Gliotoxin-Induced Cytotoxicity Proceeds via Apoptosis and Is Mediated by Caspases and Reactive Oxygen Species in LLC-PK1 Cells

Xiaoming Zhou*,1, Aiping Zhao*, Gertrud Goping{dagger} and Przemyslaw Hirszel*

* Division of Nephrology, Department of Medicine; {dagger} Department of Anatomy and Cell Biology, Uniformed Services University, Bethesda, Maryland 20814

Received September 3, 1999; accepted November 10, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Renal failure associated with aspergillosis is caused by pathogenic fungi. Gliotoxin is a toxic epipolythiodioxopiperazine metabolite produced by the pathogens. The present study investigated the cytotoxicity and underlying mechanisms induced by gliotoxin in LLC-PK1 cells, a porcine renal proximal tubular cell line. Gliotoxin at 100 ng/ml did not show a cytotoxic effect, but unmasked a dose-dependent cell death induced by TNF-{alpha}. TNF-{alpha}–induced cell death in the presence of gliotoxin was associated with hypodiploid nuclei and activation of caspase-3–like proteases. Blockade of caspases by boc-aspartyl (OMe)-fluoromethylketone and z-DEVD.fmk inhibited TNF-{alpha}–induced cell death. As the concentrations of gliotoxin were increased, gliotoxin killed the cells directly in a dose-dependent manner. Further analyses of DNA fragmentation, hypodiploid nuclei, mitochondrial membrane potential, and plasma membrane integrity revealed that cell death proceeded via apoptosis. Gliotoxin-induced apoptosis was associated with dose-dependent and time-dependent activation of caspase-3–like proteases. Boc-aspartyl (OMe)-fluoromethylketone attenuated the killing effect. Gliotoxin also increased the intracellular levels of reactive oxygen species as measured by flow cytometry. N-acetylcysteine, a well-known antioxidant, completely abolished the gliotoxin-induced caspase-3–like activity, cytotoxicity, and reactive oxygen species. In conclusion, (1) gliotoxin at 100 ng/ml unmasks the ability of TNF-{alpha}–induced apoptosis, and the effect of TNF-{alpha} is mediated by caspase-3–like proteases; and (2) at higher concentrations gliotoxin itself induces cell death, which is via apoptosis and dependent on caspase-3–like activity and reactive oxygen species.

Key Words: gliotoxin; cytotoxicity; apoptosis; LLC-PK1 cells; TNF-{alpha}; caspase; reactive oxygen species.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gliotoxin is one of the better-known members of the epipolythiodioxopiperazine class of the metabolites produced by a number of Aspergillus and Penicillium species as well as Gliocladium, Thermoascus, and Candida. Although a routine and sensitive assay for detection of gliotoxin in human tissues has yet to be established, it becomes clear that gliotoxin has a definite potential to affect human and animal health through accidental ingestion or in situ production during fungal infections (Waring and Beaver, 1996Go). Epipolythiodioxopiperazines have been unambiguously linked to facial eczema caused in animals grazing in pastures contaminated with the fungus Pithomyces chartarum. Gliotoxin has been isolated from vaginal samples of woman infected with Candida albicans and detected in strains of Eurotium isolated from meat products and in fungal isolates of aspergillus species from human axillary hairs (Waring and Beaver, 1996Go). Perhaps the most dangerous effect of gliotoxin is its potential role in opportunistic infections such as aspergillosis. Aspergillosis is often seen in immunocompromised patients. Gliotoxin has been identified in tissues of mice following development of invasive aspergillosis (Waring and Beaver, 1996Go). Invasive aspergillosis accounts for 30% of mycotic infections and carries a very high rate of mortality, with only 5–20% survival (Pahl et al., 1996Go). Renal failure is frequently associated with invasive aspergillosis (Lorf et al., 1999Go; Singh et al., 1997Go). Recently, acute renal failure resulting from isolated renal aspergillosis in immunocompetent hosts has been also reported (Krishnamurthy et al., 1998Go; Sud et al., 1998Go). However, the pathophysiology underlying aspergillosis-induced renal failure is completely unknown.

Apoptosis is a form of programmed cell death that accompanies a variety of diseases and disease models including renal diseases (Davis and Ryan, 1998Go). Gliotoxin has been shown to induce apoptosis in thymocytes, peripheral lymphocytes, macrophages, and L929 fibroblasts in vitro, and in thymus, spleen, and mesenteric nodes in vivo (Waring and Beaver, 1996Go). Whether gliotoxin also induces apoptosis in renal cells or tissue has not been examined.

One of the major differences that distinguish apoptosis from necrosis is that the plasma membrane remains intact during apoptosis. Central to apoptosis is a group of well-conserved cysteine in active sites, aspartic acid-specific proteases, caspases. At least 13 members of the caspase family have been identified (Schwartz, 1998Go; Thornberry and Lazebnik, 1998Go). Depending on their roles in apoptosis, this family of proteases can be divided into apoptosis initiators (including caspase-8, -9, -10), which participate in transduction of death signals, and apoptosis effectors (such as caspase-3, -6, -7, or caspase-3–like proteases), which execute death signals. Among the members of the caspase family, caspase-3, -6, and -7 are the closest homologues of the Caenorhabditis elegans ced-3 gene product shown to be essential for apoptosis of 131 cells out of 1090 cells born during nematodal development. Apoptosis is a well-orchestrated operation, initiated by a diverse array of proapoptotic stimuli, and culminating in activation of caspase-3–like proteases. These enzymes are either directly or indirectly involved in cutting off contacts with surrounding cells, rearranging the structure of cytoskeleton, shutting down DNA replication and repair, interrupting RNA splicing, destroying DNA, disrupting the nuclear structure, inducing the cell to display signals that mark it for phagocytosis, and disintegrating the cell into membrane-packaged apoptotic bodies (Schwartz, 1998Go; Thornberry and Lazebnik, 1998Go). Given the critical functions that caspases have, inhibition of caspases, especially caspase-3–like proteases, has been shown to prevent the appearance of apoptosis in a wide variety of cells and tissues (Schwartz, 1998Go; Thornberry and Lazebnik, 1998Go).

One of the most important properties of epipolythiodioxopiperazines is their ability to go through a redox cycle in the presence of an appropriate reducing agent. Reduced gliotoxin has been specifically identified in cells following exposure to the toxin (Waring and Beaver, 1996Go). It has been proposed that the reduced gliotoxin undergoes a redox cycling that generates superoxide anion under aerobic conditions; during the conversion of the dithiol derivative to the disulfide form, the superoxide anion is then quickly dismutated to hydrogen peroxide (Waring and Beaver, 1996Go). Hydrogen peroxide, as well as other reactive oxygen species produced by gliotoxin during the redox cycle in a cell-free system, directly damage plasmid and cellular DNA (Eichner et al., 1988Go). Whether gliotoxin-induced reactive oxygen species account for its toxic effect on renal cells remains completely unknown. The present study examined the cytotoxicity induced by gliotoxin, the mode of cell death, and the roles of caspase-3–like activity and reactive oxygen species in cell death in LLC-PK1 cells, a hog renal proximal tubular cell line.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
Gliotoxin, N-acetylcysteine, crystal violet dye and trypan blue dye, and 2'7'-dichlorofluorescin diacetate (DCFH-DA) were purchased from Sigma (St. Louis, MO). Boc-aspartyl (OMe)-fluoromethylketone (BAF) and z-DEVD.fmk were obtained from Enzyme Systems (Livermore, CA). TNF-{alpha} was purchased from Boehringer Mannheim (Gaithersburg, MD). 5, 5', 6, 6'-Tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) was purchased from Molecular Probes (Eugene, OR).

Cell culture and treatment.
LLC-PK1 cells were purchased from American Tissue Culture Collection (Rockville, MD) and used between passage 1 to passage 15 after the original passage number 194. The cells were kept in Medium 199 plus 3% fetal bovine serum in a 37°C incubator supplied with 5% CO2. For assays of cell viability, the cells were plated down at 6 x 104 cells per well (confluent) in a 96-well plate and grew for 18–22 h before treatment. The cells were preincubated with 100 ng/ml gliotoxin for 30 min prior to addition of TNF-{alpha}.

Crystal violet assay.
Cells were stained with 0.5% crystal violet in methanol for 8–10 min at 22°C, then washed three times with 1X phosphate-buffered saline solution. The absorption measured at 550 nm was used as an index for cell viability (Wang et al., 1996Go).

DNA fragmentation assay.
The fragmented DNA in cytoplasma was detected according to Sei et al. (1998). Briefly, after treatment until 80% of the cells detached from dishes, the cells were lysed in 10 mM EDTA, 0.5% Triton X-100, and 5 mM Tris–HCl (pH 8.0), and centrifuged at 10,000 x g for 30 min. The supernatant was digested with 0.1 mg/ml proteinase K at 50°C for 60 min, extracted with phenol/chloroform and precipitated with two volumes of 100% ethanol. The pellet was treated with 0.5 mg/ml RNase. The DNA was resolved in 1.8% agarose gel. The fragmented DNA in the cytosol was also quantified according to Burton (Burton, 1956Go). Briefly, the cell lysates were mixed with 1.5% diphenylamine reactive solution and incubated at 30°C for 24 h. The absorption was measured at A600.

Caspase activity assay.
The activity of caspase-3–like proteases was measured as increases in hydrolysis of fluorogenic tetrapeptide substrate, Ac-DEVD-7-amino-4-methylcoumarin (Ac-DEVD-AMC), according to the manufacturer's instructions (BIOMOL, Plymouth Meeting, PA). Because caspase-7 also cleaves the substrate, the activity obtained here is referred as caspase-3–like activity. Briefly, the cells were lysed in 25 mM HEPES Buffer (pH 7.5) containing 5 mM EDTA, 2 mM DTT, 0.1% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), and 0.1% Triton X-100 at 22°C for 10 min, then the supernatants were taken for measurement of hydrolysis of Ac-DEVD-AMC as a function of time at 22°C.

Cytofluorometric analyses of propidium iodide staining.
The cells were plated at 1 x 106/well in a 6-well plate. Cytofluorometric analyses of propidium iodide staining were performed according to Nicoletti et al. (1991). Briefly, both detached and attached cells were collected and incubated in a hypotonic fluorochrome solution (propidium iodide 0.5 mg/ml in 0.1% sodium citrate plus 0.1% Triton X-100) overnight at 4°C. The propidium iodide fluorescence of each individual nucleus was measured with excitation of 488 nm and emission of 620 nm. The cell debris was excluded from analysis by appropriately raising the forward scatter threshold. Hypodiploid nuclei due to the condensation of nuclear chromatin appeared at sub G0/G1 position.

Mitochondrial membrane potential measurement.
Briefly, the cells were loaded with 3 µg/ml JC-1 at 37°C for 30 min. After gating out small-sized debris, the red and green emitted fluorescence were collected through 575/40 nm (FL2) and 525/40 nm (FL1) bandpass filters, respectively (Salvioliet et al., 1997).

Reactive oxygen species measurement.
The cells were placed at 1 x 106/well in a 6-well plate. The cells were preloaded with 20 µM freshly prepared DCFH-DA in the buffer containing (in mM) 145 NaCl, 5 KCl, 1 Na2HPO4, 1 CaCl2, 0.5 MgSO4, 5 glucose, and 10 HEPES, pH 7.4 at 37°C for 15 min, then treated for 40 min. The cells were analyzed by flow cytometry (Robinson et al., 1997Go).

All numerical data are expressed as means of three experiments +/- standard errors, unless indicated otherwise. Statistical analyses were performed by unpaired t test or analyses of variance (ANOVA) as appropriate. Multiple post comparisons were made by Dunnett analyses.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Low Concentration of Gliotoxin Potentiates TNF-{alpha}–induced Cytolysis, Which Is Mediated by Apoptosis and Caspase-3–like Proteases
Gliotoxin at 100 ng/ml did not elicit cell death up to 24 h incubation (Fig. 1BGo). However, this concentration of gliotoxin unmasked the ability of TNF-{alpha}–induced cytotoxicity as determined by crystal violet assays. In the absence of gliotoxin, TNF-{alpha} did not show a cytotoxic effect up to 100 ng/ml incubated for 24 h. In contrast, in the presence of gliotoxin, TNF-{alpha} exhibited a dose-dependent cytotoxicity. Approximately 78% of the cells were not viable following incubation with 100 ng/ml TNF-{alpha} plus 100 ng/ml gliotoxin for 24 h (Fig. 1CGo). Figure 1AGo shows the morphology of cell death. The cells were placed down at subconfluence in order to enhance the results of phase contrast. Further analyses with flow cytometry revealed that cell death was associated with hypodiploid nuclei, the important characteristic of apoptosis (Fig. 2Go). TNF-{alpha}–induced cytotoxicity in the presence of gliotoxin via an apoptotic pathway was further substantiated by the observation that the effect of TNF-{alpha} was almost completely abolished by 100 µM BAF, a general caspase inhibitor, and by 200 µM z-DEVD.fmk, a tetrapeptide-specific inhibitor of caspase-3–like proteases (Fig. 3Go). In BAF- and z-DEVD.fmk-treated groups, the cells were challenged with 10 ng/ml TNF-{alpha} and 100 ng/ml gliotoxin for 12 h. If the cells were insulted for more than 12 h or with a higher concentration of TNF-{alpha} (30 ng/ml) for 12 h, neither BAF nor z-DEVD.fmk prevented the cells from dying (data not shown). Measurement of hydrolysis of Ac-DEVD-AMC demonstrated that the activity of the caspase-3–like proteases increased by 2.9-fold when the cells were treated with 100 ng/ml TNF-{alpha} plus 100 ng/ml gliotoxin for 4 h (Fig. 4AGo). BAF inhibited the increases in caspase-3–like activity (Fig. 4BGo). Interestingly, TNF-{alpha} alone also increased the hydrolysis of Ac-DEVD-AMC (Fig. 4BGo).



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FIG. 1. Gliotoxin at 100 ng/ml unmasked TNF-{alpha}–induced cytotoxicity and killed the cells directly at 1200 ng/ml. (A) Morphology of cell death. Top panel: no treatment (control). Bottom left panel: gliotoxin (100 ng/ml) and TNF-{alpha} (50 ng/ml) for 1.5 h. Bottom right panel: gliotoxin (1200 ng/ml) for 40 min. The phase-contrast pictures were taken through Olympus IMT-2 microscope. (B) Gliotoxin at 100 ng/ml had no significant effect on cell viability. (C) In the absence of gliotoxin, TNF-{alpha} did not induce cell death (square). However, in the presence of 100 ng/ml gliotoxin, TNF-{alpha} showed a dose-dependent cell-killing effect (diamond). The cell viability was determined by crystal violet assays at 24 h posttreatment. Each experiment was performed in duplicate. a p < 0.05. b p < 0.01 as compared with the group treated with gliotoxin alone shown in Figure 1BGo (n = 5, ANOVA).

 


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FIG. 2. Cytometric analyses revealed that few hypodiploid nuclei were present in controls. However, there was a significant amount of hypodiploid nuclei associated with TNF-{alpha}- or gliotoxin-induced death. The cells (106) were treated with TNF-{alpha} (50 ng/ml) plus gliotoxin (100 ng/ml) or gliotoxin (1200 ng/ml) until 60–80% of the cells detached from the bottom of the well. Both detached and attached cells were collected. The DNA contents were analyzed with propidium iodide-based flow cytometry. Each panel is a representative of five independent experiments. At least 20,000 cells were analyzed in each experiment.

 


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FIG. 3. BAF and z-DEVD.fmk inhibited TNF-{alpha}-induced cytotoxicity in the presence of gliotoxin. The cells were pretreated with 100 ng/ml gliotoxin and BAF or z-DEVD.fmk for 30 min prior to addition of TNF-{alpha}, then incubated for 12 h. The cell viability was determined by crystal violet assays (n = 3). Each experiment was performed in duplicate. a p < 0.01 as compared with the group with no treatment (unpaired t test). b p < 0.05 as compared with the group treated with TNF-{alpha} and gliotoxin (ANOVA).

 


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FIG. 4. TNF-{alpha} increased a dose-dependent hydrolysis of Ac-DEVD-AMC. (A, n = 3). BAF inhibited the increases in hydrolysis of Ac-DEVD-AMC induced by TNF-{alpha} and gliotoxin (B, n = 3). After treatment for 4 h, the cells were lysed, then the supernatants were taken for measurement of hydrolysis of Ac-DEVD-AMC as a function of time at 22°C with fluorometry. Each experiment was performed in duplicate. a p < 0.01 as compared with gliotoxin alone (1.0 +/- 0.0). b p< 0.01 as compared with the group treated with TNF-{alpha} and gliotoxin (A, ANOVA; B, unpaired t test).

 
High Concentration of Gliotoxin Kills the Cells Directly, Which Also Proceeds via Apoptosis and Is Involved with Caspases
Gliotoxin alone induced cell death in a dose-dependent manner with only 24% survival after treatment with 1200 ng/ml gliotoxin for 2.5 h (Fig. 5AGo). Cell death occurred with the presence of dose-dependent DNA fragmentation (Figs. 5B and CGo), hypodiploid nuclei (Fig. 2Go), and decreases in mitochondrial membrane potential (Fig. 6Go), indicating that the death was via apoptosis. Trypan blue exclusion, as well as dose-response and time-course LDH release assays, revealed that the plasma membrane remained largely intact within 2.5 h at examined doses, further substantiating that the cell death did not start with the necrotic pathway (Fig. 7Go). The permeability of plasma membrane increased after the cells were treated with 1200 ng/ml for 6 h, possibly resulting from secondary necrosis (data not shown). It appears that gliotoxin increased total LDH activity. However, the important point is that gliotoxin did not increase release of LDH to the medium. Gliotoxin-induced death was associated with dose-dependent and time-dependent increases in caspase-3–like activity (Figs. 8A and BGo). BAF significantly inhibited cell death, suggesting that the effect of gliotoxin was mediated via caspases (p < 0.01, Fig. 8CGo).



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FIG. 5. Gliotoxin killed the cells in a dose-dependent manner following incubation for 2.5 h (A, n = 4), which was associated with a dose-dependent increase in DNA fragmentation (B and C). The cell viability was determined by crystal violet assays. The DNA fragmentation in the cytoplasma was resolved in 1.8 % agarose gel and quantified with diphenylamine assays (n = 4). Each experiment was performed in duplicate. a p < 0.05. b p < 0.01 as compared with the control: no gliotoxin (ANOVA).

 


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FIG. 6. Gliotoxin decreased mitochondrial membrane potential. After treatment for 1 h, the cells (106) were stained with JC-1 for 30 min and then analyzed with flow cytometry. Each panel is representative of four independent experiments. At least 20,000 cells were analyzed in each experiment.

 


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FIG. 7. Trypan blue exclusion (A, n = 6) and lactate dehydrogenase (LDH) release assays (B and C: square, n = 3) revealed that gliotoxin did not significantly increase blue cells and release of LDH to the medium within 2.5 h at examined doses. The cells were stained with 0.05% trypan blue dye after treatment with 800 ng/ml gliotoxin for 2.5 h (A). At least 200 cells were counted for each assay. LDH assays were performed according to the manufacturer's instructions (Sigma, St. Louis, MO). Total LDH activity (B: diamond) was measured after the cells were treated with 0.1% Triton X-100. The rate of decrease in absorption at 340 nm in the first no gliotoxin or time 0 experiment was arbitrarily set as 1. The rates from rest of experiments were normalized with this experiment. Each experiment was performed in duplicate (B and C).

 


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FIG. 8. Gliotoxin induced dose-dependent and time-dependent hydrolysis of Ac-DEVD-AMC (A and B). BAF inhibited gliotoxin-induced cell death (C). A and C: the cells were treated for 2.5 h. B: the concentration of gliotoxin was 1200 ng/ml. Each experiment was performed in duplicate. a p < 0.01 as compared with the control: no gliotoxin (A, ANOVA, n = 3). b p < 0.05. c p < 0.01 as compared with the control: time 0. d p < 0.01 as compared with the group treated with 800 ng/ml gliotoxin.

 
Gliotoxin-induced Cytotoxicity Is Entirely Dependent on Reactive Oxygen Species
Cytometric analyses with DCFH-DA, a selective fluorescent probe to hydrogen peroxide (Robinson et al., 1997Go), demonstrated that the levels of reactive oxygen species increased upon exposure to gliotoxin (Fig. 9BGo) as the position of the curve shifted toward the right side as compared with the control (Fig. 9AGo). N-acetylcysteine (Fig. 9CGo) dramatically reduced the levels of reactive oxygen species. N-acetylcysteine decreased the levels of reactive oxygen species below control levels, most likely due to reduction of the endogenous reactive oxygen species levels. The antioxidant also totally abolished the cytotoxicity induced by gliotoxin (1200 ng/ml) at as little as 5 mM (Fig. 10AGo), which was associated with a complete inhibition of caspase-3–like activity (Fig. 10BGo). N-acetylcysteine also abolished TNF-{alpha}–induced cytotoxicity in the presence of gliotoxin as well (data not shown). These data indicate that reactive oxygen species mediated gliotoxin-induced caspase-3–like activity and cytotoxicity.



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FIG. 9. Gliotoxin increased intracellular production of reactive oxygen species (B) as compared with the group with no treatment (A). N-acetylcysteine (C) inhibited the production of reactive oxygen species induced by gliotoxin. The cells (106) were preloaded with 20 µM freshly prepared DCFH-DA at 37°C for 15 min, then treated for 40 min before being analyzed. The data are representative of four independent experiments. At least 10,000 cells were analyzed in each experiment.

 


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FIG. 10. N-acetylcysteine (NAC) inhibited gliotoxin (1200 ng/ml)-induced cytotoxicity in a dose-dependent manner (A) and gliotoxin-induced hydrolysis of Ac-DEVD-AMC (B). After treatment with or without NAC for 2.5 h, the cell viability was determined by crystal violet assays, and the hydrolysis of Ac-DEVD-AMC was measured as a function of time at 22°C with fluorometry. Each experiment was performed in duplicate. a p < 0.001 as compared with the group with no NAC (A) or no treatment (B) (A: ANOVA, n = 4, B: unpaired t test, n = 3).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study has shown that gliotoxin induced cytotoxicity via apoptosis in LLC-PK1 cells. Gliotoxin at 100 ng/ml unmasked TNF-{alpha}–induced dose-dependent cell death with morphologic and biochemical hallmarks of apoptosis (Figs. 1, 2, and 4AGoGoGo). During the course of this study, a similar mechanism has also been demonstrated in human granulocytes (Ward et al., 1999Go). At higher doses the toxin killed the cells directly. Whether these observations represent a mechanism that underlies renal failure induced by aspergillosis needs to be further investigated.

Despite the fact that TNF-{alpha} is a well-known potent inducer of apoptosis, LLC-PK1 cells are insensitive to the cytokine, being in accordance with the insensitivity of kidney in vivo (Bohlinger et al., 1996Go). The mechanism underlying the potentiating effect of gliotoxin appears to be due to its inhibitory effect on the activation of a transcription factor, NF-{kappa}B (Pahl et al., 1996Go; Ward et al., 1999Go; Zhou et al., 1998Go). The NF-{kappa}B is a survival signal demonstrated in a variety of cells and tissues (Magnusson and Vaux, 1999Go; Ward et al., 1999Go; Wang et al., 1996Go; Zhou et al., 1998Go).

The mechanisms that underlie gliotoxin-induced apoptosis remain largely unclear. A recent study on thymocytes from Waring's group has shed light on this issue (Waring et al., 1997Go). Gliotoxin induced phosphorylation of histone H3, thus increasing sensitivity of chromatin to nuclease digestion. The effect of gliotoxin is mediated by protein kinase A, as gliotoxin raised cyclic AMP levels and the activity of protein kinase A, and its effects on H3 phosphorylation and apoptosis were inhibited by a number of specific inhibitors of protein kinase A (Waring et al., 1997Go). The identity of the nuclease responsible for gliotoxin-induced apoptosis remains unknown. An endonuclease identified to be responsible for internucleosomal DNA degradation is caspase dependent (Sakahira et al., 1998Go). Based on the inhibitory effect of z-VAD, a general inhibitor of caspases, Ward et al (1999) have suggested that gliotoxin-induced apoptosis in the human granulocytes is mediated by caspases. It is noteworthy that the complexity of the mechanisms underlying caspase-3, apoptosis, and cell death has not been well understood. A caspase inhibitor may inhibit apoptosis but may not block cell death (Deas et al., 1998Go; Trapani et al., 1998Go). The present study has provided direct evidence that caspase-3 was involved in cell death potentiated or induced by gliotoxin. Moreover, inhibition of caspases by BAF significantly reduced cell death (Figs. 3, 4, and 8GoGoGo), although the cytoprotective effect diminished as the cells were treated with gliotoxin for a long period of time or by a high dose of insult (Figs. 3 and 8CGoGo).

This study found that TNF-{alpha} alone also increased hydrolysis of DEVD-AMC, even though the cells appeared perfectly healthy. The activation of caspase-3 without apoptosis is not a unique phenomenon observed only here. Wilhelm et al. (1998) have shown that T lymphocytes acquired high intracellular caspase-3–like activity upon activation by mitogens and IL-2 without evidence of apoptosis. Whether a checkpoint exists further downstream in the apoptotic pathway or the caspases have a role outside cell death remains to be further investigated.

A variety of toxins and chemicals induce cytotoxicity via reactive oxygen species (Savolainen et al., 1998Go). Gliotoxin is not exceptional (Figs. 9 and 10GoGo). Although the cell death was accompanied by a rise in reactive oxygen species levels, it is difficult to prove that such a rise was the cause rather than a consequence of cell death. However, in view of the ability of gliotoxin to go through redox cycle and the completely inhibitory effect of N-acetylcysteine, we believe that the increases in the levels of reactive oxygen species entirely mediated the cell death. N-acetylcysteine also inhibited the caspase-3–like activity, suggesting that reactive oxygen species activate caspase-3–like proteases, then apoptosis and cell death follow. However, the effect of reactive oxygen species is not solely mediated via caspases, as the cytoprotection promoted by N-acetylcysteine was more potent than that of BAF.


    ACKNOWLEDGMENTS
 
Authors are greatly indebted to Drs. Sonia Doi and Donald Sellitti (Uniformed Services University) for their generous help and thoughtful discussions during the course of the study. Authors appreciate Drs. Douglas Fambrough (The Johns Hopkins University) and Maurice Burg (NIH) for their discussions. Authors also thank Ms. Karen Wolcott (Uniformed Services University) for her expertise assistance in flow cytometry assays, and Drs. Aki Iwagaki and Matthew Pollack (Uniformed Services University) for their help and discussions.


    NOTES
 
1 To whom correspondence should be addressed at Department of Medicine, Uniformed Services University, 4301 Jones Bridge Road, Bethesda, MD 20814. Fax: (301) 295–3557. E-mail: xiazhou{at}usuhs.mil. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bohlinger, I., Leist, M., Gantner, F., Angermuller, S., Tiegs, G., and Wendel, A. (1996). DNA fragmentation in mouse organs during endotoxic shock. Am. J. Pathol. 149, 1381–1393.[Abstract]

Burton, K. (1956). A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 315–323.[ISI]

Davis, M. A., and Ryan, D. H. (1998). Apoptosis in the kidney. Toxicol. Pathol. 26, 810–825.[ISI][Medline]

Deas, O., Dumont, C., MacFarlane, M., Rouleau, M., Hebib, C., Harper, F., Hirsch, F., Charpentier, B., Cohen, G. M., and Senik, A. (1998). Caspase-independent cell death induced by anti-CD2 or staurosporine in activated human peripheral T lymphocytes. J. Immunol. 161, 3375–3383.[Abstract/Free Full Text]

Eichner, R. D., Waring, P., Geue, A. M., Braithwaite, A. W., and Mullbacher, A. (1988). Gliotoxin causes oxidative damage to plasmid and cellular DNA. J. Biol. Chem. 263, 3772–3777.[Abstract/Free Full Text]

Krishnamurthy, R., Aparajitha, C., Abraham, G., Shroff, S., Sekar, U., Kuruvilla, S. (1998). Renal aspergillosis giving rise to obstructive uropathy and recurrent anuric renal failure. Geriatr. Nephrol. Urol. 8, 137–139.[Medline]

Lorf, T., Braun, F., Ruchel, R., Muller, A., Sattler, B., and Ringe, B. (1999). Systemic mycoses during prophylactical use of liposomal amphotericin B (Ambisome) after liver transplantation. Mycoses 42, 47–53.[ISI][Medline]

Magnusson, C., and Vaux, D. L. (1999). Signalling by CD95 and TNF receptors: not only life and death. Immunol. Cell. Biol. 77, 41–46.[ISI][Medline]

Nicoletti, I., Migliorati, G., Pagliacci, M. C., Grignani, F., and Riccardi, C. (1991). A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods 139, 271–279.[ISI][Medline]

Pahl, H. L., Krauss, B., Schulze-Osthoff, K., Decker, T., Traenckner, E. B., Vogt, M., Myers, C., Parks, T., Warring, P., Muhlbacher, A., Czernilofsky, A. P., and Baeuerle, P. A. (1996). The immunosuppressive fungal metabolite gliotoxin specifically inhibits transcription factor NF-{kappa}B. J. Exp. Med. 183, 1829–1840.[Abstract]

Robinson, J. P. (1997). Oxidative metabolism. In Current Protocols in Cytometry (J. P. Robinson, Z. Darzynkiewicz, P. N. Dean, L. G. Dressler, P. S. Rabinovitch, C. C. Stewart, H. J. Tanke, and L. L. Wheeless, Eds.) John Wiley & Sons, Inc., New York.

Sakahira, H., Enari, M., and Nagata, S. (1998). Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 391, 96–99.[ISI][Medline]

Salvioli, S., Ardizzoni, A., Franceschi, C., and Cossarizza, A. (1997). JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess delta psi changes in intact cells: implications for studies on mitochondrial functionality during apoptosis. FEBS Lett. 411, 77–82.[ISI][Medline]

Savolainen, K. M., Loikkanen, J., Eerikainen, S., and Naarala, J. (1998). Interactions of excitatory neurotransmitters and xenobiotics in excitotoxicity and oxidative stress: glutamate and lead. Toxicol. Lett. 102-103, 363–367.

Schwartz, S. M. (1998). Cell death and the caspase cascade. Circulation 97, 227–229.[Free Full Text]

Sei, Y., Fossom, L., Goping, G., Skolnick, P., and Basile, A. S. (1998). Quinolinic acid protects rat cerebellar granule cells from glutamate-induced apoptosis. Neurosci. Lett. 241, 180–184.[ISI][Medline]

Singh, N., Arnow, P. M., Bonham, A., Dominguez, E., Paterson, D. L., Pankey, G. A., Wagener, M. M., and Yu, V. L. (1997). Invasive aspergillosis in liver transplant recipients in the 1990s. Transplantation 64, 716–720.[ISI][Medline]

Sud, K., D'Cruz, S., Kohli, H. S., Jha, V., Gupta, K. L., Chakrabarti, A., Joshi, K., and Sakhuja, V. (1998). Isolated bilateral renal aspergillosis: an unusual presentation in an immunocompetent host. Ren. Fail. 20, 839–843.[ISI][Medline]

Trapani, J. A., Jans, D. A., Jans, P. J., Smyth, M. J., Browne, K. A., and Sutton, V. R. (1998). Efficient nuclear targeting of granzyme B and the nuclear consequences of apoptosis induced by granzyme B and perforin are caspase-dependent, but cell death is caspase-independent. J. Biol. Chem. 273, 27934–27938.[Abstract/Free Full Text]

Thornberry, N. A., and Lazebnik, Y. (1998). Caspases: enemies within. Science 281, 1312–1316.[Abstract/Free Full Text]

Wang, C. Y., Mayo, M. W., and Baldwin, A. S., Jr. (1996). TNF-{alpha} and cancer therapy-induced apoptosis: potentiation by inhibition of NF-{kappa}B. Science 274, 784–787.[Abstract/Free Full Text]

Ward, C., Chilvers, E. R., Lawson, M. F., Pryde, J. G., Fujihara, S., Farrow, S. N., Haslett, C., and Rossi, A. G. (1999). NF-kappaB activation is a critical regulator of human granulocyte apoptosis in vitro. J. Biol. Chem. 274, 4309–4318.[Abstract/Free Full Text]

Waring, P., and Beaver, J. (1996). Gliotoxin and related epipolythiodioxopiperazines. Gen. Pharmacol. 27, 1311–1316.[Medline]

Waring, P., Khan, T., and Sjaarda, A. (1997). Apoptosis induced by gliotoxin is preceded by phosphorylation of histone H3 and enhanced sensitivity of chromatin to nuclease digestion. J. Biol. Chem. 272, 17929–17936.[Abstract/Free Full Text]

Wilhelm, S., Wagner, H., and Hacker, G. (1998). Activation of caspase-3-like enzymes in non-apoptotic T cells. Eur. J. Immunol. 28, 891–900.[ISI][Medline]

Zhou, X., Doi, S., Iwagaki, A., and Hirszel, P. (1998). Inactivation of nuclear factor-{kappa}B (NF-{kappa}B) Potentiates tumor necrosis factor-{alpha} (TNF-{alpha})-induced cytotoxicity in a renal proximal tubular cell line. J. Am. Soc. Nephrol. 9, A2297. (Abstract).