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Synergetic signaling for apoptosis in vitro by ethanol and acetaminophen
Alcohol 27 (2002) 89–98
Synergetic signaling for apoptosis in vitro by ethanol and acetaminophen Manuela G. Neumana,b,* a
In Vitro Toxicology Laboratory, Division of Clinical Pharmacology, Sunnybrook and Women’s College Health Sciences Centre, Room E235, 2075 Bayview Ave., Toronto, Ontario, Canada, M4N 3M5 b Department of Pharmacology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada, M5S 1A8 Received 31 July 2001; received in revised form 27 January 2002; accepted 1 February 2002
Abstract In vitro, ethanol in combination with acetaminophen induces hepatocyte apoptosis resembling immune-mediated fulminant hepatic failure in human beings. Intracellular pathways originating at the mitochondria are linked to apoptosis. I studied ethanol-induced apoptosis and hepatocytotoxicity after using an in vitro model of normal human primary hepatocytes that were exposed to 5 or 10 mM acetaminophen, 40 or 100 mM ethanol, 40 mM ethanol 5 mM acetaminophen, or 40 mM ethanol 10 mM acetaminophen, or nonexposed (control; plain medium). Transmission electron microscopy was performed at different time points after exposure to the various treatments. Apoptosis, as assessed by transmission electron microscopy, was increased in a time-dependent manner after exposure to ethanol acetaminophen. In the ethanol acetaminophen model, mitochondrial injury was associated with apoptosis of hepatocytes. Ultrastructural damage and induction of apoptosis were seen in response to N-acetyl-benzoquinone-imine plus ethanol, supporting the suggestion that the damage was due to the active metabolite of acetaminophen. The modulation of mitochondrial damage in vitro may have implications for the development of new therapeutic strategies to prevent apoptosis. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Alcohol; Acetaminophen; In vitro; Cytotoxicity; Ethanol; Normal human primary hepatocytes; Hep G2 cells
1. Introduction Although reactive oxygen species may produce a variety of liver lesions, only a few of these lesions can actually lead to apoptosis (Zhao et al., 1997). Many drugs are transformed by the cytochrome P450 enzymatic system into reactive metabolites. The xenobiotics may reach the liver by means of the systemic circulation. First, endothelial cells of the hepatic sinusoids, which possess fenestrae, allow different proteins to come into direct contact with hepatocytes and enable the liver either to eliminate the toxic metabolites or to catalyze them further. Microsomal and cytosolic epoxide hydrolases are known to hydrate reactive epoxides into diols, whereas glutathione-S-transferases catalyze the conjugation of electrophilic metabolites with glutathione (GSH). Drugs may be transformed into free radicals (Jacobson, 1996). Decreases in intracellular GSH have been shown to be an early event in apoptosis (Zamzami et al., 1995). The functional and causative role of GSH depletion in the induction and execution of apoptosis has been elucidated (Zamzami et al., 1996). Cells can be rescued from apoptosis when extrusion of GSH is inhibited (Neuman et al., 1999b; Zamzami et al., 1998). The same effect is achieved when inhibitors of * Corresponding author. Tel.: 1-416-480-6100, ext. 3503; fax: 1416-480-6025. E-mail address: [email protected] (M.G. Neuman). Editor: T.R. Jerrells
macromolecular synthesis shunt cysteine from protein to GSH synthesis (Banki et al., 1996). Glutathione works as a redox sensor and regulates two important factors in apoptosis: mitochondrial function and sphingomyelinase activity (Kroemer et al., 1997; Liu et al., 1998). Mitochondria are the source of massive reactive oxygen species production and apoptogenic molecule release [e.g., cytochrome c (Bernardi, 1996) and apoptosis-inducing factor (AIF), a protease involved in the activation of caspases]. Chronic alcohol consumption is associated with the formation of reactive oxygen species and the presence of oxidative stress in the liver (Lettéron et al., 1993; Lieber, 1983; Mansouri et al., 1997; Neuman et al., 1993). Ethanol is known to induce the cytochrome P450 isoform CYP2E1, resulting in the production of free radicals (Ingelman-Sundberg et al., 1993; Lieber, 1994; Neuman et al., 1995). Fas ligand messenger RNA has been detected by in situ hybridization in the hepatocytes of patients with alcoholic liver damage (Galle et al., 1995). At the same time, Fas was overexpressed in some hepatocytes. Conditions, which increase reactive oxygen intermediates, may thus induce Fas ligand expression by hepatocytes (Müller et al., 1997; Neuman et al., 1999b). At the same time, the increased formation of reactive oxygen intermediates might damage DNA, cause overexpression of p53, and increase Fas expression by hepatocytes (Cameron & Neuman, 1999; Hug et al., 1997; Strand et al., 1998). The Fas ligand of a first hepatocyte may
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then interact with the Fas receptor on another hepatocyte, causing fratricidal killing. In normal human primary hepatocytes (NHPH), ethanol signaling for apoptosis is initiated by Fas ligand (Neuman, 2001; Pastorino & Hoek, 2000; Shear et al., 1995). Treatment of hepatocytes with ethanol causes oxidative stress, leading to tumor necrosis factor-alpha (TNF-) expression (Neuman et al., 1998; Pastorino & Hoek, 2000). My colleagues and I have shown that treatment of Hep G2 cells with ethanol at low concentrations (40 mM) does not cause toxicity. However, addition of 80 mM ethanol can produce toxic effects, such as changes in mitochondria and endoplasmic reticulum (ER) as well as accumulation of abundant lipid vesicles (Shear et al., 1995, 1999). Repeated exposure to ethanol causes apoptosis (Neuman et al., 1999b). Toxicity is increased when ethanol is combined with other drugs, such as acetaminophen (Shear et al., 1995) or methotrexate (Neuman et al., 1999a). Acetaminophen is a widely used analgesic and antipyretic with very few side effects at its usual therapeutic doses (Chiu & Bhakthan, 1978; Spooner & Harvey, 1976). However, when taken at high doses (Mitchell et al., 1973) or in combination with other drugs such as alcohol, acetaminophen is known to cause hepatic necrosis (Seeff et al., 1986) and, in some cases, liver failure that may necessitate liver transplantation or lead to death (Zimmerman & Maddrey, 1995). Primarily, glucuronidation and sulphation processes detoxify acetaminophen (Ballet et al., 1984; Manautou et al., 1996). Selective protein arylation may contribute to toxicity (Emeigh Hart et al., 1996; Manautou et al., 1996; Tarloff et al., 1996). N-acetyl-benzoquinone-imine, the toxic metabolite of acetaminophen, is mainly generated by activation of CYP2E1 and acts as oxidizing agent of GSH, reducing it to its oxidized form (GSSG), which leads to hepatocytotoxicity. In previous work, my colleagues and I have shown that apoptotic processes, characterized by disruption of the cytoskeleton, blebbing of the cells, and nuclear chromatin condensation, appear in Hep G2 cells and skin cells treated with acetaminophen in the presence of low doses of ethanol (Neuman et al., 1999a). The present study was designed to evaluate the effect of acetaminophen on NHPH in the presence or absence of ethanol and to examine the way that this drug combination signals for apoptosis. 2. Materials and methods 2.1. Drugs and chemicals Ethanol (96% purity) was purchased from Alcohol Ltd. (Toronto, Ontario, Canada). Plain -minimal essential medium (-MEM) and Hanks’ balanced salt 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. Acetaminophen was obtained from Sigma Chemical Company (St.
Louis, MO, USA). Tetrazolium salt: MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diophenyltetrazolium bromide] that was used for the cytoviability MTT assay, and hematoxylineosin stain that was used for light microscopy, were purchased from Sigma Chemical Company (St. Louis, MO, USA). Dimethylsulfoxide (DMSO) was obtained from Fisher Chemical (Fisher Scientific, Toronto, Ontario, Canada). Phosphate-buffered saline (PBS) (without Ca or Mg) was used to wash cells and to remove medium. All plastic ware for cell cultures was obtained from Falcon (Becton Dickinson, Oxnard, CA, USA). Araldine resin was purchased from Electron Microscopy Sciences (Fort Washington, PA, USA). All remaining reagents were of analytical grade and obtained from Sigma Chemical Company (St. Louis, MO, USA). N-acetyl-benzoquinone-imine (purity 80%) was a kind gift from Dr. Steve Leader (Division of Clinical Pharmacology and Toxicology, Children Mercy Hospital and Clinics, Kansas City, MO, USA). 2.2. Hep G2 cell line Hep G2 cells were obtained from Wistar Institute (Philadelphia, PA, USA). Cells were seeded in flasks (1 106 cells per milliliter) (Neuman et al., 1993). The cell counts were monitored with the use of a Coulter counter (Coulter Electronics Inc., Hialeah, FL, USA). Cells in long-term cultures were grown in -MEM supplemented with 10% (vol./ vol.) heat-inactivated fetal bovine serum (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 PBS, and fresh serum-free medium was used as base for all treatments. Cell viability was not altered when cultured in serum-free medium up to 6 days (Neuman et al., 1993; Shear et al., 1995). The concentration of ethanol in culture media was monitored by analyzing the media with the use of a liquid chromatographic method and was shown to decrease by only 20% over 24 h (Neuman et al., 1995). Hep G2 cells express 4 pmol/106 cells of CYP1A1, 2.5 pmol/106 cells of CYP1A2, 6 pmol/106 cells of CYP2E1, and 1 pmol/106 cells of CYP3A5 (Neuman & Tiribelli, 1995). 2.3. Primary human hepatocytes culture The hepatocytes cultures were developed from normal liver tissue that was cultivated in a highly enriched medium. This tissue was obtained after lobectomy from organ donors, in which case part of the liver was used for split-transplantation in children and the rest of the liver was used for research purposes (within the ethics approval of The Toronto General Hospital, Toronto, Ontario, Canada). My method of preparing hepatocyte suspensions was originally based on that of Ballet et al. (1984) and Kalayoglu et al. (1988). The liver graft was perfused with ice-cold University of Wisconsin solution (Neuman & Tiribelli, 1995) and kept on ice (2–10 h) until cells were isolated. The material was washed with N-2-hydroxyethyl-piperazine-N-2-ethane-
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sulfonic acid (HEPES) medium. The hepatocytes were isolated by using the two-step perfusion technique. The average yield was 1 billion cells per preparation, and the average viability, assessed by trypan blue exclusion test, was 94%. The yield of the cells from the graft decreased only 10% during the 10-h maintenance in solution, whereas their phenotype was unchanged (Neuman & Tiribelli, 1995). Hepatocytes were washed three times in a culture medium (1/1 Ham F12 and Leibovitz L-15 supplemented with glucose at 5 mmol/l, penicillin at 50 U/ml, streptomycin at 50 g/l, and insulin 10 -8 mol/l). Cells were then plated (1 106/ml) in collagen-coated Falcon flasks (3 g/cm2) and cultured under conventional conditions in medium supplemented with 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 medium was changed in the presence of FBS. Non-hepatocellular cells were not present, as confirmed by screening the cells both by light and electron microscopic examination. Cells were treated with -MEM with or without the addition of ethanol or acetaminophen, as described in the experimental design. The dilutions were done in medium in the absence of FBS. My colleagues and I have been able to maintain the cells exhibiting typical morphological characteristics of hepatocytes in culture continuously for 10 days (Neuman et al., 1993). For toxicity experiments, cells were plated directly in 96-well plates not coated with collagen. The NHPH express 8 pmol/106 cells of CYP1A1, 7.5 pmol/106 cells of CYP1A2, 10 pmol/106 cells of CYP2E1, and 4 pmol/106 cells of CYP3A5 (Neuman & Tiribelli, 1995). 2.4. Experimental design Control cells were plated in 96-well plates and in 75 Falcon flasks incubated with -MEM. Cells were also incubated with medium and either ethanol at 40 mmol/l or acetaminophen at 5 mmol/l for 24 h. These levels of ethanol and acetaminophen were reported previously as being nontoxic to cells (Neuman et al., 1993; Shear et al., 1995). Acetaminophen at 10 mmol/l and ethanol at 100 mmol/l were used as positive controls. Treated cells were incubated for 24 h with medium and N-acetyl-benzoquinone-imine at 0.5 mmol/l; acetaminophen at 5 mmol/l ethanol at 40 mmol/l; or ethanol at 40 mmol/l N-acetyl-benzoquinone-imine at 0.2 mmol/l. All components were filter-sterilized, and the entire procedure was conducted under aseptic conditions. The cells were routinely maintained in a humidified incubator in 95% air and 5% CO2, at 37C. 2.5. Measurement of apoptosis After storage for 18–20 h at –20C, the cells underwent lysis and were exposed to the Digoxigenin detector antibody for 1 h at room temperature. The method distinguishes low-molecular DNA breaks associated with histone at 180– 200 bp. The cells were washed and incubated for another 30
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min with streptavidin-horseradish peroxidase conjugate. The chromogene reaction was stopped, and the absorbance was read by 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, six wells per plate in five different plates were quantitated. The results are reported as percentage of apoptosis versus control, with nontreated cells taken as 0% apoptosis. For the standard curve, replicates of six in each plate were run with the use of two different plates. The sensitivity of the assay was measured by assaying the nontreated cells at time zero. The mean signal and the standard deviation (S.D.) were calculated. The assay can distinguish 0.3% from zero. The Maxline Microplate Reader, from Molecular Device Corporation (Menlo Park, CA, USA), was connected to a computer with the use of SOFT MAX software 2.3 for Windows (Molecular Devices Corporation, Menlo Park, CA, USA), which allowed me to template the plate according to the experimental needs and perform the statistical analysis directly on the template format. 2.6. Glutathione measurement For GSH determination, the cells were collected (Neuman et al., 1995) on HEPES kalium phosphate buffer [pH 7.4, 1 mM ethylenediaminetetraacetic acid (EDTA) and 0.1% bovine serum albumin]. Initially, the pellet was homogenized on ice, with the use of a Branson model B-12 sonicator (2 10 s). All solutions were made the day the assay was run. After discarding the 3,000-rpm fraction, the rotor was accelerated to 16,000 rpm to obtain the mitochondrial pellet with the use of a JS-20 rotor, with a refrigerating ultracentrifuge (Optima L7 Beckman Instruments Inc., Fullerton, CA, USA). The supernatant was centrifuged again at 45,000 rpm (TJ-70 rotor) for 60 min at 4C; this supernatant was considered the cytosolic fraction. The GSH was determined by using the recycling assay of Tietze (1969). After isolating the organelles by centrifugation at high speeds, I measured the specific biochemical markers that characterized the major function for each entity (Dixon & Webb, 1979; Kirstensen & Horder, 1983). To check the fractions for cross-contamination, I determined specific activities of succinate dehydrogenase (SDH; EC. 1.3.99.1) for mitochondrion (Sottocasa et al., 1967), lactic dehydrogenase (LDH; EC. 1.1.1.27) for cytosol (Henderson, 1983), and glucose6-phosphatase (G6Pase; EC. 3.1.3.9) for microsomes (Belfield & Goldberg, 1969; Yasmineh et al., 1992). Enzyme activities were measured at 37C, 30C, and 37C, respectively. Succinate dehydrogenase activity was measured by reduction of 2,6-dichlorophenol/indophenol. The final mitochondrial preparation was typically enriched in SDH versus homogenate, 3.2 times. The G6Pase activity was measured by coupling to a glucose oxidase system (Henderson, 1983). The G6Pase activity in microsomes was found to be 0.3 0.015 M P/min/mg of protein, whereas in the mitochon-
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drial fraction the activity was 0.001 0.0005 M P/min/mg of protein (a cross-contamination of 0.33%). Cytosolic GSH and mitochondrial GSH were determined after correction for recovery of each specific marker enzyme. Protein content was measured by using a Biorad kit (Baumgarten, 1985) with the use of bovine serum albumin as standard. 2.7. Cytotoxicity assay Briefly, for the MTT assay, Hep G2 and NHPH cells were seeded directly into 96-well plates at a density of 106 per well. The MTT (100 l of a 1-mg/ml solution) was added to each well of the 96-well plate and incubated for 1 h at 37C while being protected from light. At the end of the incubation, the untransformed MTT was removed from the well by aspiration. Next, 100 l of DMSO was added to each well, and cells were incubated in darkness for 1 h. The plate was then shaken vigorously (Microshaker II, Dynatech, Dyna-Med, Toronto, Ontario, Canada) at speed setting 10 s/min to ensure the full solubility of the blue formazan. The optical density of each well was measured by using the two wavelength mode (560 and 690 nm) with the use of the automatic multiwell microplate spectrophotometer. Cytoviability was expressed as the percentage of SDH activity in the treated cells as compared with findings for the controls. Each measurement was done in sextuplets (Neuman et al., 1993). 2.8. Statistical analysis All data are expressed as means S.D. Differences between groups were analyzed by using an analysis of variance (ANOVA) for repeated measurements with a Bonferroni test to correct for multiple comparisons. All statistical analyses were performed with the statistical software package Microcal Origin 6.1 (OriginLab Corporation, Northhampton, MA, USA). 2.9. Light and electron microscopy Cells were prepared for light and transmission electron microscopy studies by using a standard procedure as outlined below (Neuman et al., 1993). Six flasks of either NHPH or Hep G2 cells were used for each group: -MEM only, ethanol at 40 mmol/l, acetaminophen at 5 mmol/l, N-acetyl-benzoquinone-imine at 0.5 mmol/l, or acetaminophen at 5 mmol/l ethanol at 40 mmol/l. After the period of incubation, the medium was removed, and cells were washed twice with PBS. Five milliliters of 1% trypsin was added to each flask for 2 min. Cells were washed again with PBS and then resuspended in plain medium. Cell suspensions were centrifuged at 50g for 10 min. Pellets were immediately fixed in 2.5% (vol./vol.) glutaraldehyde for a minimum of 24 h. Blocks of cells were separated, postfixed in 1% (vol./vol.) osmium tetroxide, dehydrated with a graded series of acetone concentrations, and embedded in Araldine resin. Sections (1 m thick) were viewed by light microscopy. For light microscopy studies an Olympus mi-
croscope equipped with Leco 2005 Image Processing and Analysis System (Leco Instr., Toronto, Ontario, Canada) with Microsoft Visual Basic software was 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 ultrathin sectioning, and stained with uranyl acetate and lead citrate for transmission electron microscopy. Electron micrographs were photographed with a transmission electron microscope JEOL 1200 E x II (JOEL Institute Inc., Boston, MA, USA). Ultrastructural findings were examined in five different grids per flask in each experiment. On each grid, 200–400 cells were examined. An average of 9,000 cells (300 cells per grid number of grids per flask six flasks per treatment) were analyzed for each treatment. I used standard criteria for the morphological identification of cellular structures (Phillips et al., 1987). When cells were assessed by electron microscopy, cell shrinkage, electron dark cytoplasm, and apoptotic bodies were considered criteria for classic apoptosis (Cameron et al., 1998; Müller et al., 1997; Neuman et al., 1995). 2.10. Morphometric analysis Only intact hepatocytes with nuclei were assessed both for light microscopy and transmission electron microscopy. The system used for light microscopy morphometry was a modulator high-performance image processing and analysis system, extended with high-resolution camera, which gave a true-color image processing. For each block, five slides were studied, and the 60 hepatocytes per slide were measured. The morphological dimensions (particle sizing) were implemented by a combination of hardware and software to ensure an optimized performance of Microsoft Visual Basic.
3. Results 3.1. Toxicity I measured the percentage of toxicity of acetaminophen or acetaminophen ethanol on NHPH and on Hep G2 cells with acetaminophen ethanol. The results are shown in Fig. 1. Treatment with acetaminophen at 10 mmol/l resulted in toxicity to NHPH, but was considered nontoxic to Hep G2 cells. Values for percentage of toxicity under 12% were considered nontoxic. Treatment with ethanol at 40 mmol/l alone was not toxic to either NHPH or Hep G2 cells, but there was a significant difference in the viability between the two cell types (Fig. 1). The same was true for treatment with acetaminophen at 5 mmol/l, with higher viability for Hep G2 cells compared with findings for NHPH (P .05). Treatment with acetaminophen ethanol resulted in significantly higher toxicity to both cell types (P .01) compared with findings for acetaminophen alone; toxicity for Hep G2 cells was lower in this case than for NHPH (P
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Fig. 1. Effect of ethanol on acetaminophen-induced hepatocytotoxicity. Cells were seeded in 96-well plates and exposed to different combinations of drugs for 24 h. The percentage of toxicity for normal human primary hepatocytes (NHPH) was significantly higher than the percentage of toxicity for Hep G2 cells in all cases (P .05). For both NHPH and Hep G2 cells, the percentage of toxicity to ethanol in combination with 10 mM acetaminophen was significantly greater (*) than the percentage of toxicity to ethanol in combination with 5 mM acetaminophen (P .05). EtOH ethanol; APAP acetaminophen.
.05) (Fig. 1). I investigated the effect of treating the cells up to 24 h with drug combinations. The NHPH treated 2 h with the direct toxic metabolite N-acetyl-benzoquinone-imine at 0.5 mmol/l showed 25% toxicity as measured by MTT assay, whereas the cytotoxicity in Hep G2 cells treated 2 h with N-acetyl-benzoquinoneimine at 0.5 mmol/l was 18%. No statistical difference in cytotoxicity between Hep G2 cells and NHPH was observed. Interestingly, exposure to N-acetyl-benzoquinone-imine at 0.5 mmol/l began to produce apoptosis in NHPH after 2 h (15%), whereas in Hep G2 cells, apoptosis began after 5 h (26%). Percentage of apoptosis remained low ( 3%) when cells were exposed to either ethanol or acetaminophen for the same period. However, it was the deadly combination of acetaminophen ethanol that produced the largest percentage of apoptosis: 22% and 18% for NHPH and Hep G2 cells, respectively. The percentage of apoptosis was significantly higher for cells exposed for 24 h to acetaminophen ethanol than for cells exposed to acetaminophen alone (P .001). In marked contrast to the results observed in NHPH, the human hepatoblastoma line (i.e., Hep G2 cells) was remarkably resistant, with less apoptosis occurring in Hep G2 cells than in NHPH (P .05). 3.2. Mitochondrial glutathione depletion One pathway by which these drugs may cause hepatotoxicity and apoptosis may be the reduction of intracellular GSH. Therefore I measured cytosolic GSH and mitochon-
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drial GSH in cells exposed to ethanol or acetaminophen and the combination ethanol acetaminophen and ethanol N-acetyl-benzoquinone-imine. The results of this experiment are presented in Table 1. There were no statistical differences between cytosolic or mitochondrial GSH in control cells (Hep G2 cells and NHPH). Exposure of the cells for 24 h to N-acetyl-benzoquinone-imine at 0.5 mM significantly reduced both the concentrations of cytosolic GSH (P .05) and the concentrations of mitochondrial GSH (in Hep G2 cells and in NHPH) (P .001) as compared with findings for controls. There was a significant difference (P .05) between the reduction of mitochondrial GSH in Hep G2 cells versus NHPH (delta 0.71 nmol/mg of protein in Hep G2 cells and 1.00 nmol/mg of protein in NHPH). Cells exposed to 40 mM ethanol for 24 h or 5 mM acetaminophen did not present differences in cytosolic GSH compared with findings for controls after exposure for the same period, neither in Hep G2 cells nor in NHPH. In contrast, 24 h of exposure to 10 mM acetaminophen reduced the mitochondrial GSH concentration significantly in Hep G2 cells (P .05 vs. control) and in NHPH (P .001 vs. control). Mitochondrial GSH was further reduced in Hep G2 cells (P .05) and in NHPH (P .05) when cells were exposed to a concomitant dose of 10 mM acetaminophen with 40 mM ethanol. There was a significant difference (P .05 vs. control cells) between the reduction of mitochondrial GSH in Hep G2 cells versus NHPH (delta 0.53 nmol/mg of protein in Hep G2 cells and 1.05 nmol/mg of protein in NHPH). The latter treatment led to a significant decrease in cytosolic GSH ver-
Table 1 Effect of treatment on cytosolic and mitochondrial GSH depletion
Treatment
Cell type
Cytosolic GSH (nmol/mg of protein)
-MEM (Control)
Hep G2 NHPH Hep G2 NHPH Hep G2 NHPH Hep G2 NHPH Hep G2 NHPH Hep G2 NHPH
14.06 0.60 15.44 0.80 12.44 0.03* 12.58 0.02* 14.24 0.80 15.64 0.60 14.00 0.80 15.64 0.6 14.00 0.70 15.28 0.50 12.80 0.07* 10.00 0.06*
NAPQI (5 M) Acetaminophen (5 mM) Ethanol (40 mM) Acetaminophen (10 mM) Ethanol (40 mM) acetaminophen (10 mM)
Mitochondrial GSH (nmol/mg of protein) 1.58 0.07 1.80 0.01 0.87 0.008** 0.80 0.01** 1.48 0.06 1.84 0.12 1.62 0.06 1.84 0.12 1.28 0.08* 1.00 0.05** 1.05 0.06**^ 0.75 0.05**^
*P .05 as compared with findings for control. **P .001 as compared with findings for control. **^P .001 as compared with findings for control and P .05 as compared with exposure to acetaminophen at 10 mM. -MEM Alpha-minimal essential medium; GSH glutathione; Hep G2 Hep G2 cells; NHPH normal human primary hepatocytes; NAPQI N-acetyl-benzoquinone-imine.
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Fig. 2. Transmission electron micrograph of Hep G2 cells. Cells were plated in Petri dishes 106 cells per milliliter and were incubated in plain medium for 24 h. After the medium was removed, the cells were prepared for transmission electron microscopy as described in Materials and Methods. The electron micrograph shows normal nucleus (N), normal mitochondria (M), and vesiculation of endoplasmic reticulum (er). A bile canaliculus-like structure (BC) is observed at the junction of four cells. The tight junctions (TJ) are well preserved. (Magnification 4,425.)
sus findings for control (P .05) in Hep G2 cells and in NHPH (P .05). 3.3. Transmission electron microscopy morphometric studies Cells that were not been exposed to drugs showed normal organelles: abundant mitochondria, rough and smooth ER, and occasional small lipid vesicles. With the addition of either ethanol at 40 mmol/l or acetaminophen at 5 mmol/l, cells did not change greatly (Figs. 2 and 3). When cells were
Fig. 3. Transmission electron micrograph of normal human primary hepatocytes exposed to 40 mM ethanol for 24 h. The hepatocytes show normal mitochondria (M), prominent lipid droplets (LD), and normal endoplasmic reticulum (er). A bile canaliculus-like structure (BC) can be observed between hepatocytes. Tight junctions (TJ) are preserved. (Magnification 4,425.)
treated with acetaminophen at 10 mmol/l, an enlargement of some cells was seen, and mitochondria were visibly swollen. Endoplasmic reticulum underwent vesiculation, with either slight or more extensive enlargement, respectively (Fig. 4). Lipid accumulation was seen. Lipid vacuoles were seen as rounded inclusions with a smooth homogeneous surface of low electron density, and these exhibited a sharp delimitation. Exposure to ethanol at 40 mmol/l plus acetaminophen at 10 mmol/l increased the number of lipid droplets and the mitochondrial damage. Some cells maintained a normal nucleus but exhibited giant mitochondria with fewer cristae. Tight junctions, however, were preserved (Fig. 5). When cells were exposed to ethanol at 40 mM in combination with N-acetyl-benzoquinone-imine at 0.5 mM, the metabolite of acetaminophen, morphological changes suggestive of apoptosis were observed by electron microscopy (Fig. 6). Although mitochondria were normal, the cells showed enlarged ER, condensed cytoplasm, and a pyknotic nucleus with condensed chromatin (Fig. 6). 3.4. Light microscopy morphometric studies The image processing of slides with the use of the field measurements of intact cells showed no significant differences between cells treated with acetaminophen, ethanol, or plain medium only, but revealed significant differences between the area of cells exposed to acetaminophen ethanol (5,725 215 m2) versus findings for the cells exposed only to plain medium (4,425 525 m2) (P .05). Cells treated with acetaminophen ethanol showed a cell surface of 3,555 215 m2). In the intact cells used in the morphometric studies, nuclear area was not changed significantly (i.e., 860 122 m2 in control cells vs. 780 135 m2 in cells exposed to acetaminophen ethanol). The ratio of cell surface to nuclear surface in control cells was 5.145, whereas in treated cells the ratio was 7.339 (P .05).
Fig. 4. Transmission electron micrograph of treated Hep G2 cells (10 mM acetaminophen for 24 h) showing cells with normal nuclei (N), enlarged mitochondria (M), enlarged endoplasmic reticulum (er), and multiple lipid droplets (LD). (Magnification 4,425.)
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Fig. 5. Transmission electron micrograph shows normal human primary hepatocytes treated with a dose of ethanol at 40 mM acetaminophen at 10 mM/24 h presenting a large number of big lipid droplets (LD) and enlargement of endoplasmic reticulum (er). Giant mitochondria (M), 3 m in length, are observed. Again, cells preserve their tight junctions (TJ). (Magnification 7,080.)
4. Discussion My colleagues and I have previously reported that the Hep G2 cell line and NHPH are reliable in vitro models for the study of drug- and alcohol-induced hepatotoxicity (Neuman et al., 1993, 1995). In the present work, I studied factors that may contribute to acetaminophen-induced cytotoxicity. In human beings and in animals in vivo, toxic injury depends on the balance between the toxification (i.e., production of active metabolite, which in the case of acetaminophen is N-acetyl-benzoquinone-imine) and detoxification (i.e., conjugation leading to glucuronate or sulphate or GSH conjugate of the toxic metabolite). N-acetyl-benzoquinoneimine produces cytotoxicity to both types of cells (Hep G2 cells and NHPH) almost immediately, whereas acetaminophen has to be first metabolized by the cells and transformed into the toxic metabolite to produce cytotoxicity. Therefore I exposed the cells to the toxic metabolite only for a short period (2 h), whereas exposure to acetaminophen alone or in combination with ethanol was for a longer period (24 h). In the present work, the damage produced by acetaminophen was most severe when cells were exposed to ethanol at 40 mmol/l acetaminophen at 10 mmol/l for 24 h. The toxicity may be partly due to the direct effect of acetaminophen. Ethanol, an inducer of the cytochrome P450 isoform CYP2E1, may also be responsible, as it can enhance the production of reactive oxygen species that may contribute to the cytotoxicity. My colleagues and I (Katz et al., 2001) have shown that ethanol at 40 mM induces cytochrome P450, CYP2E1 activity that may be blocked by preexposure to CYP2E1 inhibitors or to CYP2E1 antibodies. A concomitant exposure to ethanol at 40 mM (which was nontoxic) and to a nontoxic dose of valproic acid resulted in cytotoxicity to NHPH. This toxicity was reduced by inhibitors
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Fig. 6. Transmission electron micrograph of one Hep G2 cell treated with 40 mM ethanol and 0.5 mM N-acetyl-benzoquinone-imine for 2 h. The cell presents a preserved cellular membrane. The apoptotic cell exhibits enlarged endoplasmic reticulum (er), lipid droplets (LD), and normal mitochondria (M). The cytoplasm is more condensed. The pyknotic nucleus (N) presents very condensed chromatin. (Magnification 7,080.)
of CYP2E1, therefore demonstrating the role of CYP2E1 inducibility in the NHPH system (Neuman et al., 2001). In terms of ultrastructural changes, cells exposed to acetaminophen ethanol shared a specific trait: Mitochondria were enlarged and appeared swollen or elongated with disrupted cristae, lacking normal organization (Neuman & Tiribelli, 1995). The combination of 10 mM acetaminophen and 40 mM ethanol was synergistic in producing mitochondrial damage after 24 h. Giant mitochondria, 3 m in length, were observed within cells exposed to this drug combination. Interestingly, the giant mitochondrion did not preserve any of its cristae and was therefore not functional. Apoptosis was observed when cells were treated with N-acetyl-benzoquinone-imine, the metabolite of acetaminophen, in combination with ethanol. Cell membranes were preserved, whereas chromatin and cytoplasm were condensed. I have therefore demonstrated that the combination of ethanol and the active metabolite of acetaminophen induces cell death in vitro. In vivo, Mitchell’s group (Mitchell et al., 1973) suggested that liver damage from acetaminophen occurs when the ability of the cell to detoxify the toxic metabolite N-acetyl-benzoquinone-imine is exceeded. The same group suggested that phenobarbital enhances the hepatotoxic effect of acetaminophen by enhancing the activity of P450 system and therefore increasing the likelihood of hepatic injury (Mitchell & Jollow, 1975). Ethanol seems to enhance the toxicity of acetaminophen by induction of the cytochrome P450 isoform CYP2E1. Furthermore, in human beings, depletion of GSH as a result of inhibition of GSH synthesis by alcohol consumption and by the life style–imposed malnutrition of the alcoholic individual enhances susceptibility to acetaminophen toxicity (Zimmerman & Maddrey, 1995). The findings on GSH content supported my hypothesis that metabolic factors
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may be partly responsible for cytotoxicity caused by the combination of acetaminophen ethanol. The mitochondrial pool of GSH is an important cellular defense and is necessary for the maintenance of vital cell functions (Fernandez-Checa et al., 1996; Garcia-Ruiz et al., 1994, 1995a, 1995b; Hirano et al., 1992; Kaplowitz et al., 1996; Kaplowitz & Tsukamoto, 1996; Neuman et al., 1995; Shear et al., 1999). In the aforecited studies, it has been suggested that the depletion in mitochondrial GSH content could be a result of impaired transport from cytosol (Cohen et al., 1998; Neuman, 2001; Neuman & Tiribelli, 1995). Previously, my colleagues and I described structural and functional changes in mitochondria exposed to ethanol in an in vitro model in Hep G2 cells and in NHPH (Neuman et al., 1995; Neuman & Tiribelli, 1995). No differences in cytoviability were observed when cells were incubated with ethanol at 40 mmol/l or acetaminophen at 5 mmol/l (Fig. 1). High cytoviability also corresponded with normal cytosolic GSH and mitochondrial GSH. However, a significant reduction in cell viability was observed after 24 h of exposure to acetaminophen ethanol, which corresponded with a significant reduction in mitochondrial GSH. The importance of mitochondrial GSH in the development and progression of acetaminophen ethanol–induced damage is supported by the notable cytotoxicity that occurs when mitochondrial GSH is reduced by 24%, as well as by a decrease in cytosolic GSH. The present results point to an increased sensitivity to ethanol when GSH was depleted. I suggest that acetaminophen toxicity to cells could be due partially to its role in depleting mitochondrial GSH in cells. Mitochondrial GSH stabilizes the mitochondrial membrane and therefore reduces the mitochondrial permeability transition. When mitochondrial GSH is depleted, apoptotic signaling is switched on. To my knowledge, this is the first report in which nontoxic doses of ethanol have triggered acetaminophen-induced apoptosis in vitro. Normal human primary hepatocytes are more sensitive to both acetaminophen and ethanol when compared with Hep G2 cells, a phenomenon that may be due to a higher content of cytochrome P450 CYP2E1 found in NHPH (Neuman & Tiribelli, 1995). Concomitant exposure to acetaminophen ethanol may induce toxicity and may be related to the induction of cytochrome P450 CYP2E1 and the production of reactive oxygen species, which, together with a decrease of mitochondrial GSH, can contribute to cell damage. One should exercise caution in extrapolating from data obtained in an in vitro model to in vivo conditions. This article presents findings that may be considered as acute intervention as opposed to most in vivo results in which a daily acute injury is superimposed on a chronic ethanol–induced injury. However, the results of the present study may indicate that when concomitant risk factors are coupled with circumstances not directly related to ethanol, such as prolonged fasting (treatment with serum-free medium which, per se, may reduce GSH deposits), such circumstances might also
enhance susceptibility to acetaminophen-induced hepatotoxicity. Therefore an increase in mitochondrial GSH may play a beneficial role in the prevention and treatment of liver damage induced by multiple medications (e.g., chronic use of acetaminophen in conjunction with chronic alcohol consumption). This work contributes to the understanding of the relation between acetaminophen and alcohol in the pathogenesis of liver damage. The article presents human-derived cells as a model to reproduce some of the, or all, morphological and biochemical changes that may be encountered in biopsy samples of liver tissue and serum samples obtained from patients with alcohol/acetaminophen “misadventure”. Acknowledgments This work is dedicated to my teacher and mentor Prof. Hyman J. Zimmerman whose life-time work on the multifactorial phenomenon of “hepatocytotoxicity” was and will always be inspirational. I thank Lois Lanes from the Department of Pathology, The Hospital for Sick Children, Toronto, Ontario, Canada, for excellent technical assistance in preparing the electron microscopy. Thanks also to Julia Haber and Gady Katz for their help in the preparation of this manuscript and Janice Jerrells ELS, for her expert editorial work on the paper. Part of this work was presented at the ESBRA 2001: 8th Congress of the European Society for Biomedical Research on Alcoholism (Paris, September 16, 2001). References Ballet, F., Bouma, M. E., Wang, S. R., Amit, N., Marais, J., & Infante, R. (1984). Isolation, culture and characterization of adult human hepatocytes from surgical liver biopsies. Hepatology 4, 849–854. Banki, K., Hutter, E., Colombo, E., Gonchoroff, N. J., & Perl, A. (1996). Glutathione levels and sensitivity to apoptosis are regulated by changes in transaldolase expression. J Biol Chem 271, 32994–33001. Baumgarten, H. (1985). A simple microplate assay for the determination of cellular protein. J Immunol Methods 82, 25–37. Belfield, A., & Goldberg, D. M. (1969). Enzyme diversion applied to the kinetic estimation of glucose-6-phosphatase activity. Life Sci 8, 129–135. Bernardi, P. (1996). The permeability transition pore: control points of a cyclosporin A–sensitive mitochondrial channel involved in cell death. Biochim Biophys Acta 1275, 5–9. Cameron, R. G., & Neuman, M. G. (1999). Novel morphologic findings in alcoholic liver disease. Clin Biochem 32, 579–584. Cameron, R. G., Neuman, M. G., Shear, N. H., Katz, G., Bellentani, S., & Tiribelli, C. (1998). Modulation of liver specific cellular response to ethanol in vitro in Hep G2 cells. In Vitro Toxicology 12, 111–122. Chiu, S., & Bhakthan, N. M. G. (1978). Experimental acetaminopheninduced hepatic necrosis: biochemical and electron microscopic study of cysteamine protection. Lab Invest 39, 193–203. Cohen, S. D., Hoivik, D. J., & Khairallah, E. A. (1998). Acetaminopheninduced hepatoxicity. In G. L. Plaa, & W. Hewitt (Eds.), Toxicology of the Liver (2nd ed.) (pp. 159–186). New York: Raven Press. Dixon, M., & Webb, E. C. (1979). Enzymes (3rd ed.). New York: Academic Press. Emeigh Hart, S. G., Wyand, D. S., Khairallah, E. A., & Cohen, S. D. (1996). Acetaminophen nephrotoxicity in the CD-1 mouse. II. Protection by probenecid and AT-125 without diminution of renal covalent binding. Toxicol Appl Pharmacol 136, 161–169.
A.K. Halladay et al. / Alcohol 27 (2002) 89–98 Fernandez-Checa, J. C., Yi, J. R., Garcia Ruiz, C., Ookhtens, M., & Kaplowitz, N. (1996). Plasma membrane and mitochondrial transport of hepatic reduced glutathione. Semin Liver Dis 16, 147–158. Galle, P. R., Hofmann, W. J., Walczak, H., Schaller, H., Otto, G., Stremmel, W., Krammer, P. H., & Runkel, L. (1995). Involvement of the CD95 (APO-1/Fas) receptor and ligand in liver damage. J Exp Med 182, 1223–1230. Garcia-Ruiz, C., Colell, A., Morales, A., Kaplowitz, N., & FernandezCheca, J. C. (1995a). Role of oxidative stress generated from the mitochondrial electron transport chain and mitochondrial glutathione status in loss of mitochondrial function and activation of transcription factor nuclear factor-kappa B: studies with isolated mitochondria and rat hepatocytes. Mol Pharmacol 48, 825–834. Garcia-Ruiz, C., Morales, A., Ballesta, A., Rodes, J., Kaplowitz, N., & Fernandez-Checa, J. C. (1994). Effect of chronic ethanol feeding on glutathione and functional integrity of mitochondria in periportal and perivenous rat hepatocytes. J Clin Invest 94, 193–201. Garcia-Ruiz, C., Morales, A., Colell, A., Ballesta, A., Rodes, J., Kaplowitz, N., & Fernandez-Checa, J. C. (1995b). Feeding S-adenosyl-L-methionine attenuates both ethanol-induced depletion of mitochondrial glutathione and mitochondrial dysfunction in periportal and perivenous rat hepatocytes. Hepatology 21, 207–214. Henderson, A. R. (1983). Lactate dehydrogenase isoenzymes. In H. U. Bergmeyer, J. Bergmeyer, & M. Grasse (Eds.), Methods of Enzymatic Analysis (3rd ed.) (pp. 138–155). Weinheim: Verlag Chemie. Hirano, T., Kaplowitz, N., Tsukamoto, H., Kamimura, S., & FernandezCheca, J. C. (1992). Hepatic mitochondrial glutathione depletion and progression of experimental alcoholic liver disease in rats. Hepatology 16, 1423–1427. Hug, H., Strand, S., Grambihler, A., Galle, J., Hack, V., Stremmel, W., Krammer, P. H., & Galle, P. R. (1997). Reactive oxygen intermediates are involved in the induction of CD95 ligand mRNA expression by cytostatic drugs in hepatoma cells. J Biol Chem 272, 28191–28193. Ingelman-Sundberg, M., Johansson, I., Yin, H., Terelius, Y., Eliasson, E., Clot, P., & Albano, E. (1993). Ethanol-inducible cytochrome P4502E1: genetic polymorphism, regulation, and possible role in the etiology of alcohol-induced liver disease. Alcohol 10, 447–452. Jacobson, M. D. (1996). Reactive oxygen species and programmed cell death. Trends Biochem Sci 21, 83–86. Kalayoglu, M., Sollinger, H. W, Stratta, R. J., D’Alessandro, A. M., Hoffmann, R. M., Pirsch, J. D., & Belzer, F. O. (1988). Extended preservation of the liver from clinical transplantation. Lancet 1, 617–619. Kaplowitz, N., Fernandez-Checa, J. C., Kannan, R., Garcia-Ruiz, C., Ookhtens, M., & Yi, J. R. (1996). Glutathione transporters: molecular characterization and role in glutathione homeostasis. Biol Chem Hoppe Seyler 377, 267–273. Kaplowitz, N., & Tsukamoto, H. (1996). Oxidative stress and liver disease. Prog Liver Dis 14, 131–159. Katz, G. G., Shear, N. H., Malkiewicz, I. M., Valentino, K., & Neuman, M. G. (2001). Signaling for ethanol-induced apoptosis and repair in vitro. Clin Biochem 34, 219–227. Kirstensen, S. R., & Horder, M. (1983). Release and turnover of intracellular enzymes. In H. A. Homburger (Ed.), Clinical and Analytical Concepts in Enzymology (pp. 1–13). Skokie, IL: College of American Pathologists. Kroemer, G., Zamzami, N., & Susin, S. A. (1997). Mitochondrial control of apoptosis. Immunol Today 18, 44–51. Lettéron, P., Duchatelle, V., Berson, A., Fromenty, B., Fisch, C., Degott, C., Benhamou, J. P., & Pessayre, D. (1993). Increased ethane exhalation, an in vivo index of lipid peroxidation, in alcohol-abusers. Gut 34, 409–414. Lieber, C. S. (1983). Microsomal ethanol oxidizing system (MEOS): interaction with ethanol, drugs and carcinogens. Pharmacol Biochem Behav 18 Suppl 1, 181–187. Lieber, C. S. (1994). Alcohol and the liver: 1994 update. Gastroenterology 106, 1085–1105. Liu, B., Andrieu-Abadie, N., Levade, T., Zhang, P., Obeid, L. M., & Han-
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nun, Y. A. (1998). Glutathione regulation of neutral sphingomyelinase in tumor necrosis factor-alpha-induced cell death. J Biol Chem 273, 11313–11320. Manautou, J. E., Emeigh Hart, S. G., Khairallah, E. A., & Cohen, S. D. (1996). Protection against acetaminophen hepatotoxicity by a single dose of clofibrate: effects on selective protein arylation and glutathione depletion. Fundam Appl Toxicol 29, 229–237. Mansouri, A., Fromenty, B., Berson, A., Robin, M. A., Grimbert, S., Beaugrand, M., Erlinger, S., & Pessasyre, D. (1997). Multiple hepatic mitochondrial DNA deletions suggest premature oxidative aging in alcoholic patients. J Hepatol 27, 96–102. Mitchell, J. R., & Jollow, D. J. (1975). Progress in hepatology. Metabolic activation of drugs to toxic substances. Gastroenterology 68, 392–410. Mitchell, J. R., Jollow, D. J., Potter, W. Z., Davis, D. C., Gillette, J. R., & Brodie, B. B. (1973). Acetaminophen-induced hepatic necrosis. I. Role of drug metabolism. J Pharmacol Exp Ther 187, 185–194. Müller, M., Strand, S., Hug, H., Heinemann, E. M., Walczak, H., Hofmann, W. J., Stremmel, W., Krammer, P. H., & Galle, P. R. (1997). Drug-induced apoptosis in hepatoma cells is mediated by the CD95 (APO-1/Fas) receptor/ligand system and involves activation of wildtype p53. J Clin Invest 99, 403–413. Neuman, M. G. (2001). Apoptosis in diseases of the liver. Crit Rev Clin Lab Sci 38, 109–166. Neuman, M. G., Cameron, R. G., Haber, J. A., Katz, G., Malkiewicz, I. M., & Shear, N. H. (1999a). Inducers of cytochrome P450 2E1 enhance methotrexate-induced hepatocytoxicity. Clin Biochem 32, 519–536. Neuman, M. G., Cameron, R. G., Shear, N. H., Bellentani, S., & Tiribelli, C. (1995). Effect of tauroursodeoxycholic and ursodeoxycholic acid on ethanol-induced cell injuries in the human Hep G2 cell line. Gastroenterology 109, 555–563. Neuman, M. G., Koren, G., & Tiribelli, C. (1993). In vitro assessment of the ethanol-induced hepatotoxicity on Hep G2 cell line. Biochem Biophys Res Commun 197, 932–941. Neuman, M. G., Shear, N. H., Bellentani, S., & Tiribelli, C. (1998). Role of cytokines in ethanol-induced cytotoxicity in vitro in Hep G2 cells. Gastroenterology 115, 157–166. Neuman, M. G., Shear, N. H., Cameron, R. G., Katz, G., & Tiribelli, C. (1999b). Ethanol-induced apoptosis in vitro. Clin Biochem 32, 547– 555. Neuman, M. G., Shear, N. H., Jacobson-Brown, P. M., Katz, G. G., Neilson, H. K., Malkiewicz, I. M., Cameron, R. G., & Abbott, F. (2001). CYP2E1-mediated modulation of valproic acid–induced hepatocytotoxicity. Clin Biochem 34, 211–218. Neuman, M. G., & Tiribelli, C. (1995). Drugs - are they predictable in preclinical stage? Models and techniques. In J. Holoman, J. Glasa, P. R. Bechtel, & C. Tiribelli (Eds.), Liver and Drugs ‘94. Progress in Hepato-Pharmacology, Vol. I (pp. 24–32). Bratislava, Slovakia: BoArt. Pastorino, J. G., & Hoek, J. B. (2000). Ethanol potentiates tumor necrosis factor-alpha cytotoxicity in hepatoma cells and primary rat hepatocytes by promoting induction of the mitochondrial permeability transition. Hepatology 31, 1141–1152. Phillips, M. J., Poucell, S., Pattersson, J., & Valencia, P. (1987). The Liver: An Atlas and Text of Ultrastructural Pathology. New York: Raven Press. Seeff, L. B., Cuccherini, B. A., Zimmerman, H. J., Adler, E., & Benjamin, S. B. (1986). Acetaminophen hepatotoxicity in alcoholics: a therapeutic misadventure. Ann Intern Med 104, 399–404. Shear, N. H., Landau, M., Malkiewicz, I., Katz, G. G., & Neuman, M. G. (1999). Ethanol-modulated cytokine production and expression in skin cells exposed to methotrexate. Skin Pharmacol Appl Skin Physiol 12, 64–78. Shear, N. H., Malkiewicz, I. M, Klein, D., Koren, G., Randor, S., & Neuman, M. G. (1995). Acetaminophen-induced toxicity to human epidermoid cell line A431 and hepatoblastoma cell line, Hep G2, in vitro, is diminished by silymarin. Skin Pharmacol 8, 279–291. Sottocasa, G. L., Kuylenstierna, B., Ernster, L., & Bergstrand, L. (1967). An electron-transport system associated with the outer membrane of
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Zamzami, N., Marchetti, P., Castedo, M., Decaudin, D., Macho, A., Hirsch, T., Susin, S. A., Petit, P. X., Mignotte, B., & Kroemer, G. (1995). Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J Exp Med 182, 367–377. Zamzami, N., Marzo, I., Susin, S. A., Brenner, C., Larochette, N., Marchetti, P., Reed, J., Kofler, R., & Kroemer, G. (1998). The thiol crosslinking agent diamide overcomes the apoptosis-inhibitory effect of Bcl-2 by enforcing mitochondrial permeability transition. Oncogene 16, 1055–1063. Zamzami, N., Susin, S. A., Marchetti, P., Hirsch, T., Gomez-Monterrey, I., Castedo, M., & Kroemer, G. (1996). Mitochondrial control of nuclear apoptosis. J Exp Med 183, 1533–1544. Zhao, M., Laissue, J. A., & Zimmermann, A. (1997). Hepatocyte apoptosis in hepatic iron overload diseases. Histol Histopathol 12, 367–374. Zimmerman, H. J., & Maddrey, W. C. (1995). Acetaminophen (paracetamol) hepatotoxicity with regular intake of alcohol: analysis of instances of therapeutic misadventure. Hepatology 22, 767–773.
Synergetic signaling for apoptosis in vitro by ethanol and acetaminophen Manuela G. Neumana,b,* a
In Vitro Toxicology Laboratory, Division of Clinical Pharmacology, Sunnybrook and Women’s College Health Sciences Centre, Room E235, 2075 Bayview Ave., Toronto, Ontario, Canada, M4N 3M5 b Department of Pharmacology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada, M5S 1A8 Received 31 July 2001; received in revised form 27 January 2002; accepted 1 February 2002
Abstract In vitro, ethanol in combination with acetaminophen induces hepatocyte apoptosis resembling immune-mediated fulminant hepatic failure in human beings. Intracellular pathways originating at the mitochondria are linked to apoptosis. I studied ethanol-induced apoptosis and hepatocytotoxicity after using an in vitro model of normal human primary hepatocytes that were exposed to 5 or 10 mM acetaminophen, 40 or 100 mM ethanol, 40 mM ethanol 5 mM acetaminophen, or 40 mM ethanol 10 mM acetaminophen, or nonexposed (control; plain medium). Transmission electron microscopy was performed at different time points after exposure to the various treatments. Apoptosis, as assessed by transmission electron microscopy, was increased in a time-dependent manner after exposure to ethanol acetaminophen. In the ethanol acetaminophen model, mitochondrial injury was associated with apoptosis of hepatocytes. Ultrastructural damage and induction of apoptosis were seen in response to N-acetyl-benzoquinone-imine plus ethanol, supporting the suggestion that the damage was due to the active metabolite of acetaminophen. The modulation of mitochondrial damage in vitro may have implications for the development of new therapeutic strategies to prevent apoptosis. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Alcohol; Acetaminophen; In vitro; Cytotoxicity; Ethanol; Normal human primary hepatocytes; Hep G2 cells
1. Introduction Although reactive oxygen species may produce a variety of liver lesions, only a few of these lesions can actually lead to apoptosis (Zhao et al., 1997). Many drugs are transformed by the cytochrome P450 enzymatic system into reactive metabolites. The xenobiotics may reach the liver by means of the systemic circulation. First, endothelial cells of the hepatic sinusoids, which possess fenestrae, allow different proteins to come into direct contact with hepatocytes and enable the liver either to eliminate the toxic metabolites or to catalyze them further. Microsomal and cytosolic epoxide hydrolases are known to hydrate reactive epoxides into diols, whereas glutathione-S-transferases catalyze the conjugation of electrophilic metabolites with glutathione (GSH). Drugs may be transformed into free radicals (Jacobson, 1996). Decreases in intracellular GSH have been shown to be an early event in apoptosis (Zamzami et al., 1995). The functional and causative role of GSH depletion in the induction and execution of apoptosis has been elucidated (Zamzami et al., 1996). Cells can be rescued from apoptosis when extrusion of GSH is inhibited (Neuman et al., 1999b; Zamzami et al., 1998). The same effect is achieved when inhibitors of * Corresponding author. Tel.: 1-416-480-6100, ext. 3503; fax: 1416-480-6025. E-mail address: [email protected] (M.G. Neuman). Editor: T.R. Jerrells
macromolecular synthesis shunt cysteine from protein to GSH synthesis (Banki et al., 1996). Glutathione works as a redox sensor and regulates two important factors in apoptosis: mitochondrial function and sphingomyelinase activity (Kroemer et al., 1997; Liu et al., 1998). Mitochondria are the source of massive reactive oxygen species production and apoptogenic molecule release [e.g., cytochrome c (Bernardi, 1996) and apoptosis-inducing factor (AIF), a protease involved in the activation of caspases]. Chronic alcohol consumption is associated with the formation of reactive oxygen species and the presence of oxidative stress in the liver (Lettéron et al., 1993; Lieber, 1983; Mansouri et al., 1997; Neuman et al., 1993). Ethanol is known to induce the cytochrome P450 isoform CYP2E1, resulting in the production of free radicals (Ingelman-Sundberg et al., 1993; Lieber, 1994; Neuman et al., 1995). Fas ligand messenger RNA has been detected by in situ hybridization in the hepatocytes of patients with alcoholic liver damage (Galle et al., 1995). At the same time, Fas was overexpressed in some hepatocytes. Conditions, which increase reactive oxygen intermediates, may thus induce Fas ligand expression by hepatocytes (Müller et al., 1997; Neuman et al., 1999b). At the same time, the increased formation of reactive oxygen intermediates might damage DNA, cause overexpression of p53, and increase Fas expression by hepatocytes (Cameron & Neuman, 1999; Hug et al., 1997; Strand et al., 1998). The Fas ligand of a first hepatocyte may
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then interact with the Fas receptor on another hepatocyte, causing fratricidal killing. In normal human primary hepatocytes (NHPH), ethanol signaling for apoptosis is initiated by Fas ligand (Neuman, 2001; Pastorino & Hoek, 2000; Shear et al., 1995). Treatment of hepatocytes with ethanol causes oxidative stress, leading to tumor necrosis factor-alpha (TNF-) expression (Neuman et al., 1998; Pastorino & Hoek, 2000). My colleagues and I have shown that treatment of Hep G2 cells with ethanol at low concentrations (40 mM) does not cause toxicity. However, addition of 80 mM ethanol can produce toxic effects, such as changes in mitochondria and endoplasmic reticulum (ER) as well as accumulation of abundant lipid vesicles (Shear et al., 1995, 1999). Repeated exposure to ethanol causes apoptosis (Neuman et al., 1999b). Toxicity is increased when ethanol is combined with other drugs, such as acetaminophen (Shear et al., 1995) or methotrexate (Neuman et al., 1999a). Acetaminophen is a widely used analgesic and antipyretic with very few side effects at its usual therapeutic doses (Chiu & Bhakthan, 1978; Spooner & Harvey, 1976). However, when taken at high doses (Mitchell et al., 1973) or in combination with other drugs such as alcohol, acetaminophen is known to cause hepatic necrosis (Seeff et al., 1986) and, in some cases, liver failure that may necessitate liver transplantation or lead to death (Zimmerman & Maddrey, 1995). Primarily, glucuronidation and sulphation processes detoxify acetaminophen (Ballet et al., 1984; Manautou et al., 1996). Selective protein arylation may contribute to toxicity (Emeigh Hart et al., 1996; Manautou et al., 1996; Tarloff et al., 1996). N-acetyl-benzoquinone-imine, the toxic metabolite of acetaminophen, is mainly generated by activation of CYP2E1 and acts as oxidizing agent of GSH, reducing it to its oxidized form (GSSG), which leads to hepatocytotoxicity. In previous work, my colleagues and I have shown that apoptotic processes, characterized by disruption of the cytoskeleton, blebbing of the cells, and nuclear chromatin condensation, appear in Hep G2 cells and skin cells treated with acetaminophen in the presence of low doses of ethanol (Neuman et al., 1999a). The present study was designed to evaluate the effect of acetaminophen on NHPH in the presence or absence of ethanol and to examine the way that this drug combination signals for apoptosis. 2. Materials and methods 2.1. Drugs and chemicals Ethanol (96% purity) was purchased from Alcohol Ltd. (Toronto, Ontario, Canada). Plain -minimal essential medium (-MEM) and Hanks’ balanced salt 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. Acetaminophen was obtained from Sigma Chemical Company (St.
Louis, MO, USA). Tetrazolium salt: MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diophenyltetrazolium bromide] that was used for the cytoviability MTT assay, and hematoxylineosin stain that was used for light microscopy, were purchased from Sigma Chemical Company (St. Louis, MO, USA). Dimethylsulfoxide (DMSO) was obtained from Fisher Chemical (Fisher Scientific, Toronto, Ontario, Canada). Phosphate-buffered saline (PBS) (without Ca or Mg) was used to wash cells and to remove medium. All plastic ware for cell cultures was obtained from Falcon (Becton Dickinson, Oxnard, CA, USA). Araldine resin was purchased from Electron Microscopy Sciences (Fort Washington, PA, USA). All remaining reagents were of analytical grade and obtained from Sigma Chemical Company (St. Louis, MO, USA). N-acetyl-benzoquinone-imine (purity 80%) was a kind gift from Dr. Steve Leader (Division of Clinical Pharmacology and Toxicology, Children Mercy Hospital and Clinics, Kansas City, MO, USA). 2.2. Hep G2 cell line Hep G2 cells were obtained from Wistar Institute (Philadelphia, PA, USA). Cells were seeded in flasks (1 106 cells per milliliter) (Neuman et al., 1993). The cell counts were monitored with the use of a Coulter counter (Coulter Electronics Inc., Hialeah, FL, USA). Cells in long-term cultures were grown in -MEM supplemented with 10% (vol./ vol.) heat-inactivated fetal bovine serum (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 PBS, and fresh serum-free medium was used as base for all treatments. Cell viability was not altered when cultured in serum-free medium up to 6 days (Neuman et al., 1993; Shear et al., 1995). The concentration of ethanol in culture media was monitored by analyzing the media with the use of a liquid chromatographic method and was shown to decrease by only 20% over 24 h (Neuman et al., 1995). Hep G2 cells express 4 pmol/106 cells of CYP1A1, 2.5 pmol/106 cells of CYP1A2, 6 pmol/106 cells of CYP2E1, and 1 pmol/106 cells of CYP3A5 (Neuman & Tiribelli, 1995). 2.3. Primary human hepatocytes culture The hepatocytes cultures were developed from normal liver tissue that was cultivated in a highly enriched medium. This tissue was obtained after lobectomy from organ donors, in which case part of the liver was used for split-transplantation in children and the rest of the liver was used for research purposes (within the ethics approval of The Toronto General Hospital, Toronto, Ontario, Canada). My method of preparing hepatocyte suspensions was originally based on that of Ballet et al. (1984) and Kalayoglu et al. (1988). The liver graft was perfused with ice-cold University of Wisconsin solution (Neuman & Tiribelli, 1995) and kept on ice (2–10 h) until cells were isolated. The material was washed with N-2-hydroxyethyl-piperazine-N-2-ethane-
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sulfonic acid (HEPES) medium. The hepatocytes were isolated by using the two-step perfusion technique. The average yield was 1 billion cells per preparation, and the average viability, assessed by trypan blue exclusion test, was 94%. The yield of the cells from the graft decreased only 10% during the 10-h maintenance in solution, whereas their phenotype was unchanged (Neuman & Tiribelli, 1995). Hepatocytes were washed three times in a culture medium (1/1 Ham F12 and Leibovitz L-15 supplemented with glucose at 5 mmol/l, penicillin at 50 U/ml, streptomycin at 50 g/l, and insulin 10 -8 mol/l). Cells were then plated (1 106/ml) in collagen-coated Falcon flasks (3 g/cm2) and cultured under conventional conditions in medium supplemented with 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 medium was changed in the presence of FBS. Non-hepatocellular cells were not present, as confirmed by screening the cells both by light and electron microscopic examination. Cells were treated with -MEM with or without the addition of ethanol or acetaminophen, as described in the experimental design. The dilutions were done in medium in the absence of FBS. My colleagues and I have been able to maintain the cells exhibiting typical morphological characteristics of hepatocytes in culture continuously for 10 days (Neuman et al., 1993). For toxicity experiments, cells were plated directly in 96-well plates not coated with collagen. The NHPH express 8 pmol/106 cells of CYP1A1, 7.5 pmol/106 cells of CYP1A2, 10 pmol/106 cells of CYP2E1, and 4 pmol/106 cells of CYP3A5 (Neuman & Tiribelli, 1995). 2.4. Experimental design Control cells were plated in 96-well plates and in 75 Falcon flasks incubated with -MEM. Cells were also incubated with medium and either ethanol at 40 mmol/l or acetaminophen at 5 mmol/l for 24 h. These levels of ethanol and acetaminophen were reported previously as being nontoxic to cells (Neuman et al., 1993; Shear et al., 1995). Acetaminophen at 10 mmol/l and ethanol at 100 mmol/l were used as positive controls. Treated cells were incubated for 24 h with medium and N-acetyl-benzoquinone-imine at 0.5 mmol/l; acetaminophen at 5 mmol/l ethanol at 40 mmol/l; or ethanol at 40 mmol/l N-acetyl-benzoquinone-imine at 0.2 mmol/l. All components were filter-sterilized, and the entire procedure was conducted under aseptic conditions. The cells were routinely maintained in a humidified incubator in 95% air and 5% CO2, at 37C. 2.5. Measurement of apoptosis After storage for 18–20 h at –20C, the cells underwent lysis and were exposed to the Digoxigenin detector antibody for 1 h at room temperature. The method distinguishes low-molecular DNA breaks associated with histone at 180– 200 bp. The cells were washed and incubated for another 30
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min with streptavidin-horseradish peroxidase conjugate. The chromogene reaction was stopped, and the absorbance was read by 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, six wells per plate in five different plates were quantitated. The results are reported as percentage of apoptosis versus control, with nontreated cells taken as 0% apoptosis. For the standard curve, replicates of six in each plate were run with the use of two different plates. The sensitivity of the assay was measured by assaying the nontreated cells at time zero. The mean signal and the standard deviation (S.D.) were calculated. The assay can distinguish 0.3% from zero. The Maxline Microplate Reader, from Molecular Device Corporation (Menlo Park, CA, USA), was connected to a computer with the use of SOFT MAX software 2.3 for Windows (Molecular Devices Corporation, Menlo Park, CA, USA), which allowed me to template the plate according to the experimental needs and perform the statistical analysis directly on the template format. 2.6. Glutathione measurement For GSH determination, the cells were collected (Neuman et al., 1995) on HEPES kalium phosphate buffer [pH 7.4, 1 mM ethylenediaminetetraacetic acid (EDTA) and 0.1% bovine serum albumin]. Initially, the pellet was homogenized on ice, with the use of a Branson model B-12 sonicator (2 10 s). All solutions were made the day the assay was run. After discarding the 3,000-rpm fraction, the rotor was accelerated to 16,000 rpm to obtain the mitochondrial pellet with the use of a JS-20 rotor, with a refrigerating ultracentrifuge (Optima L7 Beckman Instruments Inc., Fullerton, CA, USA). The supernatant was centrifuged again at 45,000 rpm (TJ-70 rotor) for 60 min at 4C; this supernatant was considered the cytosolic fraction. The GSH was determined by using the recycling assay of Tietze (1969). After isolating the organelles by centrifugation at high speeds, I measured the specific biochemical markers that characterized the major function for each entity (Dixon & Webb, 1979; Kirstensen & Horder, 1983). To check the fractions for cross-contamination, I determined specific activities of succinate dehydrogenase (SDH; EC. 1.3.99.1) for mitochondrion (Sottocasa et al., 1967), lactic dehydrogenase (LDH; EC. 1.1.1.27) for cytosol (Henderson, 1983), and glucose6-phosphatase (G6Pase; EC. 3.1.3.9) for microsomes (Belfield & Goldberg, 1969; Yasmineh et al., 1992). Enzyme activities were measured at 37C, 30C, and 37C, respectively. Succinate dehydrogenase activity was measured by reduction of 2,6-dichlorophenol/indophenol. The final mitochondrial preparation was typically enriched in SDH versus homogenate, 3.2 times. The G6Pase activity was measured by coupling to a glucose oxidase system (Henderson, 1983). The G6Pase activity in microsomes was found to be 0.3 0.015 M P/min/mg of protein, whereas in the mitochon-
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drial fraction the activity was 0.001 0.0005 M P/min/mg of protein (a cross-contamination of 0.33%). Cytosolic GSH and mitochondrial GSH were determined after correction for recovery of each specific marker enzyme. Protein content was measured by using a Biorad kit (Baumgarten, 1985) with the use of bovine serum albumin as standard. 2.7. Cytotoxicity assay Briefly, for the MTT assay, Hep G2 and NHPH cells were seeded directly into 96-well plates at a density of 106 per well. The MTT (100 l of a 1-mg/ml solution) was added to each well of the 96-well plate and incubated for 1 h at 37C while being protected from light. At the end of the incubation, the untransformed MTT was removed from the well by aspiration. Next, 100 l of DMSO was added to each well, and cells were incubated in darkness for 1 h. The plate was then shaken vigorously (Microshaker II, Dynatech, Dyna-Med, Toronto, Ontario, Canada) at speed setting 10 s/min to ensure the full solubility of the blue formazan. The optical density of each well was measured by using the two wavelength mode (560 and 690 nm) with the use of the automatic multiwell microplate spectrophotometer. Cytoviability was expressed as the percentage of SDH activity in the treated cells as compared with findings for the controls. Each measurement was done in sextuplets (Neuman et al., 1993). 2.8. Statistical analysis All data are expressed as means S.D. Differences between groups were analyzed by using an analysis of variance (ANOVA) for repeated measurements with a Bonferroni test to correct for multiple comparisons. All statistical analyses were performed with the statistical software package Microcal Origin 6.1 (OriginLab Corporation, Northhampton, MA, USA). 2.9. Light and electron microscopy Cells were prepared for light and transmission electron microscopy studies by using a standard procedure as outlined below (Neuman et al., 1993). Six flasks of either NHPH or Hep G2 cells were used for each group: -MEM only, ethanol at 40 mmol/l, acetaminophen at 5 mmol/l, N-acetyl-benzoquinone-imine at 0.5 mmol/l, or acetaminophen at 5 mmol/l ethanol at 40 mmol/l. After the period of incubation, the medium was removed, and cells were washed twice with PBS. Five milliliters of 1% trypsin was added to each flask for 2 min. Cells were washed again with PBS and then resuspended in plain medium. Cell suspensions were centrifuged at 50g for 10 min. Pellets were immediately fixed in 2.5% (vol./vol.) glutaraldehyde for a minimum of 24 h. Blocks of cells were separated, postfixed in 1% (vol./vol.) osmium tetroxide, dehydrated with a graded series of acetone concentrations, and embedded in Araldine resin. Sections (1 m thick) were viewed by light microscopy. For light microscopy studies an Olympus mi-
croscope equipped with Leco 2005 Image Processing and Analysis System (Leco Instr., Toronto, Ontario, Canada) with Microsoft Visual Basic software was 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 ultrathin sectioning, and stained with uranyl acetate and lead citrate for transmission electron microscopy. Electron micrographs were photographed with a transmission electron microscope JEOL 1200 E x II (JOEL Institute Inc., Boston, MA, USA). Ultrastructural findings were examined in five different grids per flask in each experiment. On each grid, 200–400 cells were examined. An average of 9,000 cells (300 cells per grid number of grids per flask six flasks per treatment) were analyzed for each treatment. I used standard criteria for the morphological identification of cellular structures (Phillips et al., 1987). When cells were assessed by electron microscopy, cell shrinkage, electron dark cytoplasm, and apoptotic bodies were considered criteria for classic apoptosis (Cameron et al., 1998; Müller et al., 1997; Neuman et al., 1995). 2.10. Morphometric analysis Only intact hepatocytes with nuclei were assessed both for light microscopy and transmission electron microscopy. The system used for light microscopy morphometry was a modulator high-performance image processing and analysis system, extended with high-resolution camera, which gave a true-color image processing. For each block, five slides were studied, and the 60 hepatocytes per slide were measured. The morphological dimensions (particle sizing) were implemented by a combination of hardware and software to ensure an optimized performance of Microsoft Visual Basic.
3. Results 3.1. Toxicity I measured the percentage of toxicity of acetaminophen or acetaminophen ethanol on NHPH and on Hep G2 cells with acetaminophen ethanol. The results are shown in Fig. 1. Treatment with acetaminophen at 10 mmol/l resulted in toxicity to NHPH, but was considered nontoxic to Hep G2 cells. Values for percentage of toxicity under 12% were considered nontoxic. Treatment with ethanol at 40 mmol/l alone was not toxic to either NHPH or Hep G2 cells, but there was a significant difference in the viability between the two cell types (Fig. 1). The same was true for treatment with acetaminophen at 5 mmol/l, with higher viability for Hep G2 cells compared with findings for NHPH (P .05). Treatment with acetaminophen ethanol resulted in significantly higher toxicity to both cell types (P .01) compared with findings for acetaminophen alone; toxicity for Hep G2 cells was lower in this case than for NHPH (P
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Fig. 1. Effect of ethanol on acetaminophen-induced hepatocytotoxicity. Cells were seeded in 96-well plates and exposed to different combinations of drugs for 24 h. The percentage of toxicity for normal human primary hepatocytes (NHPH) was significantly higher than the percentage of toxicity for Hep G2 cells in all cases (P .05). For both NHPH and Hep G2 cells, the percentage of toxicity to ethanol in combination with 10 mM acetaminophen was significantly greater (*) than the percentage of toxicity to ethanol in combination with 5 mM acetaminophen (P .05). EtOH ethanol; APAP acetaminophen.
.05) (Fig. 1). I investigated the effect of treating the cells up to 24 h with drug combinations. The NHPH treated 2 h with the direct toxic metabolite N-acetyl-benzoquinone-imine at 0.5 mmol/l showed 25% toxicity as measured by MTT assay, whereas the cytotoxicity in Hep G2 cells treated 2 h with N-acetyl-benzoquinoneimine at 0.5 mmol/l was 18%. No statistical difference in cytotoxicity between Hep G2 cells and NHPH was observed. Interestingly, exposure to N-acetyl-benzoquinone-imine at 0.5 mmol/l began to produce apoptosis in NHPH after 2 h (15%), whereas in Hep G2 cells, apoptosis began after 5 h (26%). Percentage of apoptosis remained low ( 3%) when cells were exposed to either ethanol or acetaminophen for the same period. However, it was the deadly combination of acetaminophen ethanol that produced the largest percentage of apoptosis: 22% and 18% for NHPH and Hep G2 cells, respectively. The percentage of apoptosis was significantly higher for cells exposed for 24 h to acetaminophen ethanol than for cells exposed to acetaminophen alone (P .001). In marked contrast to the results observed in NHPH, the human hepatoblastoma line (i.e., Hep G2 cells) was remarkably resistant, with less apoptosis occurring in Hep G2 cells than in NHPH (P .05). 3.2. Mitochondrial glutathione depletion One pathway by which these drugs may cause hepatotoxicity and apoptosis may be the reduction of intracellular GSH. Therefore I measured cytosolic GSH and mitochon-
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drial GSH in cells exposed to ethanol or acetaminophen and the combination ethanol acetaminophen and ethanol N-acetyl-benzoquinone-imine. The results of this experiment are presented in Table 1. There were no statistical differences between cytosolic or mitochondrial GSH in control cells (Hep G2 cells and NHPH). Exposure of the cells for 24 h to N-acetyl-benzoquinone-imine at 0.5 mM significantly reduced both the concentrations of cytosolic GSH (P .05) and the concentrations of mitochondrial GSH (in Hep G2 cells and in NHPH) (P .001) as compared with findings for controls. There was a significant difference (P .05) between the reduction of mitochondrial GSH in Hep G2 cells versus NHPH (delta 0.71 nmol/mg of protein in Hep G2 cells and 1.00 nmol/mg of protein in NHPH). Cells exposed to 40 mM ethanol for 24 h or 5 mM acetaminophen did not present differences in cytosolic GSH compared with findings for controls after exposure for the same period, neither in Hep G2 cells nor in NHPH. In contrast, 24 h of exposure to 10 mM acetaminophen reduced the mitochondrial GSH concentration significantly in Hep G2 cells (P .05 vs. control) and in NHPH (P .001 vs. control). Mitochondrial GSH was further reduced in Hep G2 cells (P .05) and in NHPH (P .05) when cells were exposed to a concomitant dose of 10 mM acetaminophen with 40 mM ethanol. There was a significant difference (P .05 vs. control cells) between the reduction of mitochondrial GSH in Hep G2 cells versus NHPH (delta 0.53 nmol/mg of protein in Hep G2 cells and 1.05 nmol/mg of protein in NHPH). The latter treatment led to a significant decrease in cytosolic GSH ver-
Table 1 Effect of treatment on cytosolic and mitochondrial GSH depletion
Treatment
Cell type
Cytosolic GSH (nmol/mg of protein)
-MEM (Control)
Hep G2 NHPH Hep G2 NHPH Hep G2 NHPH Hep G2 NHPH Hep G2 NHPH Hep G2 NHPH
14.06 0.60 15.44 0.80 12.44 0.03* 12.58 0.02* 14.24 0.80 15.64 0.60 14.00 0.80 15.64 0.6 14.00 0.70 15.28 0.50 12.80 0.07* 10.00 0.06*
NAPQI (5 M) Acetaminophen (5 mM) Ethanol (40 mM) Acetaminophen (10 mM) Ethanol (40 mM) acetaminophen (10 mM)
Mitochondrial GSH (nmol/mg of protein) 1.58 0.07 1.80 0.01 0.87 0.008** 0.80 0.01** 1.48 0.06 1.84 0.12 1.62 0.06 1.84 0.12 1.28 0.08* 1.00 0.05** 1.05 0.06**^ 0.75 0.05**^
*P .05 as compared with findings for control. **P .001 as compared with findings for control. **^P .001 as compared with findings for control and P .05 as compared with exposure to acetaminophen at 10 mM. -MEM Alpha-minimal essential medium; GSH glutathione; Hep G2 Hep G2 cells; NHPH normal human primary hepatocytes; NAPQI N-acetyl-benzoquinone-imine.
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Fig. 2. Transmission electron micrograph of Hep G2 cells. Cells were plated in Petri dishes 106 cells per milliliter and were incubated in plain medium for 24 h. After the medium was removed, the cells were prepared for transmission electron microscopy as described in Materials and Methods. The electron micrograph shows normal nucleus (N), normal mitochondria (M), and vesiculation of endoplasmic reticulum (er). A bile canaliculus-like structure (BC) is observed at the junction of four cells. The tight junctions (TJ) are well preserved. (Magnification 4,425.)
sus findings for control (P .05) in Hep G2 cells and in NHPH (P .05). 3.3. Transmission electron microscopy morphometric studies Cells that were not been exposed to drugs showed normal organelles: abundant mitochondria, rough and smooth ER, and occasional small lipid vesicles. With the addition of either ethanol at 40 mmol/l or acetaminophen at 5 mmol/l, cells did not change greatly (Figs. 2 and 3). When cells were
Fig. 3. Transmission electron micrograph of normal human primary hepatocytes exposed to 40 mM ethanol for 24 h. The hepatocytes show normal mitochondria (M), prominent lipid droplets (LD), and normal endoplasmic reticulum (er). A bile canaliculus-like structure (BC) can be observed between hepatocytes. Tight junctions (TJ) are preserved. (Magnification 4,425.)
treated with acetaminophen at 10 mmol/l, an enlargement of some cells was seen, and mitochondria were visibly swollen. Endoplasmic reticulum underwent vesiculation, with either slight or more extensive enlargement, respectively (Fig. 4). Lipid accumulation was seen. Lipid vacuoles were seen as rounded inclusions with a smooth homogeneous surface of low electron density, and these exhibited a sharp delimitation. Exposure to ethanol at 40 mmol/l plus acetaminophen at 10 mmol/l increased the number of lipid droplets and the mitochondrial damage. Some cells maintained a normal nucleus but exhibited giant mitochondria with fewer cristae. Tight junctions, however, were preserved (Fig. 5). When cells were exposed to ethanol at 40 mM in combination with N-acetyl-benzoquinone-imine at 0.5 mM, the metabolite of acetaminophen, morphological changes suggestive of apoptosis were observed by electron microscopy (Fig. 6). Although mitochondria were normal, the cells showed enlarged ER, condensed cytoplasm, and a pyknotic nucleus with condensed chromatin (Fig. 6). 3.4. Light microscopy morphometric studies The image processing of slides with the use of the field measurements of intact cells showed no significant differences between cells treated with acetaminophen, ethanol, or plain medium only, but revealed significant differences between the area of cells exposed to acetaminophen ethanol (5,725 215 m2) versus findings for the cells exposed only to plain medium (4,425 525 m2) (P .05). Cells treated with acetaminophen ethanol showed a cell surface of 3,555 215 m2). In the intact cells used in the morphometric studies, nuclear area was not changed significantly (i.e., 860 122 m2 in control cells vs. 780 135 m2 in cells exposed to acetaminophen ethanol). The ratio of cell surface to nuclear surface in control cells was 5.145, whereas in treated cells the ratio was 7.339 (P .05).
Fig. 4. Transmission electron micrograph of treated Hep G2 cells (10 mM acetaminophen for 24 h) showing cells with normal nuclei (N), enlarged mitochondria (M), enlarged endoplasmic reticulum (er), and multiple lipid droplets (LD). (Magnification 4,425.)
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Fig. 5. Transmission electron micrograph shows normal human primary hepatocytes treated with a dose of ethanol at 40 mM acetaminophen at 10 mM/24 h presenting a large number of big lipid droplets (LD) and enlargement of endoplasmic reticulum (er). Giant mitochondria (M), 3 m in length, are observed. Again, cells preserve their tight junctions (TJ). (Magnification 7,080.)
4. Discussion My colleagues and I have previously reported that the Hep G2 cell line and NHPH are reliable in vitro models for the study of drug- and alcohol-induced hepatotoxicity (Neuman et al., 1993, 1995). In the present work, I studied factors that may contribute to acetaminophen-induced cytotoxicity. In human beings and in animals in vivo, toxic injury depends on the balance between the toxification (i.e., production of active metabolite, which in the case of acetaminophen is N-acetyl-benzoquinone-imine) and detoxification (i.e., conjugation leading to glucuronate or sulphate or GSH conjugate of the toxic metabolite). N-acetyl-benzoquinoneimine produces cytotoxicity to both types of cells (Hep G2 cells and NHPH) almost immediately, whereas acetaminophen has to be first metabolized by the cells and transformed into the toxic metabolite to produce cytotoxicity. Therefore I exposed the cells to the toxic metabolite only for a short period (2 h), whereas exposure to acetaminophen alone or in combination with ethanol was for a longer period (24 h). In the present work, the damage produced by acetaminophen was most severe when cells were exposed to ethanol at 40 mmol/l acetaminophen at 10 mmol/l for 24 h. The toxicity may be partly due to the direct effect of acetaminophen. Ethanol, an inducer of the cytochrome P450 isoform CYP2E1, may also be responsible, as it can enhance the production of reactive oxygen species that may contribute to the cytotoxicity. My colleagues and I (Katz et al., 2001) have shown that ethanol at 40 mM induces cytochrome P450, CYP2E1 activity that may be blocked by preexposure to CYP2E1 inhibitors or to CYP2E1 antibodies. A concomitant exposure to ethanol at 40 mM (which was nontoxic) and to a nontoxic dose of valproic acid resulted in cytotoxicity to NHPH. This toxicity was reduced by inhibitors
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Fig. 6. Transmission electron micrograph of one Hep G2 cell treated with 40 mM ethanol and 0.5 mM N-acetyl-benzoquinone-imine for 2 h. The cell presents a preserved cellular membrane. The apoptotic cell exhibits enlarged endoplasmic reticulum (er), lipid droplets (LD), and normal mitochondria (M). The cytoplasm is more condensed. The pyknotic nucleus (N) presents very condensed chromatin. (Magnification 7,080.)
of CYP2E1, therefore demonstrating the role of CYP2E1 inducibility in the NHPH system (Neuman et al., 2001). In terms of ultrastructural changes, cells exposed to acetaminophen ethanol shared a specific trait: Mitochondria were enlarged and appeared swollen or elongated with disrupted cristae, lacking normal organization (Neuman & Tiribelli, 1995). The combination of 10 mM acetaminophen and 40 mM ethanol was synergistic in producing mitochondrial damage after 24 h. Giant mitochondria, 3 m in length, were observed within cells exposed to this drug combination. Interestingly, the giant mitochondrion did not preserve any of its cristae and was therefore not functional. Apoptosis was observed when cells were treated with N-acetyl-benzoquinone-imine, the metabolite of acetaminophen, in combination with ethanol. Cell membranes were preserved, whereas chromatin and cytoplasm were condensed. I have therefore demonstrated that the combination of ethanol and the active metabolite of acetaminophen induces cell death in vitro. In vivo, Mitchell’s group (Mitchell et al., 1973) suggested that liver damage from acetaminophen occurs when the ability of the cell to detoxify the toxic metabolite N-acetyl-benzoquinone-imine is exceeded. The same group suggested that phenobarbital enhances the hepatotoxic effect of acetaminophen by enhancing the activity of P450 system and therefore increasing the likelihood of hepatic injury (Mitchell & Jollow, 1975). Ethanol seems to enhance the toxicity of acetaminophen by induction of the cytochrome P450 isoform CYP2E1. Furthermore, in human beings, depletion of GSH as a result of inhibition of GSH synthesis by alcohol consumption and by the life style–imposed malnutrition of the alcoholic individual enhances susceptibility to acetaminophen toxicity (Zimmerman & Maddrey, 1995). The findings on GSH content supported my hypothesis that metabolic factors
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may be partly responsible for cytotoxicity caused by the combination of acetaminophen ethanol. The mitochondrial pool of GSH is an important cellular defense and is necessary for the maintenance of vital cell functions (Fernandez-Checa et al., 1996; Garcia-Ruiz et al., 1994, 1995a, 1995b; Hirano et al., 1992; Kaplowitz et al., 1996; Kaplowitz & Tsukamoto, 1996; Neuman et al., 1995; Shear et al., 1999). In the aforecited studies, it has been suggested that the depletion in mitochondrial GSH content could be a result of impaired transport from cytosol (Cohen et al., 1998; Neuman, 2001; Neuman & Tiribelli, 1995). Previously, my colleagues and I described structural and functional changes in mitochondria exposed to ethanol in an in vitro model in Hep G2 cells and in NHPH (Neuman et al., 1995; Neuman & Tiribelli, 1995). No differences in cytoviability were observed when cells were incubated with ethanol at 40 mmol/l or acetaminophen at 5 mmol/l (Fig. 1). High cytoviability also corresponded with normal cytosolic GSH and mitochondrial GSH. However, a significant reduction in cell viability was observed after 24 h of exposure to acetaminophen ethanol, which corresponded with a significant reduction in mitochondrial GSH. The importance of mitochondrial GSH in the development and progression of acetaminophen ethanol–induced damage is supported by the notable cytotoxicity that occurs when mitochondrial GSH is reduced by 24%, as well as by a decrease in cytosolic GSH. The present results point to an increased sensitivity to ethanol when GSH was depleted. I suggest that acetaminophen toxicity to cells could be due partially to its role in depleting mitochondrial GSH in cells. Mitochondrial GSH stabilizes the mitochondrial membrane and therefore reduces the mitochondrial permeability transition. When mitochondrial GSH is depleted, apoptotic signaling is switched on. To my knowledge, this is the first report in which nontoxic doses of ethanol have triggered acetaminophen-induced apoptosis in vitro. Normal human primary hepatocytes are more sensitive to both acetaminophen and ethanol when compared with Hep G2 cells, a phenomenon that may be due to a higher content of cytochrome P450 CYP2E1 found in NHPH (Neuman & Tiribelli, 1995). Concomitant exposure to acetaminophen ethanol may induce toxicity and may be related to the induction of cytochrome P450 CYP2E1 and the production of reactive oxygen species, which, together with a decrease of mitochondrial GSH, can contribute to cell damage. One should exercise caution in extrapolating from data obtained in an in vitro model to in vivo conditions. This article presents findings that may be considered as acute intervention as opposed to most in vivo results in which a daily acute injury is superimposed on a chronic ethanol–induced injury. However, the results of the present study may indicate that when concomitant risk factors are coupled with circumstances not directly related to ethanol, such as prolonged fasting (treatment with serum-free medium which, per se, may reduce GSH deposits), such circumstances might also
enhance susceptibility to acetaminophen-induced hepatotoxicity. Therefore an increase in mitochondrial GSH may play a beneficial role in the prevention and treatment of liver damage induced by multiple medications (e.g., chronic use of acetaminophen in conjunction with chronic alcohol consumption). This work contributes to the understanding of the relation between acetaminophen and alcohol in the pathogenesis of liver damage. The article presents human-derived cells as a model to reproduce some of the, or all, morphological and biochemical changes that may be encountered in biopsy samples of liver tissue and serum samples obtained from patients with alcohol/acetaminophen “misadventure”. Acknowledgments This work is dedicated to my teacher and mentor Prof. Hyman J. Zimmerman whose life-time work on the multifactorial phenomenon of “hepatocytotoxicity” was and will always be inspirational. I thank Lois Lanes from the Department of Pathology, The Hospital for Sick Children, Toronto, Ontario, Canada, for excellent technical assistance in preparing the electron microscopy. Thanks also to Julia Haber and Gady Katz for their help in the preparation of this manuscript and Janice Jerrells ELS, for her expert editorial work on the paper. Part of this work was presented at the ESBRA 2001: 8th Congress of the European Society for Biomedical Research on Alcoholism (Paris, September 16, 2001). References Ballet, F., Bouma, M. E., Wang, S. R., Amit, N., Marais, J., & Infante, R. (1984). Isolation, culture and characterization of adult human hepatocytes from surgical liver biopsies. Hepatology 4, 849–854. Banki, K., Hutter, E., Colombo, E., Gonchoroff, N. J., & Perl, A. (1996). Glutathione levels and sensitivity to apoptosis are regulated by changes in transaldolase expression. J Biol Chem 271, 32994–33001. Baumgarten, H. (1985). A simple microplate assay for the determination of cellular protein. J Immunol Methods 82, 25–37. Belfield, A., & Goldberg, D. M. (1969). Enzyme diversion applied to the kinetic estimation of glucose-6-phosphatase activity. Life Sci 8, 129–135. Bernardi, P. (1996). The permeability transition pore: control points of a cyclosporin A–sensitive mitochondrial channel involved in cell death. Biochim Biophys Acta 1275, 5–9. Cameron, R. G., & Neuman, M. G. (1999). Novel morphologic findings in alcoholic liver disease. Clin Biochem 32, 579–584. Cameron, R. G., Neuman, M. G., Shear, N. H., Katz, G., Bellentani, S., & Tiribelli, C. (1998). Modulation of liver specific cellular response to ethanol in vitro in Hep G2 cells. In Vitro Toxicology 12, 111–122. Chiu, S., & Bhakthan, N. M. G. (1978). Experimental acetaminopheninduced hepatic necrosis: biochemical and electron microscopic study of cysteamine protection. Lab Invest 39, 193–203. Cohen, S. D., Hoivik, D. J., & Khairallah, E. A. (1998). Acetaminopheninduced hepatoxicity. In G. L. Plaa, & W. Hewitt (Eds.), Toxicology of the Liver (2nd ed.) (pp. 159–186). New York: Raven Press. Dixon, M., & Webb, E. C. (1979). Enzymes (3rd ed.). New York: Academic Press. Emeigh Hart, S. G., Wyand, D. S., Khairallah, E. A., & Cohen, S. D. (1996). Acetaminophen nephrotoxicity in the CD-1 mouse. II. Protection by probenecid and AT-125 without diminution of renal covalent binding. Toxicol Appl Pharmacol 136, 161–169.
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