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Tracing of labile zinc in live fish hepatocytes using FluoZin-3

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Abstract

Intracellular zinc levels are homeostatically regulated and although most is bound, a pool of labile Zn(II) is present in cells. We show here that the zinc probe FluoZin-3 is useful to monitor zinc fluxes during fluorescent imaging of the trout hepatic cell line D11. Nuclei and bulk cytosol appeared to lack detectable labile zinc, while the punctuate staining pattern colocalized with a lysosome-specific probe. Applying extracellular zinc alone resulted in vesicular sequestration of the metal ion. Together with Na-pyrithione a delayed and toxic rise in cellular fluorescence was triggered. When using another ionophore, 4-Br A23187, a zinc buffering effect of the vesicular pools was evident. Secondly, N-ethylmaleimide induced a homogeneous fluorescence rise, which was strongly enhanced by addition of Zn-pyrithione and disappeared after TPEN washing. This suggests the involvement of thiol residues in controlling available cytosolic zinc. Our observations have implications for the interpretation of calculated intracellular Zn2+ concentrations.

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References

  • W Ahne (1985) ArticleTitleStudies on the use of fish tissue-cultures for toxicity tests in order to reduce and replace the fish tests Zentralbl Bakteriol Mikrobiol Hyg B 180 480–504 Occurrence Handle4024776 Occurrence Handle1:CAS:528:DyaL2MXltFOlsrY%3D

    PubMed  CAS  Google Scholar 

  • DH Alsop CM Wood (2000) ArticleTitleKinetic analysis of zinc accumulation in the gills of juvenile rainbow trout: effects of zinc acclimation and implications for biotic ligand modeling Environ Toxicol Chem 19 1911–1918 Occurrence Handle1:CAS:528:DC%2BD3cXkt1agsr0%3D Occurrence Handle10.1897/1551-5028(2000)019<1911:KAOZAI>2.3.CO;2

    Article  CAS  Google Scholar 

  • P Arslan F Divirgilio M Beltrame RY Tsien T Pozzan (1985) ArticleTitleCytosolic Ca-2+ homeostasis in Ehrlich and Yoshida carcinomas – A new, membrane-permeant chelator of heavy metals reveals that these ascites tumor cell lines have normal cytosolic free Ca2+ J Biol Chem 260 2719–2727 Occurrence Handle3919006 Occurrence Handle1:CAS:528:DyaL2MXhsVKqs78%3D

    PubMed  CAS  Google Scholar 

  • NA Begum M Kobayashi Y Moriwaki et al. (2002) ArticleTitleMycobacterium bovis BCG cell wall and lipopolysaccharide induce a novel gene, BIGM103, encoding a 7-TM protein: Identification of a new protein family having Zn-transporter and Zn-metalloprotease signatures Genomics 80 630–645 Occurrence Handle12504855 Occurrence Handle1:CAS:528:DC%2BD38Xps1Ons7o%3D Occurrence Handle10.1006/geno.2002.7000

    Article  PubMed  CAS  Google Scholar 

  • IA Brand J Kleineke (1996) ArticleTitleIntracellular zinc movement and its effect on the carbohydrate metabolism of isolated rat hepatocytes J Biol Chem 271 1941–1949 Occurrence Handle8567642 Occurrence Handle1:CAS:528:DyaK28XnsF2lsA%3D%3D Occurrence Handle10.1074/jbc.271.4.1941

    Article  PubMed  CAS  Google Scholar 

  • SC Burdette GK Walkup B Spingler RY Tsien SJ Lippard (2001) ArticleTitleFluorescent sensors for Zn2+ based on a fluorescein platform: Synthesis, properties and intracellular distribution J Am Chem Soc 123 7831–7841 Occurrence Handle11493056 Occurrence Handle1:CAS:528:DC%2BD3MXltlSntrw%3D Occurrence Handle10.1021/ja010059l

    Article  PubMed  CAS  Google Scholar 

  • NR Bury PA Walker CN Glover (2003) ArticleTitleNutritive metal uptake in teleost fish J Exp Biol 206 11–23 Occurrence Handle12456693 Occurrence Handle1:CAS:528:DC%2BD3sXhtVWgsrk%3D Occurrence Handle10.1242/jeb.00068

    Article  PubMed  CAS  Google Scholar 

  • LMT Canzoniero DM Turetsky DW Choi (1999) ArticleTitleMeasurement of intracellular free zinc concentrations accompanying zinc-induced neuronal death J Neurosci 19 RC31 Occurrence Handle10493776 Occurrence Handle1:STN:280:DC%2BD3c3ns1enuw%3D%3D

    PubMed  CAS  Google Scholar 

  • CJ Chang J Jaworski EM Nolan M Sheng SJ Lippard (2004) ArticleTitleA tautomeric zinc sensor for ratiometric fluorescence imaging: Application to nitric oxide-induced release of intracellular zinc Proc Natl Acad Sci USA 101 1129–1134 Occurrence Handle14734801 Occurrence Handle1:CAS:528:DC%2BD2cXhtlWjsbY%3D Occurrence Handle10.1073/pnas.0308079100

    Article  PubMed  CAS  Google Scholar 

  • RA Colvin CP Fontaine M Laskowski D Thomas (2003) ArticleTitleZn2+ transporters and Zn2+ homeostasis in neurons Eur J Pharmacol 479 171–185 Occurrence Handle14612148 Occurrence Handle1:CAS:528:DC%2BD3sXovVGntb0%3D Occurrence Handle10.1016/j.ejphar.2003.08.067

    Article  PubMed  CAS  Google Scholar 

  • P Coyle PD Zalewski JC Philcox et al. (1994) ArticleTitleMeasurement of zinc in hepatocytes by using a fluorescent probe, Zinquin: Relationship to metallothionein and intracellular zinc Biochem J 303 781–786 Occurrence Handle7980447 Occurrence Handle1:CAS:528:DyaK2cXmsFShsbk%3D

    PubMed  CAS  Google Scholar 

  • KE Dineley LM Malaiyandi IJ Reynolds (2002) ArticleTitleA reevaluation of neuronal zinc measurements: Artifacts associated with high intracellular dye concentration Mol Pharmacol 62 618–627 Occurrence Handle12181438 Occurrence Handle1:CAS:528:DC%2BD38XmsVKmurs%3D Occurrence Handle10.1124/mol.62.3.618

    Article  PubMed  CAS  Google Scholar 

  • CJ Frederickson EJ Kasarskis D Ringo RE Frederickson (1987) ArticleTitleA quinoline fluorescence method for visualizing and assaying the histochemically reactive zinc (bouton zinc) in the brain J Neurosci Methods 20 91–103 Occurrence Handle3600033 Occurrence Handle1:CAS:528:DyaL2sXkslels70%3D Occurrence Handle10.1016/0165-0270(87)90042-2

    Article  PubMed  CAS  Google Scholar 

  • F Gagné C Blaise (1996) ArticleTitleAvailable intracellular Zn as a potential indicator of heavy metal exposure in rainbow trout hepatocytes Environ Toxicol Water Qual 11 319–325 Occurrence Handle10.1002/(SICI)1098-2256(1996)11:4<319::AID-TOX6>3.0.CO;2-C

    Article  Google Scholar 

  • LA Gaither DJ Eide (2001) ArticleTitleEukaryotic zinc transporters and their regulation Biometals 14 251–270 Occurrence Handle11831460 Occurrence Handle1:CAS:528:DC%2BD38XpsFWrtw%3D%3D Occurrence Handle10.1023/A:1012988914300

    Article  PubMed  CAS  Google Scholar 

  • CN Glover C Hogstrand (2002) ArticleTitleIn vivo characterisation of intestinal zinc uptake in freshwater rainbow trout J Exp Biol 205 141–150 Occurrence Handle11818420 Occurrence Handle1:CAS:528:DC%2BD38XhtlGluro%3D

    PubMed  CAS  Google Scholar 

  • G Grynkiewicz M Poenie RY Tsien (1985) ArticleTitleA new generation of Ca2+ indicators with greatly improved fluorescence properties J Biol Chem 260 3440–3450 Occurrence Handle3838314 Occurrence Handle1:CAS:528:DyaL2MXitVSmu7s%3D

    PubMed  CAS  Google Scholar 

  • H Haase D Beyersmann (2002) ArticleTitleIntracellular zinc distribution and transport in C6 rat glioma cells Biochem Biophys Res Commun 296 923–928 Occurrence Handle12200136 Occurrence Handle1:CAS:528:DC%2BD38XmsV2rsrw%3D Occurrence Handle10.1016/S0006-291X(02)02017-X

    Article  PubMed  CAS  Google Scholar 

  • R Haugland (2002) Handbook of Fluorescent Probes and Research Products Molecular Probes La Jolla

    Google Scholar 

  • LH Ho RN Ratnaike PD Zalewski (2000) ArticleTitleInvolvement of intracellular labile zinc in suppression of DEVD-caspase activity in human neuroblastoma cells Biochem Biophys Res Commun 268 148–154 Occurrence Handle10652229 Occurrence Handle1:CAS:528:DC%2BD3cXnsVWhsw%3D%3D Occurrence Handle10.1006/bbrc.2000.2090

    Article  PubMed  CAS  Google Scholar 

  • C Hogstrand CM Wood (1995) ArticleTitleMechanisms for zinc acclimation in fresh-water rainbow-trout Mar Environ Res 39 131–135 Occurrence Handle1:CAS:528:DyaK2MXkvFehsbc%3D Occurrence Handle10.1016/0141-1136(94)00040-V

    Article  CAS  Google Scholar 

  • C Hogstrand CM Wood (1996) The physiology and toxicology of zinc in fish EW Tayler (Eds) Toxicology of Aquatic Pollution: Physiological, Cellular and Molecular Approaches Cambridge University Press Cambridge 61–84

    Google Scholar 

  • JHR Kägi (1991) ArticleTitleOverview of metallothionein Methods Enzymol 205 613–626 Occurrence Handle1779825 Occurrence Handle10.1016/0076-6879(91)05145-L

    Article  PubMed  Google Scholar 

  • T Kambe Y Yamaguchi-Iwai R Sasaki M Nagao (2004) ArticleTitleOverview of mammalian zinc transporters Cell Mol Life Sci 61 49–68 Occurrence Handle14704853 Occurrence Handle1:CAS:528:DC%2BD2cXjt1yhur4%3D Occurrence Handle10.1007/s00018-003-3148-y

    Article  PubMed  CAS  Google Scholar 

  • JW Kleineke IA Brand (1997) ArticleTitleRapid changes in intracellular Zn2+ in rat hepatocytes J Pharmacol Toxicol Methods 38 181–187 Occurrence Handle9566441 Occurrence Handle1:CAS:528:DyaK1cXisVKgt7s%3D Occurrence Handle10.1016/S1056-8719(97)00094-4

    Article  PubMed  CAS  Google Scholar 

  • Krezel A, Wojcik J, Maciejczyk M, Bal W. 2003 May GSH and L-His contribute to intracellular binding of zinc? Thermodynamic and solution structural study of a ternary complex. Chem Commun, 704–705

  • HL Ley ML Failla DS Cherry (1983) ArticleTitleIsolation and characterization of hepatic metallothionein from rainbow trout (Salmo gairdneri) Comp Biochem Physiol B 74 507–513 Occurrence Handle6839717 Occurrence Handle10.1016/0305-0491(83)90219-5

    Article  PubMed  Google Scholar 

  • C Lopez-Garcia E Varea JJ Palop et al. (2002) ArticleTitleCytochemical techniques for zinc and heavy metals localization in nerve cells Microsc Res Tech 56 318–331 Occurrence Handle11877810 Occurrence Handle1:CAS:528:DC%2BD38Xit1entbc%3D Occurrence Handle10.1002/jemt.10037

    Article  PubMed  CAS  Google Scholar 

  • M Maracine H Segner (1998) ArticleTitleCytotoxicity of metals in isolated fish cells: Importance of the cellular glutathione status Comp Biochem Physiol A 120 83–88 Occurrence Handle10.1016/S1095-6433(98)10013-2

    Article  Google Scholar 

  • W Maret (2003) ArticleTitleCellular zinc and redox states converge in the metallothionein/thionein pair J Nutr 133 1460S–1462S Occurrence Handle12730443 Occurrence Handle1:CAS:528:DC%2BD3sXjs1Oqsr0%3D

    PubMed  CAS  Google Scholar 

  • AA Michalczyk J Allen RC Blomeley ML Ackland (2002) ArticleTitleConstitutive expression of hZnT4 zinc transporter in human breast epithelial cells Biochemical J 364 105–113 Occurrence Handle1:CAS:528:DC%2BD38XktFWiu7g%3D

    CAS  Google Scholar 

  • MS Nasir CJ Fahrni DA Suhy et al. (1999) ArticleTitleThe chemical cell biology of zinc: structure and intracellular fluorescence of a zinc-quinolinesulfonamide complex J Biol Inorg Chem 4 775–783 Occurrence Handle10631609 Occurrence Handle1:CAS:528:DC%2BD3cXhslWnsQ%3D%3D Occurrence Handle10.1007/s007750050350

    Article  PubMed  CAS  Google Scholar 

  • CE Outten TV O’Halloran (2001) ArticleTitleFemtomolar sensitivity of metalloregulatory proteins controlling zinc homeostasis Science 292 2488–2492 Occurrence Handle11397910 Occurrence Handle1:CAS:528:DC%2BD3MXkvFWlsrk%3D Occurrence Handle10.1126/science.1060331

    Article  PubMed  CAS  Google Scholar 

  • RD Palmiter TB Cole SD Findley (1996a) ArticleTitleZnT-2, a mammalian protein that confers resistance to zinc by facilitating vesicular sequestration Embo J 15 1784–1791 Occurrence Handle1:CAS:528:DyaK28XivVant7s%3D

    CAS  Google Scholar 

  • RD Palmiter TB Cole CJ Quaife SD Findley (1996b) ArticleTitleZnT-3, a putative transporter of zinc into synaptic vesicles Proc Natl Acad Sci USA 93 14934–14939 Occurrence Handle1:CAS:528:DyaK28XnsVOrsrw%3D Occurrence Handle10.1073/pnas.93.25.14934

    Article  CAS  Google Scholar 

  • KB Pierson (1985) ArticleTitleOccurrence and synthesis of a non-thionein, zinc-binding protein in the rainbow-trout (Salmo gairdneri) Comp Biochem Physiol C 81 71–75 Occurrence Handle2861061 Occurrence Handle1:STN:280:BiqB38bjvFc%3D Occurrence Handle10.1016/0742-8413(85)90093-3

    Article  PubMed  CAS  Google Scholar 

  • A Qiu M Shayeghi C Hogstrand (2005) ArticleTitleMolecular cloning and␣functional characterization of a high-affinity zinc importer (DrZIP1) from zebrafish (Danio rerio) Biochem J 388 745–754 Occurrence Handle15683366 Occurrence Handle1:CAS:528:DC%2BD2MXkvVGhtrk%3D Occurrence Handle10.1042/BJ20041807

    Article  PubMed  CAS  Google Scholar 

  • G Ranaldi G Perozzi A Truong-Tran P Zalewski C Murgia (2002) ArticleTitleIntracellular distribution of labile Zn(II) and zinc transporter expression in kidney and MDCK cells Am J Physiol 283 F1365–F1375 Occurrence Handle1:CAS:528:DC%2BD38XpvVWit7o%3D

    CAS  Google Scholar 

  • MJ Salguiero M Zubillaga A Lysionek et al. (2000) ArticleTitleZinc as an essential micronutrient: A review Nutr Res 20 737–755 Occurrence Handle10.1016/S0271-5317(00)00163-9

    Article  Google Scholar 

  • GR Sauer N Watabe (1989) ArticleTitleUltrastructural and histochemical aspects of zinc accumulation by fish scales Tissue & Cell 21 935–943 Occurrence Handle1:CAS:528:DyaK3cXhvFegt7c%3D Occurrence Handle10.1016/0040-8166(89)90044-X

    Article  CAS  Google Scholar 

  • SL Sensi D Ton-That PG Sullivan et al. (2003a) ArticleTitleModulation of mitochondrial function by endogenous Zn2+ pools Proc Natl Acad Sci USA 100 6157–6162 Occurrence Handle1:CAS:528:DC%2BD3sXjvFOlurw%3D Occurrence Handle10.1073/pnas.1031598100

    Article  CAS  Google Scholar 

  • SL Sensi D Ton-That JH Weiss A Rothe KR Gee (2003b) ArticleTitleA new mitochondrial fluorescent zinc sensor Cell Calcium 34 281–284 Occurrence Handle1:CAS:528:DC%2BD3sXlvVCrtrY%3D Occurrence Handle10.1016/S0143-4160(03)00122-2

    Article  CAS  Google Scholar 

  • CF Shaw LB He A Munoz et al. (1997) ArticleTitleKinetics of reversible N-ethylmaleimide alkylation of metallothionein and the subsequent metal release J Biol Inorg Chem 2 65–73 Occurrence Handle1:CAS:528:DyaK2sXhvVGku7s%3D Occurrence Handle10.1007/s007750050107

    Article  CAS  Google Scholar 

  • ZL Tang KJ Wasserloos XH Liu et al. (2002) ArticleTitleNitric oxide decreases the sensitivity of pulmonary endothelial cells to LPS-induced apoptosis in a zinc-dependent fashion Mol Cell Biochem 234 211–217 Occurrence Handle12162436 Occurrence Handle10.1023/A:1015930927407

    Article  PubMed  Google Scholar 

  • B Turan H Fliss M Desilets (1997) ArticleTitleOxidants increase intracellular free Zn2+ concentration in rabbit ventricular myocytes Am J Physiol 41 H2095–H2106

    Google Scholar 

  • BL Vallee KH Falchuk (1993) ArticleTitleThe biochemical basis of zinc physiology Physiol Rev 73 79–118 Occurrence Handle8419966 Occurrence Handle1:CAS:528:DyaK3sXhvVyhtrY%3D

    PubMed  CAS  Google Scholar 

  • PD Zalewski IJ Forbes WH Betts (1993) ArticleTitleCorrelation of apoptosis with change in intracellular labile Zn(II) using Zinquin [(2-methyl-8-P-toluenesulphonamido-6-quinolyloxy)acetic acid], a new specific fluorescent-probe for Zn(II) Biochem J 296 403–408 Occurrence Handle8257431 Occurrence Handle1:CAS:528:DyaK2cXjvVer

    PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Biology, University of Antwerp, Groenenborgerlaan 171, B-2020, Antwerp, Belgium

    Frederik A. R. Muylle, Wim De Coen & Ronny Blust

  2. Department of Biomedical Sciences, University of Antwerp, Groenenborgerlaan 171, B-2020, Antwerp, Belgium

    Dirk Adriaensen & Jean-Pierre Timmermans

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  1. Frederik A. R. Muylle
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  2. Dirk Adriaensen
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Correspondence to Frederik A. R. Muylle.

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Muylle, F.A.R., Adriaensen, D., De Coen, W. et al. Tracing of labile zinc in live fish hepatocytes using FluoZin-3. Biometals 19, 437–450 (2006). https://doi.org/10.1007/s10534-005-4576-y

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  • DOI: https://doi.org/10.1007/s10534-005-4576-y

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