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Free radical–induced megamitochondria formation and apoptosis
Free Radical Biology & Medicine, Vol. 26, Nos. 3/4, pp. 396 – 409, 1999 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/99/$–see front matter
PII S0891-5849(98)00209-3
Original Contribution FREE RADICAL–INDUCED MEGAMITOCHONDRIA FORMATION AND APOPTOSIS MARIUSZ KARBOWSKI,* CHIEKO KURONO,† MICHAL WOZNIAK,‡ MARIUSZ OSTROWSKI,* MASAAKI TERANISHI,* YUJI NISHIZAWA,* JIRO USUKURA,* TSUYOSHI SOJI,† and TAKASHI WAKABAYASHI* (Received 11 May 1998; Revised 23 July 1998; Accepted 23 July 1998) *Department of Cell Biology and Molecular Pathology, Nagoya University School of Medicine, Showa-ku, Nagoya, Japan, † Nagoya City University Medical School, Mizuho-ku, Nagoya, Japan, ‡Department of Biochemistry, Medical University of Gdansk, Gdansk, Poland
Abstract—Pathophysiological meaning and the mechanism of the formation of megamitochondria (MG) induced under physiological and pathological conditions remain obscure. We now provide evidence suggesting that the MG formation may be a prerequisite for free radical-mediated apoptosis. MG were detected in primary cultured rat hepatocytes, rat liver cell lines RL-34 and IAR-20 and kidney cell line Cos-1 treated for 22 h with various chemicals known to generate free radicals: hydrazine, chloramphenicol, methyl-glyoxal-bis-guanylhydrazone, indomethacin, H2O2, and erythromycin using a fluorescent dye Mito Tracker Red CMXRos (CMXRos) for confocal laser microscopy and also by electron microscopy. Remarkable elevations of the intracellular level of reactive oxygen species (ROS), monitored by staining of cells with a fluorescent dye carboxy-H2-DCFDA, were detected before MG were formed. Prolongation of the incubation time with various chemicals, specified above, for 36 h or longer has induced distinct structural changes of the cell, which characterize apoptosis: condensation of nuclei, the formation of apoptotic bodies, and the ladder formation. Cells treated with the chemicals for 22 h were arrested in G1 phase, and apoptotic sub-G1 populations then became gradually increased. The membrane potential of MG induced by chloramphenicol detected by CMXRos for flow cytometry was found to be decreased compared to that of mitochondria in control cells. Rates of the generation of H2O2 and O22 from MG isolated from the liver of rats treated with chloramphenicol or hydrazine were found to be lower than those of mitochondria of the liver of control animals. We suggest, based on the present results together with our previous findings, that the formation of MG may be an adaptive process at a subcellular level to unfavorable environments: when cells are exposed to excess amounts of free radicals mitochondria become enlarged decreasing the rate of oxygen consumption. Decreases in the oxygen consumption of MG may result in decreases in the rate of ROS production as shown in the present study. This will at the same time result in decreases in ATP production from MG. If cells are exposed to a large amount of free radicals beyond a certain period of time, lowered intracellular levels of ATP may result in apoptotic changes of the cell. © 1998 Elsevier Science Inc. Keywords—Megamitochondria, Free radicals, Cell culture, Apoptosis, Flow cytometry
INTRODUCTION
such as deprivation of tropic factors [4,5], heat shock [6], and various cytotoxic substances [7,8]. Reports have been accumulated that mitochondria are deeply involved in the regulation of apoptosis: bcl-2 is located mainly on the outer membrane of mitochondria [9]; bcl-2 protein has antioxidant properties and inhibits apoptosis by suppressing the formation or effects of reactive oxygen species (ROS) [10,11]; cytochrome c, localized in the inner membranes of mitochondria, is released from mitochondria of pre-apoptotic cells and can trigger the activation of CED-3 family proteases [12,13]; bcl-2 was found to prevent the release of cytochrome c from mi-
Apoptosis (programmed cell death) plays a crucial role in the normal development and differentiation of multicellular organisms and is essential for embryogenesis and metamorphosis [1–3]. Apoptosis is also induced by various pathological conditions and a variety of agents Address correspondence to: Takashi Wakabayashi, Department of Cell Biology and Molecular Pathology, Nagoya University School of Medicine, 65, Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan; Tel: 052-744-2028; Fax: 052-744-2041; E-Mail: [email protected]. nagoya-u.ac.jp. 396
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tochondria [13]; collapse of the mitochondrial transmembrane potential was reported to be an early, irreversible symptom of apoptosis occurring before the nuclear sign of cell death [14 –16]. It can, therefore, be deduced from these data that mitochondria play a crucial role in apoptosis. One of the important factors implicated in the different steps of apoptosis is the generation of ROS [17]. Oxidative stress is a common element of apoptosis induced by various stimuli that often do not exert a direct oxidant action [8,18,19]. Substances with antioxidative properties have been reported to inhibit apoptosis induced by various chemicals [14,20 –23]. We have been studying the mechanism of the formation of megamitochondria (MG) using hydrazine and ethanol as experimental models [24 –27]. We have found that free radicals are intimately related to the mechanism of the formation of MG in the cases of chloramphenicol (CP) [28], hydrazine [29], and ethanol [30]. We have succeeded in suppressing the MG formation induced by these chemicals by a-tocopherol [31], coenzyme Q10 [32] and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1oxyl(4-OH-TEMPO) [28,29,33], a spin trapping agent and at the same time a scavenger for free radicals. We have proposed that free radicals in some way modify mitochondrial membranes favorable for the membrane fusion resulting in the formation of MG based on changes in physicochemical and biochemical properties of mitochondrial membranes during the formation process of MG [28,29,34 –36]. The present study, demonstrates that the formation of MG caused by various inducers of free radicals in cultured cells precedes apoptosis. According to the classic definition of apoptosis, structural changes of mitochondria during the process of apoptosis have not been stressed except for the swelling at an early stage of the process [15,37]. In this article, we have tried to correlate the formation of MG to free radical-mediated apoptotic processes.
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with 1% heat-inactivated calf serum, 4 mM L-glutamate, 0.45% glucose and kanamycin (60 mg/L). Treatment of cells with various chemicals The following chemicals were tested in the present study: hydrazine (0.5–5.0 mM); CP (100 –500 mg/ml); H2O2 (0.1– 0.5 mM); erythromycin (100 –200 mg/ml); methylglyoxal-bis-guanylhydrazone (M-GAG) (50 mg/ ml); indomethacin (0.1– 0.5 mM). Visualization of mitochondria by confocal laser microscopy A cell-permeant, mitochondria-specific fluorescent dye, Mito Tracker Red CMXRos (CMXRos) (Molecular Probes Inc., Eugene, OR, USA), was used to visualize mitochondria. Staining of mitochondria was performed by the method of Poot et al. [42]. Cells growing on coverslips (10 3 10 mm) were incubated with CMXRos at a final concentration of 500 mM for 30 min in the culture chamber. Coverslips were then rinsed with the culture medium followed by fixation with a fixative containing 2% glutaraldehyde, 2% formaldehyde, and 0.1 M Na-cacodylate, pH 7.4. Specimens were examined under a confocal laser microscope Bio-Rad MRC 1024. Flow cytometric analysis of mitochondrial membrane potential Cells growing on 10-cm culture dishes (Falcon) were incubated with 500 nM CMXRos in the atmosphere of 5% CO2 at 37oC. After 30 min of incubation cells were washed in prewarmed PBS, collected and resuspended in 1 ml of a freshly prepared culture medium and immediately analyzed by flow cytometry using Coulter Elite FACSCAN (Coulter Corporation, Miami, FL) according to the method of Poot et al. [42]. Forward and side scatters were used to establish size-gates and to exclude cellular debris and apoptotic cells from the analysis.
MATERIALS AND METHODS
Cell culture Hepatocytes were isolated by a collagenase perfusion technique from phenobarbital-pretreated or control male Wistar rats weighing 100 to 120 g as described before [38]. Rat liver cell lines RL-34 cells (JCRB 0247) [39], IAR-20 cells (JCRB 0610) [40], and monkey kidney cell line COS-1 cells (JCRB 9082) [41] were obtained from the Health Science Research Resources Bansk (HSRRB) (Osaka, Japan). RL-34 cell and COS-1 cells were maintained in DMEM, IAR-20 cells in EMEM, supplemented
Assay for free radical production Visualization of intracellular levels of free radicals by confocal laser microscopy. Detection of the overall intracellular generation of free radicals was done essentially by the dichlorodihydrofluorescein diacetate (H2DCFDA) staining method [43] as described by Garland and Halestrap [44]. H2-DCFDA was replaced by carboxy-H2-DCFDA in the present study because the latter was expected to have enhanced retention inside the cell due to two negative charges at physiological pH. Cells cultured on glass coverslips were incubated for 60 min at
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37°C with 5 mM carboxy-H2-DCFDA dissolved in the culture medium in the culture chamber. Coverslips were then fixed with a fixative containing 2% glutaraldehyde and 2% formaldehyde dissolved in PBS, and analyzed under a confocal laser microscope using a FITC barrier filter. The laser intensities and photodetector gains were held constant to allow comparisons of relative fluorescence intensities of cells between the control and experimental cells. Flow cytometric analysis of intracellular levels of free radicals. Cells grown on 10-cm culture dishes were stained with 5 mM carboxy-H2-DCFDA and were kept for 60 min in the culture chamber, as described previously, and then harvested by trypsinization. Cells were then washed with PBS, resuspended in PBS and submitted immediately to analysis using a Coulter Elite FACSCAN (Coulter Corporation) with excitation and emission settings of 495 and 525 nm, respectively. Gating was performed to remove apoptotic cells and cellular debris before data were collected. Detection of free radicals generated from mitochondria. The rate of the generation of ROS from mitochondria was examined besides the estimation of the overall intracellular levels of free radicals, described previously, to elucidate functional aspects of MG. For this purpose, we have adopted in vivo experimental model for the induction of MG using hydrazine, because data are already available concerning the rate of oxygen consumption, phosphorylating abilities, and the rate of lipid peroxidation of hydrazine-induced MG [24,25,29,31,34,36]. Male Wistar rats aged 4 weeks were fed a hydrazine-diet as described before [24]. After 3 and 7 days, mitochondria were isolated from the liver. The inner mitochondrial membrane fraction was prepared as described subsequently: mitochondria obtained from the control and experimental animals were suspended in 3 mM phosphate buffer, pH 7.0. The suspension was centrifuged for 10 min at 20,000g. The residue obtained was resuspended in 3 mM phosphate buffer, and sonicated 3 times using a Branson sonifier (setting 3.5–5.0) for 30 s at each time. Sonicated samples were centrifuged for 20 min at 25,000g. The supernatant, therefore, obtained was centrifuged for 30 min at 100,000g, and the resultant pellet was suspended in 3 mM phosphate buffer, pH 7.0, and used as the submitochondrial fraction. Generation of O22 from the submitochondrial fraction was measured essentially according to the method of Kakinuma and Minakami [45] using acetylated cytochrome c. Generation of H2O2 from the submitochondrial fraction was measured essentially by the method of Loschen and Flohe [46] using fluorescent dye scopoletin.
Cell cycle and DNA analyses Cell cycle analysis was performed by flow cytometry using nuclei essentially according to the method of Mancini et al. [47]. DNA staining was obtained with 500 ml of a solution containing 100 mg/ml of propidium iodide (Molecular Probes, Inc.), 0.1% Triton X-100 and 1% fetal calf serum for 30 min at 4°C in the dark, followed by flow cytometric analysis using a Coulter Epics XL flow cytometer (Coulter Corporation). DNA was extracted according to the method of Garland and Halestrap [44]. Electron microscopy An equal volume of a fixative containing 4% glutaraldehyde, 4% formaldehyde and 0.2 M Na-cacodylate, pH 7.4, was added directly to the culture medium to fix detached and attached cells on culture dishes. After fixation with the aldehyde solution, sample were processed for electron microscopy as described before [38]. Thin sections were stained with lead citrate and examined in a Hitachi 7000 electron microscope operated at 75 kV. RESULTS
Confocal laser microscopic and electron microscopic observations of mitochondria of cultured cells treated with inducers of free radicals A variety of chemicals that are known to generate free radicals directly or indirectly was tested in the present study: CP, M-GAG, hydrazine, H2O2, erythromycin, and indomethacin. Cells used were: rat hepatocytes, RL-34 cells, IAR-20 Cells, and COS-1 cells. All chemicals tested in the present study invariably induced remarkable structural changes of mitochondria in four kinds of cells, specified above, treated for 22 h. Examples of confocal laser micrographs are shown in Fig. 1. In control cells [RL-34 cells (A), IAR-20 cells (B), rat hepatocytes (C)], filamentous mitochondria stained with CMXRos are discerned clearly whereas mitochondria in cells treated with various chemicals for 22 h became granular. Chemicals tested in the present study showed essentially the same results regardless of the sources of cells. Structural changes of mitochondria in cells treated with various chemicals were discerned more clearly under the electron microscope (Figs. 2 and 3). Cells of the three different cell lines used in the present study were characterized by the presence of mitochondria much smaller in size and number per cell compared to those of rat hepatocytes (Fig. 2, A: RL-34 cells; B: IAR-20 cells; C: COS -1 cells; D: rat hepatocytes). When they were cultured for 22 h in the presence of CP, for example, mitochondria became enlarged dis-
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Fig. 1. Induction of megamitochondria in culture cells by inducers of free radicals-confocal laser micrographs. (A) RL-34 cells. A1: control; A2: chloramphenicol (CP); A3: methylglyoxal-bis-guanylhydrazone (M-GAG). (B) IAR-20 cells. B1: control; B2: CP; B3: erythromycin. (C) Rat hepatocytes. C1: control; C2: CP; C3: hydrazine. Cultivation time: 22 h. Concentration of chemicals: CP, 300 mg/ml; erythromycin, 200 mg/ml; hydrazine, 2 mM. Bars: 10 mm (A, B), 10 mm (C).
tinctly. Enlarged mitochondria were often bizarre in shapes (B2, C2) and pale in their matrix (A2). Extremely elongated mitochondria were sometimes encountered (C3). In the case of rat hepatocytes cultured in the presence of hydrazine for 22 h (D2), the density of the matrix of enlarged mitochondria was similar to that of control mitochondria. When the cultivation time of these cells in the presence of CP or hydrazine was prolonged to 72 h, nuclei of these cells became condensed to various degrees, which were characterisics of apoptotic cells (Fig. 3). Enlarged mitochondria with vesicular cristae or those almost devoid of cristae were seen in these cells suggesting that they became degenerative.
Changes in the structure of mitochondria and apoptotic changes of the cell, described previously, were seen in all cases of chemicals tested in the present study. When the concentration of each chemical was raised within the range, specified in Materials and Methods, these changes became more distinct. PI staining of nuclei of CP- and M-GAG- treated RL-34 cells Because it has turned out that inducers of free radicals tested in the present study cause apoptotic changes of the cell, we examined changes in nuclei by
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Fig. 2. Induction of megamitochondria in culture cells treated with inducers of free radicals for 22 h— electron micrographs. (A): RL-34 cells. A1: control; A2: CP. (B) IAR-20 cells. B1: control; B2: CP. (C) COS-1 cells. C1: control; C2, C3: CP. (D) Hepatocytes. D1: control; D2: hydrazine. Concentrations of chemicals: CP, 300 mg/ml; hydrazine, 2 mM. Magnification of micrographs: 310,000.
PI staining. When RL-34 cells were treated for 48 h with CP or M-GAG, nuclei of these cells revealed highly fluorescent, irregular shaped structures with PI staining while those of the control cells were stained weakly with the dye keeping their round contours (Fig. 4).
Cell cycle analysis on CP- and M-GAG-treated RL-34 cells In the present study, we have also carried out flow cytometric analysis on PI-stained DNA obtained from
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Fig. 2. Continued
cells treated with chemicals for various lengths of time hoping to detect apoptotic cells quantitatively. Examples are shown in Fig. 5 using CP- and M-GAG-treated RL-34 cells. In the case of CP-treated cells, the population of cells in G1 phase of cell cycle reached 62% after 24 h whereas that in the control cells was 55%. The
prolongation of the treatment of cells with CP for up to 48 h has disclosed a sub-G1 peak, which is indicative of apoptotic cells. The population of cells in sub-G1 phase continued to increase thereafter. DNA obtained from cells treated with M-GAG showed similar results to those of the case of CP, described previously.
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Fig. 2. Continued
Detection of sub-G1 peak, described previously, commonly recognized as the landmark of apoptotic change of the cell has prompted us to examine the existence of oligonucleosome-sized genomic DNA fragmentation, which has been observed in many cases of physiological and drug-induced apoptotic changes of the cell. Fig. 6 shows the time sequence of DNA fragmentation and formations of a ladder-like pattern in RL-34 cells treated with CP or M-GAG. Treatment of cells with CP for 22 h when the formation of MG was already evident did not give any noticeable changes in DNA patterns (lane 2) whereas the ladder formation became evident in cells treated with CP for 48 h and 72 h (lanes 3 and 4, respectively). The ladder formation was not evident in cells treated with M-GAG-for up to 48 h (lane 5, 22 h; lane 6, 48 h) and it became evident after 72 h (lane 7). Similar results were obtained with erythromycin: the ladder formation was detected after 72 h, although it was less distinct compared to the case of CP or M-GAG (lane 9, 22 h; lane 10, 48 h; lane 11, 72 h).
[10]. In the present study, we have adopted carboxy-H2DCFDA, an analog of H2-DCFDA, to estimate levels of ROS in CP- and M-GAG-treated cells. In Fig. 7A, typical confocal laser micrographs were obtained from RL-34 cells cultured for 22 h in the absence (A1), or presence of CP (300 mg/ml) (A2) or M-GAG (50 mg/ml) (A3). It is evident from the figure that the amount of ROS generated in experimental cells has definitely increased compared to that of the control cells. A typical histogram of carboxy-DCF fluorescence of cells treated with CP or M-GAG for 22 h is shown in Fig. 7B. It is evident from the figure that peaks in CP- and M-GAG-treated cells, especially the latter, shifted to the right indicating increased ROS generation in a large population of these cells. To clarify the correlation between the formation of MG and the generation of ROS by inducers of free radicals, the amount of ROS generated inside the cell was plotted against the duration of time after the treatment of cells with CP (Fig. 7C). It should be stressed here that there was already a distinct increase in the level of ROS in cells treated with CP for 10 h when MG were not yet formed, and an additional increase was observed after 22 h when MG were induced.
Intracellular levels of free radicals in CP- and M-GAG-treated RL-34 cells
Rate of generation of ROS from mitochondria of hydrazine-treated rat livers
The nonfluoresent dye H2-DCFDA passively enters the cell and on oxidation forms fluorescent DCF, which is a reporter of ROS generation at a level of a single cell
To analyze the role of MG in cellular oxidative stress, generations of O22 and H2O2 from mitochondria were examined besides the estimation of overall production of
Electrophoretic patterns of nuclear DNA extracted from CP-, M-GAG-, and erythromycin-treated cells
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Fig. 3. Apoptotic changes of culture cells treated with inducers of free radicals for 72 h— electron micrographs. A1, A2: CP-treated RL-34 cells. B1, B2: CP-treated IAR-20 cells. C: CP-treated COS-1 cells. D: hydrazine-treated rat hepatocytes. Concentrations of chemicals: CP, 300 mg/ml; hydrazine, 2 mM. Magnifications of micrographs: 310,000.
intracellular ROS, as described. Table 1 summarizes the rates of generation of O22 and H2O2 from mitochondria isolated from the liver of rats that were fed a hydrazinediet for 3 d and 7 d, respectively. The rate of generation of H2O2 from mitochondria of animals treated with hydrazine for 3 d when MG were not yet formed was
definitely increased compared to that of the control animals. The rate of the generation of O22 from mitochondria of animals treated with hydrazine for 3 d had a tendency to be increased compared to that of the control animals although there was not a statistically significant difference between them. However, rates of generation
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Fig. 3. Continued
of both H2O2 and O22 from mitochondria of rats treated with hydrazine for 7 d became decreased significantly compared to those of the control. Time sequence of changes in the membrane potential of mitochondria in CP-treated RL-34 cells In the present study, we adopted CMXRos as a fluorescent probe to visualize mitochondria by confocal laser microscopy, as described. We also used this dye to detect changes in the mitochondrial membrane potential for flow cytometry (Fig. 8). For this purpose, RL-34 cells were treated with CP (300 mg/ml) for various lengths of time up to 36 h, stained with CMXRos, and were applied to flow cytometry. It is evident from the figure that the peak of cells treated with CP for 22 h when MG were
formed shifted to the left compared to that treated with CP for 12 h, indicating that the membrane potential of mitochondria in the major portion of the former cells became decreased compared to that in the latter. In addition, another small peak with low fluorescence intensity was detected in mitochondria of cells treated with CP for 36 h indicating that a certain population of cells had mitochondria with collapsed membrane potential. In control cells, there was essentially no difference in the peak of the fluorescence intensity between cells cultured for 12 h and those cultured for 22 h, and the peak shifted to the left in cells cultured for 36 h. Cells treated with valinomycin (1 mM/ml), mitochondrial transmembrane potential disrupting agent, showed distinct decreases in CMXRos fluorescence and were used as a positive control in our experiments.
Fig. 4. Propidium iodide staining of RL-34 cells cultured for 48 h in the presence of CP or M-GAG. A: control; B: CP (300 mg/ml); C: M-GAG) (50 mg/ml). Details in the experimental conditions are described in the Materials and Methods section.
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Fig. 6. Electrophoresis of nuclear DNA extracted from RL-34 cells cultured in the presence of various inducers of free radicals. Control: lane 1; CP (300 mg/ml): lanes 2– 4; M-GAG (50 mg/ml): lanes 5–7; erythromycin (200 mg/ml): lanes 8 –10. Cultivation time: 22 h: lanes 2, 5, 8; 48 h: lanes 3, 6, 9; 72 h: lanes 1, 4, 7, 10; DNA size markers: lane 11. Details in the experimental conditions are described in the Materials and Methods section.
Fig. 5. Cell cycle analysis on CP- and M-GAG-treated RL-34 cells. Cells were cultured in the absence (A1–A3) or presence of CP (300 mg/ml) (B1–B3) or M-GAG (50 mg/ml) (C1—C3). Cultivation time: 24 h (A1, B1, C1); 48 h (A2, B2, C2); 72 h (A3, B3, C3). Nuclear preparations were prepared from asynchronously growing cells. Details in the experimental conditions are described in the Materials and Methods section.
cells. (c) The rate of the generation of ROS from mitochondria of the liver of rats placed on a hydrazine-diet for 3 d when MG were not formed yet was elevated compared to that of control animals, whereas that of animals placed on a hydrazine-diet for 7 d when MG were formed was distinctly decreased compared to that of the control animals. (d) Apoptotic changes of the cell were observed in the cells treated with various chemicals for 36 h or longer. (e) Decreases in the membrane potential of mitochondria were observed in RL-34 cells treated with CP for 32 h when MG were formed. Then, the following serious questions are raised: is the formation of MG a coincidence, consequence, or prerequisite for free radical-mediated apoptosis, and how do free radicals induce MG?
Table 1. Rates of Generation of O22 and H2O2 in Hydrazine-Treated Rat Liver Mitochondrial Inner Membranesa DISCUSSION
We have shown that the formation of MG precedes apoptotic changes of the cell in the present experimental conditions. The series of events occurring in the cells treated with various free radical-generating chemicals tested in the present study for various lengths of time are summarized as follows: (a) MG were induced in various cells treated for 22 h by various free radical-inducers. (b) The formation of MG was preceded by remarkable increases in the intracellular level of ROS in CP-treated
Animals Control Hydrazine 3d 7d
O22 (nmol/min/mg protein)
H2O2 (nmol/min/mg protein)
1.25 6 0.02
0.44 6 0.01
1.40 6 0.05 0.62 6 0.03b
0.63 6 0.05c 0.32 6 0.03c
a Inner membrane fractions were obtained from the livers of rats fed with a 1% hydrazine-diet for 3 d and 7 d, respectively. Values are expressed as the means 6 SE of three different experiments. Data on experimental animals are statistically different from those of the control at: b( p , .001); c.02 , p , .05).
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Fig. 7. Levels of reactive oxygen species (ROS) in RL-34 cells cultured in the presence of CP or M-GAG. (A) Confocal micrographs. Cells were cultured for 22 h in the absence (A1) and presence of CP (300 mg/ml) (A2) or M-GAG (50 mg/ml) (A3). Cell were stained with carboxy-H2-DCFDA. (B) Flow cytometry. Cells of the control and experimental groups cultured in the same conditions as those described for the legend (A) were stained with carboxy-H2-DCFDA. Cell were then harvested by trypsinization. (C) Changes in the intracellular levels of ROS after the addition of CP. Cells were cultured for various lengths of time in the absence or presence of CP (300 mg/ml). Cells were assayed for ROS productions by the method described in the Materials and Methods section. Relative ROS productions in CP-treated cells are expressed as a percentage of the cell population with higher carboxy-DCF fluorescence intensities than the cell with the highest fluorescence intensity among the control cells. The fluorescence intensities of the dye in CP-treated cells were plotted taking that of the control as 0.
Pathophysiological meaning of the MG formation with respect to free radical-mediated apoptosis Phosphorylating abilities and rates of oxygen consumption of MG induced by various experimental conditions or obtained from human biopsied materials are often lower to various degrees compared to those of the control [25,32,48 –50]. We assume that in physiological conditions enzymatic and non-enzymatic defence systems against free radicals work effectively within the cell so that cells are devoid of free radicalmediated various injuries. However, when the level of free radicals exceeds a certain level as in the cases of the present experimental conditions, mitochondria become enlarged to various degrees lowering the rate of oxygen consumption and phosphorylating abilities.
Mitochondrial respiration accounts for about 90% of cellular uptake and 1–2% of the oxygen consumed is converted to ROS [51,52]. MG may generate less amounts of ROS because of lower rates of respiration. We have shown in the present study, that the rate of the generation of ROS from hydrazine-induced MG is actually decreased compared to that from control mitochondria. Cells then stop growing due to a shortage of ATP supply from MG. We have shown previously that the intracellular level of ATP in hepatocytes and the ability of MG to synthesize ATP are actually remarkably decreased in rats treated chronically with ethanol [32]. If free radicals are removed from the cell, MG may return to normal, functionally and structurally. However, if cells are exposed additional to excess
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need for mitochondria in the cell of hibernating animals to respire actively resulting in the generation of large amounts of ROS. If this is the case, the MG formation could be regarded as an adaptive process at a level of subcellular organelles to unfavorable environments. To test this hypothesis, we measured the total amount of ROS generated inside the cell and the level of ROS generated from mitochondria before and after the formation of MG (confer Fig. 7 and Table 1). We have shown previously that distinct changes in biochemical and physicochemical properties of mitochondrial membranes with remarkable increases in the level of lipid peroxides in mitochondria take place in the liver of rats fed a hydrazine-diet for 3 d when MG are not yet formed [34,36]. We have shown in the present study that intracellular levels of ROS in CPtreated cells become distinctly elevated before MG are formed, and become additionally elevated after MG are formed. However, the rate of the generation of ROS from hydrazine-induced MG was decreased compared to that from mitochondria of control animals. We speculate that the level of ROS per cell in the liver of animals treated with hydrazine for 7 d was kept high because the amount of ROS derived from hydrazine metabolism was far larger than that derived from MG although the rate of ROS generation from MG was decreased. Therefore, additional studies are essential to additionally test the hypothesis, and to clarify the role of MG in free radical-mediated apoptotic process of the cell.
Fig. 8. Changes in the mitochondrial membrane potential of CP-treated RL-34 cells. Cells cultured in the presence of CP (300 mg/ml) for 12 h, 22 h, and 36 h were stained with CMXRos. Numbers of cells were plotted against the fluorescence intensity of the dye. To establish a positive control, cells were treated with valinomycin (1 mM/ml), which is known to decrease the mitochondrial membrane potential, for 60 min, and then stained with the dye (shown at the bottom). A total of 10,000 cells were assayed for flow cytometry in each group.
amounts of free radicals, nuclear condensation and DNA fragmentation, which characterize apoptotic changes of the cell, may become evident. If the level of free radicals exceeds that which cells cannot accommodate themselves to, cells may become necrotic without forming MG. It is well known that MG are formed in various tissues of cold-blooded animals during hibernation [53]. Mitochondria in the cell of various tissues of such animals are required to generate a certain amount of ATP, which is sufficient for the maintenance of the basal metabolism to keep animals alive during hibernation. Therefore, there may be no
Role of the membrane fusion in the induction of free radical–induced formation of MG Previously, we have proposed that MG are formed by the fusion of adjacent mitochondria based on the observation that physicochemical and biochemical changes of mitochondrial membranes during the formation processes of hydrazine-, ethanol-, and CP-induced-MG take place [25, 28–30,35,36]. All these changes are in agreement with those occurring in cell membranes during the cell-to-cell fusion process. Distinct decreases in the number of mitochondria per cell in cells treated with various chemicals when they are revealed on one plane of section for electron microscopy will indirectly support the hypothesis that the membrane fusion may play a key role in the induction of MG (for example, number of mitochondria per cell [mean 6 SE]: hepatocytes cultured for 22 h in the presence of 2 mM hydrazine, 80.8 6 3.4; control, 184.2 6 8.7 [p , .001]). However, it must be noticed here that MG demonstrated in the present study were often characterized by their extremely pale matrix. Therefore, swelling or disturbances
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in protein synthesis in mitochondria or both must be taken into account as a secondary factor contributing to the enlargement of mitochondria besides a possible contribution of the membrane fusion to the mechanism. Besides abovementioned factors, there are many open questions concerning the molecular regulation of the MG formation, like the role of calcium cycling, lipid peroxidation, or changes in the enzyme activities. We have shown in the present study, that the formation of MG precedes apoptosis. However, it should be stressed that free radical-mediated apoptosis is not always preceded by the formation of MG. For example, we have failed to induce MG by carbon tetrachloride in culture cells and in rat or mouse livers (Karbowski and Wakabayashi, unpublished observation). There may be other cases in which free radical-mediated apoptosis is not preceded by the MG formation. Exact reason for this is not clear at the present time. There must be several factors that must be taken into account: the nature of free radicals generated from chemicals, activities of the cell to metabolize chemicals to generate free radicals, the amount of free radicals to which the cell is exposed at one time, etc. For example, if cells are exposed to a large amount of free radicals at one time, the mitochondrial membranes might be damaged severely so that there is no chance for the mitochondrial membranes to alter their physicochemical and biochemical properties into those that are favorable for the membrane fusion, and cells may become apoptotic without forming MG or in some cases may become even necrotic. The fact that it requires a certain period of time until MG are formed (20–22 h) after cells are exposed to inducers of free radicals might partly support this hypothesis. However, additional studies are definitely required.
[7]
[8]
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[13]
[14]
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[16]
[17] [18]
Acknowledgements — The authors deeply thank Dr. Bernard Tandler, Visiting Professor of Department of Oral Anatomy, Kyushu Dental College, Kitakyushu, Japan, for his valuable discussion and advice. Authors also thank Mrs. K. Yoshioka for the preparation of the manuscript. This work was supported in part by a grant from the Ministry of Education, Science and Culture of Japan (08670169).
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PII S0891-5849(98)00209-3
Original Contribution FREE RADICAL–INDUCED MEGAMITOCHONDRIA FORMATION AND APOPTOSIS MARIUSZ KARBOWSKI,* CHIEKO KURONO,† MICHAL WOZNIAK,‡ MARIUSZ OSTROWSKI,* MASAAKI TERANISHI,* YUJI NISHIZAWA,* JIRO USUKURA,* TSUYOSHI SOJI,† and TAKASHI WAKABAYASHI* (Received 11 May 1998; Revised 23 July 1998; Accepted 23 July 1998) *Department of Cell Biology and Molecular Pathology, Nagoya University School of Medicine, Showa-ku, Nagoya, Japan, † Nagoya City University Medical School, Mizuho-ku, Nagoya, Japan, ‡Department of Biochemistry, Medical University of Gdansk, Gdansk, Poland
Abstract—Pathophysiological meaning and the mechanism of the formation of megamitochondria (MG) induced under physiological and pathological conditions remain obscure. We now provide evidence suggesting that the MG formation may be a prerequisite for free radical-mediated apoptosis. MG were detected in primary cultured rat hepatocytes, rat liver cell lines RL-34 and IAR-20 and kidney cell line Cos-1 treated for 22 h with various chemicals known to generate free radicals: hydrazine, chloramphenicol, methyl-glyoxal-bis-guanylhydrazone, indomethacin, H2O2, and erythromycin using a fluorescent dye Mito Tracker Red CMXRos (CMXRos) for confocal laser microscopy and also by electron microscopy. Remarkable elevations of the intracellular level of reactive oxygen species (ROS), monitored by staining of cells with a fluorescent dye carboxy-H2-DCFDA, were detected before MG were formed. Prolongation of the incubation time with various chemicals, specified above, for 36 h or longer has induced distinct structural changes of the cell, which characterize apoptosis: condensation of nuclei, the formation of apoptotic bodies, and the ladder formation. Cells treated with the chemicals for 22 h were arrested in G1 phase, and apoptotic sub-G1 populations then became gradually increased. The membrane potential of MG induced by chloramphenicol detected by CMXRos for flow cytometry was found to be decreased compared to that of mitochondria in control cells. Rates of the generation of H2O2 and O22 from MG isolated from the liver of rats treated with chloramphenicol or hydrazine were found to be lower than those of mitochondria of the liver of control animals. We suggest, based on the present results together with our previous findings, that the formation of MG may be an adaptive process at a subcellular level to unfavorable environments: when cells are exposed to excess amounts of free radicals mitochondria become enlarged decreasing the rate of oxygen consumption. Decreases in the oxygen consumption of MG may result in decreases in the rate of ROS production as shown in the present study. This will at the same time result in decreases in ATP production from MG. If cells are exposed to a large amount of free radicals beyond a certain period of time, lowered intracellular levels of ATP may result in apoptotic changes of the cell. © 1998 Elsevier Science Inc. Keywords—Megamitochondria, Free radicals, Cell culture, Apoptosis, Flow cytometry
INTRODUCTION
such as deprivation of tropic factors [4,5], heat shock [6], and various cytotoxic substances [7,8]. Reports have been accumulated that mitochondria are deeply involved in the regulation of apoptosis: bcl-2 is located mainly on the outer membrane of mitochondria [9]; bcl-2 protein has antioxidant properties and inhibits apoptosis by suppressing the formation or effects of reactive oxygen species (ROS) [10,11]; cytochrome c, localized in the inner membranes of mitochondria, is released from mitochondria of pre-apoptotic cells and can trigger the activation of CED-3 family proteases [12,13]; bcl-2 was found to prevent the release of cytochrome c from mi-
Apoptosis (programmed cell death) plays a crucial role in the normal development and differentiation of multicellular organisms and is essential for embryogenesis and metamorphosis [1–3]. Apoptosis is also induced by various pathological conditions and a variety of agents Address correspondence to: Takashi Wakabayashi, Department of Cell Biology and Molecular Pathology, Nagoya University School of Medicine, 65, Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan; Tel: 052-744-2028; Fax: 052-744-2041; E-Mail: [email protected]. nagoya-u.ac.jp. 396
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tochondria [13]; collapse of the mitochondrial transmembrane potential was reported to be an early, irreversible symptom of apoptosis occurring before the nuclear sign of cell death [14 –16]. It can, therefore, be deduced from these data that mitochondria play a crucial role in apoptosis. One of the important factors implicated in the different steps of apoptosis is the generation of ROS [17]. Oxidative stress is a common element of apoptosis induced by various stimuli that often do not exert a direct oxidant action [8,18,19]. Substances with antioxidative properties have been reported to inhibit apoptosis induced by various chemicals [14,20 –23]. We have been studying the mechanism of the formation of megamitochondria (MG) using hydrazine and ethanol as experimental models [24 –27]. We have found that free radicals are intimately related to the mechanism of the formation of MG in the cases of chloramphenicol (CP) [28], hydrazine [29], and ethanol [30]. We have succeeded in suppressing the MG formation induced by these chemicals by a-tocopherol [31], coenzyme Q10 [32] and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1oxyl(4-OH-TEMPO) [28,29,33], a spin trapping agent and at the same time a scavenger for free radicals. We have proposed that free radicals in some way modify mitochondrial membranes favorable for the membrane fusion resulting in the formation of MG based on changes in physicochemical and biochemical properties of mitochondrial membranes during the formation process of MG [28,29,34 –36]. The present study, demonstrates that the formation of MG caused by various inducers of free radicals in cultured cells precedes apoptosis. According to the classic definition of apoptosis, structural changes of mitochondria during the process of apoptosis have not been stressed except for the swelling at an early stage of the process [15,37]. In this article, we have tried to correlate the formation of MG to free radical-mediated apoptotic processes.
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with 1% heat-inactivated calf serum, 4 mM L-glutamate, 0.45% glucose and kanamycin (60 mg/L). Treatment of cells with various chemicals The following chemicals were tested in the present study: hydrazine (0.5–5.0 mM); CP (100 –500 mg/ml); H2O2 (0.1– 0.5 mM); erythromycin (100 –200 mg/ml); methylglyoxal-bis-guanylhydrazone (M-GAG) (50 mg/ ml); indomethacin (0.1– 0.5 mM). Visualization of mitochondria by confocal laser microscopy A cell-permeant, mitochondria-specific fluorescent dye, Mito Tracker Red CMXRos (CMXRos) (Molecular Probes Inc., Eugene, OR, USA), was used to visualize mitochondria. Staining of mitochondria was performed by the method of Poot et al. [42]. Cells growing on coverslips (10 3 10 mm) were incubated with CMXRos at a final concentration of 500 mM for 30 min in the culture chamber. Coverslips were then rinsed with the culture medium followed by fixation with a fixative containing 2% glutaraldehyde, 2% formaldehyde, and 0.1 M Na-cacodylate, pH 7.4. Specimens were examined under a confocal laser microscope Bio-Rad MRC 1024. Flow cytometric analysis of mitochondrial membrane potential Cells growing on 10-cm culture dishes (Falcon) were incubated with 500 nM CMXRos in the atmosphere of 5% CO2 at 37oC. After 30 min of incubation cells were washed in prewarmed PBS, collected and resuspended in 1 ml of a freshly prepared culture medium and immediately analyzed by flow cytometry using Coulter Elite FACSCAN (Coulter Corporation, Miami, FL) according to the method of Poot et al. [42]. Forward and side scatters were used to establish size-gates and to exclude cellular debris and apoptotic cells from the analysis.
MATERIALS AND METHODS
Cell culture Hepatocytes were isolated by a collagenase perfusion technique from phenobarbital-pretreated or control male Wistar rats weighing 100 to 120 g as described before [38]. Rat liver cell lines RL-34 cells (JCRB 0247) [39], IAR-20 cells (JCRB 0610) [40], and monkey kidney cell line COS-1 cells (JCRB 9082) [41] were obtained from the Health Science Research Resources Bansk (HSRRB) (Osaka, Japan). RL-34 cell and COS-1 cells were maintained in DMEM, IAR-20 cells in EMEM, supplemented
Assay for free radical production Visualization of intracellular levels of free radicals by confocal laser microscopy. Detection of the overall intracellular generation of free radicals was done essentially by the dichlorodihydrofluorescein diacetate (H2DCFDA) staining method [43] as described by Garland and Halestrap [44]. H2-DCFDA was replaced by carboxy-H2-DCFDA in the present study because the latter was expected to have enhanced retention inside the cell due to two negative charges at physiological pH. Cells cultured on glass coverslips were incubated for 60 min at
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37°C with 5 mM carboxy-H2-DCFDA dissolved in the culture medium in the culture chamber. Coverslips were then fixed with a fixative containing 2% glutaraldehyde and 2% formaldehyde dissolved in PBS, and analyzed under a confocal laser microscope using a FITC barrier filter. The laser intensities and photodetector gains were held constant to allow comparisons of relative fluorescence intensities of cells between the control and experimental cells. Flow cytometric analysis of intracellular levels of free radicals. Cells grown on 10-cm culture dishes were stained with 5 mM carboxy-H2-DCFDA and were kept for 60 min in the culture chamber, as described previously, and then harvested by trypsinization. Cells were then washed with PBS, resuspended in PBS and submitted immediately to analysis using a Coulter Elite FACSCAN (Coulter Corporation) with excitation and emission settings of 495 and 525 nm, respectively. Gating was performed to remove apoptotic cells and cellular debris before data were collected. Detection of free radicals generated from mitochondria. The rate of the generation of ROS from mitochondria was examined besides the estimation of the overall intracellular levels of free radicals, described previously, to elucidate functional aspects of MG. For this purpose, we have adopted in vivo experimental model for the induction of MG using hydrazine, because data are already available concerning the rate of oxygen consumption, phosphorylating abilities, and the rate of lipid peroxidation of hydrazine-induced MG [24,25,29,31,34,36]. Male Wistar rats aged 4 weeks were fed a hydrazine-diet as described before [24]. After 3 and 7 days, mitochondria were isolated from the liver. The inner mitochondrial membrane fraction was prepared as described subsequently: mitochondria obtained from the control and experimental animals were suspended in 3 mM phosphate buffer, pH 7.0. The suspension was centrifuged for 10 min at 20,000g. The residue obtained was resuspended in 3 mM phosphate buffer, and sonicated 3 times using a Branson sonifier (setting 3.5–5.0) for 30 s at each time. Sonicated samples were centrifuged for 20 min at 25,000g. The supernatant, therefore, obtained was centrifuged for 30 min at 100,000g, and the resultant pellet was suspended in 3 mM phosphate buffer, pH 7.0, and used as the submitochondrial fraction. Generation of O22 from the submitochondrial fraction was measured essentially according to the method of Kakinuma and Minakami [45] using acetylated cytochrome c. Generation of H2O2 from the submitochondrial fraction was measured essentially by the method of Loschen and Flohe [46] using fluorescent dye scopoletin.
Cell cycle and DNA analyses Cell cycle analysis was performed by flow cytometry using nuclei essentially according to the method of Mancini et al. [47]. DNA staining was obtained with 500 ml of a solution containing 100 mg/ml of propidium iodide (Molecular Probes, Inc.), 0.1% Triton X-100 and 1% fetal calf serum for 30 min at 4°C in the dark, followed by flow cytometric analysis using a Coulter Epics XL flow cytometer (Coulter Corporation). DNA was extracted according to the method of Garland and Halestrap [44]. Electron microscopy An equal volume of a fixative containing 4% glutaraldehyde, 4% formaldehyde and 0.2 M Na-cacodylate, pH 7.4, was added directly to the culture medium to fix detached and attached cells on culture dishes. After fixation with the aldehyde solution, sample were processed for electron microscopy as described before [38]. Thin sections were stained with lead citrate and examined in a Hitachi 7000 electron microscope operated at 75 kV. RESULTS
Confocal laser microscopic and electron microscopic observations of mitochondria of cultured cells treated with inducers of free radicals A variety of chemicals that are known to generate free radicals directly or indirectly was tested in the present study: CP, M-GAG, hydrazine, H2O2, erythromycin, and indomethacin. Cells used were: rat hepatocytes, RL-34 cells, IAR-20 Cells, and COS-1 cells. All chemicals tested in the present study invariably induced remarkable structural changes of mitochondria in four kinds of cells, specified above, treated for 22 h. Examples of confocal laser micrographs are shown in Fig. 1. In control cells [RL-34 cells (A), IAR-20 cells (B), rat hepatocytes (C)], filamentous mitochondria stained with CMXRos are discerned clearly whereas mitochondria in cells treated with various chemicals for 22 h became granular. Chemicals tested in the present study showed essentially the same results regardless of the sources of cells. Structural changes of mitochondria in cells treated with various chemicals were discerned more clearly under the electron microscope (Figs. 2 and 3). Cells of the three different cell lines used in the present study were characterized by the presence of mitochondria much smaller in size and number per cell compared to those of rat hepatocytes (Fig. 2, A: RL-34 cells; B: IAR-20 cells; C: COS -1 cells; D: rat hepatocytes). When they were cultured for 22 h in the presence of CP, for example, mitochondria became enlarged dis-
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Fig. 1. Induction of megamitochondria in culture cells by inducers of free radicals-confocal laser micrographs. (A) RL-34 cells. A1: control; A2: chloramphenicol (CP); A3: methylglyoxal-bis-guanylhydrazone (M-GAG). (B) IAR-20 cells. B1: control; B2: CP; B3: erythromycin. (C) Rat hepatocytes. C1: control; C2: CP; C3: hydrazine. Cultivation time: 22 h. Concentration of chemicals: CP, 300 mg/ml; erythromycin, 200 mg/ml; hydrazine, 2 mM. Bars: 10 mm (A, B), 10 mm (C).
tinctly. Enlarged mitochondria were often bizarre in shapes (B2, C2) and pale in their matrix (A2). Extremely elongated mitochondria were sometimes encountered (C3). In the case of rat hepatocytes cultured in the presence of hydrazine for 22 h (D2), the density of the matrix of enlarged mitochondria was similar to that of control mitochondria. When the cultivation time of these cells in the presence of CP or hydrazine was prolonged to 72 h, nuclei of these cells became condensed to various degrees, which were characterisics of apoptotic cells (Fig. 3). Enlarged mitochondria with vesicular cristae or those almost devoid of cristae were seen in these cells suggesting that they became degenerative.
Changes in the structure of mitochondria and apoptotic changes of the cell, described previously, were seen in all cases of chemicals tested in the present study. When the concentration of each chemical was raised within the range, specified in Materials and Methods, these changes became more distinct. PI staining of nuclei of CP- and M-GAG- treated RL-34 cells Because it has turned out that inducers of free radicals tested in the present study cause apoptotic changes of the cell, we examined changes in nuclei by
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Fig. 2. Induction of megamitochondria in culture cells treated with inducers of free radicals for 22 h— electron micrographs. (A): RL-34 cells. A1: control; A2: CP. (B) IAR-20 cells. B1: control; B2: CP. (C) COS-1 cells. C1: control; C2, C3: CP. (D) Hepatocytes. D1: control; D2: hydrazine. Concentrations of chemicals: CP, 300 mg/ml; hydrazine, 2 mM. Magnification of micrographs: 310,000.
PI staining. When RL-34 cells were treated for 48 h with CP or M-GAG, nuclei of these cells revealed highly fluorescent, irregular shaped structures with PI staining while those of the control cells were stained weakly with the dye keeping their round contours (Fig. 4).
Cell cycle analysis on CP- and M-GAG-treated RL-34 cells In the present study, we have also carried out flow cytometric analysis on PI-stained DNA obtained from
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Fig. 2. Continued
cells treated with chemicals for various lengths of time hoping to detect apoptotic cells quantitatively. Examples are shown in Fig. 5 using CP- and M-GAG-treated RL-34 cells. In the case of CP-treated cells, the population of cells in G1 phase of cell cycle reached 62% after 24 h whereas that in the control cells was 55%. The
prolongation of the treatment of cells with CP for up to 48 h has disclosed a sub-G1 peak, which is indicative of apoptotic cells. The population of cells in sub-G1 phase continued to increase thereafter. DNA obtained from cells treated with M-GAG showed similar results to those of the case of CP, described previously.
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Fig. 2. Continued
Detection of sub-G1 peak, described previously, commonly recognized as the landmark of apoptotic change of the cell has prompted us to examine the existence of oligonucleosome-sized genomic DNA fragmentation, which has been observed in many cases of physiological and drug-induced apoptotic changes of the cell. Fig. 6 shows the time sequence of DNA fragmentation and formations of a ladder-like pattern in RL-34 cells treated with CP or M-GAG. Treatment of cells with CP for 22 h when the formation of MG was already evident did not give any noticeable changes in DNA patterns (lane 2) whereas the ladder formation became evident in cells treated with CP for 48 h and 72 h (lanes 3 and 4, respectively). The ladder formation was not evident in cells treated with M-GAG-for up to 48 h (lane 5, 22 h; lane 6, 48 h) and it became evident after 72 h (lane 7). Similar results were obtained with erythromycin: the ladder formation was detected after 72 h, although it was less distinct compared to the case of CP or M-GAG (lane 9, 22 h; lane 10, 48 h; lane 11, 72 h).
[10]. In the present study, we have adopted carboxy-H2DCFDA, an analog of H2-DCFDA, to estimate levels of ROS in CP- and M-GAG-treated cells. In Fig. 7A, typical confocal laser micrographs were obtained from RL-34 cells cultured for 22 h in the absence (A1), or presence of CP (300 mg/ml) (A2) or M-GAG (50 mg/ml) (A3). It is evident from the figure that the amount of ROS generated in experimental cells has definitely increased compared to that of the control cells. A typical histogram of carboxy-DCF fluorescence of cells treated with CP or M-GAG for 22 h is shown in Fig. 7B. It is evident from the figure that peaks in CP- and M-GAG-treated cells, especially the latter, shifted to the right indicating increased ROS generation in a large population of these cells. To clarify the correlation between the formation of MG and the generation of ROS by inducers of free radicals, the amount of ROS generated inside the cell was plotted against the duration of time after the treatment of cells with CP (Fig. 7C). It should be stressed here that there was already a distinct increase in the level of ROS in cells treated with CP for 10 h when MG were not yet formed, and an additional increase was observed after 22 h when MG were induced.
Intracellular levels of free radicals in CP- and M-GAG-treated RL-34 cells
Rate of generation of ROS from mitochondria of hydrazine-treated rat livers
The nonfluoresent dye H2-DCFDA passively enters the cell and on oxidation forms fluorescent DCF, which is a reporter of ROS generation at a level of a single cell
To analyze the role of MG in cellular oxidative stress, generations of O22 and H2O2 from mitochondria were examined besides the estimation of overall production of
Electrophoretic patterns of nuclear DNA extracted from CP-, M-GAG-, and erythromycin-treated cells
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Fig. 3. Apoptotic changes of culture cells treated with inducers of free radicals for 72 h— electron micrographs. A1, A2: CP-treated RL-34 cells. B1, B2: CP-treated IAR-20 cells. C: CP-treated COS-1 cells. D: hydrazine-treated rat hepatocytes. Concentrations of chemicals: CP, 300 mg/ml; hydrazine, 2 mM. Magnifications of micrographs: 310,000.
intracellular ROS, as described. Table 1 summarizes the rates of generation of O22 and H2O2 from mitochondria isolated from the liver of rats that were fed a hydrazinediet for 3 d and 7 d, respectively. The rate of generation of H2O2 from mitochondria of animals treated with hydrazine for 3 d when MG were not yet formed was
definitely increased compared to that of the control animals. The rate of the generation of O22 from mitochondria of animals treated with hydrazine for 3 d had a tendency to be increased compared to that of the control animals although there was not a statistically significant difference between them. However, rates of generation
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Fig. 3. Continued
of both H2O2 and O22 from mitochondria of rats treated with hydrazine for 7 d became decreased significantly compared to those of the control. Time sequence of changes in the membrane potential of mitochondria in CP-treated RL-34 cells In the present study, we adopted CMXRos as a fluorescent probe to visualize mitochondria by confocal laser microscopy, as described. We also used this dye to detect changes in the mitochondrial membrane potential for flow cytometry (Fig. 8). For this purpose, RL-34 cells were treated with CP (300 mg/ml) for various lengths of time up to 36 h, stained with CMXRos, and were applied to flow cytometry. It is evident from the figure that the peak of cells treated with CP for 22 h when MG were
formed shifted to the left compared to that treated with CP for 12 h, indicating that the membrane potential of mitochondria in the major portion of the former cells became decreased compared to that in the latter. In addition, another small peak with low fluorescence intensity was detected in mitochondria of cells treated with CP for 36 h indicating that a certain population of cells had mitochondria with collapsed membrane potential. In control cells, there was essentially no difference in the peak of the fluorescence intensity between cells cultured for 12 h and those cultured for 22 h, and the peak shifted to the left in cells cultured for 36 h. Cells treated with valinomycin (1 mM/ml), mitochondrial transmembrane potential disrupting agent, showed distinct decreases in CMXRos fluorescence and were used as a positive control in our experiments.
Fig. 4. Propidium iodide staining of RL-34 cells cultured for 48 h in the presence of CP or M-GAG. A: control; B: CP (300 mg/ml); C: M-GAG) (50 mg/ml). Details in the experimental conditions are described in the Materials and Methods section.
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Fig. 6. Electrophoresis of nuclear DNA extracted from RL-34 cells cultured in the presence of various inducers of free radicals. Control: lane 1; CP (300 mg/ml): lanes 2– 4; M-GAG (50 mg/ml): lanes 5–7; erythromycin (200 mg/ml): lanes 8 –10. Cultivation time: 22 h: lanes 2, 5, 8; 48 h: lanes 3, 6, 9; 72 h: lanes 1, 4, 7, 10; DNA size markers: lane 11. Details in the experimental conditions are described in the Materials and Methods section.
Fig. 5. Cell cycle analysis on CP- and M-GAG-treated RL-34 cells. Cells were cultured in the absence (A1–A3) or presence of CP (300 mg/ml) (B1–B3) or M-GAG (50 mg/ml) (C1—C3). Cultivation time: 24 h (A1, B1, C1); 48 h (A2, B2, C2); 72 h (A3, B3, C3). Nuclear preparations were prepared from asynchronously growing cells. Details in the experimental conditions are described in the Materials and Methods section.
cells. (c) The rate of the generation of ROS from mitochondria of the liver of rats placed on a hydrazine-diet for 3 d when MG were not formed yet was elevated compared to that of control animals, whereas that of animals placed on a hydrazine-diet for 7 d when MG were formed was distinctly decreased compared to that of the control animals. (d) Apoptotic changes of the cell were observed in the cells treated with various chemicals for 36 h or longer. (e) Decreases in the membrane potential of mitochondria were observed in RL-34 cells treated with CP for 32 h when MG were formed. Then, the following serious questions are raised: is the formation of MG a coincidence, consequence, or prerequisite for free radical-mediated apoptosis, and how do free radicals induce MG?
Table 1. Rates of Generation of O22 and H2O2 in Hydrazine-Treated Rat Liver Mitochondrial Inner Membranesa DISCUSSION
We have shown that the formation of MG precedes apoptotic changes of the cell in the present experimental conditions. The series of events occurring in the cells treated with various free radical-generating chemicals tested in the present study for various lengths of time are summarized as follows: (a) MG were induced in various cells treated for 22 h by various free radical-inducers. (b) The formation of MG was preceded by remarkable increases in the intracellular level of ROS in CP-treated
Animals Control Hydrazine 3d 7d
O22 (nmol/min/mg protein)
H2O2 (nmol/min/mg protein)
1.25 6 0.02
0.44 6 0.01
1.40 6 0.05 0.62 6 0.03b
0.63 6 0.05c 0.32 6 0.03c
a Inner membrane fractions were obtained from the livers of rats fed with a 1% hydrazine-diet for 3 d and 7 d, respectively. Values are expressed as the means 6 SE of three different experiments. Data on experimental animals are statistically different from those of the control at: b( p , .001); c.02 , p , .05).
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Fig. 7. Levels of reactive oxygen species (ROS) in RL-34 cells cultured in the presence of CP or M-GAG. (A) Confocal micrographs. Cells were cultured for 22 h in the absence (A1) and presence of CP (300 mg/ml) (A2) or M-GAG (50 mg/ml) (A3). Cell were stained with carboxy-H2-DCFDA. (B) Flow cytometry. Cells of the control and experimental groups cultured in the same conditions as those described for the legend (A) were stained with carboxy-H2-DCFDA. Cell were then harvested by trypsinization. (C) Changes in the intracellular levels of ROS after the addition of CP. Cells were cultured for various lengths of time in the absence or presence of CP (300 mg/ml). Cells were assayed for ROS productions by the method described in the Materials and Methods section. Relative ROS productions in CP-treated cells are expressed as a percentage of the cell population with higher carboxy-DCF fluorescence intensities than the cell with the highest fluorescence intensity among the control cells. The fluorescence intensities of the dye in CP-treated cells were plotted taking that of the control as 0.
Pathophysiological meaning of the MG formation with respect to free radical-mediated apoptosis Phosphorylating abilities and rates of oxygen consumption of MG induced by various experimental conditions or obtained from human biopsied materials are often lower to various degrees compared to those of the control [25,32,48 –50]. We assume that in physiological conditions enzymatic and non-enzymatic defence systems against free radicals work effectively within the cell so that cells are devoid of free radicalmediated various injuries. However, when the level of free radicals exceeds a certain level as in the cases of the present experimental conditions, mitochondria become enlarged to various degrees lowering the rate of oxygen consumption and phosphorylating abilities.
Mitochondrial respiration accounts for about 90% of cellular uptake and 1–2% of the oxygen consumed is converted to ROS [51,52]. MG may generate less amounts of ROS because of lower rates of respiration. We have shown in the present study, that the rate of the generation of ROS from hydrazine-induced MG is actually decreased compared to that from control mitochondria. Cells then stop growing due to a shortage of ATP supply from MG. We have shown previously that the intracellular level of ATP in hepatocytes and the ability of MG to synthesize ATP are actually remarkably decreased in rats treated chronically with ethanol [32]. If free radicals are removed from the cell, MG may return to normal, functionally and structurally. However, if cells are exposed additional to excess
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need for mitochondria in the cell of hibernating animals to respire actively resulting in the generation of large amounts of ROS. If this is the case, the MG formation could be regarded as an adaptive process at a level of subcellular organelles to unfavorable environments. To test this hypothesis, we measured the total amount of ROS generated inside the cell and the level of ROS generated from mitochondria before and after the formation of MG (confer Fig. 7 and Table 1). We have shown previously that distinct changes in biochemical and physicochemical properties of mitochondrial membranes with remarkable increases in the level of lipid peroxides in mitochondria take place in the liver of rats fed a hydrazine-diet for 3 d when MG are not yet formed [34,36]. We have shown in the present study that intracellular levels of ROS in CPtreated cells become distinctly elevated before MG are formed, and become additionally elevated after MG are formed. However, the rate of the generation of ROS from hydrazine-induced MG was decreased compared to that from mitochondria of control animals. We speculate that the level of ROS per cell in the liver of animals treated with hydrazine for 7 d was kept high because the amount of ROS derived from hydrazine metabolism was far larger than that derived from MG although the rate of ROS generation from MG was decreased. Therefore, additional studies are essential to additionally test the hypothesis, and to clarify the role of MG in free radical-mediated apoptotic process of the cell.
Fig. 8. Changes in the mitochondrial membrane potential of CP-treated RL-34 cells. Cells cultured in the presence of CP (300 mg/ml) for 12 h, 22 h, and 36 h were stained with CMXRos. Numbers of cells were plotted against the fluorescence intensity of the dye. To establish a positive control, cells were treated with valinomycin (1 mM/ml), which is known to decrease the mitochondrial membrane potential, for 60 min, and then stained with the dye (shown at the bottom). A total of 10,000 cells were assayed for flow cytometry in each group.
amounts of free radicals, nuclear condensation and DNA fragmentation, which characterize apoptotic changes of the cell, may become evident. If the level of free radicals exceeds that which cells cannot accommodate themselves to, cells may become necrotic without forming MG. It is well known that MG are formed in various tissues of cold-blooded animals during hibernation [53]. Mitochondria in the cell of various tissues of such animals are required to generate a certain amount of ATP, which is sufficient for the maintenance of the basal metabolism to keep animals alive during hibernation. Therefore, there may be no
Role of the membrane fusion in the induction of free radical–induced formation of MG Previously, we have proposed that MG are formed by the fusion of adjacent mitochondria based on the observation that physicochemical and biochemical changes of mitochondrial membranes during the formation processes of hydrazine-, ethanol-, and CP-induced-MG take place [25, 28–30,35,36]. All these changes are in agreement with those occurring in cell membranes during the cell-to-cell fusion process. Distinct decreases in the number of mitochondria per cell in cells treated with various chemicals when they are revealed on one plane of section for electron microscopy will indirectly support the hypothesis that the membrane fusion may play a key role in the induction of MG (for example, number of mitochondria per cell [mean 6 SE]: hepatocytes cultured for 22 h in the presence of 2 mM hydrazine, 80.8 6 3.4; control, 184.2 6 8.7 [p , .001]). However, it must be noticed here that MG demonstrated in the present study were often characterized by their extremely pale matrix. Therefore, swelling or disturbances
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in protein synthesis in mitochondria or both must be taken into account as a secondary factor contributing to the enlargement of mitochondria besides a possible contribution of the membrane fusion to the mechanism. Besides abovementioned factors, there are many open questions concerning the molecular regulation of the MG formation, like the role of calcium cycling, lipid peroxidation, or changes in the enzyme activities. We have shown in the present study, that the formation of MG precedes apoptosis. However, it should be stressed that free radical-mediated apoptosis is not always preceded by the formation of MG. For example, we have failed to induce MG by carbon tetrachloride in culture cells and in rat or mouse livers (Karbowski and Wakabayashi, unpublished observation). There may be other cases in which free radical-mediated apoptosis is not preceded by the MG formation. Exact reason for this is not clear at the present time. There must be several factors that must be taken into account: the nature of free radicals generated from chemicals, activities of the cell to metabolize chemicals to generate free radicals, the amount of free radicals to which the cell is exposed at one time, etc. For example, if cells are exposed to a large amount of free radicals at one time, the mitochondrial membranes might be damaged severely so that there is no chance for the mitochondrial membranes to alter their physicochemical and biochemical properties into those that are favorable for the membrane fusion, and cells may become apoptotic without forming MG or in some cases may become even necrotic. The fact that it requires a certain period of time until MG are formed (20–22 h) after cells are exposed to inducers of free radicals might partly support this hypothesis. However, additional studies are definitely required.
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Acknowledgements — The authors deeply thank Dr. Bernard Tandler, Visiting Professor of Department of Oral Anatomy, Kyushu Dental College, Kitakyushu, Japan, for his valuable discussion and advice. Authors also thank Mrs. K. Yoshioka for the preparation of the manuscript. This work was supported in part by a grant from the Ministry of Education, Science and Culture of Japan (08670169).
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