Biological Trace Element Research

, Volume 150, Issue 1–3, pp 178–194 | Cite as

Cd, Cu, Zn, Se, and Metallothioneins in Two Amphibians, Necturus maculosus (Amphibia, Caudata) and Bufo bufo (Amphibia, Anura)

  • Katarina Dobrovoljc
  • Ingrid Falnoga
  • Magda Tušek Žnidarič
  • Darja Mazej
  • Janez Ščančar
  • Boris Bulog


The accumulation of cadmium, its affinity for metallothioneins (MTs), and its relation to copper, zinc, and selenium were investigated in the experimental mudpuppy Necturus maculosus and the common toad Bufo bufo captured in nature. Specimens of N. maculosus were exposed to waterborne Cd (85 μg/L) for up to 40 days. Exposure resulted in tissue-dependent accumulation of Cd in the order kidney, gills > intestine, liver, brain > pancreas, skin, spleen, and gonads. During the 40-day exposure, concentrations increased close to 1 μg/g in kidneys and gills (0.64–0.95 and 0.52–0.76; n = 4), whereas the levels stayed below 0.5 in liver (0.14–0.29; n = 4) and other organs. Cd exposure was accompanied by an increase of Zn and Cu in kidneys and Zn in skin, while a decrease of Cu was observed in muscles and skin. Cytosol metallothioneins (MTs) were detected as Cu,Zn–thioneins in liver and Zn,Cu–thioneins in gills and kidney, with the presence of Se in all cases. After exposure, Cd binding to MTs was clearly observed in cytosol of gills as Zn,Cu,Cd–thionein and in pellet extract of kidneys as Zn,Cu,Cd–thioneins. The results indicate low Cd storage in liver with almost undetectable Cd in liver MT fractions. In field trapped Bufo bufo (spring and autumn animals), Cd levels were followed in four organs and found to be in the order kidney > liver (0.56–5.0 μg/g >0.03–0.72 μg/g; n = 11, spring and autumn animals), with no detectable Cd in muscle and skin. At the tissue level, high positive correlations between Cd, Cu, and Se were found in liver (all r > 0.80; α = 0.05, n = 5), and between Cd and Se in kidney (r = 0.76; n = 5) of autumn animals, possibly connected with the storage of excess elements in biologically inert forms. In the liver of spring animals, having higher tissue level of Cd than autumn ones, part of the Cd was identified as Cu,Zn,Cd–thioneins with traces of Se. As both species are special in having liver Cu levels higher than Zn, the observed highly preferential Cd load in kidney seems reasonable. The relatively low Cd found in liver can be attributed to its excretion through bile and its inability to displace Cu from MTs. The associations of selenium observed with Cd and/or Cu (on the tissue and cell level) point to selenium involvement in the detoxification of excessive cadmium and copper through immobilization.


Amphibia Cadmium Copper Metallothionein Selenium Zinc 



The authors are grateful to Dr. Anthony R. Byrne for critical reading of the manuscript. This study was supported by a grant from the Ministry of Higher Education, Science and Technology, Republic of Slovenia (through research program P1-0143 and through the PhD project of one of the authors—K. Dobrovoljc). The study was approved by the Veterinary Administration of the Republic of Slovenia and the Ministry of the Environment and Spatial Planning, and complies with the National and European Union guidelines for the care and treatment of laboratory animals.


  1. 1.
    Martelli A, Rousselet E, Dycke C et al (2006) Cadmium toxicity in animal cells by interference with essential metals. Biochimie 88:1807–1814PubMedCrossRefGoogle Scholar
  2. 2.
    Kägi JHR (1993) Evolution, structure and chemical activity of class I metallothioneins: an overview. In: Imura N, Kimura M, Suzuki KT (eds) Metallothionein III. Birkhauser, Basel, pp 29–56Google Scholar
  3. 3.
    Coyle P, Philcox JC, Carey LC, Rofe AM (2002) Metallothionein: the multipurpose protein. Review Cell Mol Life Sci 59:627–647CrossRefGoogle Scholar
  4. 4.
    Bremner I (1993) Metallothionein in copper deficiency and toxicity. In: Anke M, Meissner D, Mills CF (Eds) Trace elements in man and animals, vol 8. NRC Research Press, Ottawa pp. 507-515Google Scholar
  5. 5.
    Maret W (2008). Thiol reactivity as a central aspect of metallothionein's mechanism of action. In: Zatta P (Ed) Metallothioneins in biochemistry and pathology. World Scientific, New Jersey, pp. 28-45.Google Scholar
  6. 6.
    Dobrovoljc K, Falnoga I, Bulog B, Tušek Žnidarič M, Ščančar J (2003) Hepatic metallothionein in two neoteric salamanders, Proteus anguinus and Necturus maculosus (Amphibia, Caudata). Comp Biochem Physiol 135C:285–294Google Scholar
  7. 7.
    Suzuki KT, Kawamura R (1984) Metallothionein present or induced in the three species of frogs Bombina orientalis, Bufo bufo japonicas and Hyla arborea japonica. Comp Biochem Physiol 79C:255–260Google Scholar
  8. 8.
    Goldfisher S, Schiller B, Sternlieb I (1970) Copper in hepatic lysosomes of the toad, Bufo marinus L. Nat 228:172–73CrossRefGoogle Scholar
  9. 9.
    Pasanen S, Koskela P (1974) Seasonal changes in calcium, magnesium, copper and zinc content in the liver of the common frog Rana temporaria L. Comp Biochem Physiol 48:27–36CrossRefGoogle Scholar
  10. 10.
    Suzuki KT, Itoh N, Ohta K, Sunaga H (1986) Amphibian metallothionein. Induction in the frogs Rana japonica, R. nigromaculata and Rhacophorus schlegelii. Comp Biochem Physiol 83C:253–259Google Scholar
  11. 11.
    Linde AR, Klein D, Summer KH (2005) Phenomenon of hepatic overload of Cu in Mugil cephalus: role of metallothioneins and patterns of copper distribution. Basic Clin Pharmacol Toxicol 97:230–235PubMedCrossRefGoogle Scholar
  12. 12.
    Köherle J, Brigelius-Flohe R, Bock A, Gartner R, Meyer O, Flohe L (2000) Review: Selenium in biology: facts and medical perspectives. Biol Chem 381:849–864Google Scholar
  13. 13.
    Brown DD, Cai L (2007) Amphibian metamorphosis. Dev Biol 306:20–33PubMedCrossRefGoogle Scholar
  14. 14.
    Lobanov AV, Hatfield DL, Gladyshev VN (2008) Reduced reliance of the trace element selenium during evolution of mammals. Genome Biol 9:R62PubMedCrossRefGoogle Scholar
  15. 15.
    Sasakura S, Suzuki KT (1998) Biological interaction between transition metals (Ag, Cd and Hg), selenide/sulfide and selenoprotein P. J Inorganic Biochem 71:159–162CrossRefGoogle Scholar
  16. 16.
    Burk RF, Hill KE (2005) Selenoprotein P: an extracellular protein with unique physical characteristics and a role in selenium homeostasis. Annu Rev Nutr 25:215–235PubMedCrossRefGoogle Scholar
  17. 17.
    Hill CH (1975) Interrelationships of selenium with other trace elements. Fed Proc 34:2096–100PubMedGoogle Scholar
  18. 18.
    Curvin-Aralar MLA, Furness RW (1991) Mercury and selenium: a review. Ecotoxicol Environ Saf 21:348–364CrossRefGoogle Scholar
  19. 19.
    Berry JP, Zhang L, Galle P (1995) Interaction of selenium with copper, silver, and gold salts. Electron microprobe study. J Submicrosc Cytol Pathol 27:21–28PubMedGoogle Scholar
  20. 20.
    Diplock AT, Watkins WJ, Hewison M (1986) Selenium and heavy metals. Annals Clin Res 18:55–66Google Scholar
  21. 21.
    Toman R, Golian J, Šiška B, Massanyi P, Lukáč N, Adamkovičevá M (2009) Cadmium and selenium in animal tissues and their interactions after an experimental administration to rats. Slovak J Anim Sci 42:115–118Google Scholar
  22. 22.
    Byrne AR, Kosta L, Stegnar P (1975) The occurrence of mercury in Amphibia. Environ Lett 8:147–155PubMedCrossRefGoogle Scholar
  23. 23.
    Gailer J (2007) Arsenic–selenium and mercury–selenium bonds in biology. Coord Chem Rev 251:234–254CrossRefGoogle Scholar
  24. 24.
    Viljoen AJ, Tappel AL (1988) Interactions of selenium and cadmium with metallothionein-like and other cytosolic proteins of rat kidney and liver. J Inorg Biochem 34:277–290PubMedCrossRefGoogle Scholar
  25. 25.
    Takatera K, Osaki N, Yamaguchi H, Watanabe T (1994) HPLC/ICP mass spectrometric study of the selenium incorporation into cyanobacterial metallothionein induced under heavy-metal stress. Anal Sci 10:567–572CrossRefGoogle Scholar
  26. 26.
    Falnoga I, Tušek Žnidarič M, Mazej D, Stibilj V, Dobrovoljc K (2005) Selenium and metallothionein association. Abstracts, The Fifth International Conference on Metallothioneins, Beijing, China, Oct. 8–12, p 21Google Scholar
  27. 27.
    Bulog B, Mihajl K, Jeran Z, Toman MJ (2002) Trace element concentrations in the tissues of Proteus anguinus (Amphibia, Caudata) and the surrounding environment. Water Air Soil Pollut 136:147–163CrossRefGoogle Scholar
  28. 28.
    Stibilj V, Mazej D, Falnoga I (2003) A study of low level selenium determination by hydride generation atomic fluorescence spectrometry in water soluble protein and peptide fractions. Anal Bioanal Chem 337:1175–1183CrossRefGoogle Scholar
  29. 29.
    Mazej D, Falnoga I, Veber M, Stibilj V (2006) Determination of selenium species in plant leaves by HPLC–UV–HG–AFS. Talanta 68:558–568PubMedCrossRefGoogle Scholar
  30. 30.
    Klein D, Lichtmanegger J, Heizmann U, Müler-Höcker J, Summer KH (1999) Fate of copper and metallothionein in the liver of LEC rats. In: Klassen C (ed) Metallothionein IV. Birkhäuser, Basel, pp 403–412CrossRefGoogle Scholar
  31. 31.
    Wong CK, Wong MH (2000) Morphological and biochemical changes in the gills of Tilapia (Oreochromis mossambicus) to ambient cadmium exposure. Aquat Toxicol 48:517–525PubMedCrossRefGoogle Scholar
  32. 32.
    Smith PN, Cobb GP, Godard-Codding C, Hoff D, McMurray SC, Rainwater TR, Reynolds KD (2007) Contaminant exposure in terrestrial vertebrates. Environ Poll 150:41–64CrossRefGoogle Scholar
  33. 33.
    Vogiatzis AK, Loumbourdis NS (1997) Uptake, tissue distribution and depuration of cadmium (Cd) in the frog Rana ridibunda. Bull Environ Contam Toxicol 59:770–776PubMedCrossRefGoogle Scholar
  34. 34.
    Vogiatzis AK, Loumbourdis NS (1998) Cadmium accumulation in liver and kidneys and hepatic metallothionein and glutathione levels in Rana ridibunda, after exposure to CdCl2. Arch Environ Contam Toxicol 34:64–68PubMedCrossRefGoogle Scholar
  35. 35.
    Mihajl K (1998). Tracing the microelements in the tissues and environments of Proteus anguinus (Urodela, Amphibia). Graduation thesis, University of Ljubljana (in Slovene)Google Scholar
  36. 36.
    Tjälve H, Henriksson J (1999) Uptake of metals in the brain via olfactory pathways. Neurotoxicol 20:181–196Google Scholar
  37. 37.
    Gu C, Chen S, Xu X, Zheng L, Li Y, Wu K, Liu J, Qi Z, Han D, Chen G, Huo X (2009) Lead and cadmium synergistically enhance the expression of divalent metal transporter 1 protein in central system of developing rats. Neurochem Res 34:1150–1156PubMedCrossRefGoogle Scholar
  38. 38.
    James SM, Little EE, Semlitch RD (2004) The effect of soil composition and hydration on the bioavailability and toxicity of cadmium to hibernating juvenile American toads (Bufo americanus). Environ Pollut 132:523–532PubMedCrossRefGoogle Scholar
  39. 39.
    Stawarz R, Fromicki G, (2003) Copper, zinc, cadmium and lead levels in fat bodies of adult female Rana esculenta L. During hibernation. Rizikové faktory potravového ret'azca III. Nitra, 133–38Google Scholar
  40. 40.
    Mihajl K (2002) Copper, zinc, and cadmium binding metallothionein in the liver of Proteus anguinus (Amphibia: Caudata). Master of Science thesis, University of Ljubljana (in Slovene)Google Scholar
  41. 41.
    Papadimitriou E, Loumbourdis LS (2003) Copper kinetics and hepatic metallothionein levels in the frog Rana ridibunda, after exposure to CuCl2. BioMetals 16:271–277PubMedCrossRefGoogle Scholar
  42. 42.
    Sichel G, Scalia M, Corsaro C (2002) Amphibia Kupffer cells. Microscopy Res Tech 57:477–490CrossRefGoogle Scholar
  43. 43.
    Barni S, Vaccarone R, Bertone V, Fraschini A, Bernini F, Fenoglio C (2002) Mechanisms of changes to the liver pigmentary component during the annual cycle (activity and hibernation) of Rana esculenta L. J Anat 200:185–194PubMedCrossRefGoogle Scholar
  44. 44.
    Gallone A, Sagliano A, Guida G, Ito S, Wakamatsu K, Capozzi V, Perna G, Zanna P, Cicero R (2007) The melanogenic system of the liver pigmented macrophages of Rana esculenta L.—tyrosinase activity. Histol Histopathol 22:1065–75PubMedGoogle Scholar
  45. 45.
    Hong L, Simon JD (2007) Current understanding of the binding sites, capacity, affinity, and biological significance of metals in melanin. J Phys Chem B 111:7938–7947PubMedCrossRefGoogle Scholar
  46. 46.
    Balamurogan K, Hua H, Georgiev O, Schaffner W (2009) Mercury and cadmium trigger expression of the copper importer Ctr1B, which enables Drosophila to thrive on heavy metal-loaded food. Biol Chem 320:109–13Google Scholar
  47. 47.
    Gaetke LM, Chow CK (2003) Copper toxicity, oxidative stress, and antioxidant nutrients. Toxicol 189:147–63CrossRefGoogle Scholar
  48. 48.
    Viarengo A (1989) Heavy metals in marine invertebrates: mechanisms of regulation and toxicity at the cellular level. CRC Crit Rev Aquat Sci 1:295–317Google Scholar
  49. 49.
    Berry JP, Galle P (1994) Selenium–arsenic interaction in renal cells: role of lysosomes. Electron microprobe study. J Submicrosc Cytol Pathol 26:203–106PubMedGoogle Scholar
  50. 50.
    Falnoga I, Tušek Žnidarič M (2007) Selenium–mercury interactions in man and animals. Biol Trace Elem Res 3:212–220CrossRefGoogle Scholar
  51. 51.
    Loumburdis NS, Vogiatzis AK (2002) Impact of liver pigmentary system of the frog Rana ridibunda. Ecotoxicol Environmen 53:52–58CrossRefGoogle Scholar
  52. 52.
    Ghost P, Thomas P (1995) Binding of metals to red drum vitellogenin and incorporation into oocytes. Mar Environ Res 39:165–168CrossRefGoogle Scholar
  53. 53.
    Sunderman FW Jr, Katarzyna A, Antonijczuk A (1995) Xenopus lipovitellin1 is an Zn2+- and Cd2+-binding protein. Mol Reprod Dev 42:180–187PubMedCrossRefGoogle Scholar
  54. 54.
    Olsson PE, Zafarullah M, Gedamu L (1989) A role of metallothionein in zinc regulation after estradiol induction of vitogellin synthesis in rainbow trout, Salmo gardneri. Biochem J 257:555–559PubMedGoogle Scholar
  55. 55.
    Fabisiak JP, Tyurin VA, Tyurina YY, Borisenko GG, Korotaeva A, Pitt BR, Lazo JS, Kagan VE (1999) Redox regulation of copper–metallothionein. Arch Biochem Biophys 363:171–181PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Katarina Dobrovoljc
    • 2
  • Ingrid Falnoga
    • 1
  • Magda Tušek Žnidarič
    • 1
  • Darja Mazej
    • 1
  • Janez Ščančar
    • 1
  • Boris Bulog
    • 2
  1. 1.Department of Environmental SciencesJožef Stefan InstituteLjubljanaSlovenia
  2. 2.Department of Biology, Biotechnical FacultyUniversity of LjubljanaLjubljanaSlovenia

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