Abstract
There is now a wealth of information regarding the apoptotic mode of cell death and its importance in toxicological studies in many mammalian organs including the liver. In this study, we investigated the modulatory effects of the heavy metal Zn2+ on transforming growth factor-β1 (TGF-β1)-induced apoptosis in primary rat hepatocytes. Apoptosis induced by TGF-β1 (1 ng/ml) in hepatocytes was accompanied by nuclear condensation as assessed morphologically by staining with Hoechst 33258 and DNA cleavage as detected biochemically by in situ end-labeling, field inversion and conventional gel electrophoresis. Pretreatment with 100 μmol/L Zn2+ abrogated the nuclear condensation, in situ end-labeling, and DNA laddering in TGF-β1-treated hepatocytes. Surprisingly, Zn2+ did not inhibit the formation of high-molecular-weight DNA fragments (30–50 kbp to 250–300 kbp). These data provide evidence that Zn2+ exerts its effects on the endonucleases that act downstream in the execution phase of TGF-β1-induced apoptosis in hepatocytes.
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Beere HM, Chresta CM, Alejo-Herberg A, et al. Investigation of the mechanisms of higher order chromatin fragmentation observed in drug-induced apoptosis. Mol Pharmacol. 1995;45:986–96.
Bray TM, Bettger WJ. The physiological role of zinc as an antioxidant. Free Radic Biol Med. 1990;8:281–91.
Brown DG, Sun X-M, Cohen GM. Dexamethasone-induced apoptosis involves cleavage of DNA to large fragments prior to internucleosomal fragmentation. J Biol Chem. 1993;268:3037–9.
Bursch W, Paffe S, Putz B, Barthel G, Schulte-Hermann R. Determination of the length of the histological stages of apoptosis in rat liver and altered hepatic foci. Carcinogenesis. 1990;11:847–53.
Cain K, Inayat-Hussain SH, Wolfe JT, Cohen GM. DNA fragmentation into 200–250 and/or 30–50 kilobase pair fragments in rat liver nuclei is stimulated by Mg2+ alone and Ca2+/Mg2+ but not by Ca2+ alone. FEBS Lett. 1994;349:385–91.
Cain K, Inayat-Hussain SH, Couet C, Qin H-M, Oberhammer FA. A novel method for detecting apoptosis shows that hepatocytes undergo a time dependent increase in DNA cleavage and chromatin condensation which is augmented after TGF-β1 treatment. Cytometry. 1996;23:312–21.
Cohen GM, Sun X-M, Snowden RT, Dinsdale D, Skilleter DN. Key morphological features of apoptosis may occur in the absence of internucleosomal DNA fragmentation. Biochem J. 1992;286:331–4.
Cohen GM, Sun X-M, Fearnhead H, et al. Formation of large molecular weight fragments is a key committed step of apoptosis in thymocytes. J Immunol. 1994;153:507–16.
Cohen JJ, Duke RC. Glucocorticoid activation of a calcium-dependent endonuclease in thymocyte nuclei leads to cell death. J Immunol. 1984;132:38–42.
Coleman JE. Zinc proteins: enzymes, storage proteins, transcription factors and replication proteins. Annu Rev Biochem. 1992;61:897–946.
Duke RC, Chervenak R, Cohen JJ. Endogenous endonuclease-induced DNA fragmentation: an early event in cell-mediated cytolysis. Proc Natl Acad Sci USA. 1983;80:6361–5.
Filipski J, Leblanc J, Youdale T, Sikorska M, Walker PR. Periodicity of DNA folding in high order chromatin structures. EMBO J. 1990;9:1319–27.
Fraker PJ, Telford WG. A reappraisal of the role of zinc in life and death decisions of cells. Proc Soc Exp Biol Med. 1997;215:229–36.
Hara S, Halicka HD, Bruno S, Gong J, Traganos F, Darzynkiewicz Z. Effect of protease inhibitors on early events of apoptosis. Exp Cell Res. 1996;223:372–84.
Inayat-Hussain SH, Couet C, Cohen GM, Cain K. Processing/activation of CPP32-like proteases is involved in transforming growth factor beta1-induced apoptosis in rat hepatocytes. Hepatology. 1997;25:1516–26.
Inayat-Hussain SH, Cohen GM, Cain K. Mn2+ activates DNA cleavage in rat liver nuclei. Asia Pacific J Mol Biol Biotech. 1998;6:67–73.
Kwo P, Patel T, Bronk SF, Gores GJ. Nuclear serine protease activity contributes to bile acid-induced apoptosis in hepatocytes. Am J Physiol. 1995;268:G613–21.
Lazebnik YA, Takahashi A, Moir RD, et al. Studies of the lamin proteinase reveal multiple parallel biochemical pathways during apoptotic execution. Proc Natl Acad Sci USA. 1995;26:9042–6.
Leccia MT, Richard MJ, Favier A, Beani JC. Zinc protects against ultraviolet A1-induced DNA damage and apoptosis in cultured human fibroblasts. Biol Trace Elem Reg. 1999;69:177–90.
McCabe Jr MJ, Jiang SA, Orrenius S. Chelation of intracellular zinc triggers apoptosis in mature thymocytes. Lab Invest. 1993;69:101–10.
Ni R, Tomita Y, Matsuda K, et al. Fas-mediated apoptosis in primary cultured mouse hepatocytes. Exp Cell Res. 1994;215:332–7.
Oberhammer FA, Pavelka M, Sharma S, et al. Induction of apoptosis in cultured hepatocytes and in regressing liver by transforming growth factor β1. Proc Natl Acad Sci USA. 1992;89:5408–12.
Oberhammer FA, Fristch G, Schmeid M, et al. Condensation of the chromatin at the membrane of an apoptotic nucleus is not associated with activation of an endonuclease. J Cell Sci. 1993a;104:317–26.
Oberhammer FA, Wilson JW, Dive C, et al. Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO J. 1993b;12:3679–84.
Oberhammer FA, Froschl G, Tiefenbacher R, Inayat-Hussain SH, Cain K, Stopper H. A topical issue of microscopy research and technique. Microsci Res Tech. 1996;34:247–58.
Ogasawara J, Watanabe-Fukunaga R, Adachi M, et al. Lethal effect of the anti-Fas antibody in mice. Nature. 1993;364:806–9.
Patel T, Bronk SF, Gores GJ. Increases of intracellular magnesium promote glycodeoxycholate-induced apoptosis in rat hepatocytes. J Clin Invest. 1994;94:2183–92.
Perry DK, Smith MJ, Stennicke HR, et al. Zinc is a potent inhibitor of the apoptotic protease, caspase-3. A novel target for zinc in the inhibition of apoptosis. J Biol Chem. 1997;272:18530–3.
Sanchez V, Lucas M, Sanz A, Gobernam R. Decreased protein kinase C activity is associated with programmed cell death (apoptosis) in freshly isolated hepatocytes. Biosci Rep. 1992;12:199–206.
Sanchez A, Alvarez AM, Benito M, Fabregat I. Cycloheximide prevents apoptosis, reactive oxygen species production, and glutathione depletion induced by transforming growth factor beta in fetal rat hepatocytes in primary culture. Hepatology. 1997;26:935–43.
Schulte-Hermann R, Bursch W, Grasl-Kraupp B. Active cell death (apoptosis) in liver biology and disease. Prog Liver Dis. 1995;13:1–35.
Shinagawa T, Yoshioka K, Kakuma S et al. Apoptosis in cultured hepatocytes: the effects of tumor necrosis factor α and interferon γ. Pathology. 1991;165:247–53.
Steinebach OM, Wolterbeek HT. Effects of zinc on rat hepatoma HTC cells and primary cultured rat hepatocytes. Toxicol Appl Pharmacol. 1993;118:245–54.
Sunderman FW Jr. The influence of zinc on apoptosis. Ann Clin Lab Sci. 1995;25:134–42.
Takahashi A, Alnemri ES, Lazebnik YA, et al. 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. 1996;6:8395–400.
Walker PR, Smith C, Youdale T, Leblanc J, Whitfield JF, Sikorska M. Topoisomerase II-reactive chemotherapeutic drugs induce apoptosis in thymocytes. Cancer Res. 1991;51:1078–85.
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Inayat-Hussain, S., Cohen, G. & Cain, K. A reappraisal of the role of Zn2+ in TGF-β1-induced apoptosis in primary hepatocytes. Cell Biol Toxicol 15, 381–387 (1999). https://doi.org/10.1023/A:1007606016901
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DOI: https://doi.org/10.1023/A:1007606016901