Archives of Toxicology

, Volume 87, Issue 2, pp 311–321 | Cite as

Metallothionein blocks oxidative DNA damage in vitro

Immunotoxicology

Abstract

The role of metallothionein (MT) in mitigation of oxidative DNA damage (ODD) induced by either cadmium (Cd) or the direct oxidant hydrogen peroxide (H2O2) was systematically examined using MT-I/II double knockout (MT-null) or MT-competent wild-type (WT) cells. Both toxicants were much more lethal to MT-null cells (Cd LC50 = 6.6 μM; H2O2 LC50 = 550 μM) than to WT cells (Cd LC50 = 16.5 μM; H2O2 LC50 = 930 μM). Cd induced concentration-related MT increases in WT cells, while the basal levels were undetectable and not increased by Cd in MT-null cells. ODD, measured by the immuno-spin trapping method, was minimally induced by sub-toxic Cd levels (1 or 5 μM; 24 h) in WT cells, but markedly increased in MT-null cells (>430 %). Similarly, ODD was induced to higher levels by lower concentrations of H2O2 in MT-null cells than WT cells. Transfection of MT-I into MT-null cells reduced both Cd- and H2O2-induced cytolethality and ODD. Cd increased the expression of the oxidant defense genes, HO-1, and GSTa2 to a much greater extent in MT-null cells than in WT. Cd or H2O2 exposure increased the expression of key transport genes, Mrp1 and Mrp2, in WT cells but not in MT-null cells. MT protects against Cd- and H2O2-induced ODD in MT-competent cells possibly by multiple mechanisms, potentially including direct metal ion sequestration and sequestration of oxidant radicals by MT. MT-deficient cells appear to adapt to Cd primarily by turning on oxidant response systems, while MT-competent cells activate MT and transport systems.

Keywords

DNA Cadmium Oxidative damage Metallothionein Cancer 

Abbreviations

Cd

Cadmium

CdCl2

Cadmium chloride

DMPO

5,5-dimethyl-1-pyrroline N-oxide

GST

Glutathione S-transferase

GST-π

Glutathione S-transferase pi 1

GSTα2

Glutathione S-transferase-α2

HO-1

Heme oxygenase 1

H2O2

Hydrogen peroxide

IST

Immuno-spin trapping

LC50

Lethal concentration 50 %

Mrp1

Multidrug resistance-related protein 1

Mrp2

Multidrug resistance-related protein 2

MT

Metallothionein

MT-null

MT-I/II knockout

ODD

Oxidative DNA damage

RT-PCR

Reverse transcription-polymerase chain reaction

ROS

Reactive oxygen species

WT

Wild type

Notes

Acknowledgments

The authors thank Drs. Rachel Person, Katie Pelch, John Bucher, and Nigel Walker for critical review of this manuscript. The authors also thank Matthew W. Bell for aid with the graphics. This article may be the work product of an employee or a group of employees of the NIEHS, NIH; however, the statements contained herein do not necessarily represent the statements, opinions, or conclusions of the NIEHS, NIH. or the United States Government. The content of this publication does not necessarily reflect the views or the policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

Conflict of interest

None.

References

  1. Bertin G, Averbeck D (2006) Cadmium: cellular effects, modifications of biomolecules, modulation of DNA repair and genotoxic consequences. Biochimie 88:1549–1559CrossRefPubMedGoogle Scholar
  2. Braithwaite EK, Mattie MD, Freedman JH (2010) Activation of metallothionein transcription by 4-hydroxynonenal. J Biochem Mol Toxicol 24:330–334CrossRefPubMedGoogle Scholar
  3. Carrière P, Mantha M, Champagne-Paradis S, Jumarie C (2011) Characterization of basolateral-to-apical transepithelial transport of cadmium in intestinal TC7 cell monolayers. Biometals 24:857–874CrossRefPubMedGoogle Scholar
  4. Chiaverini N, Ley MD (2010) Protective effect of metallothionein on oxidative stress-induced DNA damage. Free Radic Res 44:605–613CrossRefPubMedGoogle Scholar
  5. Colangelo D, Mahboobi H, Viarengo A, Osella D (2004) Protective effect of metallothioneins against oxidative stress evaluated on wild type and MT-null cell lines by means of flow cytometry. Biometals 17:365–370CrossRefPubMedGoogle Scholar
  6. Coogan TP, Bare RM, Waalkes MP (1992) Cadmium-induced DNA strand damage in cultured liver cells: reduction in cadmium genotoxicity following zinc pretreatment. Toxicol Appl Pharmacol 113:227–233CrossRefPubMedGoogle Scholar
  7. Cuypers A, Plusquin M, Remans T, Jozefczak M, Keunen E, Gielen H, Opdenakker K, Nair AR, Munters E, Artois TJ, Nawrot T, Vangronsveld J, Smeets K (2010) Cadmium stress: an oxidative challenge. Biometals 23:927–940CrossRefPubMedGoogle Scholar
  8. Filipic M, Hei TK (2004) Mutagenicity of cadmium in mammalian cells: implication of oxidative DNA damage. Mutat Res 546:81–91CrossRefPubMedGoogle Scholar
  9. Fridovich I (1986) Biological effects of the superoxide radical. Arch Biochem Biophys 247:1–11CrossRefPubMedGoogle Scholar
  10. Fujishiro H, Kubota K, Inoue D, Inoue A, Yanagiya T, Enomoto S, Himeno S (2011) Cross-resistance of cadmium-resistant cells to manganese is associated with reduced accumulation of both cadmium and manganese. Toxicology 280:118–125CrossRefPubMedGoogle Scholar
  11. Gioacchino MD, Petrarca C, Perrone A, Martino S, Esposito DL, Lotti LV, Mariani-Costantini R (2008) Autophagy in hematopoietic stem/progenitor cells exposed to heavy metals: biological implications and toxicological relevance. Autophagy 4:537–539PubMedGoogle Scholar
  12. Halliwell B, Clement MV, Long LH (2000) Hydrogen peroxide in the human body. FEBS Lett 486:10–13CrossRefPubMedGoogle Scholar
  13. Hoffmann U, Kroemer HK (2004) The ABC transporters MDR1 and MRP2: multiple functions in disposition of xenobiotics and drug resistance. Drug Metab Rev 36:669–701CrossRefPubMedGoogle Scholar
  14. International Agency for Research on Cancer (IARC) (2012) Monographs on the evaluation of the carcinogenic risks to humans; arsenic, metals and fibers; volume 100C “Arsenic and Arsenic Compounds”, Lyon, France, pp 41–93 (in press; download available online)Google Scholar
  15. Jomova K, Valko M (2011) Advances in metal-induced oxidative stress and human disease. Toxicology 10:65–87CrossRefGoogle Scholar
  16. Joseph P, Muchnok TK, Klishis ML, Roberts JR, Antonini JM, Whong WZ, Ong T (2001) Cadmium-induced cell transformation and tumorigenesis are associated with transcriptional activation of c-fos, c-jun, and c-myc proto-oncogenes: role of cellular calcium and reactive oxygen species. Toxicol Sci 61:295–303CrossRefPubMedGoogle Scholar
  17. Jungsuwadee P, Nithipongvanitch R, Chen Y, Oberley TD, Butterfield DA, St Clair DK, Vore M (2009) Mrp1 localization and function in cardiac mitochondria after doxorubicin. Mol Pharmacol 75:1117–1126CrossRefPubMedGoogle Scholar
  18. Keshava N, Zhou G, Hubbs AF, Ensell MX, Ong T (2000) Transforming and carcinogenic potential of cadmium chloride in BALB/c-3T3 cells. Mutat Res 448:23–28CrossRefPubMedGoogle Scholar
  19. Klaassen CD, Liu J, Choudhuri S (1999) Metallothionein: an intracellular protein to protect against cadmium toxicity. Annu Rev Pharmacol Toxicol 39:267–294CrossRefPubMedGoogle Scholar
  20. Klaassen CD, Liu J, Diwan BA (2009) Metallothionein protection of cadmium toxicity. Toxicol Appl Pharmacol 238:215–220CrossRefPubMedGoogle Scholar
  21. Knipp M (2009) Metallothioneins and platinum(II) anti-tumor compounds. Curr Med Chem 16:522–537CrossRefPubMedGoogle Scholar
  22. Lazo JS, Kondo Y, Dellapiazza D, Michalska AE, Choo KH, Pitt BR (1995) Enhanced sensitivity to oxidative stress in cultured embryonic cells from transgenic mice deficient in metallothionein I and II genes. J Biol Chem 270:5506–5510CrossRefPubMedGoogle Scholar
  23. Leslie EM, Liu J, Klaassen CD, Waalkes MP (2006) Acquired cadmium resistance in metallothionein-I/II(-/-) knockout cells: role of the T-type calcium channel Cacnalpha1G in cadmium uptake. Mol Pharmacol 69:629–639CrossRefPubMedGoogle Scholar
  24. Lim SC, Hahm KS, Lee SH, Oh SH (2010) Autophagy involvement in cadmium resistance through induction of multidrug resistance-associated protein and counterbalance of endoplasmic reticulum stress WI38 lung epithelial fibroblast cells. Toxicology 276:18–26CrossRefPubMedGoogle Scholar
  25. Liu J, Cheng ML, Yang Q, Shan KR, 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 115:1101–1106CrossRefPubMedGoogle Scholar
  26. Mates JM, Sanchez-Jimenez FM (2000) Role of reactive oxygen species in apoptosis: implications for cancer therapy. Int J Biochem Cell Biol 32:157–170CrossRefPubMedGoogle Scholar
  27. Misra RR, Smith GT, Waalkes MP (1998) Evaluation of the direct genotoxic potential of cadmium in four different rodent cell lines. Toxicology 126:103–114CrossRefPubMedGoogle Scholar
  28. Nawrot TS, Van Hecke E, Thijs L, Richart T, Kuznetsova T, Jin Y, Vangronsveld J, Roels HA, Staessen JA (2008) Cadmium-related mortality and long-term secular trends in the cadmium body burden of an environmentally exposed population. Environ Health Perspect 116:1620–1628CrossRefPubMedGoogle Scholar
  29. Nemmiche S, Chabane-Sari D, Kadri M, Guiraud P (2011) Cadmium chloride-induced oxidative stress and DNA damage in the human Jurkat T cell line is not linked to intracellular trace elements depletion. Toxicol In Vitro 25:191–198CrossRefPubMedGoogle Scholar
  30. Nzengue Y, Steiman R, Rachidi W, Favier A, Guiraud P (2012) Oxidative stress induced by cadmium in the c6 cell line: role of copper and zinc. Biol Trace Elem Res 146:410–419CrossRefPubMedGoogle Scholar
  31. O’Brien P, Salacinski HJ (1998) Evidence that the reactions of cadmium in the presence of metallothionein can produce hydroxyl radicals. Arch Toxicol 72:690–700CrossRefPubMedGoogle Scholar
  32. Pabla N, Dong Z (2008) Cisplatin nephrotoxicity: mechanisms and renoprotective strategies. Kidney Int 73:994–1007CrossRefPubMedGoogle Scholar
  33. Park JD, Liu Y, Klaassen CD (2001) Protective effect of metallothionein against the toxicity of cadmium and other metals. Toxicology 163:93–100CrossRefPubMedGoogle Scholar
  34. Qu W, Diwan BA, Liu J, Goyer RA, Dawson T, Horton JL, Cherian MG, Waalkes MP (2002) The metallothionein-null phenotype is associated with heightened sensitivity to lead toxicity and an inability to form inclusion bodies. Am J Pathol 160:1047–1056CrossRefPubMedGoogle Scholar
  35. Qu W, Diwan BA, Reece JM, Bortner CD, Pi J, Liu J, Waalkes MP (2005) Cadmium-induced malignant transformation in rat liver cells: role of aberrant oncogene expression and minimal role of oxidative stress. Int J Cancer 114:346–355CrossRefPubMedGoogle Scholar
  36. Quaife CJ, Cherne RL, Newcomb TG, Kapur RP, Palmiter RD (1999) Metallothionein overexpression suppresses hepatic hyperplasia induced by hepatitis B surface antigen. Toxicol Appl Pharmacol 155:107–116CrossRefPubMedGoogle Scholar
  37. Ramirez DC, Gomez-Mejiba SE, Mason RP (2006) Immuno-spin trapping of DNA radicals. Nat Methods 3:123–127CrossRefPubMedGoogle Scholar
  38. Ramirez DC, Gomez-Mejiba SE, Mason RP (2007) Immuno-spin trapping analyses of DNA radicals. Nat Protoc 2:512–522CrossRefPubMedGoogle Scholar
  39. Report on Carcinogens (2011) National Toxicology Program, 12th edn. Department of Health and Human Services, Research Triangle Park, NCGoogle Scholar
  40. Sekine S, Ito K, Horie T (2006) Oxidative stress and Mrp2 internalization. Free Radic Biol Med 40:2166–2174CrossRefPubMedGoogle Scholar
  41. Shiraishi N, Hochadel JF, Coogan TP, Koropatnick J, Waalkes MP (1995) Sensitivity to cadmium-induced genotoxicity in rat testicular cells is associated with minimal expression of the metallothionein gene. Toxicol Appl Pharmacol 130:229–236CrossRefPubMedGoogle Scholar
  42. St Croix CM, Wasserloos KJ, Dineley KE, Reynolds IJ, Levitan ES, Pitt BR (2002) Nitric oxide-induced changes in intracellular zinc homeostasis are mediated by metallothionein/thionein. Am J Physiol Lung Cell Mol Physiol 282:L185–L192Google Scholar
  43. Suzuki JS, Nishimura N, Zhang B, Nakatsuru Y, Kobayashi S, Satoh M, Tohyama C (2003) Metallothionein deficiency enhances skin carcinogenesis induced by 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate in metallothionein-null mice. Carcinogenesis 24:1123–1132CrossRefPubMedGoogle Scholar
  44. Thévenod F (2003) Nephrotoxicity and proximal tubule: insights from cadmium. Nephron Physiol 93:87–93CrossRefGoogle Scholar
  45. Thévenod F (2009) Cadmium and cellular signaling cascades: to be or not to be? Toxicol Appl Pharmacol 238:221–239CrossRefPubMedGoogle Scholar
  46. Waalkes MP, Klaassen CD (1985) Concentration of metallothionein in major organs of rats after administration of various metals. Fundam Appl Toxicol 5:473–477CrossRefPubMedGoogle Scholar
  47. Waalkes MP, Liu J (2009) Metallothionein in inorganic carcinogenesis, Chapter 13. In: Sigel A, Sigel H, Sigel RKO (eds) Metal ions in life science, vol 5. Royal Society of Chemistry, London, pp 399–412Google Scholar
  48. Waalkes MP, Rehm S (1994) Chronic toxic and carcinogenic effects of cadmium chloride in male DBA/2NCr and NFS/NCr mice: strain-dependent association with tumors of the hematopoietic system, injection site, liver, and lung. Fundam Appl Toxicol 23:21–31CrossRefPubMedGoogle Scholar
  49. Waalkes MP, Liu J, Kasprzak KS, Diwan BA (2006) Hypersusceptibility to cisplatin carcinogenicity in metallothionein-I/II double knockout mice: production of hepatocellular carcinoma at clinically relevant doses. Int J Cancer 119:28–32CrossRefPubMedGoogle Scholar
  50. Waisberg M, Joseph P, Hale B, Beyersmann D (2003) Molecular and cellular mechanisms of cadmium carcinogenesis. Toxicology 192:95–117CrossRefPubMedGoogle Scholar
  51. Zuo P, Qu W, Cooper RN, Goyer RA, Diwan BA, Waalkes MP (2009) Potential role of alpha-synuclein and metallothionein in lead-induced inclusion body formation. Toxicol Sci 111:100–108CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag (outside the USA) 2012

Authors and Affiliations

  1. 1.Inorganic Toxicology Group, National Toxicology Program Laboratory, Division of the National Toxicology ProgramNational Institute of Environmental Health SciencesResearch Triangle ParkUSA
  2. 2.Division of Translational BiologyThe Hamner Institutes for Health SciencesResearch Triangle ParkUSA

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