Abstract
Varying intracellular concentrations of zinc in laryngeal Hep-2 cells in relation to changing cultivation conditions in vitro were determined by atomic absorption spectrophotometry. Upon standard cultivation in DMEM with 10% serum, the mean concentration of zinc was determined at 0.88 ± 0.09 μg/mg protein, with substantially decreased values in the cells exposed to a low-serum medium. Next, the study of the effects of a series of physiological and supraphysiological concentrations of ZnSO4 on laryngeal cells and their correlation with determined intracellular concentrations of zinc was performed. It was found that zinc concentrations above 100 μM were toxic to Hep-2 cells, inducing cell death in the interval of 96 h as determined by videomicroscopy, selective nuclear staining, and immunofluorescence detection of caspase-3 and specific cytokeratin 18 fragment. Both types of cell death were observed, with apoptosis being induced at moderately toxic zinc concentration of 150 μM and necrosis at higher zinc concentrations of 300 μM and 750 μM, respectively. Lower concentrations (1.5–100 μM), on the other hand, did not produce any measurable changes in cell morphology and function in the same time interval. Zinc at concentration of 1.5 μM was found to slightly enhance proliferation of Hep-2 cells up to the certain time point, which seemed to correlate with maximal tolerable momentary intracellular level of zinc. These results illustrate the importance of determining the intracellular levels of zinc when trying to characterize the effect of exogenous zinc on life and death of laryngeal cells.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Ahn YH, Kim YH, Hong S, Koh JY. 1998 Depletion of intracellular zinc induces protein synthesis-dependent neuronal apoptosis in mouse cortical culture. Exp Neurol 1, 47-56.
Aiuchi T, Mihara S, Nakaya M, Masuda Y, Nakajo S, Nakaya K. 1998 Zinc ions prevent processing of caspase-3 during apoptosis induced by geranylgeraniol in HL-60 cells. J Biochem (Tokyo) 2, 300-303.
Bradford MM. 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 248-254.
Cario E, Jung S, Harder D'Heureuse J, Schulte C, Sturm A, Wiedenmann B, Goebell H, Dignass AU. 2000 Effects of exogenous zinc supplementation on intestinal epithelial repair in vitro. Eur J Clin Invest 5, 419-428.
Catimel G. 1996 Head and neck cancer: guidelines for chemotherapy. Drugs 1, 73-88.
Chai F, Truong-Tran AQ, Ho LH, Zalewski PD. 1999 Regulation of caspase activation and apoptosis by cellular zinc fluxes and zinc deprivation: a review. Immunol Cell Biol 3, 272-278.
Chimienti F, Seve M. Richard S, Mathieu J, Favier A. 2001 Role of cellular zinc in programmed cell death: temporal relationship between zinc depletion, activation of caspases, and cleavage of Sp family transcription factors. Biochem Pharmacol 1, 51-62.
Choi DW, Yokoyama M, Koh J. 1988 Zinc neurotoxicity in cortical cell culture. Neuroscience 1, 67-79.
Cousins RJC. 1996 Zinc. In: Present Knowledge in Nutrition. International Life Sciences Institute Nutrition Foundation: 293-306.
Fraker PJ, Telford WG. 1997 A reappraisal of the role of zinc in life and death decisions of cells. Proc Soc Exp Biol Med 3, 229-236.
Haase H, Beyersmann D. 1999 Uptake and intracellular distribution of labile and total Zn (II) in C6 rat glioma cells investigated with fluorescent probes and atomic absoption. Biometals 12, 247-254.
Haase H, Watjen W, Beyersmann D. 2001 Zinc induces apoptosis that can be suppressed by lanthanum in C6 rat glioma cells. Biol Chem 8, 1227-1234.
Huang X, Cuajungco MP, Atwood CS, Moir RD, Tanzi RE, Bush AI. 2000 Alzheimer's disease, beta-amyloid protein and zinc. J Nutr 5S Suppl, 1488S-1492S.
Hyun HJ, Sohn J, Ahn YH, Shin HC, Koh JY, Yoon YH. 2000 Depletion of intracellular zinc induces macromolecule synthesisand caspase-dependent apoptosis of cultured retinal cells. Brain Res 1-2, 39-48.
Kaji T, Fujiwara Y, Yamamoto C, Sakamoto M, Kozuka H. 1994 Stimulation by zinc of cultured vascular endothelial cell proliferation: possible involvement of endogenous basic fibroblast growth factor. Life Sci 23, 1781-1787.
Kim YH, Kim EY, Gwag BJ, Sohn S, Koh JY. 1999 Zinc-induced cortical neuronal death with features of apoptosis and necrosis: mediation by free radicals. Neurosci. 1, 175-182.
Kulkarni GV, McCulloch CAG. 1994 Serum deprivation induces apoptotic cell death in a subset of Balb/c 3T3 fibroblasts. J Cell Sci 107, 1169-1179.
Leure du Pree AE, McClain CJ. 1982 The effect of severe zinc defi-ciency on the morphology of the rat retinal pigment epithelium. Invest Ophthalmol Vis Sci 4, 425-434.
Lewis DJ. 1991 Morphological assessment of pathological changes within the rat larynx. Toxicol Pathol 4Pt 1, 352-357.
Liang JY, Liu YY, Zou J, Franklin RB, Costello LC, Feng P. 1999 Inhibitory effect of zinc on human prostatic carcinoma cell growth. Prostate 3, 200-207.
Lohmann RD, Beyersmann D. 1993 Cadmium and zinc mediated changes of the Ca(2+)-dependent endonuclease in apoptosis. Biochem Biophys Res Commun 190, 1097-1103.
Marini M, Musiani D. 1998 Micromolar zinc affects endonucleolytic activity in hydrogen peroxide-mediated apoptosis. Exp Cell Res 2, 393-398.
McCabe MJ, Jiang SA, Orrenius S. 1993 Chelation of intracellular zinc triggers apoptosis in mature thymocytes. Lab Invest 69, 101-110.
Meerarani P, Ramadass P, Toborek M, Bauer HC, Bauer H, Hennig B. 2000 Zinc protects against apoptosis of endothelial cells induced by linoleic acid and tumor necrosis factor alpha. Am J Clin Nutr 1, 81-87.
Nakatani T, Tawaramoto M, Kennedy DO, Kojima A, Matsui-Yuasa I. 2000 Apoptosis induced by chelation of intracellular zinc is associated with depletion of cellular reduced glutathione level in rat hepatocytes. Chem-Biol Interact 3, 151-163.
O'Dell BL. 2000 Role of zinc in plasma membrane function. J Nutr 5S Suppl, 1432S-1436S.
Provinciali M, Di Stefano G, Fabris N. 1995 Dose-dependent opposite effect of zinc on apoptosis in mouse thymocytes. Int J Immunopharmacol 9, 735-744.
Smart TG. 1992 A novel modulatory binding site for zinc on the GABAA receptor complex in cultured rat neurones. J Physiol (Lond) 447, 587-625.
Szuster-Ciesielska A, Stachura A, Slotwinska M, Kaminska T, Sniezko R, Paduch R, Abramczyk D, Filar J, Kandefer-Szerszen M. 2000 The inhibitory effect of zinc on cadmium-induced cell apoptosis and reactive oxygen species (ROS) production in cell cultures. Toxicology 2-3, 159-171.
Szyfter K, Banaszewski J, Jaloszynski P, Pabiszczak M, Szyfter W, Szmeja Z. 1999 Carcinogen: DNA adducts in tobacco smokeassociated cancer of the upper respiratory tract. Acta Biochim Pol 2, 275-287.
Takahashi A, Alnemri ES, Lazebnik YA, Fernandes-Alnemri T, Litwack G, Moir RD, Goldman RD, Poirier GG, Kaufmann SH, Earnshaw WC. 1996 Cleavage of lamin A by Mch2 alpha but not CPP32: multiple interleukin 1 beta-converting enzyme-related proteases with distinct substrate recognition properties are active in apoptosis. Proc Natl Acad Sci USA 16, 8395-8400.
Truong Tran AQ, Carter J, Ruffin R, Zalewski PD. 2001 New insights into the role of zinc in the respiratory epithelium. Immunol Cell Biol 2, 170-177.
Vallee BL, Falchuk KH. 1993 The biochemical basis of zinc physiology. Physiol Rev 1, 79-118.
Wolf CM, Eastman A. 1999 The temporal relationship between protein phosphatase, mitochondrial cytochrome c release, and caspase activation in apoptosis. Exp Cell Res 2, 505-513.
Wolf CHM, Morana SJ, Eastman A. 1997 Zinc inhibits apoptosis upstream of ICE/CED-3 proteases rather than at the level of an endonuclease. Cell Death and Differentiation 4, 125-129.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Rudolf, E., Rudolf, K., Radocha, J. et al. The role of intracellular zinc in modulation of life and death of Hep-2 cells. Biometals 16, 295–309 (2003). https://doi.org/10.1023/A:1020603110255
Issue Date:
DOI: https://doi.org/10.1023/A:1020603110255