Division of Toxicology, Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana
Received April 1, 1999; accepted June 29, 1999
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ABSTRACT |
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Key Words: rotenone; apoptosis; mitochondrial permeability transition; liver cells.
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INTRODUCTION |
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The consistent characteristics of apoptosis, produced by a variety of stimuli and in a number of different cell types, appear to indicate a common effector pathway(s). The consistent observation of mitochondrial dysfunction prior to the nuclear changes associated with apoptotic cell death implies that it may be a critical regulator of the metabolic events involved in the apoptotic cascade (Deckwerth and Johnson, 1993; Jacobson et al., 1994
; Newmeyer et al., 1994
; Petit et al., 1995
; Schulze-Osthoff et al., 1994
; Vayssiere et al., 1995
; Zamzami et al., 1995a
,b
, 1996a
). Furthermore, in cell-free systems, mitochondria are a necessary component of the cytosolic fraction to produce apoptotic features in isolated nuclei (Newmeyer et al., 1994
). Subsequent evidence revealed only mitochondria undergoing the mitochondrial membrane permeability transition (MPT) are pro-apoptotic in this system (Zamzami et al., 1996a
).
The MPT involves the formation of a non-specific pore across the inner mitochondrial membrane permitting the free distribution of ions, solutes, and small-molecular-weight molecules (<1500 Da) across the membrane (Bernardi et al., 1994; Zoratti and Szabo, 1995
). The collapse of the mitochondrial membrane potential (
m) and uncoupling of the electron transport chain from ATP production have been shown to promote MPT (Bernardi et al., 1994
; Kroemer et al., 1995
). Additionally, consequential to the disruption or collapse of the
m and the induction of the MPT, is the loss of matrix Ca2+ and glutathione, increased oxidation of thiols, and further depolarization of the inner mitochondrial membrane, which increase the gating potential for the MPT pore. As the consequences of the MPT are also involved in the induction of the MPT, the MPT may function as a self-amplifying "switch" that, once activated, irreversibly commits the cell to apoptosis (Bernardi et al., 1994
; Kroemer et al., 1995
; Zoratti and Szabo, 1994
). Additionally, a variety of known apoptogens, including the oxidizing agents menadione and hydrogen peroxide, thiol agents such as diamide, adenine nucleotide-translocating ligands such as atractyloside, adenine nucleotide-depleting agents, and mitochondrial transmembrane-potential (
m) reducing agents such as rotenone and mClCCP induce the MPT (Zoratti and Szabo, 1995
).
Several agents, including cyclosporin A, prevent opening of the MPT pore. Cyclosporin A binds to cyclophilin, a peptidyl proylyl cis-trans-isomerase located in the inner mitochondrial matrix (Broekemeier et al., 1989; Halestrap and Davidson, 1990
). It has been proposed that when bound by cyclosporin A, cyclophilin remains inactive, thereby maintaining the MPT pore in a closed state (Broekemeier et al., 1989
; Halestrap and Davidson, 1990
). Treatment with inhibitors of the permeability transition pore and subsequently inhibition of the MPT prevent apoptosis in a variety of cell types, following treatment with a variety of apoptogenic stimuli (Kroemer et al., 1995
; Zamzami et al., 1996b
). Therefore, inhibition of the MPT by cyclosporin A appears to prevent the cascade of events leading to apoptotic cell death.
The naturally occurring pesticide, rotenone, is derived from the Derris and Lonchorcarpus species root and bark. The pesticidal activity of rotenone is attributed to irreversible binding and inactivation of complex I of the mitochondrial electron transport chain, thereby inhibiting oxidative phosphorylation (Lindahl and Oberg, 1961). Rotenoids have demonstrated anticancer activity in chemically induced preneoplastic lesions in mammary organ culture and inhibition of papillomas in the 2-stage mouse-skin model (Gerhauser et al., 1995
). Furthermore, the most abundant rotenoid, rotenone, demonstrated anticancer activity against hepatic tumor formation in B6C3F1 mice following chronic treatment (Abdo et al., 1988
) and in an initiation-promotion protocol (Isenberg et al., 1997
). Previous studies by our group attributed the anticancer activity of rotenone to the induction of apoptosis (Isenberg et al., 1997
).
Mitochondrial respiratory chain inhibitors induce apoptosis in a variety of cell types including primary cultured hepatocytes (Pastorino et al., 1995b, 1993
; Wolventang et al., 1994; Zamzami et al., 1996a
). Wolventang et al (1994) examined the effect of several inhibitors of mitochondrial energy metabolism on apoptosis in several cell lines. Following treatment with inhibitors of mitochondrial respiration and uncoupling agents, apoptosis was induced in these cell lines (Wolventang et al., 1994). Inhibition of the electron transport chain following treatment with rotenone, anoxic conditions, cyanide, or 1-methyl-4-phenylpyridinium (MPP+) demonstrated that MPT is critical in the killing of cultured hepatocytes (Pastorino et al., 1993
; Seaton et al., 1998
; Synder et al., 1993). Cyclosporin A has been shown to prevent the MPT (Pastorino et al., 1995b
) and apoptosis induced by anoxia, rotenone, MPP+ or cyanide (Pastorino et al., 1995b
, 1993
; Seaton et al., 1998
). These studies indicate inhibitors of mitochondrial energy metabolism alter mitochondrial function (reduce the
m) and this alteration appears to be an important event in the induction of apoptosis in these cells.
Reduced energy metabolism has been linked to a decrease in the m (Lindahl and Oberg, 1961
; Pastorino et al., 1993
, 1995b
). Furthermore, inhibition of the electron transport chain by mitochondrial poisons, such as rotenone, has been shown to reduce the
m (Zamzami et al., 1995b
). Maintenance of the
m is necessary for cell survival and the disruption or collapse of the
m is consistently observed in preapoptotic cells following treatment with mitochondrial poisons (Deckwerth and Johnson, 1993
; Kroemer et al., 1995
; Petit et al., 1995
; Zamzami et al, 1995a
,b
). In addition to mitochondrial poison-induced apoptosis, reduction of the
m has been observed prior to the characteristic nuclear morphology associated with apoptosis in nerve growth factor (NGF)-deprived sympathetic neurons, tumor necrosis factor-alpha (TNF-
)-stimulated U937 cells, Fas ligation, ceramide treatment and gamma irradiation (Deckwerth and Johnson, 1993
; Garcia-Ruiz et al., 1997
; Scaife (1966); Zamzami et al., 1995a
,b
). Thereby further implicating the role for mitochondrial dysfunction as a central element in the execution of apoptotic cell death.
To further characterize the anti-carcinogenic effect of rotenone through the induction of apoptosis, the present study examined the effect of rotenone on apoptosis in rat liver cells. Since rotenone is a mitochondrial poison and mitochondrial dysfunction has been observed in preapoptotic cells, the mechanism of rotenone-induced apoptosis was examined through evaluation of the MPT.
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MATERIALS AND METHODS |
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Cell Culture
WB-F344 cells, a rat-liver cell line, were plated at 1 x 106/60 mm2 culture dish, with a glass coverslip for apoptosis and MitoTracker Red studies and without coverslips in cytofluorometric studies. Cells were allowed to attach and were grown in asynchronous culture for 24 h in DMEM/F12 media containing 5% FBS, dexamethasone, insulin, and antibiotics. The media were then replaced with fresh serum-free media containing the test compounds, for the selected treatment duration. In all experiments involving cyclosporin-A treatment, the media were removed one h prior to treatment and were replaced with fresh serum-free media with or without cyclosporin A. Following one-h pretreatment with cyclosporin A, the media were removed and replaced with fresh serum-free media containing the test compounds, cyclosporin A or cyclosporin A and the test compounds, for the selected treatment duration. All test compounds were dissolved in DMSO and DMSO was examined as a solvent control. Cultures were maintained at 37°C in a humidified atmosphere at 95% O2 and 5% CO2.
Measurement of Apoptosis
Apoptosis was quantified by the detection of condensed chromatin with the fluorochrome, Hoechst 33258, as previously described (Bayly et al., 1995; James and Roberts, 1997) with modifications. To assess the effect of rotenone, atractyloside, and cyclosporin A on liver-cell death by apoptosis, fresh serum-free media with or without the test compounds were added to culture dishes containing glass coverslips. Following the selected treatment duration, liver cells were fixed with 4% paraformaldehyde (pH 7.4) for 10 min. Cells were then stained with Hoechst 33258 (5 ng/ml physiological saline) for 5 min and washed twice in distilled water. The coverslips were removed and mounted on glass microscope slides with Supermount® (Biogenex, San Ramon, CA) to achieve optimal fluorescence. The slides were examined on a Nikon Diaphot inverted microscope utilizing a 350-nm excitation and a 460-nm emission-fluorescent filter. Apoptotic cells were identified by brightly staining condensed chromatin and morphological appearance under phase-contrast conditions. The number of apoptotic cells or apoptotic bodies (cells that generated multiple apoptotic bodies were scored as one) was divided by the total number of cells observed and multiplied by 100 to achieve an apoptotic index. At least 30005000 cells per treatment group were counted.
Measurement of Mitochondrial Membrane Potential (m)
MitoTracker Red measurement of the mitochondrial membrane potential (m).
The m was quantified by microscopic examination of cells stained with the fluorochrome MitoTracker Red (CMXRos) and confirmed by cytofluorometric analysis of cells stained with DiOC6(3). Briefly, CMXRos is an aldehyde fixable cationic lipophilic fluorochrome that passively diffuses through the plasma membrane of viable cells and is selectively sequestered in mitochondria with an active
m and permits the examination of the
m in adherent cells (Haugland (1996). To assess the effect of rotenone, atractyloside, and cyclosporin A on mitochondrial function in liver cells, fresh serum-free media, with or without the test compounds, was added to culture dishes containing glass coverslips. Fifteen min prior to the duration of each treatment, CMXRos (500 nm) was added to each medium and incubated at 37°C in a humidified atmosphere at 95% O2 and 5% CO2. At the conclusion of each treatment duration, the media containing the test compound(s) and CMXRos were removed and the liver cells were fixed with 4% paraformaldehyde (pH 7.4) for 10 min. Following fixation, the cells were washed twice in distilled water and the coverslips were removed and mounted on glass microscope slides with Supermount® (Biogenex, San Ramon, CA) to achieve optimal fluorescence. A Nikon Diaphot inverted microscope connected to a cooled CCD camera was utilized in each treatment to examine the area of CMXRos staining in liver cells. Fluorescent micrographic images were obtained (ex. 579 nm, em. 599 nm) and BDS Imaging software (Oncor, Inc., Gaithersberg, MD) was utilized to quantify the area of CMXRos staining in each cell. A total of 5001000 cells per treatment were examined for the determination of the area of CMXRos staining as a measure of mitochondrial function. A reduction in the area of CMXRos staining is indicative of a cell that has mitochondria with reduced membrane potential.
Cytofluorometric measurement of them.
To confirm the CMXRos studies, cytofluorometric analysis of the m#in liver cells was performed. Liver cells were treated with various concentrations of the test compounds for the selected treatment duration. Fifteen min prior to the conclusion of each treatment duration, in vitro labeling of liver cells was performed with 40 nm DiOC6(3) and incubated at 37°C in a humidified atmosphere at 95% O2 and 5% CO2 as previously described (Zamzami et al., 1995b
), with minor modifications. The cells were then trypsinized and centrifuged at 1500 x g for 5 min. As a control in some experiments, cells were labeled in the presence of the uncoupling agent mClCCP (50-µM). The supernatant was removed and the pellet was resuspended in physiological saline (Darzynkiewicz et al., 1997
; Deitch et al., 1982
). For analysis, a FACScan® cytofluorometer (Becton Dickinson, San Jose, CA) with argon laser excitation at 501 nm was used to analyze 10,000 cells from each sample. Comparison of the forward- and side-light scatters was utilized to gate the major population of normal-sized liver cells. Additionally, as exclusion of vital dyes provides an assessment of cell viability (Darzynkiewicz et al., 1997
), propidium iodide (5 µg/ml) staining was utilized to differentiate the live cell population from the dead cell population (data not shown) and to ensure that cells excluded from the gate were either cellular debris or non-viable cells.
Measurement of Cytolethality
To examine the effect of rotenone on non-apoptotic cell death, liver cells were exposed to increasing concentrations of rotenone (0.5, 1.0, 1.5, 2.0, 2.5, 5.0, 7.5, and 10.0 µM) in serum-free medium for 4, 6, 12, and 24 h. Following each treatment duration, 500 µL of medium was removed (sample LDH) from each culture dish and was evaluated as previously described (Ruch et al., 1989). The total LDH from each culture dish from each treatment was determined by solubilizing the plasma membrane with 1% Triton-X for 3 h followed by evaluation of LDH in the medium. To obtain the percent LDH release from each treatment, the sample LDH was divided by the LDH release following solubilization with Triton-X and multiplied by 100.
Statistics
Statistical difference (p < 0.05) from control values was determined by ANOVA followed by a Student-Newman-Keuls test (Gad and Weil, 1986). For analysis of apoptosis, 3 to 4 slides per treatment group were evaluated and the results represent the mean ± the standard deviation. For evaluation of the
m with CMXRos, a total of two slides, and a total of 5001000 individual cells per treatment were evaluated by ANOVA, followed by a Student-Newman-Keuls test. Analysis of cytofluorimetry data was based on 2 to 3 individual measurements of 10,000 cells each and statistical differences (Student's t test, p < 0.05) in the percent of DiOC6(3) positive cells were determined as previously described by (Zamzami et al., 1995b
). To determine the correlation between apoptosis and serum concentration in the media, a linear regression analysis was performed (Gad and Weil, 1986
).
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RESULTS |
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Time Course Evaluation of Rotenone-Induced Apoptosis
The induction of apoptosis following treatment with rotenone and other mitochondrial respiratory chain inhibitors has been demonstrated in several cell types (Jacobson et al., 1994; Newmeyer et al., 1994
; Pastorino et al., 1993
; Schulze-Osthoff et al., 1994
; Wolventang et al., 1994; Seaton et al., 1998
). The LDH release assay was utilized to determine the concentration of rotenone to be used in subsequent studies for the evaluation of apoptotic cell death (did not produce LDH release (cytolethality) (Fig. 1
). These studies demonstrate that following 12 h of treatment, concentrations of rotenone less than 7.5 µM did not produce LDH release. Furthermore, as the duration of rotenone exposure increased, the cytolethality of rotenone increased, such that following 24 h of continuous exposure, all concentrations of rotenone examined (except 1.0 µM) produced LDH release. Therefore, subsequent treatments were terminated following 12 h of treatment.
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Similar results were obtained following treatment with atractyloside (Figs.11 and 12). Treatment with 5.0-µM atractyloside produced 42.6%, 10.5%, 24.5%, 24.7%, and 35.1% reductions in the percent of DiOC6(3)-positive (
m-positive) liver cells when compared to DMSO-treated controls [96.8% DiOC6(3)-positive liver cells] following 15, 30, 60, 120, and 180 min of treatment, respectively (Figs.11 and 12
). Similar to the results obtained following rotenone treatment, cotreatment with 10.0-µM cyclosporin A prevented the atractyloside-induced reduction in the
m-positive cell population at all time points examined (Figs. 11 and 12
).
Taken together, these data indicate treatment with equimolar concentrations of the known MPT pore ligand, atractyloside, and rotenone produce a similar reduction in the percent of m-positive rat liver cells, which is prevented by cotreatment with cyclosporin A. These data suggest rotenone induces the MPT and the induction of the MPT appears to be consequential to the rotenone-induced inhibition of the electron transport chain.
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DISCUSSION |
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The toxicity of rotenone has been attributed to the inhibition of cell respiration by blocking the oxidation of nicotinamide adenine dinucleotide (NAD) at complex I of the electron transport chain, thus maintaining a high NADH/NAD+ (Lindahl and Oberg, 1961; Pastorino et al., 1995a
). This may result in the collapse of the mitochondrial proton motive force necessary to generate the
m and in turn produce ATP (Lindahl and Oberg, 1961
; Pastorino et al., 1995a
). Disruption of the
m has been observed prior to the demonstration of nuclear apoptosis in a variety of cell types (Deckwerth and Johnson, 1993
; Jacobson et al., 1994
; Newmeyer et al., 1994
; Schulze-Osthoff et al., 1994
; Petit et al., 1995
; Vayssiere et al., 1995
; Zamzami et al., 1995a
; 1995b
; 1996a
).
Additionally, the induction of cell death by rotenone in several cell types has been shown to be related to the inhibition of the electron transport chain (Pastorino et al., 1995b; Wolvetang et al., 1994
; Zamzami et al., 1995a
). This inhibition produces a de-energized mitochondria with a depolarized membrane (Deckwerth and Johnson, 1993
; Kroemer et al., 1995
; Pastorino et al., 1993
; Petit et al., 1995
; Zamzami et al., 1995a
,b
). The reduction of the
m and decreased ATP are necessary for the induction of the MPT (Deckwerth and Johnson, 1993
; Kroemer et al., 1995
; Petit et al., 1995
; Zamzami et al., 1995a
; 1995b
). Furthermore, induction of the MPT produces a further disruption of the
m and uncoupling of the respiratory chain which promotes the opening of additional MPT pores (Bernardi et al., 1994
; Kroemer et al., 1995
; Zoratti and Szabo, 1994
).
In the present study, rotenone treatment for greater than 20 min induced apoptosis. Upon induction of apoptosis by rotenone, removal of the compound or treatment with cyclosporin A did not prevent rotenone-induced apoptosis. Therefore, the mitochondrial dysfunction induced rotenone treatment (within 20 min) may be sufficient for the activation of a self-amplification pathway and the induction of further damage in the absence of rotenone in the medium. Alternatively, it is possible that rotenone persists intracellularly upon washing. Therefore, irreversible binding of rotenone to complex I (Gutman et al., 1969) may be responsible for the prolonged effect of rotenone on mitochondrial function observed even in the absence of rotenone in the culture medium. Further support for the early and irreversible nature of rotenone-induced mitochondrial dysfunction in the apoptotic process was apparent in the MitoTracker Red and cytofluorometric study results. Rotenone and atractyloside (a known inducer of the MPT) disrupted the
m within 15 min of treatment (Figs. 8, 9, and 10
). This disruption occurred prior to a measurable increase in the apoptotic index (20 min , Figs. 6 and 7
) and support the proposal that the induction of the MPT is an early, irreversible step in rotenone-induced apoptosis.
In this study, rotenone and other mitochondrial respiratory inhibitors induced apoptosis in cultured rat hepatocytes (Pastorino, 1993, 1995b), in other cultured mammalian cells (Wolventang et al., 1994; Zamzami et al., 1996a,c
), and in rat liver cells. Our observation of necrotic cell death following 24 h of treatment may be consequential to ATP depletion in liver cells. Rotenone, as an electron transport chain inhibitor, decreases the generation of ATP through oxidative phosphorylation (Simbula et al., 1997
). Several studies have demonstrated the necessity of ATP to activate the metabolic machinery involved in apoptosis, as well as the role of a reduction in ATP concentrations in the induction of apoptosis (Bossy-Wetzel et al., 1998
; Leist et al., 1997
). Although reduction of ATP concentrations have been observed during apoptosis, it appears to occur relatively late in the process (Bossy-Wetzel et al., 1998
) and is required for the downstream events in apoptosis (Eguchi et al., 1997
). Treatment with staurosporine or stimulation of the CD95 receptor in Jurkat cells resulted in a switch from apoptosis to necrosis when cells were depleted of ATP (Leist et al., 1997
). Therefore, in a state of reduced ATP generation, as observed following rotenone treatment, apoptosis may proceed until cellular ATP stores are depleted or reduced to a point where cell death becomes necrotic rather than apoptotic. Alternatively, the observed increased presence of LDH in the medium following 24-h rotenone treatment may be secondary to the ongoing apoptosis. The presence of LDH in the medium may be a result of the absence of phagocytes that would normally engulf late stage apoptotic cells with leaky plasma membranes (Cejna et al., 1994
; Raffray and Cohen, 1997
).
The present study also evaluated the role of the MPT in rotenone-induced apoptosis. The MPT is mediated by the opening of a non-specific pore across the inner mitochondrial membrane. The opening of the permeability pore and the subsequent MPT are inhibited by several agents including cyclosporin A (Bernardi et al., 1994; Crompton et al., 1987
; Pastorino et al., 1993
; Zamzami et al., 1995a
, 1996b
). Cyclosporin A binds to cyclophilin D, a peptidyl proylyl cis-trans-isomerase located in the inner mitochondrial membrane and a proposed component of the MPT pore, which maintains the permeability pore in a closed state (Broekemeier et al., 1989
; Halestrap and Davidson, 1990
). MPT pore inhibitors, including bongkrekic acid, monochlorodimane, and cyclosporin A, prevent apoptosis induced by agents that induce the MPT (Crompton et al., 1987
; Pastorino et al., 1993
; Zamzami et al., 1996b
) as well as several other apoptogens (Bernardi, 1992
; Crompton et al., 1988
; Hirsch et al., 1998
; Pastorino et al., 1993
; Yang and Cortopassi, 1998
; Zamzami et al., 1996a
,c
). Since disruption of the
m has been shown to induce and be a consequence of the MPT (Deckwerth and Johnson, 1993
; Petit et al., 1995
; Zamzami et al., 1995a
,b
), reduction of the
m, and the subsequent induction of the MPT may be critical events in the apoptotic pathway following treatment with mitochondrial respiratory chain inhibitors and uncouplers (Pastorino et al., 1995b
; Raffray and Cohen, 1997
; Wolventang et al., 1994; Zamzami et al., 1996c
).
Atractyloside, an inducer of the MPT, competitively binds and inhibits adenine nucleoside translocase, thereby preventing translocation of adenosine diphosphate (ADP) across the mitochondrial membrane and also oxidative phosphorylation (Obatomi and Bach, 1998). Previous studies have shown that atractyloside treatment promotes the MPT in isolated liver mitochondria and promotes the subsequent release of cytochrome c from the mitochondrial matrix (Kantrow and Piantadosi, 1997
). In the present study, atractyloside induced apoptosis in liver cells (Fig. 5
). However, upon cotreatment with cyclosporin A, the atractyloside-induced apoptosis was inhibited or attenuated (Fig. 5
). Similarly, following 12 h of treatment with rotenone, an increase in apoptosis was observed that was prevented by cotreatment with cyclosporin A (Fig. 4
). The similarity of these results suggests that MPT appears to be involved in the execution of apoptosis in rat liver cells following treatment with rotenone.
The consistent observation of a reduced or disrupted m in preapoptosis indicates maintenance of the
m is necessary for cell survival (Deckwerth and Johnson, 1993
; Petit et al., 1995
; Vayssiere et al., 1995
; Zamzami et al., 1995a
, 1996c
). Isolation and subsequent culture of a population of cells with a subnormal (low)
m indicated that when compared to cells with a normal
m, cells with a reduced
m proceed to an apoptotic morphology rapidly (Zamzami et al., 1995a
). Disruption of the
m has also been shown to be involved in apoptosis mediated by a variety of apoptogens, including etoposide, doxorubacin, cytosine arabinose, and ceramide (Decaudin et al., 1997
; Zamzami et al., 1996b
). Furthermore, previous studies demonstrated the reduction or collapse of the
m is involved in the induction and is a consequence of the MPT (Bernardi et al., 1994
; Kroemer et al., 1995
; Zoratti and Szabo, 1994
). Therefore, observation of mitochondria with a reduced or diminished
m appears to be associated with dysfunctional mitochondria in preapoptotic cells. In the present study, treatment with an apoptogenic concentration of rotenone or atractyloside reduced the
m in rat liver cells within 15 min of treatment. However, in the presence of cyclosporin A, the rotenone- and atractyloside-induced reductions of the
m are attenuated. Similar to these results, attenuation of the dexamethasone-, irradiation- and etoposide-induced disruption of the
m in splenocytes and thymocytes following treatment with cyclosporin A (Zamzami et al., 1995a
) and bongkrekic acid (Zamzami et al., 1996b
) was detected by cytofluorometric analysis of DiOC6(3)-stained cells. However, Pastorino et al. (1993) reported that cyclosporin A protected cultured hepatocytes from anoxia- and rotenone-induced cell death without attenuating the reduction of the
m, as determined by the release of preloaded [3H] TPP+ from the mitochondria. Therefore, the discrepancy between these results may be related to the utilization of different methods of detection.
Following treatment with a mitochondrial uncoupler, mClCCP, a rapid induction of the MPT as determined by the loss of the m was observed in the majority of mitochondria (Bernardi et al., 1993
). However, when the concentration of mClCCP was decreased, the disruption of the
m was observed in only a fraction of the mitochondria (Bernardi et al., 1993
). Similar results were obtained in the present study with rotenone. Although a reduction of the
m or induction of the MPT was not evident in the majority of cells following treatment with either rotenone or atractyloside, a concentration response was observed (Fig. 12
). Inhibition of the mitochondrial dysfunction and apoptosis by cyclosporin A also suggests the induction of the MPT is an important event in the preapoptotic mitochondrial dysfunction induced by rotenone (Figs. 4 and 12
).
Several tumor promoters have been shown to alter mitochondrial function (Cai et al., 1996; Chen et al., 1998
; Klohn et al., 1998
). Treatment with the liver-tumor promoter, ethinyl estradiol (Chen et al., 1998
), and several peroxisome proliferators, including clofibrate, perfluorooctanoic acid, and acetylsalicylic acid (Cai et al., 1996
), enhances the expression of several mitochondrial respiratory chain components, including NADH dehydrogenase (complex I) subunit I and cytochrome c oxidase subunit I. Furthermore, enhanced levels of mitochondrial respiratory chain protein expression enhanced mitochondrial respiration (Chen et al., 1998
). Enhanced mitochondrial function may provide these cells with a selective growth advantage or increased resistance to apoptosis. Recent studies by Klohn et al (1998) demonstrated that mitochondria, isolated from rats fed the carcinogen, 2-acetylaminofluorene, are more resistant to the induction of the MPT by elevated Ca2+ than mitochondria isolated from untreated rats. The present study demonstrated the role of the MPT in rotenone-induced apoptosis in rat liver cells. Since apoptosis is considered to counteract cell proliferation, and dysregulated apoptosis has been implicated in carcinogenesis (Bayly et al., 1995
; Bursch et al., 1984
; Roberts et al., 1995
; Wyllie et al., 1980
) and since rotenone induces MPT, enhanced apoptosis through induction of MPT may be the mechanism for the anti-carcinogenicity of rotenone.
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NOTES |
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REFERENCES: |
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Aoki, M., Morishita, R., Matsushita, H., Nakano, N., Hayashi, S., Tomita, N., Yamamoto, K., Moriguci, A., Higaki, J., and Ogihara, T. (1997). Serum deprivation-induced apoptosis accompanied by up-regulation of p53 and bax in human aortic vascular smooth muscle cells. Heart Vessels, 12(Suppl.), 7175.
Bayly, A. C., Roberts, R. A., and Dive, C. (1995). Suppression of liver-cell apoptosis in vitro by the nongenotoxic hepatocarcinogen and peroxisome proliferator nafenopin. J. Cell. Biol. 125, 197203.[Abstract]
Bernardi, P. (1992). Modulation of the mitochondrial cyclosporin-A-sensitive permeability transition pore by the proton electrochemical gradient: Evidence that the pore can be opened by membrane depolarization. J. Biol. Chem. 267(13), 88348839.
Bernardi, P., Broekemeier, K. M., and Pfeifer, D. R. (1994). Recent progress on regulation of the mitochondrial permeability transition pore; a cyclosporin-sensitive pore in the inner mitochondrial membrane. J. Bioenergetics and Biomembranes 26, 509517.[ISI][Medline]
Bernardi, P., Veronese, P., and Petronilli, V. (1993). Modulation of the mitochondrial cyclosporin A-sensitive permeability transition pore: I. Evidence for two separate Me2+ binding sites with opposing effects on the pore open probability. J. Biol. Chem. 268(2), 10051010.
Bossy-Wetzel, E., Newmeyer, D. D., and Green, D. R. (1998) Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. EMBO J. 17(1), 3749.
Broekemeier, K. M., Dempsey, M. E., and Pfeiffer, D. R. (1989). Cyclosporin A is a potent inhibitor of the inner membrane permeability transition in liver mitochondria. J. Biol. Chem. 264, 78267830.
Bursch, W. B., Lauer, B., Timmermann-Trosiener, I., Barthel, G., Schuppler, J., and Schulte-Hermann, R. (1984). Controlled death (apoptosis) of normal and preneoplastic cells in rat liver following withdrawal of tumor promoters. Carcinogenesis 5, 453458.[Abstract]
Cai, Y., Nelson, B. D., Li, R., Luciakova, K., and dePierre, J. W. (1996). Thyromimetic action of the peroxisome proliferators clofibrate, perfluorooctanoic acid, and acetysalicylic acid includes changes in mRNA levels for certain genes involved in mitochondrial biogenesis. Arch. Biochem. Biophys. 325(1), 107112.
Cejna, M., Fritsch, G., Printz, D., Schulte-Hermann, R., and Bursch, W. (1994). Kinetics of apoptosis and secondary necrosis in cultured rat thymocytes and S.49 mouse lymphoma and CEM human leukemia cells. Biochem. Cell Biol. 72, 677685.[ISI][Medline]
Chen, J., Gokhale, M., Li, Y., Trush, M. A., and Yager, J. D. (1998). Enhanced levels of several mitochondrial mRNA transcripts and mitochondrial superoxide production during ethinyl estradiol-induced hepatocarcinogenesis and after estrogen treatment of HepG2 cells. Carcinogenesis 19(12), 21872193.
Crompton, M., Costi, A., and Hayat, L. (1987). Evidence for the presence of a reversible Ca2+-dependent pore activated by oxidative stress in heart mitochondria. Biochem. J., 245(3), 915918.
Crompton, M., Ellinger, H., and Costi, A. (1988). Inhibition by cyclosporin A of a Ca2+-dependent pore in heart mitochondria activated by inorganic phosphate and oxidative stress. Biochem. J. 255(1), 357360.
Darzynkiewicz, Z., Juan, G., Li, X., Gorczyca, W., Murakami, T., and Traganos, F. (1997) Cytometry in cell necrobiology: Analysis of apoptosis and accidental cell death (necrosis). Cytometry 27, 120.[ISI][Medline]
Decaudin, D., Geley, S., Hirsch, T., Castedo, M., Marchetti, P., Macho, A., Kofler, R., and Kroemer, G (1997). Bcl-2 and Bcl-XL antagonize the mitochondrial dysfunction preceding nuclear apoptosis induced by chemotherapeutic agents. Cancer Res. 57(1), 6267.
Deckwerth, T. L., and Johnson, E. M. (1993). Temporal analysis of events associated with programmed cell death (apoptosis) of sympathetic neurons deprived of nerve growth factor. J. Cell Biol. 123, 12071222.[Abstract]
Deitch, A. D., Law, H., and White, R. D. (1982). A stable propidium iodide staining procedure for flow cytometry. J. Histochem. Cytochem. 30(9), 967972.
Eguchi, Y., Shimizu, S., and Tsujimoto, Y. (1997). Intracellular ATP levels determine cell death fate by apoptosis or necrosis. Cancer Res. 57, 18351840.[Abstract]
Ellis, R. E., Yuan, J., and Horvitz, H. R. (1991). Mechanisms and functions of cell death. Annu. Rev. Cell Biol. 7, 663698.[ISI]
Gad, S., and Weil, C. S. (1986). Statistics and Experimental Design for Toxicologists. Telford Press, New Jersey.
Garcia-Ruiz, C., Colell, A., Miari, M., Morales, A., Fernandez-Checa, J.C. (1997) Direct effect of ceramide on the mitochondrial electron transport chain leads to generation of reactive oxygen species. Role of mitochondrial glutathione. J. Biol. Chem., 272(17), 1136911377.
Gerhauser, C., Mar, W., Lee, S. K., Suh, N., Luo, Y., Kosmeder, J., Luyengi, L., Fong, H. H. S., Kinghorn, A. D., Moriarity, R. M., Mehta, R. G., Constantinou, A., Moon, R. C., and Pezzuto, J. M. (1995). Rotenoids mediate potent cancer chemopreventative activity through transcriptional regulation of ornithine decarboxylase. Nat. Med. 1, 260266.[ISI][Medline]
Gottlieb, E., Haffner, R., von Ruden, T., Wagner, E. F., and Oren, M. (1994). Down-regulation of wild-type p53 activity interferes with apoptosis in IL-3-dependent hematopoietic cells following IL-3 withdrawal. EMBO J. 13, 13681374.[Abstract]
Gutman, M., Singer, T. P., and Casida, J. E. (1969). Role of multiple binding sites in the inhibition of NADH oxidase by piericidin and rotenone. Biochem. Biophys. Res. Commun. 37, 615.[ISI][Medline]
Halestrap, A. P., and Davidson, A. M. (1990). Inhibition of Ca2(+)-induced large-amplitude swelling of liver and heart mitochondria by cyclosporin is probably caused by the inhibitor binding to mitochondrial-matrix peptidyl-prolyl cis-trans isomerase and preventing it interacting with the adenine nucleotide translocase. Biochem. J. 268, 153160.[ISI][Medline]
Haugland, R. P. (1996) Molecular Probes: Handbook of Fluorescent Probes and Research Chemicals, 6th Ed., pp. 266267. Molecular Probes, Inc., Eugene, OR.
Haviv, R., Lindenboim, L., Yuan, J., and Stein, R. (1998). Need for caspase-2 in apoptosis of growth-factor-deprived PC12 cells. J. Neurosci. Res. 52(5), 491497.
Hirsch, T., Susin, S.A., Marzo, I., Marchetti, P., Zamzami, N., and Kroemer, G. (1998) Mitochondrial permeability transition in apoptosis and necrosis. Cell Biol. Toxicol. 14(2), 141145.
Isenberg, J. S., Kolaja, K. L., Ayoubi, S. A., Watkins III, J. B., and Klaunig, J. E. (1997). Inhibition of Wy-14,643 induced hepatic lesion growth in mice by rotenone. Carcinogenesis 18(8), 15111519.
Jacobson, M. D., Burne, J. F., and Raff, M. C. (1994). Programmed cell death and Bcl-2 protection in the absence of a nucleus. EMBO J. 13, 18991910.[Abstract]
James, N. H., and Roberts, R. A. (1996). Species differences in response to peroxisome proliferators correlate in vitro with induction of DNA synthesis rather than suppression of apoptosis. Carcinogenesis 17(8), 16231632.
Kantrow, S. P., and Piantadosi, C. A. (1997). Release of cytochrome c from liver mitochondria during permeability transition. Biochem. Biophys. Res. Commun. 232(3), 669671.
Klohn, P.-C., Bitsch, A., and Neumann, H.-G. (1998). Mitochondrial permeability transition is altered in early stages of carcinogenesis of 2-acetylaminofluorene. Carcinogenesis 19(7), 11851190.
Kroemer, G., Petit, P. X., Zamzami, N., Vayssiere, J.-L., and Mignotte, B. (1995). The biochemistry of programmed cell death. FASEB J. 9, 12771287.
Leist, M., Single, B., Castoldi, A. F., Kuhnle, S., and Nicotera, P. (1997). Intracellular adenosine triphosphate (ATP) concentration: A switch in the decision between apoptosis and necrosis. J. Exp. Med., 185(8), 14811486.
Lindahl, P. E., and Oberg, K. E. (1961). The effect of rotenone on respiration and its point of attack. Exp. Cell Res. 23, 228237.[ISI]
Malorini, W., Giammarioli, A. M., Matarrese, P., Piettrangeli, P., Agostinelli, E., Ciaccio, A., Grassilli, E., and Mondovi, B. (1998). Protection against apoptosis by monamine oxidase A inhibitors. FEBS Lett. 426, 155159.[ISI][Medline]
Mesner, Jr., P. W., Budihardjo, I. I., and Kaufmann, S. H. (1997). Chemotherapy-induced apoptosis. Adv. Pharmacol. 41, 461499.[Medline]
Newmeyer, D. D., Farschon, D. M., and Reed, J. C. (1994). Cell-free apoptosis in Xenopus egg extracts: inhibition of Bcl-2 and requirement for an organelle fraction enriched in mitochondria. Cell 79, 353364.[ISI][Medline]
Obatomi, D. K., and Bach, P. H. (1998) Biochemistry and toxicology of the diterpenoid glycoside atractyloside. Food Chem. Toxicol. 36(4), 335346.
Pastorino, J. G., Synder, J. W., Hoek, J. B., and Farber, J. L. (1995a) Ca2+ depletion prevents anoxic death of hepatocytes by inhibiting mitochondrial permeability transition. Am. J. Physiol. 268 (Cell Physiol. 37), C676C685.
Pastorino, J. G., Snyder, J. W., Serroni, A., Hoek, J. B., and Farber, J. L. (1993). Cyclosporin and carnitine prevent the anoxic death of cultured hepatocytes by inhibiting the mitochondrial permeability transition. J. Biol. Chem. 268(19), 1379113798.
Pastorino, J. G., Wilhelm, T. J., Glascott, Jr, P. A., Kucsis, J. J., and Farber, J. L. (1995b) Dexamethasone induces resistance to the lethal consequences of electron transport inhibition in cultured hepatocytes. Arch. Biochem. Biophys. 318(1), 175181.
Petit, P. X., Lecouer, H., Zorin, E., Dauguet, C., Mignotte, B., and Gougeon, M. L. (1995). Alterations of mitochondrial structure and function are early events of dexmethasone-induced thymocyte apoptosis. J. Cell Biol. 130, 157167.[Abstract]
Raff, M. C. (1995). Social controls on cell survival and cell death. Nature 356, 397400.
Raffray, M., and Cohen, G. M. (1997). Apoptosis and necrosis in toxicology: A continuum or distinct modes of cell death. Pharmacol. Ther. 75(3), 153177.
Roberts, R. A., Soames, A. R., Gill, J. H., James, N. H., and Wheldon, E. B. (1995) Non-genotoxic hepatocarcinogens stimulate DNA synthesis and their withdrawal induces apoptosis, but in a different hepatocyte population. Carcinogenesis 16, 16931698.[Abstract]
Ruch, R. J., Christ, K. A., and Klaunig, J. E, (1989). Effects of culture duration on hydrogen peroxide-induced hepatocyte toxicity. Toxicol. Appl. Pharmacol. 100, 451464.[ISI][Medline]
Scaife, J. F. (1966). The effect of lethal doses of X-irradiation on the enzymatic activity of mitochondrial cytochrome c. Can. J. Biochem. 44, 433448.[ISI][Medline]
Schulze-Osthoff, K., Walczak, H., Droge, W., and Krammer, P. H. (1994). Cell nucleus and DNA fragmentation are not required for apoptosis. J. Cell Biol. 127, 1520.[Abstract]
Seaton, T. A., Cooper, J. M., and Schapira, A. H. V. (1998). Cyclosporin inhibition of apoptosis induced by mitochondrial complex I toxins. Brain Res. 809, 1217.[ISI][Medline]
Simbula, G., Glascott, Jr., P. A., Akita, S., Hoek, J. B., and Farber, J. L. (1997). Two mechanisms by which ATP depletion potentiates induction of the mitochondrial permeability transition. Am. J. Physiol. 273 (Cell Physiol. 42), C-479488.
Snyder, J. W., Pastorino, J. G., Attie, A. M., and Farber, J. L. (1993). Protection by cyclosporin A of cultured hepatocytes from the toxic consequences of the loss of mitochondrial energization produced by 1-methyl-4-phenylpyridinium. Biochem. Pharmacol. 44, 833835.[ISI]
Steller, H. (1995). Mechanisms and genes of cellular suicide. Science 267, 14451449.[ISI][Medline]
Vayssiere, J.-L., Petit, P. X., Risler, Y., and Mignotte, B. (1995. Commitment to apoptosis is associated with changes in mitochondrial biogenesis and activity in cell lines conditionally immortalized with a simian virus 40. Proc. Natl. Acad. Sci. U S A 91, 1175211756.
Wolvetang, E. J., Johnson, K. L., Krauer, K., Ralph, S. J., and Linnane, A. W. (1994). Mitochondrial respiratory chain inhibitors induce apoptosis. FEBS Lett. 339, 4044.[ISI][Medline]
Wyllie, A. H., Kerr, J. F. R., and Currie, A. R. (1980). Cell death: The significance of apoptosis. Int. Rev. Cytol. 68, 251306.[Medline]
Xu, Q., and Reed, J. C. (1998). BAX inhibitor-1, a mammalian apoptosis suppressor identified by functional screening in yeast. Mol. Cell. 1(3), 337346.
Yang, J. C., and Cortopassi, G. A. (1998). Induction of the mitochondrial permeability transition causes release of the apoptogenic factor cytochrome c. Free Radic. Biol. Med. 24(4), 624631.
Zamzami, N., Marchetti, P., Castedo, M., Decaudin, D., Macho, A., Hirsch, T., Susin, S. A., Petit, P. X., Mignotte, B., and Kroemer, G. (1995a). Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J. Exp. Med. 182, 367377.[Abstract]
Zamzami, N., Marchetti, P, Castedo, M., Hirsch, T., Susin, S.A., Masse, B., and Kroemer, G. (1996a). Inhibitors of permeability transition interfere with the disruption of the mitochondrial transmembrane potential during apoptosis. FEBS Lett. 384(1), 5357.
Zamzami, N., Marchetti, P., Castedo, M., Zanin, C., Vayssiere, J.-L., Petit, P. X., and Kroemer, G. (1995b). Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte cell death in vivo. J. Exp. Med. 181, 16611672.[Abstract]
Zamzami, N. Susin, S. A., Marchetti, T., Hirsch, T., Castedo, M., and Kroemer, G. (1996b) Mitochondrial control of nuclear apoptosis. J. Exp. Med. 183, 15331544.[Abstract]
Zamzami, N., Susin, S. A., Marchetti, P. Hirsch, T., Gomez-Monterrey, I., Castedo, M., and Kroemer, G. (1996c). Mitochondrial control of nuclear apoptosis. J. Exp. Med. 183, 15331544.[Abstract]
Zoratti, M., and Szabo, I. (1994). Electrophysiology of the inner mitochondrial membrane. J. Bioenerg. Biomembr. 26, 543553.[ISI][Medline]
Zoratti, M., and Szabo, I. (1995). The mitochondrial permeability transition. Biochem Biophys. Acta 1241, 139176.[ISI][Medline]