Scholar
  12. 12.
    Andres D, Cascales M (2002) Novel mechanism of Vitamin E protection against cyclosporine A cytotoxicity in cultured rat hepatocytes. Biochem Pharmacol 64:267–76PubMedCrossRefGoogle Scholar
  13. 13.
    Padh H (1990) Cellular functions of ascorbic acid. Biochem Cell Biol 68:1166–1173PubMedCrossRefGoogle Scholar
  14. 14.
    Cheng WH, Quimby FW, Lei XG (2003) Impacts of glutathione peroxidase-1 knockout on the protection by injected selenium against the pro-oxidant-induced liver aponecrosis and signaling in selenium-deficient mice. Free Radic Biol Med 34:918–927PubMedCrossRefGoogle Scholar
  15. 15.
    Naziroglu M (1999) Protective role of intraperitoneally administered vitamin E and selenium in rats anesthetized with enflurane. Biol Trace Elem Res 69:199–209PubMedCrossRefGoogle Scholar
  16. 16.
    Sjunnesson H, Sturegard E, Willen R, Wadström T (2001) High intake of selenium, β-carotene, and vitamins A, C, and E reduces growth of Helicobacter pylori in the guinea pig. Comp Med 51:418–423PubMedGoogle Scholar
  17. 17.
    Kaplowitz N (2000) Mechanisms of liver cell injury. J Hepatol 32:39–47PubMedCrossRefGoogle Scholar
  18. 18.
    Kaplowitz N (2002) Biochemical and cellular mechanisms of toxic liver injury. Semin Liver Dis 22:137–144PubMedCrossRefGoogle Scholar
  19. 19.
    Cheng CC, Etoh J, Tanimura T, Egashira Y, Ohta T, Sanada H (1996) Effects of dietary gluten on the hepatotoxic action of galactosamine and/or endotoxin in rats. Biosci Biotechnol Biochem 60:439–443PubMedCrossRefGoogle Scholar
  20. 20.
    Tran-Thi TA, Phillips J, Falk H, Decker K (1985) Toxicity of d-galactosamine for rat hepatocytes in monolayer culture. Exp Mol Path 42:89–116CrossRefGoogle Scholar
  21. 21.
    Tsutsui S, Hirasawa K, Takeda M, Itagaki S, Kawamura S, Maeda K, Mikami T, Doi K (1997) Galactosamine-induced apoptosis in the primary mouse hepatocyte cultures. Exp Toxicol Pathol 49:301–306PubMedGoogle Scholar
  22. 22.
    Siendones E, Fouad D, Abou-Elella AM, Quintero A, Barrera P, Muntane J (2003) Role of nitric oxide in d-galactosamine-induced cell death and its protection by PGE1 in cultured hepatocytes. Nitric Oxide 8:133–43PubMedCrossRefGoogle Scholar
  23. 23.
    Osawa Y, Nagaki M, Banno Y, Yamada Y, Imose M, Nozawa Y, Moriwaki H, Nakashima S (2001) Possible involvement of reactive oxygen species in d-galactosamine-induced sensitization against tumor necrosis factor-α-induced hepatocyte apoptosis. J Cell Phys 187:374–385CrossRefGoogle Scholar
  24. 24.
    Kasravi FB, Wang L, Wang XD, Molin G, Bengmark S, Jeppsson B (1996) Bacterial translocation in acute liver injury induced by d-galactosamine. Hepatology 23:97–103PubMedGoogle Scholar
  25. 25.
    Lopez-Torres M, Gredilla R, Sanz A, Barja G (2002) Influence of aging and long-term caloric restriction on oxygen radical generation and oxidative DNA damage in rat liver mitochondria. Free Radic Biol Med 32:882–889PubMedCrossRefGoogle Scholar
  26. 26.
    Slater AF, Nobel CS, Orrenius S (1995) The role of intracellular oxidants in apoptosis. Biochim Biophys Acta 1271:59–62PubMedGoogle Scholar
  27. 27.
    Deas O, Dumont C, Mollereau B, Metivier D, Pasquier C, Bernard-Poöier G, Hirsch F, Charpentier B, Senik A (1997) Thiol-mediated inhibition of FAS and CD2 apoptotic signaling in activated human peripheral T cells. Int Immunol 9:117–125PubMedCrossRefGoogle Scholar
  28. 28.
    Tapalaga D, Tiegs G, Angermüller S (2002) NFκB and caspase-3 activity in apoptotic hepatocytes of galactosamine-sensitized mice treated with TNF-α. J Histochem Cytochem 50:1599–1609PubMedGoogle Scholar
  29. 29.
    Sun F, Hayami S, Ogiri Y, Haruna S, Tanaka K, Yamada Y, Tokumaru S, Kojo S (2000) Evaluation of oxidative stress based on lipid hydroperoxide, vitamin C and vitamin E during apoptosis and necrosis caused by thioacetamide in rat liver. Biochim Biophys Acta 1500:181–185PubMedGoogle Scholar
  30. 30.
    Higuchi M, Honda T, Proske RJ, Yeh ET (1998) Regulation of reactive oxygen species-induced apoptosis and necrosis by caspase 3-like proteases. Oncogene 17:2753–2760PubMedCrossRefGoogle Scholar
  31. 31.
    Shen HM, Yang CF, Liu J, Ong CN (2000) Dual role of glutathione in selenite-induced oxidative stress and apoptosis in human hepatoma cells. Free Radic Biol Med 28:1115–1124PubMedCrossRefGoogle Scholar
  32. 32.
    Simeonova PP, Gallucci RM, Hulderman T, Wilson R, Kommineni C, Rao M, Luster MI (2001) The role of tumor necrosis factor-alpha in liver toxicity, inflammation, and fibrosis induced by carbon tetrachloride. Toxicol Appl Pharmacol 177:112–120PubMedCrossRefGoogle Scholar
  33. 33.
    Shimizu S, Eguchi Y, Kosaka H, Kamiike W, Matsuda H, Tsujimoto Y (1995) Prevention of hypoxia-induced cell death by Bcl-2 and Bcl-xL. Nature 374:811–813PubMedCrossRefGoogle Scholar
  34. 34.
    Hayama S, Ikeda K, Sun F, Tanaka K, Kojo S (1999) Increase of caspase-3 activity in rat liver and plasma by thioacetamide. Biochem Pharmacol 58:1941–1943CrossRefGoogle Scholar
  35. 35.
    Eckle VS, Buchmann A, Bursch W, Hermann-Schulte R, Schwarz M (2004) Immunohistochemical detection of activated caspases in apoptotic hepatocytes in rat liver. Toxic Path 32:9–15CrossRefGoogle Scholar
  36. 36.
    Sclafani L, Shimm P, Edelman J, Seifter E, Levenson SM, Demetriou AA (1986) Protective effect of vitamin E in rats with acute liver injury. JPEN J Parenter Enteral Nutr 10:184–187PubMedCrossRefGoogle Scholar
  37. 37.
    Asaoka Y, Sakai H, Takahashi N, Hirata A, Tsukamoto T, Yamamoto M, Yanai T, Masegi T, Tatematsu T (2005) Intraperitoneal injection of d-galactosamine provides a potent cell proliferation stimulus for the detection of initiation activities of chemicals in rat liver. J Appl Toxicol 25:554–561PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  1. 1.Department of Molecular Biology and Genetics, Faculty of Sciences and LettersIstanbul Technical UniversityMaslak, IstanbulTurkey
  2. 2.Department of Biology, Faculty of ScienceIstanbul UniversityVezneciler, IstanbulTurkey

Personalised recommendations