* Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, 4301 W. Markham St. (Mailslot 638), Little Rock, Arkansas 72205; and
Department of Pathology, University of Texas Health Science Center, Houston, Texas
Received October 25, 2001; accepted January 8, 2002
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ABSTRACT |
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Key Words: acetaminophen; liver failure; cell death; apoptosis; oncosis; necrosis; caspases.
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INTRODUCTION |
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MATERIALS AND METHODS |
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Experimental protocol.
Animals were fasted overnight before the experiments. They were injected intraperitoneally with acetaminophen (300500 mg AAP/kg) dissolved in phosphate buffered saline or vehicle (16 ml/kg). The animals were sacrificed at various time-points between 30 min and 24 h after AAP administration. As a positive control for apoptosis, mice were treated with 700 mg/kg galactosamine and 100 µg/kg endotoxin for 6 h (Jaeschke et al., 1998; Lawson et al., 1998
). A blood sample was collected from the vena cava with a heparinized syringe. Samples of each liver were fixed in phosphate-buffered formalin for histological analysis, snap-frozen in liquid nitrogen, or immediately homogenized for caspase-3 activity measurements and Western blotting.
Analytical procedures.
Plasma was used for determination of alanine aminotransferase (ALT) activity with test kit DG 159-UV (Sigma Chemical, St. Louis, MO). Caspase-3 activities were determined as described in detail (Jaeschke et al., 1998). Briefly, a liver sample was homogenized in 25 mM HEPES buffer (pH 7.5) containing 5 mM EDTA, 2 mM DTT, and 0.1% CHAPS. After centrifugation at 14,000 g, the diluted supernatant was assayed for caspase activity using the synthetic fluorogenic substrate Ac-DEVD-MCA (Acetyl-Asp-Glu-Val-Asp-4-methylcoumaryl-7-amide; Peptide Institute, Osaka, Japan) for caspase 3 (CPP32) at concentrations of 50 µM. The samples were assayed in duplicate wells, with or without 10 µM pancaspase inhibitor Z-VAD-fmk (Z-Val-Ala-Asp-fluoromethylketone, Alexis Corp., San Diego, CA). The kinetics of the proteolytic cleavage of the substrate was monitored in a fluorescence microplate reader (Cytofluor 2350, Millipore, Bedford, MA) using an excitation wavelength of 360 nm and an emission wavelength of 460 nm. Caspase activity was calculated from the slope of the recorder trace and expressed in
F/min/mg protein. All caspase activities are reported as ZVAD-inhibitable enzyme activities. Protein concentrations in the supernatant were assayed using the bicinchoninic acid kit (Sigma). Caspase-3 processing was evaluated by Western blot analysis as described (Bajt et al., 2000
, 2001
). Liver tissue was homogenized in 25 mM HEPES (pH 7.5) containing 5 mM EDTA, 2 mM DTT, 0.1% CHAPS, 1 mg/ml pepstatin, leupeptin, and aprotinin. Homogenates were centrifuged at 14,000 x g at 4°C for 20 min. Cytosolic extracts (50 µg per lane) were resolved by 420% SDSpolyacrylamide gel electrophoresis under reducing conditions. After transfer to polyvinylidine difluoride membranes (PVDF, Immobilin-P, Millipore, Bedford, MA), the membranes were first blocked with 5% milk overnight at 4°C followed by incubation with primary antibody for 2 h at room temperature. A goat anti-caspase 3 polyclonal IgG (Santa Cruz Biotechnology) was used as a primary antibody. The membranes were washed and then incubated with the secondary antibody anti-goat IgG-HRP (Santa Cruz Biotechnology). Proteins were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) according to the manufacturer's instructions.
Histology.
Formalin-fixed tissue samples were embedded in paraffin and 5-µm sections were cut. Replicate sections were either stained with hematoxylin and eosin (H&E) for evaluation of necrosis and apoptosis (Gujral et al., 2001) or stained with the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay (Roche Molecular Biochemicals, Indianapolis, IN). The numbers of apoptotic hepatocytes were counted in 10 high-power fields (x400 magnification) using a KF2 microscope (Carl Zeiss, Inc., Thornwood, NY). Preliminary counts in untreated control livers confirmed that this area contained an average of 1800 hepatocytes. Apoptotic cells were identified by morphological criteria (cell shrinkage, chromatin condensation and margination, apoptotic bodies) and by staining with the TUNEL assay. Cell necrosis was evaluated in replicate sections stained with hematoxylin and eosin. The percent of necrosis was estimated by evaluating the number of microscopic fields with necrosis compared to the entire histologic section. All histological evaluations were done in a blinded fashion by 2 investigators (A.F. and J.S.G.).
Statistics.
Data are given as mean ± SE. Comparisons between multiple groups were performed with 1-way ANOVA followed by Bonferroni t-test. If the data were not normally distributed, the Kruskal-Wallis Test (nonparametric ANOVA) followed by Dunn's Multiple Comparisons Test was performed; p < 0.05 was considered significant.
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RESULTS |
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Recently, it was reported that fed mice developed liver injury and apoptosis 12 h after injection with 300 mg/kg AAP (Zhang et al., 2000). Therefore, we repeated our experiment with fed mice and evaluated apoptosis and necrosis 12 h after AAP injection. Consistent with the feeding-dependent diurnal rhythm of liver glutathione (Jaeschke and Wendel, 1985
), fed mice, which received 300 mg/kg AAP in the evening, had extensive liver injury (ALT: 6270 ± 1395 IU/l; n = 3). In contrast, injection of AAP during the peak levels of hepatic glutathione in the morning did not result in any relevant liver injury (ALT: 140 ± 20 IU/l). Despite the difference in liver injury, there was no evidence of any significant changes in hepatic caspase-3 activity compared to controls or between the 2 AAP-treated groups (1.1 ± 0.6
F/min/mg protein). Quantitation of apoptotic hepatocytes showed a slightly higher number of apoptotic cells in the AAP-treated group with injury (3.5 ± 0.9 cells/10 HPF) compared to the one without injury (0.5 ± 0.5). However, even the elevated numbers are in the range observed with starved mice (Fig. 4
).
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DISCUSSION |
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A ladder pattern of DNA fragments on an agarose gel created by multiples of 180-bp fragments (Nagata, 2000) and leakage of DNA/histone fragments into the cytosol (Leist et al., 1995
) are characteristic features of apoptotic cells. Furthermore, DNA strand breaks, as indicated by the TUNEL assay, also occur in apoptotic cells. However, none of these assays is absolutely specific for an apoptotic cell (Dong et al., 1997
; Grasl-Kraupp et al., 1995
; Gujral et al., 2001
). DNA ladders (Dong et al., 1997
) and DNA/histone fragments in the cytosol Gujral et al., 2001
; Lawson et al., 1998
) have been observed in oncotic cells. Moreover, both apoptotic and oncotic cells can be TUNEL-positive (Grasl-Kraupp et al., 1995
; Gujral et al., 2001
). However, as clearly demonstrated in Figure 3
, the staining pattern shows a distinct nuclear staining in apoptotic hepatocytes from Gal/ET-treated livers compared to generalized staining of the entire cell during an oncotic process induced by AAP toxicity. Furthermore, a clear difference in the size of DNA fragments released into the plasma of animals was also found after Fas receptor-mediated apoptosis compared to AAP-induced necrosis (Jahr et al., 2001
). A spectrum of multiples of 180-bp fragments dominates after Fas receptor-induced apoptosis (Jahr et al., 2001
), which is characteristic for caspase-activated endonucleases (Nagata, 2000
). However, 6 h after AAP administration, the overall concentration of DNA in plasma is almost 10-fold higher compared to Fas-induced apoptosis. In addition, the majority of DNA fragments are >10,000 bp (Jahr et al., 2001
), which is characteristic for karyolysis during oncotic necrosis. Thus, DNA fragmentation and positive TUNEL staining during Fas or TNF receptor-mediated apoptosis and AAP-induced cell injury appear to be the result of different processes. These findings further question if apoptotic cell death is relevant for the pathophysiology of AAP-induced liver failure.
Extensive investigations into the signaling mechanisms of receptor-mediated apoptosis demonstrated a critical role of the caspase cascade of proteases (Cohen, 1997). Proteolytic processing of constitutively present procaspases leads to the formation of the active enzymes (Cohen, 1997
). Caspase-3 is one of the prominent downstream effector caspases. The processing of this enzyme and the increased enzyme activity are easily detectable in the liver during TNF-
(Jaeschke et al., 1998
Jaeschke et al., 2000) and Fas receptor-mediated hepatocellular apoptosis in vivo (Bajt et al., 2000
, 2001
). Furthermore, pancaspase inhibitors effectively prevent processing of this enzyme and protect against receptor-mediated apoptosis (Jaeschke et al., 1998
; 2000; Lawson et al., 1999
). In striking contrast to these findings, caspase-3 is not processed during the first 24 h after AAP administration (Fig. 6
), there is no increase in caspase-3 activity, and pancaspase inhibitors do not prevent AAP-induced liver injury (Lawson et al., 1999
). In fact, AAP treatment actually inhibits Fas receptor-mediated apoptosis (Lawson et al., 1999
), presumably by causing mitochondrial injury and interrupting the vital signal transduction through the mitochondria (Knight et al., 2001a
). Thus, AAP not only fails to induce apoptotic signaling mechanisms in hepatocytes by itself, but AAP even prevents the execution of the intracellular signaling cascade of Fas-receptor-mediated apoptosis.
Apoptosis and oncosis are not completely independent processes (Lemasters, 1999). In particular, when a large number of cells are undergoing apoptosis, the process may switch to "secondary necrosis," which then may become indistinguishable from oncotic necrosis (Levin et al., 1999
). However, as shown with the Fas antibody model, such a mechanism starts out with all the characteristic features of apoptosis, i.e., apoptotic morphology, caspase activation, and DNA fragmentation without cell content release (Bajt et al., 2000
). Only at later stages does secondary necrosis with cell lysis occur. However, many of the apoptotic features such as caspase activation are still detectable at this time (Bajt et al., 2000
). In contrast, in our detailed time course evaluation of AAP-induced liver injury, we could not find, at any time, evidence for relevant morphological or biochemical changes characteristic of apoptosis. The observations made with AAP overdose, especially when compared to the secondary necrosis seen after Fas receptor-induced apoptosis (Bajt et al., 2000
), clearly suggest that this cell injury is the result of an oncotic process and not secondary necrosis.
In summary, our data showed the time-dependent development of severe hepatocellular injury after various doses of AAP. Based on morphological evaluation, we found that the number of apoptotic hepatocytes was very limited and never exceeded 0.35% of all injured cells. These findings were corroborated by lack of caspase-3 processing and the absence of any increase in caspase-3 enzyme activities in these livers. In contrast, extensive, confluent centrilobular oncotic necrosis of up to 60% of all hepatocytes was observed. Therefore, we conclude that oncotic necrosis and not apoptosis is the predominant mode of cell death during AAP overdose in mice.
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ACKNOWLEDGMENTS |
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NOTES |
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