Biological Trace Element Research

, Volume 169, Issue 2, pp 218–229 | Cite as

Protective Effects of Zinc Against Acute Arsenic Toxicity by Regulating Antioxidant Defense System and Cumulative Metallothionein Expression

  • Renuka GangerEmail author
  • Roobee Garla
  • Biraja Prasad Mohanty
  • Mohinder Pal Bansal
  • Mohan Lal Garg


Arsenic (As), a toxic metalloid, is one of the major global concerns. The toxicity resulting from As exposure is linked to the generation of reactive oxygen intermediates during their redox cycling and metabolic activation processes that cause lipid peroxidation (LPO). Zinc (Zn), a redox-inactive metal, helps to maintain cellular functions because of its prominent role in antioxidant network through multiple mechanisms. The present study, therefore, explores the effectiveness of administered Zn to combat against acute As toxicity by analysis of antioxidant defense status, alkaline phosphatase (ALP) activity, histological profile, MT expression, and elemental status in rat liver. To achieve this goal, four experimental groups, one control and three receiving different metal supplementations, were chosen (group 1, control; group 2, Zn supplemented; group 3, As substituted; group 4, Zn + As supplemented). The levels of reduced glutathione (GSH) and activities of glutathione reductase (GR) and ALP were lowered, whereas LPO levels and activity of superoxide dismutase (SOD) were elevated with no significant change in catalase (CAT) activity. Histopathological changes were also observed in the As substituted group in comparison to the control. Particle-induced X-ray emission (PIXE) analysis showed decrease in Fe and S concentration in rat liver after As intoxication, whereas As was below detection limit, i.e., <1 ppm. Zn administration almost restored the antioxidants, ALP activity, histopathological changes, and elemental status. A cumulative increase in MT expression was found with the combined treatment of Zn and As. Also, Zn alone caused no significant change in the antioxidant defense system. It can be concluded that restoration of antioxidant activity and increased MT expression are the two independent protective mechanisms of Zn to reduce acute As toxicity.


Arsenic Zinc Metallothionein Antioxidant defense system Liver 



This work is funded by University Grants Commission (UGC), New Delhi, India, and UGC Department of Atomic Energy (UGC-DAE) Consortium for Scientific Research, Kolkatta, India. Renuka Ganger and Roobee Garla are thankful to UGC, New Delhi, and Biraja Mohanty (Research Associate) is thankful to Indian Council of Medical Research (ICMR), New Delhi, for providing financial assistance in the form of Research Fellowships.


  1. 1.
    Flora SJS (2011) Arsenic-induced oxidative stress and its reversibility. Free Radic Biol Med 51:257–281CrossRefPubMedGoogle Scholar
  2. 2.
    Tseng CH (2009) A review on environmental factors regulating arsenic methylation in humans. Toxicol Appl Pharmacol 235:338–350CrossRefPubMedGoogle Scholar
  3. 3.
    Ventura-Lima J, Bogo MR, Monserrat JM (2011) Arsenic toxicity in mammals and aquatic animals: a comparative biochemical approach. Ecotoxicol Environ Saf 74:211–218CrossRefPubMedGoogle Scholar
  4. 4.
    Halliwell B, Whiteman M (2004) Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean? Br J Pharmacol 142:231–255PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Flora SJS, Bhadauria S, Kannan GM, Singh N (2007) Arsenic induced oxidative stress and the role of antioxidant supplementation during chelation: a review. J Environ Biol 28:333–347PubMedGoogle Scholar
  6. 6.
    Shen S, Li X-F, Cullen WR, Weinfeld M, Le XC (2013) Arsenic binding to proteins. Chem Rev 113:7769–7792PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Andreini C, Bertini I (2012) A bioinformatics view of zinc enzymes. J Inorg Biochem 111:150–156CrossRefPubMedGoogle Scholar
  8. 8.
    National Institute of Health (2013) NIH. Available:
  9. 9.
    Oteiza PI (2012) Zinc and the modulation of redox homeostasis. Free Radic Biol Med 53:1748–1759PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Ruttkay-Nedecky B, Nejdl L, Gumulec J, Zitka O, Masarik M, Eckschlager T, Stiborova M, Adam V, Kizek R (2013) The role of metallothionein in oxidative stress. Int J Mol Sci 14:6044–6066PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    Romero-Isart N, Vašák M (2002) Advances in the structure and chemistry of metallothioneins. J Inorg Biochem 88:388–396CrossRefPubMedGoogle Scholar
  12. 12.
    Liu J, Cheng M-L, Yang Q, Shan K-R, Shen J, Zhou Y, Zhang X, Dill AL, Waalkes MP (2007) Blood metallothionein transcript as a biomarker for metal sensitivity: low blood metallothionein transcripts in arsenicosis patients from Guizhou, China. Environ Health Perspect 115:1101–1106PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Beklova M, Adam V, Mares P, Zeman L, Dolezal P, Pikula J, Trnkova L, Kizek R (2008) Employing voltammetry for determination of low molecular mass thiols and metallothionein in blood of pig (Sus scrofa domestica). Nat Croat 17:293–301Google Scholar
  14. 14.
    Buico A, Cassino C, Dondero F, Vergani L, Osella D (2008) Radical scavenging abilities of fish MT-A and mussel MT-10 metallothionein isoforms: an ESR study. J Inorg Biochem 102:921–927CrossRefPubMedGoogle Scholar
  15. 15.
    Toh PP, Li JJ, Yip GW, Lo SL, Guo CH, Phan TT, Bay BH (2010) Modulation of metallothionein isoforms is associated with collagen deposition in proliferating keloid fibroblasts in vitro. Exp Dermatol 19:987–993CrossRefPubMedGoogle Scholar
  16. 16.
    Kang YJ (2006) Metallothionein redox cycle and function. Exp Biol Med 231:1459–1467Google Scholar
  17. 17.
    He X, Ma Q (2009) Induction of metallothionein I by arsenic via metal-activated transcription factor 1: critical role of C-terminal cysteine residues in arsenic sensing. J Biol Chem 284:12609–12621PubMedCentralCrossRefPubMedGoogle Scholar
  18. 18.
    Li Y, Kimura T, Laity JH, Andrews GK (2006) The zinc-sensing mechanism of mouse MTF-1 involves linker peptides between the zinc fingers. Mol Cell Biol 26:5580–5587PubMedCentralCrossRefPubMedGoogle Scholar
  19. 19.
    Garla R, Ganger R, Mohanty BP, Sudarshan M, Bansal MP, Garg ML (2013) Metal stoichiometry of isolated and arsenic substituted metallothionein: PIXE and ESI-MS study. Biometals 26(6):887–896CrossRefPubMedGoogle Scholar
  20. 20.
    Kreppel H, Liu J, Liu Y, Reichl FX, Klaassen CD (1994) Zinc-induced arsenite tolerance in mice. Fundam Appl Toxicol 23:32–37CrossRefPubMedGoogle Scholar
  21. 21.
    Kumar A, Malhotra A, Nair P, Garg M, Dhawan DK (2010) Protective role of zinc in ameliorating arsenic-induced oxidative stress and histological changes in rat liver. J Environ Pathol Toxicol Oncol 29:91–100CrossRefPubMedGoogle Scholar
  22. 22.
    Wills ED (1966) Mechanisms of lipid peroxide formation in animal tissues. Biochem J 99:667–676PubMedCentralCrossRefPubMedGoogle Scholar
  23. 23.
    Moron MS, Depierre JW, Mannervik B (1979) Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochim Biophys Acta 582:67–78CrossRefPubMedGoogle Scholar
  24. 24.
    Carlberg I, Mannervik B (1985) Glutathione reductase. Methods Enzymol 113:484–490CrossRefPubMedGoogle Scholar
  25. 25.
    Luck HE (ed) (1971) Catalase. Academic press, New YorkGoogle Scholar
  26. 26.
    Kono Y (1978) Generation of superoxide radical during autoxidation of hydroxylamine and an assay for superoxide dismutase. Arch Biochem Biophys 186:189–195CrossRefPubMedGoogle Scholar
  27. 27.
    Bergmeyer HE (ed) (1963) Methods of enzymatic analysis. Academic press, New YorkGoogle Scholar
  28. 28.
    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  29. 29.
    Bremner I, Davies NT (1975) The induction of metallothionein in rat liver by zinc injection and restriction of food intake. Biochem J 149:733–738PubMedCentralCrossRefPubMedGoogle Scholar
  30. 30.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  31. 31.
    Switzer RC, Merril CR, Shifrin S (1979) A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels. Anal Biochem 98:231–237CrossRefPubMedGoogle Scholar
  32. 32.
    Suzuki JS, Kodama N, Molotkov A, Aoki E, Tohyama C (1998) Isolation and identification of metallothionein isoforms (MT-1 and MT-2) in the rat testis. Biochem J 334:695–701PubMedCentralCrossRefPubMedGoogle Scholar
  33. 33.
    Krizkova S, Adam V, Eckschlager T, Kizek R (2009) Using of chicken antibodies for metallothionein detection in human blood serum and cadmium‐treated tumour cell lines after dot‐and electroblotting. Electrophoresis 30:3726–3735CrossRefPubMedGoogle Scholar
  34. 34.
    Loo OK, Adon MY, Kqueen CY, Ismail P, Jais AMM (2010) A comparative study of metallothionein gene expression in peripheral lymphocytes and blood cadmium level among die casting male workers. Global J Health Sci 2:129–136Google Scholar
  35. 35.
    Ebrahimi-Kalan A, Roudkenar MH, Halabian R, Milan PB, Zarrintan A, Roush AM (2011) Down-regulation of metallothionein 1 and 2 after exposure to electromagnetic field in mouse testis. Iran Biomed J 15:151–156PubMedCentralPubMedGoogle Scholar
  36. 36.
    Liu J, Zhou ZX, Zhang W, Bell MW, Waalkes MP (2009) Changes in hepatic gene expression in response to hepatoprotective levels of zinc. Liver Int 29:1222–1229PubMedCentralCrossRefPubMedGoogle Scholar
  37. 37.
    Ramos O, Carrizales L, Yanez L, Mejia J, Batres L, Ortiz D, Diaz-Barriga F (1995) Arsenic increased lipid peroxidation in rat tissues by a mechanism independent of glutathione levels. Environ Health Perspect 103:85–88PubMedCentralCrossRefPubMedGoogle Scholar
  38. 38.
    Van de Casteele M, Van Pelt JF, Nevens F, Fevery J, Reichen J (2003) Low NO bioavailability in CCl4 cirrhotic rat livers might result from low NO synthesis combined with decreased superoxide dismutase activity allowing superoxide-mediated NO breakdown: a comparison of two portal hypertensive rat models with healthy controls. Comp Hepatol 2:1–8CrossRefGoogle Scholar
  39. 39.
    Yeh CT, Yen GC (2006) Induction of hepatic antioxidant enzymes by phenolic acids in rats is accompanied by increased levels of multidrug resistance-associated protein 3 mRNA expression. J Nutr 136:11–15PubMedGoogle Scholar
  40. 40.
    Ranjith PB, Babu H, Ganesan V, Mathew M (2014) In-vivo antioxidant activity of Caesalpinia mimosoides LAMK. Res J Pharm Biol Chem Sci 5:1116–1120Google Scholar
  41. 41.
    Kujawska M, Jodynis-Liebert J, Ewertowska M, Adamska T, Matlawska I, Bylka W (2007) Protective effect of Aquilegia vulgaris (L.) on carbon tetrachloride-induced oxidative stress in rats. Indian J Exp Biol 45:702–711PubMedGoogle Scholar
  42. 42.
    Evelson P, Susemihl C, Villarreal I, Llesuy S, Rodríguez R, Peredo H, Lemberg A, Perazzo J, Filinger E (2005) Hepatic morphological changes and oxidative stress in chronic streptozotocin-diabetic rats. Ann Hepatol 4:115–120PubMedGoogle Scholar
  43. 43.
    Ferzand R, Gadahi JA, Saleha S, Ali Q (2008) Histological and haematological disturbance caused by arsenic toxicity in mice model. Pak J Biol Sci 11:1405–1413CrossRefPubMedGoogle Scholar
  44. 44.
    Sharma A, Sharma MK, Kumar M (2009) Modulatory role of Emblica officinalis fruit extract against arsenic induced oxidative stress in Swiss albino mice. Chem Biol Interact 180:20–30CrossRefPubMedGoogle Scholar
  45. 45.
    Powell SR (2000) The antioxidant properties of zinc. J Nutr 130:1447S–1454SPubMedGoogle Scholar
  46. 46.
    Jomova K, Valko M (2011) Advances in metal-induced oxidative stress and human disease. Toxicology 283:65–87CrossRefPubMedGoogle Scholar
  47. 47.
    Maiti S, Chatterjee AK (2001) Effects on levels of glutathione and some related enzymes in tissues after an acute arsenic exposure in rats and their relationship to dietary protein deficiency. Arch Toxicol 75:531–537CrossRefPubMedGoogle Scholar
  48. 48.
    Ferretti L, Elviri L, Pellinghelli MA, Predieri G, Tegoni M (2007) Glutathione and N-acetylcysteinylglycine: protonation and Zn2+ complexation. J Inorg Biochem 101:1442–1456CrossRefPubMedGoogle Scholar
  49. 49.
    Chouchane S, Snow ET (2001) In vitro effect of arsenical compounds on glutathione-related enzymes. Chem Res Toxicol 14:517–522CrossRefPubMedGoogle Scholar
  50. 50.
    Rodriguez VM, Del Razo LM, Limon-Pacheco JH, Giordano M, Sanchez-Pena LC, Uribe-Querol E, Gutierrez-Ospina G, Gonsebatt ME (2005) Glutathione reductase inhibition and methylated arsenic distribution in Cd1 mice brain and liver. Toxicol Sci 84:157–166CrossRefPubMedGoogle Scholar
  51. 51.
    Pi J, Yamauchi H, Kumagai Y, Sun G, Yoshida T, Aikawa H, Hopenhayn-Rich C, Shimojo N (2002) Evidence for induction of oxidative stress caused by chronic exposure of Chinese residents to arsenic contained in drinking water. Environ Health Perspect 110:331–336PubMedCentralCrossRefPubMedGoogle Scholar
  52. 52.
    Gupta R, Dubey DK, Kannan GM, Flora SJS (2007) Concomitant administration of Moringa oleifera seed powder in the remediation of arsenic-induced oxidative stress in mouse. Cell Biol Int 31:44–56CrossRefPubMedGoogle Scholar
  53. 53.
    Sidhu P, Garg ML, Dhawan DK (2004) Protective role of zinc in nickel induced hepatotoxicity in rats. Chem Biol Interact 150:199–209CrossRefPubMedGoogle Scholar
  54. 54.
    Albores A, Koropatnick J, Cherian MG, Zelazowski AJ (1992) Arsenic induces and enhances rat hepatic metallothionein production in vivo. Chem Biol Interact 85:127–140CrossRefPubMedGoogle Scholar
  55. 55.
    Yang Y, Maret W, Vallee BL (2001) Differential fluorescence labeling of cysteinyl clusters uncovers high tissue levels of thionein. Proc Natl Acad Sci 98:5556–5559PubMedCentralCrossRefPubMedGoogle Scholar
  56. 56.
    Haq F, Mahoney M, Koropatnick J (2003) Signaling events for metallothionein induction. Mutat Res Fundam Mol Mech Mutagen 533:211–226CrossRefGoogle Scholar
  57. 57.
    Albores A, Cebrian ME, Bach PH, Connell JC, Hinton RH, Bridges JW (1989) Sodium arsenite induced alterations in bilirubin excretion and heme metabolism. J Biochem Toxicol 4:73–78CrossRefPubMedGoogle Scholar
  58. 58.
    Cebrian ME, Albores A, Connelly JC, Bridges JW (1988) Assessment of arsenic effects on cytosolic heme status using tryptophan pyrrolase as an index. J Biochem Toxicol 3:77–86CrossRefPubMedGoogle Scholar
  59. 59.
    Leonoudakis D, Gray AT, Winegar BD, Kindler CH, Harada M, Taylor DM, Chavez RA, Forsayeth JR, Yost CS (1998) An open rectifier potassium channel with two pore domains in tandem cloned from rat cerebellum. J Neurosci 18:868–877PubMedGoogle Scholar
  60. 60.
    Harrison NL, Radke HK, Tamkun MM, Lovinger DM (1993) Modulation of gating of cloned rat and human K+ channels by micromolar Zn2+. Mol Pharmacol 43:482–486PubMedGoogle Scholar
  61. 61.
    Morris DR, Levenson CW (2012) Ion channels and zinc: mechanisms of neurotoxicity and neurodegeneration. J Toxicol 2012:1–6CrossRefGoogle Scholar
  62. 62.
    William CW, Christopher MG, Hoyt DB (2007) Trauma: critical care. Volume 2. CRC Press, Taylor and Francis Group, Boca RatonGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Renuka Ganger
    • 1
    Email author
  • Roobee Garla
    • 1
  • Biraja Prasad Mohanty
    • 1
  • Mohinder Pal Bansal
    • 1
  • Mohan Lal Garg
    • 1
  1. 1.Department of BiophysicsPanjab UniversityChandigarhIndia

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