ARTICLE |
Correspondence to: Sabine Angermüller, Dept. of Anatomy and Cell Biology II, U. of Heidelberg, Im Neuenheimer Feld 307, D-69120 Heidelberg, Germany..
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Summary |
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Tumor necrosis factor (TNF) induces apoptotic death of hepatocytes in the galactosamine (GalN)-sensitized mouse liver after 5 hr. In our study, the most remarkable sign of the early stage of apoptosis was the focal rupture of the outer mitochondrial membrane. Parts of the inner membrane extended through the gap of the outer membrane, whereas the rest of the inner membrane still formed the cristae. This feature appeared in hepatocytes before chromatin condensation. With the diaminobenzidine technique for localization of cytochrome oxidase activity, the reaction product was detectable by light and electron microscopy. Ten percent of the hepatocytes were apoptotic, with condensed chromatin and high enzyme activity, 37% were pre-apoptotic, without chromatin condensation but high enzyme activity, and 53% had neither condensed chromatin nor a remarkable reaction product of cytochrome oxidase activity. Fas (APO-1, CD95) molecules on the plasma membrane of hepatocytes increased and were represented immunohistochemically in cells without chromatin condensation. DNA strand breaks were also detectable before chromatin aggregation. The results of this study indicate that mitochondria play a pivotal role in pre-apoptotic hepatocytes, together with an increase of the Fas molecule on the plasma membrane and with the occurrence of DNA strand breaks in the nucleus. (J Histochem Cytochem 46:11751183, 1998)
Key Words: hepatocytes, apoptosis, mitochondria, cytochrome oxidase, Fas, DNA strand breaks
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Introduction |
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The genetic program for apoptosis appears to be present in all mammalian cells during their entire life span. Morphological signs of apoptosis include chromatin condensation, forming crescents along the nuclear envelope, shrinkage of the cell, loss of microvilli, detachment from neighboring cells, and fragmentation into apoptotic bodies ( (
TNF-mediated hepatocellular apoptosis was observed in mice after either macrophage activation by LPS (
Recently, the participation of mitochondria in the apoptotic cascade was described: The pre-apoptotic disruption of the mitochondrial transmembrane potential occurred before cells exhibited nuclear DNA fragmentation (-mediated apoptosis in cell cultures.
The aim of this study was to investigate morphological changes and functional characteristics of mitochondria in pre-apoptotic and apoptotic mouse hepatocytes. Applying immunohistochemical methods, we examined whether Fas expression and DNA fragmentation into strand breaks occurred before chromatin condensation was seen.
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Materials and Methods |
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Recombinant murine TNF was generously provided by Dr. G.R. Adolf (Boehringer Institute; Vienna, Austria).
Animals
This study was performed in compliance with the German Animal Protection Law under a permit issued by the state government (Freiburg, Germany). Six- to 8-week-old male BALB/c mice were obtained from the animal facility of the University of Konstanz (Germany) and kept on a standard laboratory diet with free access to water. Sixteen hours before the beginning of the experiments food was withdrawn. GalN (700 mg/kg) (Roth Chemicals; Karlsruhe, Germany) was given IP in a volume of 200 µl saline. Recombinant murine TNF (10 µg/kg) was injected into the tail vein in a volume of 300 µl saline containing 0.1% HSA 20 min after GalN administration. The administration of only TNF or only GalN served as a negative control for the induction of apoptosis. Five hours after treatment, animals were anesthetized by sodium pentobarbital 150 mg/kg IP (Nembutal; Sanofi-Ceva, Hannover, Germany).
Fixation
For light and electron microscopic studies, the livers were fixed via the portal vein with a fixative containing 0.25% glutaraldehyde and 2% sucrose in 100 mM piperazine-N,N'-bis (2-ethanesulphonic acid) (Pipes) buffer at a pH value of 7.4 for 5 min. After fixation, 50-µm sections were cut with a microslicer (Dosaka EM; Kyoto, Japan) and collected in 100 mM Pipes buffer at a pH value of 7.4.
Electron Microscopy
Incubation for Cytochrome Oxidase Activity.
For demonstration of cytochrome oxidase activity we applied a modified 3,3-diaminobenzidine-tetrahydrochloride (DAB) technique (
Light Microscopy
Immunohistochemical Localization of Fas Receptor.
Epon-embedded semithin sections were etched with 10% NaOH in absolute ethanol for 3045 min (
Immunohistochemical Detection of DNA Strand Breaks
For in situ labeling of DNA breaks in nuclei, we used LR White-embedded semithin sections and applied the method of
Quantification of Pre-apoptotic and Apoptotic Cells
Animals treated only with GalN or TNF did not show pre-apoptotic or apoptotic changes. Therefore, we counted the nuclei or cells only of GalN/TNF-treated mice. In LR White-embedded semithin sections apoptotic nuclei stained by hematoxylin/eosin were determined. A total of 1400 nuclei were randomly counted and the percentage of the number of nuclei with condensed chromatin was calculated. In Epon-embedded semithin sections incubated for demonstration of cytochrome oxidase activity, pre-apoptotic and apoptotic hepatocytes were identified by the strong brown reaction product. A total of 1200 cells were counted randomly and the percentage of the number of pre-apoptotic and apoptotic hepatocytes was calculated.
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Results |
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We investigated death factor-induced hepatocellular apoptosis in the livers of GalN/TNF-treated mice cytochemically and immunohistochemically 5 hr after treatment. The liver was sparsely infiltrated by lymphocytes. Therefore, additional leukocyte mediators were unlikely to contribute to hepatocellular apoptosis. Apoptotic cell death is very rare in the healthy adult mouse liver. Approximately 15 apoptotic cells/10,000 hepatocytes are found in untreated mice (for review see
The DAB method for demonstration of cytochrome oxidase activity revealed that this last enzyme of the respiratory chain was very active in mitochondria of pre-apoptotic and apoptotic hepatocytes when we offered cytochrome c as electron donor in the incubation medium (Figure 1A and Figure 1B). Before chromatin condensation was seen, alterations occurred in mitochondria. The outer membrane of the mitochondria was scarified, and the inner membrane had expanded through this gap to form a large bulge (Figure 2BD). In that part of the mitochondrion in which the outer membrane was intact, cristae stained by the DAB reaction product were tightly folded by the inner membrane, and the bulge resembled a vacuole without or with fewer cristae (Figure 1A). When chromatin condensation had taken place, the same alterations were found in mitochondria (Figure 1B and Figure 2A). Cytochrome oxidase was very active in all mitochondria of pre-apoptotic and apoptotic hepatocytes (Figure 1A, Figure 1B, Figure 2A, Figure 3A, and Figure 4A). It should be emphasized that 47% of the hepatocytes showed these characteristic alterations. In GalN/TNF-treated animals, 53% of the hepatocytes showed normal mitochondrial morphology with less cytochrome oxidase activity (Figure 3A, Figure 3B, and Figure 4A). Neither GalN nor TNF administration caused any bulges of the inner mitochondrial membrane, but the administration induced a remarkable increase in the number of cytochrome oxidase-positive mitochondria in comparison to the above mentioned 53% with normal mitochondrial structure (Figure 3AD and Figure 4AC). In control sections incubated with NaCN together with cytochrome c, the DAB reaction product was not detected in mitochondria.
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Immunohistochemical Localization of Fas
The Fas receptor is present in cell membranes of several tissues, including normal liver (
Immunohistochemical Detection of DNA Strand Breaks
The TUNEL method end-labeled DNA double-strand breaks and single-strand breaks in hepatocellular nuclei of LR White-embedded semithin sections. Figure 6A shows the polymerization of dUTP to the 3'-hydroxyl end of fragmented DNA, resulting in a brown reaction product that was detected in most nuclei of hepatocytes (Figure 6A). The negative control of a semithin section from a treated animal incubated only with dUTP failed to show any reaction product (Figure 6B).
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Discussion |
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In this study we investigated functional characteristics and earliest morphological alterations of TNF-induced hepatocellular apoptosis in the GalN-sensitized mouse liver.
Before the known morphological signs of apoptosis, such as chromatin condensation, cell shrinkage, and fragmentation were detectable (
In contrast to the morphological description of mitochondria in apoptotic cells, their functional role has been emphasized by many authors. The reduction in the mitochondrial transmembrane potential (m) that causes opening of mitochondrial permeability transition pores is an early event in the apoptotic process (
It appears that mitochondrial apoptotic factors and all structures involved in the pre-apoptotic ruptures of the m are present in mitochondria lacking mitochondrial DNA. Therefore, apoptosis is controlled by the nuclear rather than by the mitochondrial genome (
DNA strand breaks are a typical sign of apoptosis. Using the same GalN/TNF-model as we did,
In summary, our study demonstrates characteristic morphological alterations in mitochondria as a very early hallmark of the hepatocellular apoptotic program. Before chromatin condensation occurs, the outer mitochondrial membrane is focally disrupted and the inner membrane protrudes through this gap. High levels of cytochrome oxidase activity are observed cytochemically in pre-apoptotic and apoptotic hepatocytes. Fas molecules on the plasma membrane of hepatocytes increase and are also present in cells without chromatin condensation. The appearance of DNA strand breaks is detectable before nuclear chromatin becomes aggregated in dense crescents beneath the nuclear envelope.
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Acknowledgments |
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Supported by grants An 192/1-3 and Ti 169/4-1 from the Deutsche Forschungsgemeinschaft (Bonn Bad-Godesberg).
We thank Prof H.D. Fahimi for helpful discussion. The excellent technical assistance by Ms Barbara Lenschow and Ms Annette Stradtmann is gratefully acknowledged.
Received for publication December 8, 1997; accepted May 21, 1998.
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Literature Cited |
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Angermüller S, Fahimi HD (1981) Selective cytochemical localization of peroxidase, cytochrome oxidase and catalase in rat liver with 3,3'-diaminobenzidine. Histochemistry 71:33-44[Medline]
Arends MJ, Wyllie AH (1991) Apoptosis: mechanisms and roles in pathology. Int Rev Exp Pathol 32:223-254[Medline]
Asoh S, Mori T, Hayashi JI, Ohta S (1996) Expression of the apoptosis-mediator Fas is enhanced by dysfunctional mitochondria. J Biochem 120:600-607[Abstract]
Castedo M, Macho A, Zamzami N, Hirsch T, Marchetti P, Uriel J, Kroemer G (1995) Mitochondrial perturbations define lymphocytes undergoing apoptotic depletion in vivo. Eur J Immunol 25:3277-3284[Medline]
Cossarizza A, Franceschi C, Monti D, Salvioli S, Bellesia E, Rivabene R, Biondo L, Rainaldi G, Tinari A, Malorni W (1995) Protective effect of N-acetylcysteine in tumor necrosis factor--induced apoptosis in U937 cells: the role of mitochondaria. Exp Cell Res 220:232-240[Medline]
Enari M, Hase A, Nagata S (1995) Apoptosis by a cytosolic extract from Fas-activated cells. EMBO J 14:5201-5208[Abstract]
Faa G, Ambu R, Congiu T, Costa V, LeddaColumbano GM, Coni P, Curto M, Giacomini L, Columbano A (1992) Early ultrastructural changes during thioacetamide-induced apoptosis in rat liver. J Submicrosc Cytol Pathol 24:417-424[Medline]
Feldmann G (1997) Liver apoptosis. J Hepatol 26:1-11[Medline]
Galle PR, Hofmann WJ, Walczak H, Schaller H, Otto G, Stremmel W, Krammer PH, Runkel L (1995) Involvement of the CD95 (APO-1/Fas) receptor and ligand in liver damage. J Exp Med 182:1223-1230[Abstract]
Gantner F, Leist M, Jilg S, Germann PG, Freudenberg MA, Tiegs G (1995a) Tumor necrosis factor-induced hepatic DNA fragmentation as an early marker of T cell-dependent liver inury in mice. Gastroenterology 109:166-176[Medline]
Gantner F, Leist M, Lohse AW, Germann PG, Tiegs G (1995b) Concavalin A-induced T-cell-mediated hepatic injury in mice: the role of tumor necrosis factor. Hepatology 21:190-198[Medline]
Gavrieli Y, Sherman Y, Ben-Sasson SA (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493-501[Abstract]
Gressner AM, Lahme B, Mannherz HG, Polzar B (1997) TGF-ß-mediated hepatocellular apoptosis by rat and human hepatoma cells and primary rat hepatocytes. J Hepatol 26:1079-1092[Medline]
Hiramatsu N, Hayashi N, Katayama K, Mochizuki K, Kawanishi Y, Kasahara A, Fusamoto H, Kamada T (1994) Immunohistochemical detection of Fas antigen in liver tissue of patients with chronic hepatitis C. Hepatology 19:1354-1359[Medline]
Hockenbery D (1995) Defining apoptosis. Am J Pathol 146:16-19[Medline]
Hockenbery D, Nuñez G, Milliman C, Schreiber RD, Korsmeyer SJ (1990) Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 348:334-336[Medline]
Hockenbery DM, Zutter M, Hickey W, Nahm M, Korsmeyer SJ (1991) BCL2 protein is topographically restricted in tissues characterized by apoptotic cell death. Proc Natl Acad Sci USA 88:6961-6965[Abstract]
Jacobson MD, Burne JF, King MP, Miyashita T, Reed JC, Raff MC (1993) Bcl-2 blocks apoptosis in cells lacking mitochondrial DNA. Nature 361:365-369[Medline]
Kerr JFR, Wyllie AH, Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26:239-257[Medline]
Kimura K, Sasano H, Shimosegawa T, Kato K, Noguchi T, Mochizuki S, Sawai T, Koizumi M, Toyota T, Nagura H (1997) Ultrastructural and confocal laser scanning microscopic examination of TUNEL-positive cells. J Pathol 181:235-242[Medline]
Kluck RM, BossyWetzel E, Green DR, Newmeyer DD (1997) The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275:1132-1136
Krippner A, MatsunoYagi A, Gottlieb RA, Babior BM (1996) Loss of function of cytochrome c in Jurkat cells undergoing Fas-mediated apoptosis. J Biol Chem 271:21629-21636
Kroemer G, Petit P, Zamzami N, Vayssière J-L, Mignotte B (1995) The biochemistry of programmed cell death. FASEB J 9:1277-1287
Kroemer G, Zamzami N, Susin SA (1997) Mitochondrial control of apoptosis. Immunol Today 18:44-51[Medline]
Küsters S, Gantner F, Künstle G, Tiegs G (1996) Inteferon gamma plays a critical role in T cell-dependent liver injury in mice initiated by concanavalin A. Gastroenterology 111:462-471[Medline]
Leist M, Gantner F, Bohlinger I, Germann PG, Tiegs G, Wendel A (1994) Murine hepatocyte apoptosis induced in vitro and in vivo by TNF- requires transcriptional arrest. J Immunol 153:1778-1788
Leist M, Gantner F, Bohlinger I, Tiegs G, Germann PG, Wendel A (1995) Tumor necrosis factor-induced hepatocyte apoptosis precedes liver failure in experimental murine shock models. Am J Pathol 146:1220-1234[Abstract]
Leist M, Gantner F, Künstle G, Bohlinger I, Tiegs G, Bluethmann H, Wendel A (1996) The 55-kD tumor necrosis factor receptor and CD95 independently signal murine hepatocyte apoptosis and subsequent liver failure. Mol Med 2:109-124[Medline]
Leist M, Gantner F, Naumann H, Bluethmann H, Vogt K, BrigeliusFlohé R, Nicotera P, Volk HD, Wendel A (1997) Tumor necrosis factor-induced apoptosis during the poisoning of mice with hepatotoxins. Gastroenterology 112:923-934[Medline]
Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X (1997) Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91:479-489[Medline]
Litwin JA, Yokota S, Hashimoto T, Fahimi HD (1984) Light microscopic immunocytochemical demonstration of peroxisomal enzymes in epon sections. Histochemistry 81:15-22[Medline]
Majno G, Joris I (1995) Apoptosis, oncosis, and necrosis. An overview of cell death. Am J Pathol 146:3-15[Abstract]
Marchetti P, Susin SA, Decaudin D, Gamen S, Castedo M, Hirsch T, Zamzami N, Naval J, Senik A, Kroemer G (1996) Apoptosis-associated derangement of mitochondrial function in cells lacking mitochondrial DNA. Cancer Res 56:2033-2038[Abstract]
Monaghan P, Robertson D, Amos TAS, Dyer MJS, Mason DY, Greaves MF (1992) Ultrastructural localization of Bcl-2 protein. J Histochem Cytochem 40:1819-1825
Nicholson DW, Thornberry NA (1997) Caspases: killer proteases. Trends Biochem Sci 22:299-306[Medline]
Oberhammer F, Bursch W, Tiefenbacher R, Fröschl G, Pavelka M, Purchio T, SchulteHermann R (1993) Apoptosis is induced by transforming growth factor-ß1 within 5 hours in regressing liver without significant fragmentation of the DNA. Hepatology 18:1238-1246[Medline]
O'Donnell VB, Spycher S, Azzi A (1995) Involvement of oxidants and oxidant-generating enzyme(s) in tumor-necrosis-factor--mediated apoptosis: role for lipoxygenase pathway but not mitochondrial respiratory chain. Biochem J 310:133-141[Medline]
Ogasawara J, WatanabeFukunaga R, Adachi M, Matsuzawa A, Kasugai T, Kitamura Y, Itoh N, Suda T, Nagata S (1993) Lethal effect of the anti-Fas antibody in mice. Nature 364:806-809[Medline]
Patel T, Gores GJ (1995) Apoptosis in hepatobiliary disease. Hepatology 21:1725-1741[Medline]
Petit PX, Lecoeur H, Zorn E, Dauguet C, Mignotte B, Gougeon M-L (1995) Alterations in mitochondrial structure and function are early events of dexamethasone-induced thymocyte apoptosis. J Cell Biol 130:157-167[Abstract]
SchulteHermann R, Bursch W, GraslKraupp (1995) B Active cell death (apoptosis) in liver biology and disease. In Boyer JL, Ockner RK, eds. Progress in Liver Disease. Vol 8. Philadelphia, WB Saunders, 1-35
Searle J, Harmon BV, Bishop CJ, Kerr JFR (1987) The significance of cell death by apoptosis in hepatobiliary disease. J Gastroenterol Hepatol 2:77-96
Shinagawa T, Yoshioka K, Kakumu S, Wakita T, Ishikawa T, Itoh Y, Takayanagi M (1991) Apoptosis in cultured rat hepatocytes: the effects of tumour necrosis factor and interferon
. J Pathol 165:247-253[Medline]
Susin SA, Zamzami N, Castedo M, Hirsch T, Marchetti P, Macho A, Daugas E, Geuskens M, Kroemer G (1996) Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. J Exp Med 184:1331-1341[Abstract]
Wyllie AH, Duvall E (1992) Cell injury and death. In McGee JO'D, Isaacson PG, Whright NA, eds. Oxford Textbook of Pathology. Vol 1. Principles of Pathology. Oxford, UK, Oxford University Press, 141157
Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, Peng T-I, Jones DP, Wang X (1997) Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275:1129-1132
Zamzami N, Marchetti P, Castedo M, Zanin C, Vayssière J-L, Petit PX, Kroemer G (1995) Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J Exp Med 181:1661-1672[Abstract]
Zamzami N, Susin SA, Marchetti P, Hirsch T, GómesMonterrey I, Castedo M, Kroemer G (1996) Mitochondrial control of nuclear apoptosis. J Exp Med 183:1533-1544[Abstract]
Zou H, Henzel WJ, Liu X, Lutschg A, Wang X (1997) Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90:405-413[Medline]