Swelling of Free-Radical-Induced Megamitochondria Causes Apoptosis

Experimental and Molecular Pathology 68, 104–123 (2000) doi:10.1006/exmp.1999.2288, available online at http://www.idealibrary.com on

Swelling of Free-Radical-Induced Megamitochondria Causes Apoptosis

Masa-aki Teranishi,* Jan H. Spodonik,*,1 Mariusz Karbowski,* Chieko Kurono,† Tsuyoshi Soji,† and Takashi Wakabayashi* *Department of Cell Biology and Molecular Pathology, Nagoya University School of Medicine, Nagoya 466-8550, Japan; and † Department of Anatomy, Nagoya City University Medical School, Nagoya, Japan

Received July 30, 1999

Recently, we have found that cultured cells from various sources exposed to free radicals become apoptotic in the presence of megamitochondria (MG). The purpose of the present study is to answer the following two questions: (1) Do functions obtained from the “MG fraction” isolated from normal mitochondria by a routine procedure represent the functions of MG since the fraction consists of enlarged and normal-size mitochondria? (2) What is the correlation between MG formation and apoptotic changes of the cell? In the present study the heavy fraction rich in mitochondria enlarged to varying degrees and the light fraction consisting mainly of normal-size mitochondria were isolated independently from the livers of rats treated with hydrazine for 4 days (4H animals) and 8 days (8H animals), and some functions related to apoptosis were compared. Results were as follows: (1) Mitochondria in both fractions obtained from 8H animals swelled far less in various media than those obtained from the controls, suggesting that the permeability transition pores had been opened before they were exposed to swelling media. (2) The membrane potential of mitochondria in both fractions obtained from 8H animals was distinctly decreased. (3) The rates of reactive oxygen species generation from mitochondria of both fractions in 4H animals were equally elevated, while those in 8H animals were equally decreased compared to those of controls. These results, together with morphological data obtained in the present study, suggest that enlarged and normal-size mitochondria are a part of MG and that the secondary swelling of MG causes the apoptotic changes in the cell. q 2000 Academic Press Key Words: megamitochondria; free radicals; apoptosis; permeability transition pores; swelling; reactive oxygen species.

INTRODUCTION

Accumulating data indicate that free radicals are intimately related to the mechanism of megamitochondria (MG) formation (Adachi et al., 1994, 1995; Antosiewicz et al., 1994; Karbowski et al., 1997, 1999a,b; Matsuhashi et al., 1996, 1997, 1998; Tandler et al., 1996; Teranishi et al., 1999a; Wakabayashi et al., 1997). We have shown that scavengers of free radicals, including 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, coenzyme Q10, and a-tocopherol, suppress hydrazine-, ethanol-, and chloramphenicol (CP)induced formation of MG (Adachi et al., 1995a; Antosiewicz et al., 1994; Karbowski et al., 1999a; Matsuhashi et al., 1996, 1997, 1998; Teranishi et al., 1999a; Wakabayashi et al., 1997). Furthermore, we have succeeded in inducing MG in cultured cells by hydrogen peroxide, suggesting that free radicals may be directly involved in the MG formation process (Karbowski et al., 1997). However, details of the pathophysiological meaning of the phenomenon of MG formation are still obscure. Recently, we have found that the freeradical-induced formation of MG is succeeded by apoptosis (Karbowski et al., 1999a,b). We have shown that MG induced in cultured cells treated for 22–24 h with inducers of free radicals such as CP and methylglyoxal bis(guanylhydrazone) become swollen with time and a large population of these cells becomes apoptotic after 48–72 h (ca. 48–49%

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On leave of absence from the Department of Anatomy and Neurobiology, Medical University of Gdansk, Gdansk, Poland.

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0014-4800/00 $35.00 Copyright q 2000 by Academic Press All rights of reproduction in any form reserved.

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in the case of CP) (Karbowski et al., 1999a). We have shown data suggesting that the permeability transition pores (PT) or megachannels may be opened by free radicals, resulting in swelling of MG, a decrease in the membrane potential of mitochondria, and apoptotic changes in the cells (Karbowski et al., 1999a, 1999b). Most of these results have been obtained with MG fractions isolated from livers of animals or from cultured cells from various sources treated with inducers of free radicals. However, a serious question has been raised: Does a mitochondrial fraction obtained from the liver of animals or from cultured cells treated with various inducers of free radicals really represent the MG? It is always the case that mitochondria enlarged to various degrees, sometimes reaching or even exceeding the size of the nucleus, and mitochondria that are normal-size or even smaller than those of the control coexist within individual cells. If the latter mitochondria are functionally different from the former, data obtained from the “MG fraction” isolated using the routine isolation procedure for normal mitochondria may not represent those of MG. Thus, we have tried to isolate “enlarged mitochondria” and “small or normal-size mitochondria” independently and we have compared some functional properties of the two mitochondrial fractions. We demonstrate here some evidence that mitochondria enlarged to various degrees and small or normal-size mitochondria coexisting in individual cells are functionally almost identical. We also demonstrate experimental data to indicate that the secondary swelling of MG may lead to apoptotic changes of the cell under oxidative stress.

MATERIALS AND METHODS

Treatment of Animals and of Cultured Cells with CP or Hydrazine Four-week old male Wistar rats were placed on a 1% hydrazine diet for 4–5 and 8–9 days as described previously (Wakabayashi et al., 1983). Four-week old male mice of the ddY strain were fed a 2% CP diet for 9–10 and 14–15 days as described previously (Matuhashi et al., 1996). Hepatocytes were isolated by a collagenase perfusion technique from 4week-old phenobarbital-treated male Wistar rats and cultured as described before (Karbowski et al., 1997). Cells were cultured for up to 48 h in the presence of hydrazine (2 mM) or CP (300 mg/ml).

Isolation of Mitochondria Mitochondria were isolated from the livers of animals treated with CP or hydrazine for various lengths of time, as specified above, according to two isolation procedures. In the routine isolation procedure employed in our laboratory, the liver homogenate was centrifuged for 10 min at 700g, and the resultant supernatant was centrifuged for 10 min at 7000g and washed three times in the isolation medium (designated the (700–7000g)R2- fraction). In an alternative procedure, the liver homogenate was centrifuged for 10 min at 500g, and the resultant supernatant was centrifuged for 10 min at 2000g. The pellet thus obtained was washed three times with isolation medium (the heavy mitochondrial fraction, designated as (500–2000g) R2-). The post-2000g supernatant was centrifuged for 10 min at 7000g. The pellet thus obtained was washed three times (the light mitochondrial fraction, designated as (2000–7000g) R2-).

Swelling Experiments Opening of the PT pores of the mitochondrial membranes was monitored indirectly by exposing mitochondria to various hypotonic media (Petit et al., 1996). Three different swelling media were employed in the present study. Medium A consisted of 100 mM potassium acetate, 15 mM Tris–Cl, pH 7.4, antimycin A (0.5 mg/ml), and 0.1 mM EDTA. The reaction was started by the addition of valinomycin (1 mg/ ml). Medium B consisted of 100 mM ammonium succinate, 15 mM Tris–Cl, pH 7.4, 0.1 mM EDTA, and antimycin A (0.5 mg/ml). The reaction was started by the addition of inorganic phosphate (10 mM). Medium C consisted of 15 mM KCl, 50 mM Tris–Cl, pH 7.4, 5 mM potassium phosphate, 0.1 mM EDTA, 100 mM sucrose, rotenone (1 mg/ ml), and 5 mM potassium succinate. The reaction was started by the addition of 10 mM CaCl2. Mitochondria were suspended in the reaction media at final concentrations ranging from 1.0 to 1.5 mg protein/ml.

Measurements of Cytochrome Contents of Mitochondria Cytochrome contents of mitochondria were measured from the different spectra between the reduced and oxidized forms of cytochromes using a Beckman DU 640 spectrophotometer essentially according to the method of Williams (Williams, 1964) as described before (Wakabayashi et al., 1984).

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Flow Cytometric Analyses of Changes in Volume, Rate of Reactive Oxygen Species (ROS) Generation, and Membrane Potential of Mitochondria Changes in volume of mitochondria. A mitochondrial suspension was incubated with a mitochondria-specific fluorescent dye, Mito Tracker Green FM (FM) (Molecular Probes Inc., Eugene, OR) as described before (Teranishi et al., 1999a). The suspension was incubated with 75 mM FM for 15 min at 258C, centrifuged, resuspended in 1 ml of the isolation medium, and immediately analyzed by flow cytometry using a Coulter Elite FACScan (Coulter Corp., Miami, FL) according to the method of Poot et al. (1996) as described before (Teranishi et al., 1999a). Rate of ROS generation from mitochondria. The rate of ROS generation from mitochondria was measured following the formation of carboxydichlorofluorescein (carboxyDCF), a fluorescent derivative of carboxydichlorodihydrofluorescein diacetate (carboxy-H2-DCFDA) (Molecular Probes Inc.) using flow cytometry according to the method of Garcia-Ruiz et al. (Garcia-Ruiz et al., 1997) as described before (Karbowski et al., 1999a, 1999b). A mitochondrial suspension was incubated with 5 mM carboxy-H2-DCFDA for 1 h at 258C, washed in PBS (pH 7.4), resuspended in PBS, and submitted immediately to FACScan analysis. Membrane potential of mitochondria (Dcm). A mitochondrial suspension was incubated with a mitochondrionspecific fluorescent dye, Mito Tracker CMXRos (CMXRos) (Molecular Probes Inc.) (500 nM), or with rhodamine-123 (10 mg/ml) for 15 min at 258C. The suspension was washed in PBS, resuspended in PBS, and submitted to FACScan analysis as described before (Teranishi et al., 1999a). Determination of Protein Protein was determined by Lowry’s procedure using bovine serum albumin as a standard (Lowry et al., 1951).

the same buffer which had been used for aldehyde fixation, specimens were postfixed with 1% osmium tetroxide and processed for electron microscopy. Mitochondrial suspensions were fixed in 2% glutaraldehyde containing 0.25 M sucrose and 0.05 M sodium cacodylate, pH 7.4, and processed for electron microscopy as described above. Thin sections were cut on a Reichert Ultracut N, stained with lead citrate, and examined in a Hitachi H-7000 electron microscope operated at 100 kV. Mitochondrial suspensions were also prepared for scanning electron microscopy by the method of Winslow et al. (Winslow et al., 1991). Aldehydefixed samples were pelleted, frozen in liquid Freon, and freeze-cleaved with a razor blade. After being thawed, specimens were treated with 0.1% osmium tetroxide, dehydrated, dried, coated with a gold/palladium alloy, and examined in a Hitachi Model S-450 scanning electron microscope.

RESULTS Structural Changes of Mitochondria in the Livers of Rats Treated with Hydrazine and Those of Mice Treated with CP Mitochondria in the livers of rats treated with hydrazine (Fig. 1) for 4–5 days (B) and in those of mice treated with CP for 9–10 days (E) became distinctly enlarged compared to those of the controls (A and D). The density of the matrix of these mitochondria was moderately preserved. When the duration of the feeding of these toxic diets was prolonged further, mitochondria became enlarged further (C and F). The density of the matrix of these extremely enlarged mitochondria decreased distinctly and cristae were seen only on the periphery, suggesting that they became swollen.

Electron Microscopy

Ultrastructural Appearances of Light and Heavy Mitochondria Isolated from the Livers of Rats Treated with Hydrazine

Part of the liver was cut into small pieces and treated with a fixative containing 2% glutaraldehyde, 2% formaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, as described before (Wakabayashi et al., 1984). After being washed in

In Fig. 2, electron micrographs of the (2000–7000g)R2and (500–2000g)R2- fractions obtained from the livers of rats treated with hydrazine for 4–5 and 8–9 days, respectively, are shown. The difference in the size of mitochondria

FIG. 1. Ultrastructural appearances of megamitochondria (MG) induced by hydrazine and chloramphenicol (CP). (A–C) Male Wistar rats aged 4 weeks were placed on a 1% hydrazine diet for 4 (B) or 8 days (C). Control, A. (D–F) Male mice of the ddY strain aged 4 weeks were placed on a 2% CP diet for 9 (E) or 14 days (F). Control, D. Original magnification of electron micrographs, 310,000.

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between the light and heavy mitochondrial fractions obtained from the control animals (A and D, respectively) was not evident whereas that obtained from animals treated with hydrazine for 4 (B and E) and 8 days (C and F) was distinct. It should be noted here that although MG detected in the liver of animals treated with hydrazine for 8 days were extremely swollen (see Fig. 1C) they were isolated in the condensed form, suggesting that they retained the impermeability of the inner mitochondrial membranes to sucrose. Changes in the size of mitochondria in the livers of hydrazine-treated animals were also examined by flow cytometry using a fluorescent dye, FM, to detect the volume of each mitochondrion (Fig. 3). In the case of the controls the peak fluorescence intensity of FM in (500–2000g)R2- shifted to the right distinctly compared to that in (2000–7000g)R2-, indicating that the volume of the major population of mitochondria became distinctly larger in the former than in the latter. As already shown above, electron micrographs demonstrated in Fig. 2 show the cut surface of each mitochondrion sectioned on one plane of section whereas data obtained by flow cytometric analysis on FM-stained mitochondria indicate the volume of each mitochondrion. Thus, although the difference between the two fractions in the cut surface area of mitochondria revealed by electron microscopy was small, difference between the two fractions in the size of mitochondria obtained by flow cytometry was more evident. The peak fluorescence intensity of FM of mitochondria in both (2000–7000g)R2- and (500–2000g)R2- obtained from the livers of animals treated with hydrazine for 4 days (designated 4H(2000–7000g)R2- and 4H(500–2000g)R2-, respectively, thereafter) shifted to the right compared to that of the corresponding controls. Moreover, the peak intensity of FM was definitely larger in 4H(500–2000g)R2- than in 4H(2000–7000g)R2-, indicating that a major population of mitochondria in the former fraction had definitely larger volumes than those in the latter fraction. It is obvious from the figure that a major population of mitochondria in (2000– 7000g)R2- and (500–2000g)R2- obtained from the livers of animals treated with hydrazine for 8 days (designated 8H(2000–7000g)R2- and 8H(500–2000g)R2-, respectively, thereafter) had larger volumes than those in 4H(2000– 7000g) R2- and 4H(2000–7000g)R2-.

109 Indirect Measurement of PT Pores in Hydrazine-Treated Rat Liver Mitochondria MG reduced in the livers of animals treated with hydrazine for 4 days became further enlarged, possibly by swelling, when the duration of the toxic diet was prolonged for up to 8 days as shown above. Thus, swelling experiments were carried out as described below (Fig. 4). Mitochondria in both 4H(500–2000g)R2- and 4H(2000– 7000g)R2- swelled almost to the same degree as the control mitochondria in various swelling media (Fig. 4A) whereas those in 8H(500–2000g)R2- and 8H(2000–7000g)R2swelled far less compared to those of the corresponding controls (Fig. 4B), indicating that the latter had already swollen to some degree before they were exposed to the swelling media. Namely, these results suggest that MG induced in the liver of animals treated with hydrazine for 4 days became enlarged further by swelling due to the opening of the PT pores when the toxic diet treatment was prolonged for 8–9 days.

Changes in the Rate of ROS Generation from HydrazineTreated Rat Liver Mitochondria and from Swollen Mitochondria The rates of ROS generation from mitochondria obtained from the livers of rats treated with hydrazine for 4 and 8 days, respectively, are shown in Fig. 5A. The rates of ROS generation from mitochondria in 4H(500–2000g)R2- and 4H(2000–7000g)R2- increased almost equally compared to those for the paired controls. In contrast, the rates of ROS generation from 8H(500–2000g)R2- and 8H(2000– 7000g)R2- decreased compared to those of the paired controls. To find the reason for the difference in the rate of ROS generation from mitochondria obtained from the livers of animals treated with hydrazine for different periods of time, changes in the rate of ROS generation from swollen mitochondria were examined (Fig. 5B), since mitochondria from the livers of animals treated with hydrazine for 8 days were extremely swollen, as described above. There was already a shift to the right in peak intensity of carboxy-DCF in

FIG. 2. Ultrastructure of the light mitochondrial fraction ((2000–7000g)R2-) and heavy mitochondrial fractions ((500–2000g)R2-) obtained from hydrazine-treated rat livers. (B and E) Mitochondria were isolated from the livers of animals treated with hydrazine for 4 days. (B) Light fraction; (E) heavy fraction. (C and F) Mitochondria were isolated from the livers of animals treated with hydrazine for 8 days. (C) Light fraction; (F) heavy fraction. (A and D) Mitochondria were isolated from the control animals. (A) Light fraction; (D) heavy fraction. Original magnification of electron micrographs, 310,000. For details, see Materials and Methods.

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FIG. 3. Flow cytometric analysis on the volume of mitochondria isolated from hydrazine-treated rat livers. Mitochondria were isolated from the liver of animals treated with hydrazine for 4 and 8 days, respectively, and stained with a fluorescent dye FM. For details, see Materials and Methods.

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FIG. 4. Swelling of hydrazine-treated rat liver mitochondria in various swelling media. The heavy and light mitochondrial fractions obtained from the liver of animals treated with hydrazine for 4 (A) and 8 days (B), designated 4H and 8H, respectively, were incubated with various swelling media specified in the figure at 258C. Changes in absorbance at 540 nm were plotted against time after incubation. For details, see Materials and Methods.

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FIG. 5. Flow cytometric analysis on the rate of the generation of reative oxygen species (ROS) from hydrazine-treated rat liver mitochondria and from mitochondria exposed to a swelling medium. (A) The heavy and light mitochondrial fractions obtained from the livers of animals treated with hydrazine for 4 and 8 days, respectively, were stained with a fluorescent dye carboxy-H2-DCFDA. (B) Rat liver mitochondria were incubated with a medium containing 100 mM potassium acetate, 0.1 mM EDTA, antimycin A (0.5 mg/ml), 15 mM Tris–Cl, pH 7.4, at 258C for up to 5 min. Swelling was initiated by the addition of valinomycin (2 mg).

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FIG. 5—Continued

114 mitochondria exposed for 15 s to a hypotonic medium, indicating that the rate of ROS generation from these mitochondria increased. This tendency became evident when it was measured in the presence of oxidizable substrates (succinate or glutamate plus malate). Increases in the rate of ROS generation from mitochondria were constant findings in the presence and absence of oxidizable substrates when the duration of the experiment was prolonged for up to 5 min, suggesting that decreases in the rate of ROS generation from mitochondria of animals treated with hydrazine for 8 days cannot be ascribed simply to swelling. Changes in the Membrane Potential of Mitochondria (Dcm) Isolated from Hydrazine-Treated Rat Livers and in that of Swollen Mitochondria We have shown previously that MG are formed in RL34 cells treated with CP for 22–24 h and the membrane potential of mitochondria decreases after 48 h or longer when cells become apoptotic (Karbowski et al., 1999a). Decreases in the membrane potential of mitochondria have been shown to be related to the induction of apoptotic changes of the cell (Petit et al., 1995, 1996; Vander Heiden et al., 1997; Vayssiere et al., 1994). Thus, the membrane potentials of the light and heavy mitochondrial fractions obtained from the livers of animals treated with hydrazine for 4 and 8 days, respectively, was compared (Fig. 6A). The peak fluorescence intensities of CMTMRos in mitochondria in both 4H(2000–7000g)R2- and 4H(2000–7000g)R2shifted slightly to the left compared to those of the corresponding controls, indicating that the membrane potential of mitochondria in the former two fractions had a tendency to decrease slightly. In mitochondria of 8H(2000–7000g)R2and 8H(500–2000g)R2-, another small peak besides the main tall peak appeared and the fluorescence intensity of CMTMRos of such small peaks was definitely higher than that of the controls. Previously, we have shown that the fluorescence intensity of Mito Tracker CMXRos detected in each mitochondrion reflects not only the membrane potential of the mitochondrion but also changes in its volume (Karbowski et al., 1999a). Thus, we have also measured the membrane potential of mitochondria in the presence of gramicidin, which is a typical ionophore preventing oxidative

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phosphorylation of mitochondria. There were remarkable shifts in peak intensity of CMTMRos in both the light and the heavy mitochondrial fractions in the control and experimental animals, as shown in the figure. Thus, the difference in the fluorescence intensity of CMTMRos between data obtained in the absence of gramicidin and those obtained in the presence of the ionophore indicates the H+-dependent membrane potential of mitochondria. In Table 1, the mean fluorescence intensity of CMTMRos with and without gramicidin of mitochondria in the light and heavy mitochondrial fractions obtained from control and experimental groups is shown by arbitrary fluorescence units as a typical example. In the case of the control, the differences in the mean fluorescence intensity of CMTMRos of mitochondria measured in the absence and presence of gramicidin between (2000– 7000g)R2- and (500–2000g)R2- were practically the same (720.8 and 712.1 arbitrary fluorescence units, respectively, for 4C, and 641.2 and 632.6 arbitrary fluorescence units, respectively, for 8C). Values obtained from mitochondria of 4H(2000–7000g)R2- and of (2000–7000g)R2- were almost identical (704.0 and 700.4 arbitrary fluorecence units, respectively) and were similar to those obtained from the control mitochondria described above, indicating that the membrane potential of mitochondria of the two fractions in experimental animals compared to that in control animals was equally preserved. On the other hand, values obtained from mitochondria in 8H(2000–7000g)R2- and 8H(500– 2000g)R2- were distinctly smaller than those of the corresponding controls (321.6 and 288.4 arbitrary fluorescence units, respectively), indicating that the membrane potential of mitochondria in both fractions decreased distinctly compared to that of the controls. Since one distinct difference between MG obtained from animals treated with hydrazine for 4 days and for 8 days was remarkable swelling in the latter, we have tested effects of swelling on the membrane potential of mitochondria (Fig. 6B). In this experiment the membrane potential of mitochondria was measured using mitochondria exposed to a hypotonic medium for up to 5 min. The membrane potential of mitochondria exposed to a hypotonic medium for 15 s had already decreased, especially when it was measured in the presence of oxidizable substrates. Rhodamine-123 was adopted to stain mitochondria

FIG. 6. Flow cytometric analysis on the membrane potential of mitochondria isolated from hydrazine-treated rat livers and that of rat liver mitochondria exposed to a swelling medium. (A) The heavy and light mitochondrial fractions were obtained from the animals used for Fig. 5A. Mitochondria were stained with a fluorescent dye CMTMRos in the presence and absence of gramicidin. (B) Mitochondria were exposed to the same swelling medium used for Fig. 5B at 258C for up to 5 min and stained with rhodamine-123.

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FIG. 6—Continued

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MEGAMITOCHONDRIA AND APOPTOSIS TABLE 1 Membrane Potential of Light and Heavy Mitochondrial Fractions Isolated from Hydrazine-Treated Rat Liversa Mean fluorescence intensity of CMTMRos Mitochondria

2Gramicidin(A)

1Gramicidin(B)

A-B

4C(2000–7000g) 4H(2000–7000g) 4C(500–2000g) 4H(500–2000g) 8C(2000–7000g) 8H(2000–7000g) 8C(500–2000g) 8H(500–2000g)

748.0 730.0 740.0 729.0 665.0 346.0 660.0 320.0

27.2 26.0 27.9 28.6 23.8 24.4 27.4 31.6

720.8 704.0 712.1 700.4 641.2 321.6 632.6 288.4

a The light ((2000–7000g)) and heavy ((500–2000g)) mitochondrial fractions were obtained from the livers of rats fed with a 1% hydrazine diet for 4 (4H) and 8 days (8H) and corresponding paired control animals (4C and 8C, respectively). Data are expressed as arbitrary fluorescence units of CMTMRos-stained mitochondria.

and apparent increases in the membrane potential of mitochondria due to increases in the volume of mitochondria detected using CMTMRos, described above, were not observed.

Cytochrome Contents in MG and Swollen Mitochondria In the present study we have shown that the rate of ROS generation from MG in the livers of rats treated with hydrazine for 4 days is increased compared to that for the controls, whereas that from MG in the livers of animals treated with hydrazine for 8 days is decreased. It is well known that functional states of the electron transfer chains of mitochondria control the rate of ROS generation (Boveris et al., 1972; Cardenas et al., 1977; Chandel et al., 1998; Garcia-Ruiz et al., 1995; Markossian et al., 1978; Papa et al., 1996; Pitkanen and Robinson, 1996). Thus, we have measured the content of cytochromes of MG (Fig. 7). It is evident from the figure that the content of cytochrome a 1 a3 in 4H(500–2000g)R2and 4H(2000–7000g)R2- (Fig. 7A) became equally decreased compared to those of the corresponding controls and such decreases in the content of cytochrome a 1 a3 became more evident in the mitochondria of animals treated with hydrazine for 8 days. Furthermore, the prolongation of the treatment of animals with hydrazine to 8 days caused decreases in the content of cytochrome c in the light and heavy mitochondrial fractions (Fig. 7B). We also measured changes

FIG. 7. Contents of cytochromes in the light ((2000–7000g)R2-) and heavy ((500–2000g)R2-) fractions isolated from the livers of rats treated with hydrazine for 4 (4H) (A) or 8 days (8H) (B). 4C and 8C: paired controls for 4H and 8H, respectively. Data are the averages and standard errors (mean 6 SE) of three different experiments. Data on experimental animals are statistically different from those on the corresponding controls at a (P , 0.0001), b (0.001 , P , 0.01), c (0.01 , P , 0.02), and d (0.02 , P , 0.05).

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DISCUSSION

FIG. 8. Changes in the contents of cytochromes in rat liver mitochondria exposed to a swelling medium. Mitochondria were incubated with the same swelling medium used for Fig. 5B at 258C for up to 10 min, and then the contents of cytochrome c were measured.

in the content of cytochromes in mitochondria exposed to a swelling medium for various lengths of time in order to analyze the reason for the changes in the content of cytochromes in MG, described above (Fig. 8). The content of cytochromes in mitochondria, including that of cytochrome a 1 a3 exposed to a swelling medium for up to 10 min, remained at essentially the same level as that of the control mitochondria except for that of cytochrome c. The content of cytochrome c exposed to a hypotonic medium for 15 s already decreased significantly (about 62% of the control) and the prolongation of the treatment of mitochondria with a hypotonic solution for 5 min caused further decreases in the content of cytochrome c (about 29.2% of the control).

We have presented here some evidence to suggest that the light and heavy mitochondrial fractions obtained from hydrazine-treated rat livers are similar to each other with respect to some of their functions, including changes in the membrane potential of mitochondria, the content of cytochromes, and the rate of ROS generation. We have been studying the mechanism and pathophysiological meaning of the phenomenon of MG formation and have presented data to suggest that MG are formed by the fusion of adjacent mitochondria (Adachi et al., 1992, 1994; Matsuhashi et al., 1996, 1997, 1998; Wakabayashi et al., 1984) and that free radicals are intimately related to the phenomenon as a common factor (Antosiewicz et al., 1994; Matsuhashi et al., 1996, 1997, 1998; Teranishi et al., 1999a; Wakabayashi et al., 1997). Furthermore, we have recently found that the formation of MG is followed by apoptotic changes of the cell (Karbowski et al., 1999a, 1999b). Analysis on the structural changes of MG has led us to speculate that MG formed by the fusion of adjacent mitochondria may swell secondarily by continuous exposure to free radicals as a result of the opening of “permeability transition pores” or “megachannels” (Karbowski et al., 1999a, 1999b) and the cell finally becomes apoptotic in the presence of swollen MG. Most of these data have been obtained by analyses on “isolated MG.” However, a serious question has been raised: do data on functional aspects of MG obtained using an isolated MG fraction really represent those of MG? It has been pointed out by others that it is essential to establish a mitochondria isolation procedure by which only enlarged mitochondria can be collected in order to study functions of MG, based on the assumption that enlarged mitochondria are functionally different from small ones (Tandler and Hoppel, 1986). It is always the case that, in cells treated with various inducers of free radicals including hydrazine, CP, and ethanol, mitochondria enlarged to various degrees, sometimes reaching the size of the nucleus, and normal-sized mitochondria coexist (Matsuhashi et al., 1996; Wakabayashi et al., 1983, 1984, 1991). If only the former represent MG, it might be absolutely necessary to develop a method to collect only enlarged mitochondria for the analysisof functional aspects of MG as described above.

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Functional Similarities between the Light and Heavy Mitochondrial Fractions Obtained from HydrazineTreated Rat Livers We have shown in the present study that mitochondria became distinctly enlarged in the liver of rats treated with hydrazine for 4 days, and yet they were much smaller than those induced after 8 days of hydrazine treatment. The former enlarged mitochondria, nonetheless, should be designated MG since they are more than three times larger in diameter than those of the control (Teranishi et al., 1999b). The density of their matrix was moderately preserved, suggesting that they were not yet swollen. On the other hand, the matrix of the latter MG became pale and cristae were seen only on the periphery, indicating that they became swollen. Actually, mitochondria in 4H(500–2000g)R2- and 4H(2000–7000g)R2- swelled almost to the same level as those of the corresponding controls, whereas those in 8H(500–2000g)R2- and 8H(2000–7000g)R2- swelled far less than those of the corresponding controls. These results prove indirectly that PT pores for the former fractions are closed whereas those for the latter fractions are already opened before they are exposed to a hypotonic medium (Wakabayashi et al., 1984). If this is the case, then we should detect decreases in the content of cytochrome c in the latter fractions since cytochrome c is electrostatically attached to the outer membrane side of cristae and is easily released into the cytoplasm when mitochondria are exposed to a hypotonic environment. The contents of cytochrome c in the 4H(500–2000g)R2- and 4H(2000–7000g)R2- were equally well preserved whereas those in 8H(500–2000g)R2- and 8H(2000–7000g)R- were equally decreased compared to those of the corresponding controls. Similarly, the membrane potential of mitochondria in the former two fractions was well preserved whereas that of mitochondria in the latter two fractions decreased distinctly compared to that of mitochondria in the controls. We may conclude based on these data that mitochondria enlarged to various degrees and small or normal-size mitochondria are similar to each other at least with respect to functions related to apoptotic changes of the cell. MG Formation and Apoptosis We have shown in the present study that the rates of ROS generation from 4H(500–2000g)R2- and 4H(2000– 7000g)R2- were equally enhanced compared to those of the control whereas those from 8H(500–2000g)R- and 8H(2000–7000g)R- decreased.

119 Mitochondria are the main source for ROS generation, and complexes I, II, and III of the mitochondrial electron transfer chain are the ROS generation sites (Boveris et al., 1972; Cardenas et al., 1977; Turrens and Boveris, 1980). On the other hand, complex IV does not generate ROS and serves to decrease ROS levels, mainly via its SOD activity (Markossian et al., 1978). We have shown in the present study that the content of cytochrome a 1 a3, a component of complex IV, in both 4H(500–2000g)R2- and 4H(2000– 7000g)R2-, decreased compared to that in the corresponding controls and decreased further in both 8H(500–2000g)R2and 8H(2000–7000g)R2-. Decreases in the content of cytochrome a 1 a3, together with decreases in cytochrome oxidase activity, have also been reported in MG induced by hydrazine (Wakabayashi et al., 1984), CP (Adachi et al., 1991), and ethanol (Adachi et al., 1991; Hosein et al., 1980. Schilling and Reitz, 1980). Thus, increases in the rate of ROS generation from the former two mitochondrial fractions may be due partly to decreases in the content of cytochrome a 1 a3 and in cytochrome c oxidase activity. However, the rates of ROS generation from the latter two mitochondrial fractions decreased distinctly compared to those of the corresponding controls. One of the functional changes common to MG induced under several experimental conditions is distinct decreases in the rate of oxygen consumption with lowered phosphorylating abilities (Adachi et al., 1995b; Bernstein and Pennial, 1978; Cederbaum et al., 1974; DiMauro et al., 1976; Gimeno et al., 1973; Matsuhashi et al., 1997; Quintaniella and Tampier, 1971; Schotland et al., 1976; Thayer, 1987; Wakabayashi et al., 1974, 1984). We have shown previously that the rate of oxygen consumption of MG induced by hydrazine, cuprizone, and ethanol in both state 3 and state 4 respiration is remarkably decreased compared to that of the control mitochondria (Adachi et al., 1991; Wakabayashi et al., 1974, 1987, 1991). Thus, we may speculate that decreases in the rate of oxygen consumption of MG possibly result in decreases in the rate of ROS generation from MG, overcoming expected increases in the rate due to decreases in the content of cytochrome a 1 a3 and cytochrome c oxidase activities described above. The content of cytochrome a 1 a3 remained at almost the same level in mitochondria exposed to a swelling medium and the rate of ROS generation was kept high in those swollen mitochondria. If one accepts the above-described speculation, increases in the rate of ROS generation from swollen mitochondria shown in the present study could be explained by increases in the rate of oxygen consumption by these swollen, uncoupled mitochondria.

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FIG. 9. Three-dimensional structure of megamitochondria. (A and B) Hepatocytes isolated from the rat livers were cultured for 24 h in the presence of 2 mM hydrazine (B) and stained with rhodamine 123 for confocal microscopy. A, control. Bar indicates 20 mm. (C and D) Mitochondria isolated from rats treated with hydrazine for 8 days were pelleted after aldehyde fixation, frozen in liquid nitrogen, freeze-cleaved with a razor blade, and processed for scanning electron microscopy. Original magnification of electron micrographs, 317,500.

Three-Dimensional Structure of MG in the Cell In thin-sectioned specimens for electron microscopy, round or oval mitochondria measuring 1–2 mm in diameter are scattered in the cytoplasm of hepatocytes of the liver in physiological conditions. On the other hand, mitochondria stained with fluorescent dyes such as rhodamine-123 reveal highly filamentous structures. Three-dimensional images of

mitochondria cannot be obtained from electron micrographs taken from one plane of sections for electron microscopy. This would indicate that mitochondria in the living cell are far more dynamic in their structures than expected from electron micrographs obtained by a routine thin-sectioning technique and they are often connected with each other. MG induced in the hepatocytes of the liver are, as shown in the

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present study, granular and apparently separated from each other when they are revealed by electron microscopy. However, the reconstruction of mitochondria using a serial sectioning technique for electron microscopy has shown that mitochondria enlarged to various degrees and small or normal-size mitochondria are often connected to each other (Wakabayashi et al., 1974, 1975). On the other hand, confocal micrographs obtained from hepatocytes cultured for 22–24 h in the presence of hydrazine have revealed that granular mitochondria enlarged to various degrees and filamentous ones coexist and the former and latter are often continuous (Fig. 9B), in contrast to highly filamentous mitochondria in control hepatocytes (Fig. 9A). This may suggest that small or normal-size, granular mitochondria observed among MG under electron microscopy are obtained by sections cut rectangular to the long axis of such filamentous mitochondria seen by confocal microscopy. Thus, we may deduce from these results that small or normal-size mitochondria seen on one plane of an electron microscopy section are part of MG in the sense that they are connected to the enlarged ones. If one accepts this then why the light and heavy mitochondrial fractions obtained in the present study are similar to each other with respect to their functions might be understandable. We may conclude that the MG fraction, obtained by a routine isolation procedure for normal mitochondria, can be applied to their functional analysis although they are heterogeneous with respect to their sizes. However, there still remains a problem to be taken into account: there is no method available at the moment to isolate MG exactly in their original structures in the cell. Continuity of mitochondria in the cell may be lost while they are isolated by mechanical homogenization of the tissue and differential centrifugation although it is maintained to some extent when a MG fraction is examined under the scanning electron microscope (Fig. 9D), while mitochondria isolated from the liver of control animals are separated from each other (Fig. 9C). Furthermore, some MG, especially when they are extremely swollen, may be broken or fragmented during the isolation procedure. Thus, it is suggested that functional aspects of MG should be analyzed not only at a level of isolated mitochondrial fractions but also at a level of isolated cells or perfused tissues (Adachi et al., 1995b; Teranishi et al., 1999b).

ACKNOWLEDGMENTS The authors thank Mr. M. Masaoka for his skilled technical assistance and Mrs. K. Yoshioka for preparation of the manuscript. This

work was supported in part by Grants 10470142 and 0967-152 from the Ministry of Education, Science, and Culture of Japan.

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