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Ethanol-induced apoptosis in vitro
Clinical Biochemistry, Vol. 32, No. 7, 547–555, 1999 Copyright © 1999 The Canadian Society of Clinical Chemists Printed in the USA. All rights reserved 0009-9120/99/$–see front matter
PII S0009-9120(99)00054-5
Ethanol-Induced Apoptosis In Vitro MANUELA G. NEUMAN,1 NEIL H. SHEAR,1 ROSS G. CAMERON,2 GADY KATZ,1 and CLAUDIO TIRIBELLI3 1
Division of Clinical Pharmacology, Sunnybrook and Women’s College Health Sciences Centre, Toronto, Ontario, Canada, 2Department of Pathology, Toronto Hospital, Departments of Pharmacology, Pathology and Medicine, University of Toronto, Toronto, Ontario, Canada, and 3 Centro Studi Fegato Modena-Trieste, Department of B. B. C. M., University of Trieste, Trieste, Italy Objectives: The aim is to study the apoptotic process in a human hepatocyte model for ethanol (EtOH)-induced apoptosis. Design and methods: Normal human primary hepatocytes (HPH) and Hep G2 cells were exposed to increasing EtOH. 6000 cells/ sample were analyzed by transmission electron microscopy. Results: Apoptotic cells were observed (mmol/L EtOH): 40: 6 ⫾ 0.5%, 60: 13 ⫾ 2% (p ⬍ 0.05), 80: 26 ⫾ 1% (p ⬍ 0.001) (vs. control). Two consecutive doses of 80 mmol/L for 24 h each additionally increased apoptosis 55 ⫾ 3% (p ⬍ 0.0001 vs. control and p ⬍ 0.001 vs. single dose). In response to this exposure, there is a stronger apoptotic activity in HPH when compared to Hep G2 (p ⬍ 0.05). Conclusions: In vitro, EtOH-induced apoptosis is regulated by dose level and the frequency of exposure. Copyright © 1999 The Canadian Society of Clinical Chemists
KEY WORDS: apoptosis; ethanol; Hep G2; normal human primary hepatocytes; nutritional interventions.
Introduction egulation of organ size and liver function is the result of a dynamic balance between stimulatory and inhibitory signals (1– 8). Under physiological conditions, senescent or damaged cells are removed by apoptosis, whereas the response to acute cell injury mostly involves a necrotic process (9,10). After EtOH ingestion, rat and mice models showed subcellular changes compatible with apoptosis (11, 12). We have been able to reproduce a model of EtOHinduced liver damage in human Hep G2 cells in vitro (13). Biochemical and morphological features in our model present the same pattern observed in the serum of patients with alcohol-induced liver damage and human biopsies from the same category of patients (14). Previously, we reported that in Hep G2 cells (15) and Hep G2 in co-culture with normal
R
Correspondence: M. G. Neuman, Ph.D., Division Clinical Pharmacology, E-240, Sunnybrook HSC, 2075 Bayview Avenue, Toronto, Ontario, M4N 3M5, Canada. Email: [email protected]. Manuscript received June 29, 1999; accepted June 29, 1999 CLINICAL BIOCHEMISTRY, VOLUME 32, OCTOBER 1999
human stellate cells, apoptosis can be detected and measured when exposed to 80 mmol/L EtOH (16). Hep G2 cells have inducible cytochrome P450 2E1 activity but low content of CYP 2E1 (17) and a small amount of alcohol dehydrogenase activity (ADH) (18). CYP 2E1 and ADH are associated with EtOHinduced liver damage and are thought to be critical in the hepatocellular regulation of apoptotic processes. When comparing the inducibility of cytochrome P450 2E1 and ADH activity in Hep G2 cells versus HPH treated with 80 mmol/L EtOH a significant difference was observed (17,18). Therefore, we decided to parallel our observations in Hep G2 line with studies in HPH. Cells in these HPH cultures retain morphological features of hepatocytes by both light and electron microscopy. They also retain glucose-6-phosphatase activity and secrete proteins characteristic of hepatocytes, such as albumin, alpha-fetoprotein and transferrin (19). This work deals with the differences and similarities in an apoptotic-induced cascade by EtOH in HPH in comparison with the Hep G2 cells. Detection of cell death by apoptosis, expressed by normal hepatocytes that retain liver-specific functions of differentiated hepatocytes, can be considered a new application of studying human hepatocyte reactions under EtOH stress. Methods PRIMARY
HUMAN HEPATOCYTES CULTURE
The hepatocyte cultures were developed from normal liver tissue obtained after lobectomy from organ donors where only a part of the liver was used for transplantation, by cultivating in a highly enriched medium. Our method for preparing hepatocytes suspension was based originally on that of Ballet et al. (20). The liver graft was perfused with ice-cold University of Wisconsin solution (21) and kept on ice (2–10 h) until cell isolation. The material was washed with HEPES media. The hepatocytes were 547
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isolated using the two step perfusion technique. The average yield was 1.5 billion cells/preparation and the average viability, assessed by trypan blue exclusion test was 94%. Hepatocytes were washed three times in a culture medium (1/1 Ham F12 and Leibovitz L-15 supplemented 5 mmol/L glucose, 50 U/mL penicillin, 50 g/L streptomycin, 10⫺8 mol/L insulin). Cells were then plated 1 ⫻ 106/ml in collagen coated Falcon flasks (3 g /cm2) and cultured with conventional conditions in medium supplemented with fetal bovine serum (FBS). One hour later, the medium was changed to remove the floating, unattached hepatocytes. The medium was replaced after 12 h. Every 24 h thereafter, the media was changed with a fresh one in the presence of FBS. Non-hepatocellular cells were not present. Cells were treated with media in presence or in absence of EtOH as described in the experimental design. The dilutions were done in media in absence of FBS. We have been able to maintain the cells in culture continuously for 10 days exhibiting typical morphological characteristics of hepatocytes. To address the role of nutritional factors acting as priming cells for apoptosis, in some experiments the serum free media was supplemented with choline or glycine. For enzyme-linked immunosorbent assay (ELISA), cells were plated directly in 96 well plates not coated with collagen. HEP G2
CELLS LINE
Hep G2 cells were obtained from Wistar Institute (Philadelphia, PA, USA). Cells were seeded in flasks (1 ⫻ 106 cells/mL) (13). The cell counts were monitored using a Coulter counter (Coulter Electronics Inc., Hialeah, FL, USA). Cells in long-term cultures were grown in ␣-MEM supplemented with 10% v/v heat inactivated FBS. At the beginning of the experiment, when cells reached 70% confluence, the growth medium was removed from the culture flasks, the cultures were washed twice with phosphate buffered saline (PBS) and fresh serum free medium was used as base for all the treatments. The viability of the cells not treated with EtOH was not altered by culturing them for up to 6 days serum free medium (13). DRUGS
AND CHEMICALS
EtOH was purchased from Alcohol Ltd. (Toronto, Ontario, Canada). Plain ␣-MEM, Hanks balanced sodium solution and calcium chloride were obtained from Gibco (Burlington, Ontario, Canada). Trypsin was purchased from Difco (Detroit, MI, USA) and was prepared as a 1% solution. PBS (phosphate buffered saline without Ca2⫹ or Mg2⫹ ) was used to wash cells and to remove medium. All plastic ware for cell cultures was obtained from Falcon (Becton Dickinson, Oxnard, CA, USA). All of the remaining reagents were of analytical grade, obtained from Sigma Chemical Co. (St. Louis, MO, USA). For quantitative determination of DNA frag548
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ments, we used the Nucleosome ELISA kit from Oncogene Research Products CN Biosciences (Cambridge, MA, USA). The Nucleosome kit of low molecular DNA associated with histone is considered to be a sensitive tool for DNA detection at the length of 180 –200 bp. The ELISA applies a Digoxigenin detector antibody and a Streptavidin conjugate with horseradish peroxidase (SA-HRP) Sheep antidigoxigenin antibody to quantitate the apoptotic nucleosomes. EXPERIMENTAL
DESIGN
Control cells were exposed for 24 h only to plain essential medium (␣ MEM), while the treated cells were incubated with EtOH (40, 60, 80 mmol/L). For chronic exposure, the cells were treated for 24 h with 80 mmol/L EtOH washed with PBS pH 7.4, and incubated for additional 24 h with a second dose of 80 mmol/L EtOH. Some cells were exposed to one dose or two doses of 80 mmol/L EtOH in the presence of choline 70 mol/L and 40 mol/L glycine. All components were filtered-sterilised, and the entire procedure was conducted under aseptic conditions. The cells were routinely maintained in a humidified incubator in 95% air/5% CO2, at 37° C. Further details of the individual experiments are provided in the figure legends. After a period of 18 –20 h storage at ⫺20°C, the cells underwent lysis and were exposed to the detector antibody for 1 h at room temperature. The cells were washed and incubated for another 30 min with SA-HRP conjugate. The chromogene reaction was stopped and the absorbance was read using a spectrophotometer plate reader with dual lengths 450/ 595 nm. The intensity of the yellow color is proportional to the number of nucleosomes in the sample. For each treatment, 6 wells/plate in 5 different plates were quantitated. The results are reported as % of apoptosis vs. control, non-treated taken as 0% apoptosis. For the standard curve, we ran replicates of six in each plate using two different plates. The sensitivity of the assay was measured by assaying the non-treated cells at time zero. The mean signal and the standard deviation were calculated. The assay can distinguish 0.3% from zero. The Maxline Microplate Reader, from Molecular Device Corp. (Menlo Park, CA, USA) was connected to a computer using SOFT MAX software 2.3 for Windows (Molecular Devices Corp.) permitting to template the plate according to the experimental needs and perform the statistical analysis directly on the template format. STATISTICAL
ANALYSIS
All data are expressed as means ⫾ standard deviation (SD). Differences between groups were analyzed using an ANOVA test for repeated measurements with a Bonferroni test to correct for multiple comparison. All statistical analyses were CLINICAL BIOCHEMISTRY, VOLUME 32, OCTOBER 1999
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Figure 1 — Transmission electron micrograph (TEM) of Hep G2 cells (control, non-treated). Cells were plated 75 Falcon flasks in a density of 106 cells/mL and were grown in media supplemented with FBS. At 70% confluence, cells were incubated in plain media only, for 24 h. After removing the media the cells were prepared for TEM as described in Material ), numerous normal and Methods, The EM shows normal looking cells with normal central located nucleus (N mitochondria ( m). One of the cell displays vesiculation of the cisternae of smooth endoplasmic reticulum ( er). Several small lipid droplets are also present ( ld). Tight junctions ( tj) between two adjacent cells can be observed. Magnification ⫻ 5,000.
performed with the statistical software package Microcal Origin 301, Microcal Inc. LIGHT
AND ELECTRON MICROSCOPY
The cells were prepared for light and transmission electron microscopy (TEM) studies using a standard procedure as outlined below. For each group of control (0 EtOH/24 or 48 h) and treated cells (80 mmol/L for 24 h, or 2 doses of 80 mmol/L/ 2 consecutive periods of 24 h), six flasks of either HPH or Hep G2 were used. After the period of incubation, the media was removed and cells were washed twice with PBS. Five mL of 1% trypsin was added for 2 min in each flask. Cells were washed again with PBS and then resuspended in plain media. Cell suspensions were centrifuged at 50 g for 10 min. Pellets were immediately fixed in 2.5% v/v glutaraldehyde for a minimum of 24 h. Blocks of cells were separated, post-fixed in 1% v/v osmium tetroxide, dehydrated with graded series of acetone concentraCLINICAL BIOCHEMISTRY, VOLUME 32, OCTOBER 1999
tions and embedded in Araldine. Sections (1 micron thick) were viewed by light microscopy (LM). For light microscopy studies an Olympus microscope equipped with Leco 2005, Image Processing and Analysis System (Leco Instruments, Toronto, Ontario, Canada) with Microsoft威 Visual Basic™ software were used. Cells were considered apoptotic if the classic features of pyknotic nuclei, cytoplasmic condensation and nuclear chromatin fragmentation could be observed. Representative blocks were selected, subjected to ultra-thin sectioning and stained with uranyl acetate and lead citrate for TEM. Electron micrographs were taken with a transmission electron microscope JOEL 1200 E ⫻ II (JOEL Institute Inc., Lexington, MA, USA). Ultrastructural findings were examined in five different grids/flask in each experiment. On each grid, 200 – 400 cells were examined. An average of 9000 (300 cells/grid ⫻ grids/flask ⫻ 6 flasks/treatment) cells were analyzed for each treatment. The 549
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Figure 2 — Transmission electron micrograph of Hep G2 cells exposed to 80 mmol/L EtOH for 24 h. Cells were plated in 75 Falcon flasks in a density of 106 cells/mL and were grown in media supplemented with FBS. At 70% confluence, cells were incubated in media containing 80 mmol/L ethanol. In the middle of the micrograph a is a large size cell. The nucleus (N) is normal looking and presents a normal nucleolus ( n). The cell shows considerable enlargement of mitochondria M). The cisternae of endoplasmic reticulum show vesiculation ( ( er), and dilatation ( er ). A part of the cell forms a bleb (B), which separation from the cell can be seen. In the opposite side of the cell, in the upper corner, an apoptotic body (ab ) is present. In the lower corner, an apoptotic cell in the first stage presenting a piknotic nucleus PN. The cell is not anymore linked to the others. Between the cells debris (d ) can be observed. In the upper corner a classic elongated apoptotic cell (A ) has dark condensed cytoplasm. Magnification ⫻ 5,000.
criteria for the morphological identification of cellular structures were standard (22). In assessing cells by electron microscopy, the classic shrinkage of cells, electron dark cytoplasm, and apoptotic bodies are the criteria for classical apoptosis (23). Results LIGHT
AND ELECTRON MICROSCOPY
Cells incubated with plain media show normal organelles: abundant mitochondria, rough and smooth endoplasmic reticulum and only occasional small lipid vesicles (Figure 1). Bile canaliculus-like structure can be observed at the confluence of 3 cells suggesting that the cells are functional. In previous publications (13,24), we reported that exposure to 80 mmol/L EtOH produces changes in mitochondria, endoplasmic reticulum, and in the number of lipid vesicles per cell, as well as changes in lipid content of the fat droplets (25). Figure 2 shows a large cell with a normal looking nucleus with pleomorphism of mitochondria, vesiculation, and dilatation of endoplasmic reticulum. In one part of the cells, a characteristic “bleb” can be observed. This work also de550
scribes ultrastructural changes compatible with apoptosis, e.g., some cells having intact plasma membrane and intact nuclei membrane, but being retracted from the surrounding cells (Figure 3). These cells no longer have tight junctions that can be observed between two adjacent cells in the control cultures not treated with EtOH. Some of the cells are elongated and present intact organelles (Figure 4). The cytoplasm is darker and the nucleus presents condensation of chromatin, which can be located adjacent to the nuclear membrane. Some cells are fragmented and the nuclear fragmentation occurs concomitantly. Therefore, multiple vesicles with compacted chromatin and intact cell (apoptotic bodies) can be seen. In HPH treated with 2 consecutive doses of 80 mmol/L EtOH for 24 h, a large cell presenting intact organelles, but showing changes in chromatin distribution and enlargement of endoplasmic reticulum, may undergo apoptosis (Figure 5). MORPHOMETRIC
ANALYSIS
The software we used to analyze the LM material permitted us not only to make quantitative observaCLINICAL BIOCHEMISTRY, VOLUME 32, OCTOBER 1999
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Figure 3 — Transmission electron micrograph of Hep G2 cells treated with 80 mmol/L EtOH for 2 times ⫻ 24 h. When 70% confluent, cells were washed with PBS and media containing 80 mmol/L EtOH was added (0 time). After a period of 24-h incubation, followed by aspiration of the media, a second equimolar dose of EtOH was added for a second period of 24 h. The cells formed a monolayer that has not been disrupted by the trypsinization. There is a mitochondrial pleomorphism with normal size mitochondria. Two cells with normal looking nucleus (N) can be seen in the micrograph. There are two apoptotic cells ( A) containing preserved organelles. The one in the upper center position has large mitochondria and ). The other cell shows the characteristic features of apoptotic cells including enlarged endoplasmic reticulum (er elongation, contortion, and shrinking of the cell. The integrity of organelles and of plasma membrane remains intact and they present dense chromatin indicated by the small white arrow (c) aggregated against the nuclear envelope, indicated by the white arrow (n). The cytoplasm is condensed and dark. Magnification ⫻ 5,000.
tions regarding the size of the cells and their organelles as described by our group (24), but also to identify the apoptotic cells by comparing the density of the cytoplasm between cells and the difference in electron density between intact viable cell nuclei and nuclei with condensed chromatic content compatible with apoptotic nuclei. Using morphometric measurements in non-treated Hep G2 cells, the apoptosis was 0, while in cells incubated with 80 mmol/L EtOH for 2 periods of 24 h, the apoptosis was 30%. DNA
FRAGMENTATION
After 24 h of incubation with 40 mmol/L EtOH, 6.0 ⫾ 0.5% HPH were apoptotic compared with 0.1% of control as assessed by ELISA. The process of apoptosis is dose dependent: 60 mmol/L EtOH 15 ⫾ 2% (p ⬍ 0.05), 80 mmol/L EtOH 26 ⫾ 1% (p ⬍ 0.001) apoptosis (vs. control). Two consecutive doses of 80 mmol/L EtOH for 24 h each caused 55 ⫾ 3% apoptosis (p ⬍ 0.0001 vs. control and p ⬍ 0.001 vs. previous dose) (Figure 5). DNA fragmentation showed no significant differences between HPH exposed to one CLINICAL BIOCHEMISTRY, VOLUME 32, OCTOBER 1999
dose of EtOH for 24 h and Hep G2 exposed to the same dose of EtOH for the same period of time. Incubation of hepatocytes for 2 consecutive days with 80 mmol/L EtOH results in a stronger apoptotic response (p ⬍ 0.05) than the apoptotic-induced response in Hep G2 cells (40 ⫾ 2.5%). Interestingly, the cells that have been exposed to 80 mmol/L EtOH/1 dose per 24 h in presence of 70 M choline and 40 M glycine have a lower rate of apoptosis (13 ⫾ 2.5%) than the cells grown in serum free media (p ⬍ 0.05). The HPH are more sensitive to nutrients that are Hep G2. Two doses exposure to 80 mmol/L EtOH in the presence of glycine and choline reduced apoptosis in Hep G2 cells from 40 ⫾ 2.5% to 28 ⫾ 3.5% (p ⬍ 0.05) while in HPH apoptosis was reduced from 55 ⫾ 3% to 20.5 ⫾ 3.5% (p ⬍ 0.001). Discussion The mechanism whereby EtOH produces liver damage is still a matter of considerable debate. The current study was designed to test the hypothesis that chronic EtOH exposure promotes changes in hepatocytes that lead to apoptosis. Hepatocytes con551
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Figure 4 — Transmission electron micrograph of HPH cells treated with 80 mmol/L EtOH for 2 times ⫻ 24 h. A necro-apoptotic cell can be seen in the micrograph. The integrity of plasma membrane and of organelles (mitochondria M) remains intact. There are lipid droplets (LD) and enlarged endoplasmic reticulum (er). The nucleus (N) is large and has condensed, dark clumps of chromatin. Magnification ⫻ 5,000.
tain plasma membranes as well as many other membrane-bound subcellular organelles including mitochondria, lysosomes and endoplasmic reticulum which have been as targets of injurious effects of EtOH and/or its toxic metabolites (26). There is a growing body of evidence which suggests that hepatocyte apoptosis is an important process in the pathophysiology of EtOH-induced liver injury. EtOH-fed rats and mice showed an increase in the number of apototic cells, as well as alteration and distribution of apoptotic bodies within the liver (11,12,27). These previous studies were conducted by using animal models of chronic EtOH feeding. However, there are few in vitro studies, and relatively little is known about the mechanisms by which EtOH induces hepatocyte apoptosis. Higuchi et al. (28) showed that a culture of rat hepatocytes subjected to the acute EtOH exposure of 50 mmol/L for 4 h increased the number of hepatocytes with fragmented DNA. The same group (29), using 100 mmol/L EtOH instead of 50 mmol/L for the same period of time, showed a higher number of apoptotic cells. In previous reports, we ascertain that acute EtOH intoxication promotes liver cell apoptosis in vitro, in Hep G2 cells (15), and in Hep G2 cells co-cultured 552
with normal human stellate cells (16). The fact that EtOH can induce molecular and ultrastructural changes compatible with apoptosis in a relatively short period of time (24 h) in Hep G2 cells raises the possibility that EtOH per se, or its metabolites, can induce apoptosis without the non-parenchymal liver cell support, while the co-existence of a non parenchymal normal population of liver cells enhances the apoptotic process. In the present study, we have investigated the appearance of apoptosis in isolated normal HPH that were subjected to acute EtOH exposure for 24 h, as well as chronic exposure for two consecutive periods of 24 h. The novel findings of this study relate to the effect of EtOH-induced apoptosis and the differences between the two in vitro models: a normal HPH culture and a human derived hepatoblastoma cell line (Hep G2). A high dose of EtOH (80 mmol/L) but clinically relevant was used. Exposure of cells to this dose for 24 h increases the apoptosis as measured by DNA fragmentation, suggesting apoptosis which is dose dependent both in Hep G2 and in HPH (Figure 5). Significant differences in DNA fragmentation were observed between Hep G2 and HPH when the cells CLINICAL BIOCHEMISTRY, VOLUME 32, OCTOBER 1999
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Figure 5 — EtOH-induced apoptosis in normal HPH and in Hep G2 cells. Hepatocytes plated directly in 96 well trays were exposed for 24 h only to plain medium (control) or to EtOH (40, 60, 80 mmol/L). For chronic exposure the cells were treated for 24 h with 80 mmol/L EtOH washed with PBS and incubated for additional 24 h with a second dose of 80 mmol/L EtOH. For quantitative determination of DNA fragments Nucleosome kit of low molecular DNA associated with histone was used for DNA detection at the length of 180 –200 bp. Data values for each column (treatment) are expressed as means of 30 wells (6 well/plate ⫻ 5 plates) ⫾ SD. Differences between groups were analyzed using an ANOVA for repeated measurements with a Bonferroni test to correct for multiple comparison. In the first panel are presented the values for normal primary hepatocytes and in the second panel the values for Hep G2 cells.
were treated for 2 consecutive 24 h with the 80 mmol/L EtOH. ADH activity is higher in HPH (unpublished data). Therefore, the cells metabolize larger amounts of EtOH, producing higher quantities of toxic metabolites. The differences observed in apoptotic quantitation in the two systems strongly suggested that the metabolism of EtOH by ADH leads to the occurrence of more toxic metabolites and higher oxidative stress that may be responsible for this process. Reports show the direct oxidative destruction of folic acid by acetaldehyde, the toxic product of EtOH metabolism (30). EtOH-induced alterations in folate and methionine metabolism (31) decreased availability of methyl groups for DNA methylation (9) or by altered regulation of DNA precursor metabolism (32). Choline and glycine are important nutrients for cells. Dietary choline is considered to be essential for humans (33). Choline is a source of methyl groups in the diet and a major component of phospholipids. Choline deficiency induces apoptosis both in vitro and in animal models (34,35). Glycine is required for cell growth in culture (36). Phosphatidylcholine supplementation has been postulated to attenuate the consequences of EtOHCLINICAL BIOCHEMISTRY, VOLUME 32, OCTOBER 1999
induced oxidative stress in baboons (37) and to attenuate the transformation of stellate cells in culture (38). We have shown that depletion of mitochondrial glutathione plays an important role in EtOH-induced apoptosis in Hep G2 cells, in vitro. Lack of phosphocholine precursor will disable the normal function of mitochondria. It is possible that choline and glycine supplementation will enhance mitochondrial glutathione in the cells, therefore, their ability to detoxify the toxic metabolites produced by EtOH. We investigated the influence of EtOH of cytokine release and expression in Hep G2 (39) showing that EtOH up-regulates both the release of tumor necrosis factor ␣ (TNF␣) to the media and its expression at the RNA level. Bour et al. (40) also reported that TNF␣-induced apoptosis in hepatocytes in long-term culture. In our model, both the oxidative stress and the cytokines may contribute to the process by which apoptosis occurs. The differences that can be observed between the apoptosis quantified in Hep G2 cells by DNA fragmentation (40%) and by morphometry (30%) may be due to the difficulty in having all the apoptotic cells 553
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and apoptotic bodies visualized by microscopy, and, therefore, scanned and measured. The differences between the responses in EtOH may be explained either by a tolerance to a second intoxication that Hep G2 may develop, or by a lower sensitivity to oxidative stress in the Hep G2. We conclude that, derived from normal liver tissue, the HPH cultures provide a new model system for studying the regulation of cell growth differentiated functions and death in human hepatocytes. References 1. Kerr JFR. Shrinkage necrosis: A distinct mode of cellular death. J Path 1971; 105: 13–20. 2. Kerr JFR, Wyllie AH, Currie AR. Apoptosis: A basic biological phenomenon with wide ranging implications in tissue kinetics. Br J Cancer 1972; 26: 239 –57. 3. Liston P, Roy N, Tamai K, et al. Suppression of apoptosis in mammalian cells by NAIP and a related family of IAP genes. Nature 1996; 379: 349 –53. 4. Oberhammer FA, Pavelka M, Sharma S, et al. Induction of apoptosis in cultured hepatocytes and in regressing liver by transforming growth factor ␣1. Proc Natl Acad Sci USA 1992; 89: 5408 –12. 5. Oberhammer FA, Bursch W, Tiefenbacher R, et al. Apoptosis is induced by transforming growth factor-␣1 within 5 hours in regressing liver without significant fragmentation of the DNA. Hepatology 1993; 18: 1238 – 45. 6. Oberhammer FA, Pavelka M, Sharma S, et al. Hepatocytes apoptosis: Is transforming growth factor-1 the kiss of death? Hepatology 1993; 18: 1536 –7. 7. Steller H. Mechanisms and genes of cellular suicide. Science 1995; 267: 1445–9. 8. Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science 1995; 267: 1456 – 62. 9. Hoffman RM. Unbalanced transmethylation and the perturbation of the differentiated state leading to cancer. Bioassays 1990; 12:163– 6. 10. Kinzler KW, Vogelstein B. Life (and death) in malignant tumor. Nature 1996; 379:19 –20. 11. Benedetti A, Brunelli E, Risicato R, Cilluffo T, Jezequel AM, Orlandi F. Subcellular changes and apoptosis induced by ethanol in rat liver. J Hepatol 1988; 6: 137– 43. 12. Goldin RD, Hunt NC, Clarck J, Wickramasinghe SN. Apoptotic bodies in a murine model of alcoholic liver disease: Reversibility of ethanol-induced changes. J Pathol 1993; 171: 173– 6. 13. Neuman MG, Koren G, Tiribelli C. In vitro, assessment of the ethanol-induced hepatotoxicity on Hep G2 cell line. Biochem Biophys Res Commun 1993; 197: 931– 42. 14. Neuman MG, Cameron RG, Shear NH, Bellentani S, Tiribelli C. Effect of Tauroursodeoxycholate and Ursodeoxycholic acid on ethanol-induced cell injuries in the human Hep G2 cell line. Gastroenterology 1995; 109: 555– 63. 15. Neuman MG, Cameron RG, Shear NH, Tiribelli C. Molecular pathological and electron microscopic changes in ethanol-induced apoptotic cells, in vitro. Hepatology 1996; 30: 125. 16. Neuman MG, Cameron RG, Shear NH, Casini A, Tiribelli C. Ethanol-induced apoptosis in a co-culture of Hep G2 and normal human stellate cells (abstr. 35). Cell Biol Toxicol 1996; 20: 55. 554
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membrane phosphatidylcholine and sphingomyelin accumulation of ceramide and diacylglycerol and activation of a caspase. FASEB J 1999; 15: 135– 42. 35. Holmes-McNary MQ, Loy R, Mar M.-H., Albright GD, Ziesel SH. Apoptosis is induced by choline deficiency in fetal brain and in PG12 cells. Dev Brain Res 1997; 101: 9 –16. 36. Simpson SH, Singh RP, Perani A, Goldenzon C, AlRubeai M. In hybridoma cultures, deprivation of any single amino acid leads to apoptotic death, which is suppressed by expression of the bcl-2 gene. Biotechnol Bioeng 1998; 59(1): 90 – 8.
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PII S0009-9120(99)00054-5
Ethanol-Induced Apoptosis In Vitro MANUELA G. NEUMAN,1 NEIL H. SHEAR,1 ROSS G. CAMERON,2 GADY KATZ,1 and CLAUDIO TIRIBELLI3 1
Division of Clinical Pharmacology, Sunnybrook and Women’s College Health Sciences Centre, Toronto, Ontario, Canada, 2Department of Pathology, Toronto Hospital, Departments of Pharmacology, Pathology and Medicine, University of Toronto, Toronto, Ontario, Canada, and 3 Centro Studi Fegato Modena-Trieste, Department of B. B. C. M., University of Trieste, Trieste, Italy Objectives: The aim is to study the apoptotic process in a human hepatocyte model for ethanol (EtOH)-induced apoptosis. Design and methods: Normal human primary hepatocytes (HPH) and Hep G2 cells were exposed to increasing EtOH. 6000 cells/ sample were analyzed by transmission electron microscopy. Results: Apoptotic cells were observed (mmol/L EtOH): 40: 6 ⫾ 0.5%, 60: 13 ⫾ 2% (p ⬍ 0.05), 80: 26 ⫾ 1% (p ⬍ 0.001) (vs. control). Two consecutive doses of 80 mmol/L for 24 h each additionally increased apoptosis 55 ⫾ 3% (p ⬍ 0.0001 vs. control and p ⬍ 0.001 vs. single dose). In response to this exposure, there is a stronger apoptotic activity in HPH when compared to Hep G2 (p ⬍ 0.05). Conclusions: In vitro, EtOH-induced apoptosis is regulated by dose level and the frequency of exposure. Copyright © 1999 The Canadian Society of Clinical Chemists
KEY WORDS: apoptosis; ethanol; Hep G2; normal human primary hepatocytes; nutritional interventions.
Introduction egulation of organ size and liver function is the result of a dynamic balance between stimulatory and inhibitory signals (1– 8). Under physiological conditions, senescent or damaged cells are removed by apoptosis, whereas the response to acute cell injury mostly involves a necrotic process (9,10). After EtOH ingestion, rat and mice models showed subcellular changes compatible with apoptosis (11, 12). We have been able to reproduce a model of EtOHinduced liver damage in human Hep G2 cells in vitro (13). Biochemical and morphological features in our model present the same pattern observed in the serum of patients with alcohol-induced liver damage and human biopsies from the same category of patients (14). Previously, we reported that in Hep G2 cells (15) and Hep G2 in co-culture with normal
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Correspondence: M. G. Neuman, Ph.D., Division Clinical Pharmacology, E-240, Sunnybrook HSC, 2075 Bayview Avenue, Toronto, Ontario, M4N 3M5, Canada. Email: [email protected]. Manuscript received June 29, 1999; accepted June 29, 1999 CLINICAL BIOCHEMISTRY, VOLUME 32, OCTOBER 1999
human stellate cells, apoptosis can be detected and measured when exposed to 80 mmol/L EtOH (16). Hep G2 cells have inducible cytochrome P450 2E1 activity but low content of CYP 2E1 (17) and a small amount of alcohol dehydrogenase activity (ADH) (18). CYP 2E1 and ADH are associated with EtOHinduced liver damage and are thought to be critical in the hepatocellular regulation of apoptotic processes. When comparing the inducibility of cytochrome P450 2E1 and ADH activity in Hep G2 cells versus HPH treated with 80 mmol/L EtOH a significant difference was observed (17,18). Therefore, we decided to parallel our observations in Hep G2 line with studies in HPH. Cells in these HPH cultures retain morphological features of hepatocytes by both light and electron microscopy. They also retain glucose-6-phosphatase activity and secrete proteins characteristic of hepatocytes, such as albumin, alpha-fetoprotein and transferrin (19). This work deals with the differences and similarities in an apoptotic-induced cascade by EtOH in HPH in comparison with the Hep G2 cells. Detection of cell death by apoptosis, expressed by normal hepatocytes that retain liver-specific functions of differentiated hepatocytes, can be considered a new application of studying human hepatocyte reactions under EtOH stress. Methods PRIMARY
HUMAN HEPATOCYTES CULTURE
The hepatocyte cultures were developed from normal liver tissue obtained after lobectomy from organ donors where only a part of the liver was used for transplantation, by cultivating in a highly enriched medium. Our method for preparing hepatocytes suspension was based originally on that of Ballet et al. (20). The liver graft was perfused with ice-cold University of Wisconsin solution (21) and kept on ice (2–10 h) until cell isolation. The material was washed with HEPES media. The hepatocytes were 547
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isolated using the two step perfusion technique. The average yield was 1.5 billion cells/preparation and the average viability, assessed by trypan blue exclusion test was 94%. Hepatocytes were washed three times in a culture medium (1/1 Ham F12 and Leibovitz L-15 supplemented 5 mmol/L glucose, 50 U/mL penicillin, 50 g/L streptomycin, 10⫺8 mol/L insulin). Cells were then plated 1 ⫻ 106/ml in collagen coated Falcon flasks (3 g /cm2) and cultured with conventional conditions in medium supplemented with fetal bovine serum (FBS). One hour later, the medium was changed to remove the floating, unattached hepatocytes. The medium was replaced after 12 h. Every 24 h thereafter, the media was changed with a fresh one in the presence of FBS. Non-hepatocellular cells were not present. Cells were treated with media in presence or in absence of EtOH as described in the experimental design. The dilutions were done in media in absence of FBS. We have been able to maintain the cells in culture continuously for 10 days exhibiting typical morphological characteristics of hepatocytes. To address the role of nutritional factors acting as priming cells for apoptosis, in some experiments the serum free media was supplemented with choline or glycine. For enzyme-linked immunosorbent assay (ELISA), cells were plated directly in 96 well plates not coated with collagen. HEP G2
CELLS LINE
Hep G2 cells were obtained from Wistar Institute (Philadelphia, PA, USA). Cells were seeded in flasks (1 ⫻ 106 cells/mL) (13). The cell counts were monitored using a Coulter counter (Coulter Electronics Inc., Hialeah, FL, USA). Cells in long-term cultures were grown in ␣-MEM supplemented with 10% v/v heat inactivated FBS. At the beginning of the experiment, when cells reached 70% confluence, the growth medium was removed from the culture flasks, the cultures were washed twice with phosphate buffered saline (PBS) and fresh serum free medium was used as base for all the treatments. The viability of the cells not treated with EtOH was not altered by culturing them for up to 6 days serum free medium (13). DRUGS
AND CHEMICALS
EtOH was purchased from Alcohol Ltd. (Toronto, Ontario, Canada). Plain ␣-MEM, Hanks balanced sodium solution and calcium chloride were obtained from Gibco (Burlington, Ontario, Canada). Trypsin was purchased from Difco (Detroit, MI, USA) and was prepared as a 1% solution. PBS (phosphate buffered saline without Ca2⫹ or Mg2⫹ ) was used to wash cells and to remove medium. All plastic ware for cell cultures was obtained from Falcon (Becton Dickinson, Oxnard, CA, USA). All of the remaining reagents were of analytical grade, obtained from Sigma Chemical Co. (St. Louis, MO, USA). For quantitative determination of DNA frag548
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ments, we used the Nucleosome ELISA kit from Oncogene Research Products CN Biosciences (Cambridge, MA, USA). The Nucleosome kit of low molecular DNA associated with histone is considered to be a sensitive tool for DNA detection at the length of 180 –200 bp. The ELISA applies a Digoxigenin detector antibody and a Streptavidin conjugate with horseradish peroxidase (SA-HRP) Sheep antidigoxigenin antibody to quantitate the apoptotic nucleosomes. EXPERIMENTAL
DESIGN
Control cells were exposed for 24 h only to plain essential medium (␣ MEM), while the treated cells were incubated with EtOH (40, 60, 80 mmol/L). For chronic exposure, the cells were treated for 24 h with 80 mmol/L EtOH washed with PBS pH 7.4, and incubated for additional 24 h with a second dose of 80 mmol/L EtOH. Some cells were exposed to one dose or two doses of 80 mmol/L EtOH in the presence of choline 70 mol/L and 40 mol/L glycine. All components were filtered-sterilised, and the entire procedure was conducted under aseptic conditions. The cells were routinely maintained in a humidified incubator in 95% air/5% CO2, at 37° C. Further details of the individual experiments are provided in the figure legends. After a period of 18 –20 h storage at ⫺20°C, the cells underwent lysis and were exposed to the detector antibody for 1 h at room temperature. The cells were washed and incubated for another 30 min with SA-HRP conjugate. The chromogene reaction was stopped and the absorbance was read using a spectrophotometer plate reader with dual lengths 450/ 595 nm. The intensity of the yellow color is proportional to the number of nucleosomes in the sample. For each treatment, 6 wells/plate in 5 different plates were quantitated. The results are reported as % of apoptosis vs. control, non-treated taken as 0% apoptosis. For the standard curve, we ran replicates of six in each plate using two different plates. The sensitivity of the assay was measured by assaying the non-treated cells at time zero. The mean signal and the standard deviation were calculated. The assay can distinguish 0.3% from zero. The Maxline Microplate Reader, from Molecular Device Corp. (Menlo Park, CA, USA) was connected to a computer using SOFT MAX software 2.3 for Windows (Molecular Devices Corp.) permitting to template the plate according to the experimental needs and perform the statistical analysis directly on the template format. STATISTICAL
ANALYSIS
All data are expressed as means ⫾ standard deviation (SD). Differences between groups were analyzed using an ANOVA test for repeated measurements with a Bonferroni test to correct for multiple comparison. All statistical analyses were CLINICAL BIOCHEMISTRY, VOLUME 32, OCTOBER 1999
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Figure 1 — Transmission electron micrograph (TEM) of Hep G2 cells (control, non-treated). Cells were plated 75 Falcon flasks in a density of 106 cells/mL and were grown in media supplemented with FBS. At 70% confluence, cells were incubated in plain media only, for 24 h. After removing the media the cells were prepared for TEM as described in Material ), numerous normal and Methods, The EM shows normal looking cells with normal central located nucleus (N mitochondria ( m). One of the cell displays vesiculation of the cisternae of smooth endoplasmic reticulum ( er). Several small lipid droplets are also present ( ld). Tight junctions ( tj) between two adjacent cells can be observed. Magnification ⫻ 5,000.
performed with the statistical software package Microcal Origin 301, Microcal Inc. LIGHT
AND ELECTRON MICROSCOPY
The cells were prepared for light and transmission electron microscopy (TEM) studies using a standard procedure as outlined below. For each group of control (0 EtOH/24 or 48 h) and treated cells (80 mmol/L for 24 h, or 2 doses of 80 mmol/L/ 2 consecutive periods of 24 h), six flasks of either HPH or Hep G2 were used. After the period of incubation, the media was removed and cells were washed twice with PBS. Five mL of 1% trypsin was added for 2 min in each flask. Cells were washed again with PBS and then resuspended in plain media. Cell suspensions were centrifuged at 50 g for 10 min. Pellets were immediately fixed in 2.5% v/v glutaraldehyde for a minimum of 24 h. Blocks of cells were separated, post-fixed in 1% v/v osmium tetroxide, dehydrated with graded series of acetone concentraCLINICAL BIOCHEMISTRY, VOLUME 32, OCTOBER 1999
tions and embedded in Araldine. Sections (1 micron thick) were viewed by light microscopy (LM). For light microscopy studies an Olympus microscope equipped with Leco 2005, Image Processing and Analysis System (Leco Instruments, Toronto, Ontario, Canada) with Microsoft威 Visual Basic™ software were used. Cells were considered apoptotic if the classic features of pyknotic nuclei, cytoplasmic condensation and nuclear chromatin fragmentation could be observed. Representative blocks were selected, subjected to ultra-thin sectioning and stained with uranyl acetate and lead citrate for TEM. Electron micrographs were taken with a transmission electron microscope JOEL 1200 E ⫻ II (JOEL Institute Inc., Lexington, MA, USA). Ultrastructural findings were examined in five different grids/flask in each experiment. On each grid, 200 – 400 cells were examined. An average of 9000 (300 cells/grid ⫻ grids/flask ⫻ 6 flasks/treatment) cells were analyzed for each treatment. The 549
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Figure 2 — Transmission electron micrograph of Hep G2 cells exposed to 80 mmol/L EtOH for 24 h. Cells were plated in 75 Falcon flasks in a density of 106 cells/mL and were grown in media supplemented with FBS. At 70% confluence, cells were incubated in media containing 80 mmol/L ethanol. In the middle of the micrograph a is a large size cell. The nucleus (N) is normal looking and presents a normal nucleolus ( n). The cell shows considerable enlargement of mitochondria M). The cisternae of endoplasmic reticulum show vesiculation ( ( er), and dilatation ( er ). A part of the cell forms a bleb (B), which separation from the cell can be seen. In the opposite side of the cell, in the upper corner, an apoptotic body (ab ) is present. In the lower corner, an apoptotic cell in the first stage presenting a piknotic nucleus PN. The cell is not anymore linked to the others. Between the cells debris (d ) can be observed. In the upper corner a classic elongated apoptotic cell (A ) has dark condensed cytoplasm. Magnification ⫻ 5,000.
criteria for the morphological identification of cellular structures were standard (22). In assessing cells by electron microscopy, the classic shrinkage of cells, electron dark cytoplasm, and apoptotic bodies are the criteria for classical apoptosis (23). Results LIGHT
AND ELECTRON MICROSCOPY
Cells incubated with plain media show normal organelles: abundant mitochondria, rough and smooth endoplasmic reticulum and only occasional small lipid vesicles (Figure 1). Bile canaliculus-like structure can be observed at the confluence of 3 cells suggesting that the cells are functional. In previous publications (13,24), we reported that exposure to 80 mmol/L EtOH produces changes in mitochondria, endoplasmic reticulum, and in the number of lipid vesicles per cell, as well as changes in lipid content of the fat droplets (25). Figure 2 shows a large cell with a normal looking nucleus with pleomorphism of mitochondria, vesiculation, and dilatation of endoplasmic reticulum. In one part of the cells, a characteristic “bleb” can be observed. This work also de550
scribes ultrastructural changes compatible with apoptosis, e.g., some cells having intact plasma membrane and intact nuclei membrane, but being retracted from the surrounding cells (Figure 3). These cells no longer have tight junctions that can be observed between two adjacent cells in the control cultures not treated with EtOH. Some of the cells are elongated and present intact organelles (Figure 4). The cytoplasm is darker and the nucleus presents condensation of chromatin, which can be located adjacent to the nuclear membrane. Some cells are fragmented and the nuclear fragmentation occurs concomitantly. Therefore, multiple vesicles with compacted chromatin and intact cell (apoptotic bodies) can be seen. In HPH treated with 2 consecutive doses of 80 mmol/L EtOH for 24 h, a large cell presenting intact organelles, but showing changes in chromatin distribution and enlargement of endoplasmic reticulum, may undergo apoptosis (Figure 5). MORPHOMETRIC
ANALYSIS
The software we used to analyze the LM material permitted us not only to make quantitative observaCLINICAL BIOCHEMISTRY, VOLUME 32, OCTOBER 1999
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Figure 3 — Transmission electron micrograph of Hep G2 cells treated with 80 mmol/L EtOH for 2 times ⫻ 24 h. When 70% confluent, cells were washed with PBS and media containing 80 mmol/L EtOH was added (0 time). After a period of 24-h incubation, followed by aspiration of the media, a second equimolar dose of EtOH was added for a second period of 24 h. The cells formed a monolayer that has not been disrupted by the trypsinization. There is a mitochondrial pleomorphism with normal size mitochondria. Two cells with normal looking nucleus (N) can be seen in the micrograph. There are two apoptotic cells ( A) containing preserved organelles. The one in the upper center position has large mitochondria and ). The other cell shows the characteristic features of apoptotic cells including enlarged endoplasmic reticulum (er elongation, contortion, and shrinking of the cell. The integrity of organelles and of plasma membrane remains intact and they present dense chromatin indicated by the small white arrow (c) aggregated against the nuclear envelope, indicated by the white arrow (n). The cytoplasm is condensed and dark. Magnification ⫻ 5,000.
tions regarding the size of the cells and their organelles as described by our group (24), but also to identify the apoptotic cells by comparing the density of the cytoplasm between cells and the difference in electron density between intact viable cell nuclei and nuclei with condensed chromatic content compatible with apoptotic nuclei. Using morphometric measurements in non-treated Hep G2 cells, the apoptosis was 0, while in cells incubated with 80 mmol/L EtOH for 2 periods of 24 h, the apoptosis was 30%. DNA
FRAGMENTATION
After 24 h of incubation with 40 mmol/L EtOH, 6.0 ⫾ 0.5% HPH were apoptotic compared with 0.1% of control as assessed by ELISA. The process of apoptosis is dose dependent: 60 mmol/L EtOH 15 ⫾ 2% (p ⬍ 0.05), 80 mmol/L EtOH 26 ⫾ 1% (p ⬍ 0.001) apoptosis (vs. control). Two consecutive doses of 80 mmol/L EtOH for 24 h each caused 55 ⫾ 3% apoptosis (p ⬍ 0.0001 vs. control and p ⬍ 0.001 vs. previous dose) (Figure 5). DNA fragmentation showed no significant differences between HPH exposed to one CLINICAL BIOCHEMISTRY, VOLUME 32, OCTOBER 1999
dose of EtOH for 24 h and Hep G2 exposed to the same dose of EtOH for the same period of time. Incubation of hepatocytes for 2 consecutive days with 80 mmol/L EtOH results in a stronger apoptotic response (p ⬍ 0.05) than the apoptotic-induced response in Hep G2 cells (40 ⫾ 2.5%). Interestingly, the cells that have been exposed to 80 mmol/L EtOH/1 dose per 24 h in presence of 70 M choline and 40 M glycine have a lower rate of apoptosis (13 ⫾ 2.5%) than the cells grown in serum free media (p ⬍ 0.05). The HPH are more sensitive to nutrients that are Hep G2. Two doses exposure to 80 mmol/L EtOH in the presence of glycine and choline reduced apoptosis in Hep G2 cells from 40 ⫾ 2.5% to 28 ⫾ 3.5% (p ⬍ 0.05) while in HPH apoptosis was reduced from 55 ⫾ 3% to 20.5 ⫾ 3.5% (p ⬍ 0.001). Discussion The mechanism whereby EtOH produces liver damage is still a matter of considerable debate. The current study was designed to test the hypothesis that chronic EtOH exposure promotes changes in hepatocytes that lead to apoptosis. Hepatocytes con551
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Figure 4 — Transmission electron micrograph of HPH cells treated with 80 mmol/L EtOH for 2 times ⫻ 24 h. A necro-apoptotic cell can be seen in the micrograph. The integrity of plasma membrane and of organelles (mitochondria M) remains intact. There are lipid droplets (LD) and enlarged endoplasmic reticulum (er). The nucleus (N) is large and has condensed, dark clumps of chromatin. Magnification ⫻ 5,000.
tain plasma membranes as well as many other membrane-bound subcellular organelles including mitochondria, lysosomes and endoplasmic reticulum which have been as targets of injurious effects of EtOH and/or its toxic metabolites (26). There is a growing body of evidence which suggests that hepatocyte apoptosis is an important process in the pathophysiology of EtOH-induced liver injury. EtOH-fed rats and mice showed an increase in the number of apototic cells, as well as alteration and distribution of apoptotic bodies within the liver (11,12,27). These previous studies were conducted by using animal models of chronic EtOH feeding. However, there are few in vitro studies, and relatively little is known about the mechanisms by which EtOH induces hepatocyte apoptosis. Higuchi et al. (28) showed that a culture of rat hepatocytes subjected to the acute EtOH exposure of 50 mmol/L for 4 h increased the number of hepatocytes with fragmented DNA. The same group (29), using 100 mmol/L EtOH instead of 50 mmol/L for the same period of time, showed a higher number of apoptotic cells. In previous reports, we ascertain that acute EtOH intoxication promotes liver cell apoptosis in vitro, in Hep G2 cells (15), and in Hep G2 cells co-cultured 552
with normal human stellate cells (16). The fact that EtOH can induce molecular and ultrastructural changes compatible with apoptosis in a relatively short period of time (24 h) in Hep G2 cells raises the possibility that EtOH per se, or its metabolites, can induce apoptosis without the non-parenchymal liver cell support, while the co-existence of a non parenchymal normal population of liver cells enhances the apoptotic process. In the present study, we have investigated the appearance of apoptosis in isolated normal HPH that were subjected to acute EtOH exposure for 24 h, as well as chronic exposure for two consecutive periods of 24 h. The novel findings of this study relate to the effect of EtOH-induced apoptosis and the differences between the two in vitro models: a normal HPH culture and a human derived hepatoblastoma cell line (Hep G2). A high dose of EtOH (80 mmol/L) but clinically relevant was used. Exposure of cells to this dose for 24 h increases the apoptosis as measured by DNA fragmentation, suggesting apoptosis which is dose dependent both in Hep G2 and in HPH (Figure 5). Significant differences in DNA fragmentation were observed between Hep G2 and HPH when the cells CLINICAL BIOCHEMISTRY, VOLUME 32, OCTOBER 1999
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Figure 5 — EtOH-induced apoptosis in normal HPH and in Hep G2 cells. Hepatocytes plated directly in 96 well trays were exposed for 24 h only to plain medium (control) or to EtOH (40, 60, 80 mmol/L). For chronic exposure the cells were treated for 24 h with 80 mmol/L EtOH washed with PBS and incubated for additional 24 h with a second dose of 80 mmol/L EtOH. For quantitative determination of DNA fragments Nucleosome kit of low molecular DNA associated with histone was used for DNA detection at the length of 180 –200 bp. Data values for each column (treatment) are expressed as means of 30 wells (6 well/plate ⫻ 5 plates) ⫾ SD. Differences between groups were analyzed using an ANOVA for repeated measurements with a Bonferroni test to correct for multiple comparison. In the first panel are presented the values for normal primary hepatocytes and in the second panel the values for Hep G2 cells.
were treated for 2 consecutive 24 h with the 80 mmol/L EtOH. ADH activity is higher in HPH (unpublished data). Therefore, the cells metabolize larger amounts of EtOH, producing higher quantities of toxic metabolites. The differences observed in apoptotic quantitation in the two systems strongly suggested that the metabolism of EtOH by ADH leads to the occurrence of more toxic metabolites and higher oxidative stress that may be responsible for this process. Reports show the direct oxidative destruction of folic acid by acetaldehyde, the toxic product of EtOH metabolism (30). EtOH-induced alterations in folate and methionine metabolism (31) decreased availability of methyl groups for DNA methylation (9) or by altered regulation of DNA precursor metabolism (32). Choline and glycine are important nutrients for cells. Dietary choline is considered to be essential for humans (33). Choline is a source of methyl groups in the diet and a major component of phospholipids. Choline deficiency induces apoptosis both in vitro and in animal models (34,35). Glycine is required for cell growth in culture (36). Phosphatidylcholine supplementation has been postulated to attenuate the consequences of EtOHCLINICAL BIOCHEMISTRY, VOLUME 32, OCTOBER 1999
induced oxidative stress in baboons (37) and to attenuate the transformation of stellate cells in culture (38). We have shown that depletion of mitochondrial glutathione plays an important role in EtOH-induced apoptosis in Hep G2 cells, in vitro. Lack of phosphocholine precursor will disable the normal function of mitochondria. It is possible that choline and glycine supplementation will enhance mitochondrial glutathione in the cells, therefore, their ability to detoxify the toxic metabolites produced by EtOH. We investigated the influence of EtOH of cytokine release and expression in Hep G2 (39) showing that EtOH up-regulates both the release of tumor necrosis factor ␣ (TNF␣) to the media and its expression at the RNA level. Bour et al. (40) also reported that TNF␣-induced apoptosis in hepatocytes in long-term culture. In our model, both the oxidative stress and the cytokines may contribute to the process by which apoptosis occurs. The differences that can be observed between the apoptosis quantified in Hep G2 cells by DNA fragmentation (40%) and by morphometry (30%) may be due to the difficulty in having all the apoptotic cells 553
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and apoptotic bodies visualized by microscopy, and, therefore, scanned and measured. The differences between the responses in EtOH may be explained either by a tolerance to a second intoxication that Hep G2 may develop, or by a lower sensitivity to oxidative stress in the Hep G2. We conclude that, derived from normal liver tissue, the HPH cultures provide a new model system for studying the regulation of cell growth differentiated functions and death in human hepatocytes. References 1. Kerr JFR. Shrinkage necrosis: A distinct mode of cellular death. J Path 1971; 105: 13–20. 2. Kerr JFR, Wyllie AH, Currie AR. Apoptosis: A basic biological phenomenon with wide ranging implications in tissue kinetics. Br J Cancer 1972; 26: 239 –57. 3. Liston P, Roy N, Tamai K, et al. Suppression of apoptosis in mammalian cells by NAIP and a related family of IAP genes. Nature 1996; 379: 349 –53. 4. Oberhammer FA, Pavelka M, Sharma S, et al. Induction of apoptosis in cultured hepatocytes and in regressing liver by transforming growth factor ␣1. Proc Natl Acad Sci USA 1992; 89: 5408 –12. 5. Oberhammer FA, Bursch W, Tiefenbacher R, et al. Apoptosis is induced by transforming growth factor-␣1 within 5 hours in regressing liver without significant fragmentation of the DNA. Hepatology 1993; 18: 1238 – 45. 6. Oberhammer FA, Pavelka M, Sharma S, et al. Hepatocytes apoptosis: Is transforming growth factor-1 the kiss of death? Hepatology 1993; 18: 1536 –7. 7. Steller H. Mechanisms and genes of cellular suicide. Science 1995; 267: 1445–9. 8. Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science 1995; 267: 1456 – 62. 9. Hoffman RM. Unbalanced transmethylation and the perturbation of the differentiated state leading to cancer. Bioassays 1990; 12:163– 6. 10. Kinzler KW, Vogelstein B. Life (and death) in malignant tumor. Nature 1996; 379:19 –20. 11. Benedetti A, Brunelli E, Risicato R, Cilluffo T, Jezequel AM, Orlandi F. Subcellular changes and apoptosis induced by ethanol in rat liver. J Hepatol 1988; 6: 137– 43. 12. Goldin RD, Hunt NC, Clarck J, Wickramasinghe SN. Apoptotic bodies in a murine model of alcoholic liver disease: Reversibility of ethanol-induced changes. J Pathol 1993; 171: 173– 6. 13. Neuman MG, Koren G, Tiribelli C. In vitro, assessment of the ethanol-induced hepatotoxicity on Hep G2 cell line. Biochem Biophys Res Commun 1993; 197: 931– 42. 14. Neuman MG, Cameron RG, Shear NH, Bellentani S, Tiribelli C. Effect of Tauroursodeoxycholate and Ursodeoxycholic acid on ethanol-induced cell injuries in the human Hep G2 cell line. Gastroenterology 1995; 109: 555– 63. 15. Neuman MG, Cameron RG, Shear NH, Tiribelli C. Molecular pathological and electron microscopic changes in ethanol-induced apoptotic cells, in vitro. Hepatology 1996; 30: 125. 16. Neuman MG, Cameron RG, Shear NH, Casini A, Tiribelli C. Ethanol-induced apoptosis in a co-culture of Hep G2 and normal human stellate cells (abstr. 35). Cell Biol Toxicol 1996; 20: 55. 554
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membrane phosphatidylcholine and sphingomyelin accumulation of ceramide and diacylglycerol and activation of a caspase. FASEB J 1999; 15: 135– 42. 35. Holmes-McNary MQ, Loy R, Mar M.-H., Albright GD, Ziesel SH. Apoptosis is induced by choline deficiency in fetal brain and in PG12 cells. Dev Brain Res 1997; 101: 9 –16. 36. Simpson SH, Singh RP, Perani A, Goldenzon C, AlRubeai M. In hybridoma cultures, deprivation of any single amino acid leads to apoptotic death, which is suppressed by expression of the bcl-2 gene. Biotechnol Bioeng 1998; 59(1): 90 – 8.
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37. Lieber CS. Prevention and treatment of liver fibrosis based on pathogenisis. Alcohol Clin Exp Res 1999; 23: 944 –9. 38. Poniachik J, Baraona E, Zhao J, Lieber CS. Dilinoleoylphosphatidylcholine decrease stellate cell activation. J Lab Clin Med 1999; 133: 342– 8. 39. Neuman MG, Shear NH, Bellentani S, Tiribelli C. Role of cytokines in ethanol-induced hepatocytotoxicity in Hep G2 cells. Gastroenterology 1998; 114: 157– 69. 40. Bour ES, Ward LK, Cornman GA, Isom HC. Tumor necrosis factor-␣- induced apoptosis in hepatocytes in long-term culture. Am J Pathol 1996; 148: 485–95.
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