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Metallothionein induction in cultured fibroblasts and liver of a marine flatfish, the turbot,Scophthalmus maximus

Fish Physiology and Biochemistry volume 10, pages 43–54 (1992)Cite this article

Abstract

Intraperitoneal injection of turbot with Cd induced the synthesis of a low molecular weight hepatic Cd-binding protein and a 500bp mRNA, which hybridised to a plaice metallothionein (MT) cRNA probe. The Cd-binding protein displayed cross-reactivity in a competitive ELISA with antiserum raised against rainbow trout MT and had the characteristic amino acid composition, metal stoichiometry and spectral characteristics of a Class I MT. Only one isoform was apparent on ion exchange chromatography. Southern blot analysis of DNA cleaved with four restriction enzymes suggested that only a single MT gene is present in turbot.

In an established turbot fibroblast cell line, Cd induced MT mRNA and MT levels in a dose and time-dependent manner. MT was also induced by Cu, Hg and Zn but not Pb exposure. Physiological concentrations of glucocorticoids and sex hormones did not induce MT synthesis, although at high concentrations a positive response to corticosterone, dexamethasone, hydrocortisone or progesterone was observedin vitro indicating the possible presence of a functional steroid regulatory element in the fish MT gene.

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Abbreviations

AMP:

adenosine monophosphate

CHSE:

chinook salmon embryo

DMSO:

dimethyl sulphoxide

HPLC:

high performance liquid chromatography

KB:

kenacid blue

MT:

metallothionein

Mr:

molecular weight (kDaltons)

NR:

neutral red

PEG:

polyethylene glycol

RTH:

rainbow trout hepatoma

SDS:

sodium dodecyl sulphate

SSC:

0.15M NaCl, 0.015M sodium citrate

TF:

turbot fibroblast

References cited

  • Bonham, K., Zafarullah, M. and Gedamu, L. 1987. The rainbow trout metallothioneins: Molecular cloning and characterisation of two distinct cDNA sequences. DNA. 6: 519–528.

    Google Scholar 

  • Borenfruend, E. and Puerner, J.A. 1985. Toxicity determinedin vitro by morphological alterations and neutral red absorption. Toxicol. Lett. 24: 119–124.

    Google Scholar 

  • Bracken, W.M. and Klaassen, C.D. 1987. Induction of metallothionein by steroids in rat primary hepatocyte cultures. Toxicol. Appl. Pharmacol. 87: 381–388.

    Google Scholar 

  • Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.

    Google Scholar 

  • Bremner, I. 1987. Nutritional and physiological significance of metallothionein. Experientia, Suppl. 52: 81–107.

    Google Scholar 

  • Campbell, C.M., Walsh, J.M. and Idler, D.R. 1976. Steroids in the plasma of the winter flounder (Pseudopleuronectes americanus Walbaum). A seasonal study and investigation of steroid involvement in oocyte maturation. Gen. Comp. Endocrinol. 29: 14–20.

    Google Scholar 

  • Chan, K.M., Davidson, W.S., Hew, C.L. and Fletcher, G.L. 1989. Molecular cloning of metallothionein cDNA and analysis of metallothionein gene expression in winter flounder tissues. Can. J. Zool. 67: 2520–2527.

    Google Scholar 

  • Chromczynski, P. and Sacchi, N. 1987. Single step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal. Biochem. 162: 156–159.

    Google Scholar 

  • Cousins, R.J. 1985. Absorption, transport and hepatic metabolism of copper and zinc: Special reference to metallothionein and ceruloplasmin. Physiol. Rev. 65: 238–309.

    Google Scholar 

  • Eaton, D.L. and Toal, B.F. 1982. Evaluation of the Cd/hemoglobin affinity assay for the rapid determination of metallothionein in biological tissues. Toxicol. Appl. Pharmacol. 66: 134–142.

    Google Scholar 

  • Fowler, B., Hildebrand, C.E., Kojima, Y. and Webb, M. 1987. Nomenclature of metallothioneins. Experientia, Suppl. 52: 19–22.

    Google Scholar 

  • George, S.G. 1990. Assessment of metal toxicity in marine organisms.In Metals in the Marine Environment. pp. 123–142. Edited by R.W. Furness and P.S. Rainbow. CRC Press, Boca Raton.

    Google Scholar 

  • George, S.G. and Young, P. 1986. The time course of effects of cadmium and 3-methylcholanthrene on activities of enzymes of xenobiotic metabolism and metallothionein levels in the plaice,Pleuronectes platessa. Comp. Biochem. Physiol. 83C: 37–44.

    Google Scholar 

  • George, S., Leaver, M., Frerichs, N. and Burgess, D. 1989. Fish metallothioneins: Molecular cloning studies and induction in cultured cells. Mar. Environ. Res. 28: 173–177.

    Google Scholar 

  • Hager, L.J. and Palmiter, R.D. 1981. Transcriptional regulation of mouse liver metallothionein-I gene by glucocorticoids. Nature, Lond. 291: 340–342.

    Google Scholar 

  • Hamer, D.H. 1986. Metallothionein. Ann. Rev. Biochem. 55: 913–951.

    Google Scholar 

  • Hyllner, S.J., Andersson, T., Haux, C. and Olsson, P.-E. 1989. Cortisol induction of metallothionein in primary culture of rainbow trout hepatocytes. J. Cell Physiol. 139: 24–28.

    Google Scholar 

  • Kagi, J.H.R. and Kojima, Y. 1987. Metallothionein II. Experientia Suppl. 52: 25–61.

    Google Scholar 

  • Kagi, J.H.R. and Schaffer, A. 1988. Biochemistry of metallothionein. Biochemistry 27: 8509–8515.

    Google Scholar 

  • Karin, M. 1985. Metallothioneins: Proteins in search of function. Cell 41: 9–10.

    Google Scholar 

  • Karin, M. and Herschman, H. 1980. Glucocorticoid hormone receptor mediated induction of metallothionein synthesis in HeLa cells. J. Cell. Physiol. 103: 35–40.

    Google Scholar 

  • Karin, M., Imbra, R.J., Heguy, A. and Wong, G. 1985. Interleukin-I regulates human metallothionein gene expression. Mol. Cell. Biol. 5: 2866–2869.

    Google Scholar 

  • Karin, M., Imagawa, M., Imbra, R.J., Chiu, R., Heguy, A., Haslinger, A., Cooke, T., Satyabhmama, S., Jonat, C. and Herrlich, P. 1987. Hormonal and environmental control of metallothionein gene expression.In Transcriptional Control Mechanisms. UCLA Symp. Mol. Cell. Biology. New ser., V, pp. 295–311. Alan R. Liss, New York.

  • Knox, P., Uphill, P.F., Fry, J.R., Benford, J. and Balls, M. 1986. The FRAME multicentre project onin vitro cytotoxicology. Fed. Chem. Toxic. 24: 457–463.

    Google Scholar 

  • Krieg, P.A. and Melton, D.A. 1984. Functional messenger RNAs are produced by SP6in vitro transcription of cloned cDNAs. Nucleic Acids Res. 12: 7057–7070.

    Google Scholar 

  • Leaver, M. and George, S.G. 1991. Nucleotide sequence of a metallothionein cDNA clone from the marine fish,Pleuronectes platessa. EMBL Accession # 56743.

  • Ley, H.L., Failla, M.L. and Cherry, D.S. 1983. Isolation and characterisation of hepatic metallothionein from rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol. 74B: 507–513.

    Google Scholar 

  • Mayo, K.E. and Palmiter, R. 1981. Glucocorticoid regulation of metallothionein-I mRNA synthesis in cultured mouse cells. J. Biol. Chem. 256: 2621–2624.

    Google Scholar 

  • McCardle, H.J., Kyriakou, P., Grimes, A., Mercer, J.F.B. and Danks, D.M. 1990. The effect of D-penicillamine on metallothionein mRNA levels and copper distribution in mouse hepatocytes. Chem.-Biol. Interactions, 75: 315–324.

    Google Scholar 

  • Norey, C.G., Lees, E.E., Darke, B.M., Stark, J.M., Baker, T.S., Cryer, A. and Kay, J. 1990. Immunological distinction between piscine and mammalian metallothioneins. Comp. Biochem. Physiol. 9SB: 597–601.

    Google Scholar 

  • Olsson, P.-E. and Haux, C. 1986. Increased hepatic metallothionein content correlates to cadmium accumulation in environmentally exposed perch,Perca fluviatilis. Aquat. Toxicol. 9: 231–242.

    Google Scholar 

  • Olsson, P.-E., Haux, C. and Forlin, L. 1987. Variations in hepatic metallothionein, zinc and copper levels during an annual reproductive cycle in rainbow trout,Salmo gairdneri. Fish Physiol. Biochem. 3: 39–47.

    Google Scholar 

  • Olsson, P.-E., Zafarullah, M. and Gedamu, L. 1989. A role of metallothionein in zinc regulation after oestradiol induction of vitellogenin synthesis in rainbow trout,Salmo gairdneri. Biochem. J. 257: 555–559.

    Google Scholar 

  • Overnell, J. and Coombs, T.L. 1979. Purification and properties of plaice metallothionein, a cadmium-binding protein from the liver of the plaice (Pleuronectes platessa). Biochem. J. 183: 277–283.

    Google Scholar 

  • Overnell, J., McIntosh, R. and Fletcher, T.C. 1987a. The enhanced induction of metallothionein by zinc, its half-life in the marine fishPleuronectes platessa, and the influence of stress factors on metallothionein levels. Experientia 43: 178–181.

    Google Scholar 

  • Overnell, J., McIntosh, R. and Fletcher, T.C. 1987b. The levels of liver metallothionein and zinc in the plaice,Pleuronectes platessa L., during the breeding season, and the effect of oestradiol injection. J. Fish Biol. 30: 539–546.

    Google Scholar 

  • Palmiter, R.D. 1987. Molecular biology of metallothionein gene expression. Experientia Suppl. 52: 63–80.

    Google Scholar 

  • Price-Haughey, J., Bonham, K. and Gedamu, L. 1987. Metallothionein gene expression in fish cell lines: its activation in embryonic cells by 5-azacytidine. Biochim. biophys. Acta 908: 158–168.

    Google Scholar 

  • Richards, R.I., Heguy, A. and Karin, M. 1984. Structural and functional analysis of the human metallothionein-IA gene: differential induction by metal ions and glucocorticoids. Cell 37: 263–272.

    Google Scholar 

  • Roch, M., McCarter, A., Matheson, M.C. and Olafson, R. 1982. Hepatic metallothionein in rainbow trout (Salmo gairdneri) as an indicator of metal pollution in the Campbell River system. Can. J. Fish. Aquat. Sci. 39: 1596–1601.

    Google Scholar 

  • Schmidt, C.J. and Hamer, D.H. 1986. Cell specificity and an effect of RAS on human metallothionein gene expression. Proc. Nat. Acad. Sci. U.S.A. 83: 3346–3350.

    Google Scholar 

  • Shears, M.A. and Fletcher, G.L. 1984. The relationship between metallothionein and intestinal zinc absorption in the winter flounder. Can. J. Zool. 62: 2211–2220.

    Google Scholar 

  • Thomas, P.S. 1980. Hybridisation of denatured RNA and small DNA fragments transferred to nitrocellulose. PNAS 77: 5201–5205.

    Google Scholar 

  • Thomas, D.G., Brown, M.W., Shurben, D., Solbe, J.F. de L.G., Cryer, A. and Kay, J. 1985. A comparison of the sequestration of cadmium and zinc in the tissues of rainbow trout (Salmo gairdneri) following exposure to the metals singly or in combination. Comp. Biochem. Physiol. 82C: 55–62.

    Google Scholar 

  • Varshney, U., Jahroudi, N., Foster, R. and Gedamu, L. 1986. Structure, organisation and regulation of human metallothionein 1F gene-differential and cell-type specific expression in response to heavy metals and glucocorticoids. Mol. Cell. Biol. 6: 26–37.

    Google Scholar 

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Author information

Affiliations

  1. NERC Unit of Aquatic Biochemistry, University of Stirling, Stirling, FK9 4LA, Scotland

    Stephen George, David Burgess & Michael Leaver

  2. Institute of Aquaculture, School of Natural Sciences, University of Stirling, Stirling, FK9 4LA, Scotland

    Nick Frerichs

Authors
  1. Stephen George
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  2. David Burgess
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  3. Michael Leaver
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  4. Nick Frerichs
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George, S., Burgess, D., Leaver, M. et al. Metallothionein induction in cultured fibroblasts and liver of a marine flatfish, the turbot,Scophthalmus maximus . Fish Physiol Biochem 10, 43–54 (1992). https://doi.org/10.1007/BF00004653

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Keywords

  • marine fish
  • metallothionein
  • purification
  • induction
  • metals
  • hormones
  • cell culture
  • mRNA