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Regular Articles |
From the Departments of Medicine*
and
Pharmacology and Toxicology,
University of
Louisville School of Medicine, and the Jewish Hospital Heart and Lung
Institute,
Louisville, Kentucky
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cellular machinery involved in the execution of apoptosis includes a family of cysteine proteases termed caspases.18 Although more than a dozen of caspases have been identified up to date, caspase-3 stands out because it is commonly activated in response to various death stimuli.19-21 Two general conceptual pathways have been shown to lead to caspase-3 activation: 1) death signal and receptor systems such as Fas ligand (Fas L)/Fas and tumor necrosis factor (TNF)/TNF receptor (TNFR), and 2) intracellular stress signals such as mitochondrial cytochrome c release.22,23 Although chronic ethanol administration was found to elevate caspase-3 activity and Fas L mRNA expression in the liver,24,25 the signaling pathways of ethanol-induced apoptosis remain primarily unknown.
Systemic administration of specific caspase inhibitors has been widely used to investigate the role of caspases in apoptosis. Several reports have demonstrated that intravenous injection of caspase-3 inhibitors attenuates Fas- and ischemia/reperfusion-induced apoptosis.26-29 Recently, systemic administration of a neutralizing Fas L monoclonal antibody was shown to effectively block the Fas/Fas L system and attenuate apoptosis.30,31 By using these in vivo approaches for the investigation of apoptotic signaling pathway, the present study was undertaken to determine the role of caspase-3 in ethanol-induced hepatic apoptosis and to explore the possible upstream signals. Ethanol was administrated intragastrically with or without an intravenous injection of a caspase-3 inhibitor or a neutralizing Fas L monoclonal antibody. DNA fragmentation was determined using a terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and immunogold electron microscopy. Caspase-3 activation, mitochondrial cytochrome c release, and Fas L expression were monitored by light and electron microscopy, Western blot, and enzymatic assay.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ApopTag in situ apoptosis detection kit was purchased from Intergen Co. (Purchase, NY). Monoclonal hamster anti-mouse Fas ligand (no azide/low endotoxin), monoclonal mouse anti-cytochrome c, and polyclonal rabbit anti-active caspase-3 antibodies were purchased from PharMingen (San Francisco, CA). Polyclonal rabbit anti-Fas ligand antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Normal hamster IgG was purchased from ICN Pharmaceuticals, Inc. (Aurora, Ohio). Biotinylated rabbit anti-mouse IgG and goat anti-rabbit IgG antibodies and horseradish peroxidase (HRP)-streptavidin were obtained from Zymed Laboratories, Inc. (San Francisco, CA). Gold conjugates of protein A, goat anti-mouse IgG, and sheep anti-digoxigenin were obtained from BB International (Cardiff, UK). Caspase-3 inhibitor II (Z-DEVD-FMK), caspase-3 substrate I (Ac-DEVD-pNA), and p-nitroaniline were the products of Calbiochem Corp. (La Jolla, CA). Bicinchoninic acid protein assay kit was the product of Pierce (Rockford, IL). All other chemicals and reagents were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated.
Animals
Male FVB mice, 10 weeks of age, were used in this study. The mice were housed in the animal quarters at the University of Louisville Research Resources Center. They were maintained at 22°C with a 12-hour light/dark cycle and free access to rodent chow and water. The experimental procedures were approved by the Institutional Animal Care and Use Committee, which is certified by the American Association of Accreditation of Laboratory Animal Care.
Experimental Treatments
A binge drinking model developed by Carson and Pruett32 was followed for ethanol challenge with some minor modifications. This model was designed to achieve blood alcohol levels, behavioral effects, and physiological effects comparable to human binge drinking. Animals were randomly assigned into three treatments groups (5 to 7 mice in each group) along with appropriate controls: 1) ethanol, 2) caspase-3 inhibition, and 3) Fas L neutralization. After 6 hours of fasting, animals were administrated 25% (w/v) ethanol at a total accumulative dosage of 6 g/kg body weight by four equally divided gavages in 20-minute intervals. Control mice received the same volume of water. For caspase-3 inhibition, an irreversible caspase-3 inhibitor Z-DEVD-FMK was dissolved in 1% dimethylsulfoxide (DMSO). The inhibitor was injected into the tail vein at a dosage of 20 µg/g body weight before ethanol administration. Control mice received the same amount of 1% DMSO. For Fas L neutralization, a hamster anti-mouse monoclonal antibody was injected into the tail vein at a dosage of 5 mg/kg body weight before ethanol administration. Control mice received the same amount of normal hamster IgG. Twelve hours after ethanol administration, the mice were anesthetized with pentobarbital sodium (5 µg/g body weight), and liver tissues were taken for light and electron microscopy, Western blot analysis, and enzymatic assay.
Tissue Processing for Light and Electron Microscopy
For light microscopy, liver tissues were fixed with 10% formalin
in 0.01 mol/L of phosphate-buffered saline (PBS), pH 7.4, and embedded
in paraplast. Tissue sections of 5 µm were cut and mounted on
silanized slides. For electron microscopy, liver tissues were cut into
1 mm3
and fixed in 2% freshly depolymerized
paraformaldehyde with 0.5% glutaldehyde in 0.1 mol/L of sodium
cacodylate buffer, pH 7.4, for 2 hours. After rinsing in sodium
cacodylate buffer, the samples were partially dehydrated with ethanol
and embedded in LR White resin. Ultrathin sections were cut and
collected on gold grids. Labeled ultrathin sections were observed with
a Philip transmission electron microscope.
Assessment of Apoptosis by Light and Electron Microscopic TUNEL Assay
Apoptosis was assessed by detection of DNA fragmentation using in situ TUNEL assay with both light and electron microscopes. For light microscopic TUNEL, liver slides were processed with an ApopTag in situ apoptosis detection kit (Intergen Co.) according to the manufacture’s instructions. Briefly, liver tissue slides were pretreated with proteinase K and H2O2, and incubated with the reaction mixture containing terminal deoxynucleotidyl transferase (TdT) and digoxigenin-conjugated dUTP for 1 hour at 37°C. The labeled DNA was visualized with HRP-conjugated anti-digoxigenin antibody with diaminobenzidine as the chromagen. Rat mammary gland tissue provided in the kit was used as positive control. For negative control, TdT enzyme was omitted from the reaction mixture. For electron microscopic TUNEL assay, the ultrathin sections were incubated with normal sheep serum for 30 minutes to block nonspecific reactions. The sections were then incubated in the presence of 0.25 U/µl TdT and 0.5 µmol/L of biotinylated dUTP in TdT buffer (0.5 mol/L potassium cacodylate, 2 mmol/L CoCl2, and 0.2 mmol/L dithiothreitol, pH 7.2) for 30 minutes at 37°C. After rinsing in immunogold buffer (0.01 mol/L PBS with 1% normal serum, 1% bovine serum albumin, 0.1% Tween 20, and 0.1% Na3N, pH 8.2), the ultrathin sections were labeled with 10-nm gold-conjugated sheep anti-digoxigenin for 1 hour. The ultrathin sections were then counterstained with uranyl acetate and lead citrate.
Immunoperoxidase Staining of Active Caspase-3, Cytochrome c, and Fas Ligand
After pretreatment, respectively, with 3% H2O2 and 5% normal serum, liver tissue sections were then incubated overnight at 4°C with polyclonal rabbit anti-active caspase-3 antibody, or monoclonal mouse anti-cytochrome c (clone 7H8.2C12) antibody or polyclonal rabbit anti-Fas ligand antibody. Sections were then incubated for 30 minutes in either biotinylated rabbit anti-mouse IgG antibody or biotinylated goat anti-rabbit IgG antibody, followed by incubation with HRP-streptavidin for 20 minutes. The antibody-binding sites were visualized by incubation with a diaminobenzidine-H2O2 solution using a diaminobenzidine kit. Finally, sections were counterstained with 0.5% methyl green.
Immunogold Labeling of Active Caspase-3 and Cytochrome c
Liver ultrathin sections were incubated with either monoclonal mouse anti-cytochrome c antibody or polyclonal rabbit anti-active caspase-3 antibody overnight at 4°C. After rinsing in immunogold buffer (0.01 mol/L PBS with 1% bovine serum albumin, 0.1% Tween, and 0.1% Na3N, pH 8.2), the ultrathin sections were incubated in either 10-nm gold-conjugated rabbit anti-mouse IgG antibody or 10-nm gold-conjugated protein A diluted in immunogold buffer for 1 hour. The ultrathin sections were then rinsed in distilled water and counterstained with uranyl acetate and lead citrate.
Enzymatic Assay of Caspase-3
Fresh liver tissues were homogenized with a Teflon homogenizer in the extraction buffer [25 mmol/L HEPES buffer, pH 7.4, containing 5 mmol/L ethylenediaminetetraacetic acid (EDTA), 2 mmol/L dithiothreitol, and 0.1% CHAPS]. The homogenate was centrifuged at 20,000 x g for 30 minutes. The supernatant was diluted with the assay buffer (50 mmol/L HEPES, 10 mmol/L dithiothreitol, 1.0 mmol/L EDTA, 100 mmol/L NaCl, 0.1% CHAPS, and 10% glycerol, pH 7.4) and incubated at 37°C with 200 µmol/L caspase-3 substrate I (Ac-DEVD-pNA). p-Nitroaniline was used as the standard. Cleavage of the substrate was monitored at 405 nm and the specific activity was expressed in pmol of the product, nitroaniline, per minute per mg protein. For measurement of nuclear caspase-3 activity, hepatic nuclei were isolated as described previously.33 Briefly, liver was perfused, removed, and minced in 2 volumes of ice-cold 0.32 mol/L sucrose in 25 mmol/L Tris-HCl buffer (pH 7.4, containing 3 mmol/L MgCl2, 25 mmol/L KCl, 2 mmol/L EDTA, 0.1 mmol/L spermine, 0.1% CHAPS, and 1% proteinase inhibitor cocktail). The tissue was homogenized with a glass Dounce homogenizer, and filtered through two layers of nylon mesh. The homogenate was centrifuged at 700 x g for 10 minutes. The pellet was resuspended in 2.4 mol/L of sucrose in 25 mmol/L of Tris-HCl buffer and centrifuged at 50,000 x g for 60 minutes. The nuclear pellet was washed in the extraction buffer, homogenized, and centrifuged at 20,000 x g for 30 minutes. The resulting supernatant was used for caspase-3 activity assay. Protein concentration was determined by using a Bicinchoninic acid protein assay kit (Pierce).
Western Blot Analysis of Cytochrome c
Fresh liver tissues were homogenized gently with a glass tissue grinder in a suspension buffer (20 mmol/L HEPES, pH 7.4, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 1.0 mmol/L EDTA, 1.0 mmol/L EGTA, 1.0 mmol/L dithiothreitol, and 1% proteinase inhibitor cocktail) with 0.25 mol/L of sucrose. The crude homogenate was centrifuged at 750 x g for 10 minutes at 4°C and then at 8000 x g for 20 minutes at 4°C. The 8000 x g pellet was homogenized with Teflon homogenizer in the suspension buffer without sucrose and used as the mitochondrial fraction. The 8000 x g supernatant was further centrifuged at 100,000 x g for 60 minutes at 4°C and used as a cytosolic fraction. Aliquots containing 15 µg of protein were loaded on a 15% sodium dodecyl sulfate-polyacrylamide gel. After electrophoresis, protein was transferred to nitrocellulose membrane. The membrane was blocked with 5% nonfat milk in Tris-buffered saline (pH 7.5) and probed with monoclonal mouse anti-cytochrome c. The membrane was then processed with HRP anti-mouse IgG. The protein bands were visualized by an enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL) and quantified by Bio-Rad MultiAnalyst (Bio-Rad Laboratories, Richmond, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ethanol-induced hepatic apoptosis was assessed by detection of DNA
fragmentation using TUNEL assay under both light and electron
microscopies. In comparison to the control mice (Figure 1A)
, ethanol-treated mice showed positive
staining in the liver (Figure 1B)
under light microscopy. The positive
nuclei were found mostly in the perivenous hepatocytes. The staining
intensity was different among the positive cells. The hepatocytes
closer to the central vein showed stronger staining. Under electron
microscopy, only a few gold particles were seen in the nucleus of
hepatocytes of control mice (Figure 1C)
. However, numerous gold
particles were found in the hepatic nucleus of ethanol-treated mice
(Figure 1D)
, indicating the occurrence of DNA fragmentation. Nuclear
condensation of hepatocytes in ethanol-treated mice was apparent, and
the heavy gold labeling was found in the region with a uniformed
structure (Figure 1D)
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Caspase-3 activation associated with ethanol challenge was
examined with a specific antibody to the active form of caspase-3 by
both light and electron microscopies. Light microscopic staining
demonstrated a negative result in the liver of control mice (Figure 2A)
, but a positive result in the
ethanol-treated mice (Figure 2B)
. The distribution of the positive
cells was perivenous. Electron-microscopic immunogold labeling showed
that there was no active caspase-3 in either the cytoplasm (Figure 2C)
or nucleus (Figure 2D)
in control mice. However, positive labeling was
demonstrated in both the cytoplasm (Figure 2E)
and nucleus (Figure 2F)
of the ethanol-treated mice, but mainly localized in the cytoplasm. The
nuclear distribution of active caspase-3 was found in the region with a
uniformed structure. To confirm the translocation of active caspase-3
into the nucleus, caspase-3 activity was determined in the isolated
liver nuclei. A significant increase in the nuclear caspase-3 activity
was found after acute ethanol administration (from 0.60 ± 0.41 to
1.61 ± 0.42 nmol/min/mg protein).
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, acute ethanol treatment
significantly elevated the caspase-3 activity in the liver and
administration of Z-DEVD-FMK inhibited ethanol-induced caspase-3
activation. The TUNEL assay demonstrated that Z-DEVD-FMK attenuated
ethanol-induced hepatic apoptosis (Figure 4)
.
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Cytochrome c is one of the upstream signals for
caspase-3 activation. The effect of ethanol on hepatic cytochrome
c was monitored by light and electron microscopic
immunocytochemistry and Western blot. Under light microscopy,
immunoperoxidase staining of cytochrome c showed moderate,
uniform reactivity in the control liver (Figure 5A)
and strong reactivity was found in
the ethanol-treated liver (Figure 5B)
. By immunogold labeling under
electron microscopy, cytochrome c in the hepatocytes was
found mainly in the mitochondria of hepatocytes in the control liver
(Figure 5C)
, whereas the cytosolic localization was prominent in the
ethanol-treated liver (Figure 5D)
. Using Western blot analysis,
cytosolic elevation of cytochrome c was detected in the
ethanol-treated liver (Figure 6)
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To demonstrate whether the Fas/Fas L system, another upstream
signal for caspase-3 activation, is responsible for ethanol-elevated
caspase-3 activity, Fas L protein expression in the liver was examined
by light microscopic immunoperoxidase staining. In comparison to the
weak staining in the liver of control mice (Figure 7A)
, strong Fas L immunoreactivity was
observed after acute ethanol administration (Figure 7B)
. A neutralizing
Fas L monoclonal antibody was applied to confirm the cause-and-effect
relationship between the Fas/Fas L system and casapse-3
activation-mediated apoptosis. Neutralization of Fas L not only
suppressed caspase-3 activation (Figure 8)
, but also attenuated ethanol-induced
apoptosis (Figure 9)
. Western blotting
analysis was then performed to clarify the possible linkage between the
Fas/Fas L system and mitochondrial cytochrome c release. The
result showed that neutralization of Fas L had no effect on
ethanol-induced mitochondrial cytochrome c release (Figure 10)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we observed that the most TUNEL-positive cells are located around the central vein. However, only a few positive cells showed nuclear condensation, a characteristic morphological change of apoptosis. The TUNEL-positive nuclei with condensation appearance are considered to be the late stage of apoptosis. The condensed nuclei are referred to as apoptotic bodies by conventional light or electron microscopy in human alcoholic liver disease and chronic alcohol-feeding animal models.8,9,12-14,16 The TUNEL-positive nuclei without condensation should be the early stage of apoptosis. In human alcoholic liver disease, the number of apoptotic cells detected by TUNEL assay were much more than the number of hepatocytes containing apoptotic bodies.16 The TUNEL assay identifies DNA strand breaks that are seen in apoptosis. However, DNA strand breaks may also occur late in the terminal evolution of cell necrosis. The complexity of measuring apoptosis thus involves the difficulty of distinguishing apoptosis from necrosis. A fundamental difference between the two mechanisms of cell death is the morphological alteration of the cell. The nuclear modification of the apoptotic cells is accompanied by a preservation of the cytoplasmic structures of the cell. In contrast, immediate loss of membrane integrity occurs in the necrotic cells. This distinction thus far has made the electron microscopic evaluation of morphological changes a most reliable tool for determination of apoptosis. We thus used immunogold electron microscopy to examine hepatocyte apoptosis. In addition to the ultrastructural examination, the immunogold labeling of apoptotic nuclei in hepatocytes provided further evidence of ethanol-induced apoptosis in hepatocytes. Thus both conventional TUNEL and the immunogold electron microscopy demonstrated that acute ethanol administration indeed induced apoptosis in the liver.
Caspase-3 activation in the ethanol-treated liver occurred mostly in the hepatocytes and the immunoperoxidase staining intensity is higher in the cells around the central vein, which closely correlates with the location of apoptotic hepatocytes. Immunogold labeling further detected active caspase-3 in ethanol-treated hepatocytes. Colorimetric enzymatic assay showed a fivefold increase in the caspase-3 activity in the ethanol-treated liver. Activation of caspase-3 by ethanol has been reported previously. Chronic ethanol feeding caused a 37.6-fold enhancement in caspase-3 activity with a 7.9-fold increase in apoptosis in the buccal epithelial cells.34 In the liver of chronic ethanol-treated rats, caspase-3 activity was significantly increased in three types of isolated hepatic cells, Kupffer cells, hepatocytes, and sinusoidal endothelial cells.35 A novel aspect of the present study, however, is that the systemic administration of caspase-3 inhibitor dramatically blocked ethanol-induced caspase-3 activity as well as the DNA fragmentation, indicating caspase-3 is the key mediator of ethanol-induced hepatic apoptosis. Immunogold labeling and electron microscopic examination is a unique method to monitor subcellular localization of active caspase-3. It has been shown by immunogold labeling that procaspase-3 is located in the cytosol.36 In the present study, we detected that active caspase-3 was distributed not only in cytosol but also in the nucleus of ethanol-treated hepatocytes. The nucleus localization of active caspase-3 identified by the immunogold electron microscopy was further confirmed by enzymatic assay using the nucleus fraction. The nuclear distribution of active caspase-3 provides direct evidence that caspase-3 locally cleaves nuclear substrates such as poly(ADP-ribose) polymerase.
The mitochondrial cytochrome c release-mediated caspase-3 activation pathway has been found in various cells in response to different death stimuli.23 There is also evidence that cytochrome c is dispensable for caspase-3 activation and apoptosis.37 In the present study, ethanol-induced cytosolic elevation of cytochrome c was detected by immunoperoxidase staining and immunogold labeling. Cytosolic elevation of cytochrome c has been shown to be accompanied with a decrease in mitochondrial cytochrome c concentrations.23 However, both immunogold electron microscopic and Western blot analyses demonstrated that the elevation of cytosolic cytochrome c was not accompanied with an apparent decrease in the mitochondrial pool. Immunoperoxidase staining also showed stronger staining intensity in the ethanol-treated liver, therefore, indicating that an increase in the total amount of cytochrome c might occur in the ethanol-treated liver. It is thus possible that de novo synthesis of cytochrome c may take place under the treatment of ethanol in the liver.
Growing evidence suggests that Fas/Fas L system is one of most important signaling pathways that mediates caspase-3 activation and apoptosis in liver. Experimental activation of Fas by intraperitoneal injection of a Fas antibody to mice has led to liver failure and animal death as a result of massive hepatocyte apoptosis.38,39 Studies with primary hepatocyte culture also showed that triggering the Fas/Fas L system induces hepatocyte apoptosis through processing and activation of caspase-3, -7, and -9.40,41 Intravenous administration of caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp, blocked Fas antibody-induced caspase-3 and -7 activation and hepatic apoptosis in the mouse liver.26 In the chronic ethanol-feeding animal model and human alcoholic liver disease, elevated expression of Fas and Fas L mRNA were detected in association with increased caspase-3 activity.24,25 To elucidate the cause-and-effect relationship between Fas/Fas L system and caspase-3 activation and apoptosis, a neutralizing Fas L antibody was used to block the Fas/Fas L system. This method has been shown to attenuate apoptosis in vivo in some tissues.30,31 The neutralization of Fas L in this study resulted in a significant inhibition of caspase-3 activation and attenuation of hepatic apoptosis. This result indicates that the Fas/Fas L system may act as a key signal in the ethanol-induced hepatic apoptosis. Fas/Fas L system-induced caspase-3 activation has been shown to be mediated by caspase-8.41 Because caspase-8 can also cause mitochondrial cytochrome c release,42 blocking the Fas/Fas L system might reduce the cytosolic elevation of cytochrome c. However, Western blot analysis in the present study demonstrated that mitochondrial cytochrome c release is independent of the Fas/Fas L system. In the presence of mitochondrial cytochrome c release, however, the hepatic apoptosis was attenuated by blocking the Fas/Fas L system. Therefore, mitochondrial cytochrome c release may not be an important pathway in the acute ethanol-induced hepatic apoptosis.
In conclusion, the data presented in this study indicate that the Fas/Fas L system-mediated caspase-3 activation signaling pathway plays a central role in ethanol-induced hepatic apoptosis. New therapeutic approaches may be expected based on the interference with Fas/Fas L or inhibition of caspase-3 activation.
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Footnotes |
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Supported in part by the University of Louisville Hospital and the Jewish Hospital Foundation, Louisville, Kentucky.
Accepted for publication March 29, 2001.
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