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Takashi Wakabayashi - One of the best experts on this subject based on the ideXlab platform.
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Structural changes of mitochondria during free radical-induced apoptosis.
Folia Morphologica, 2020Co-Authors: Takashi Wakabayashi, Jan Henryk SpodnikAbstract:The initial proposal for apoptosis stressed nuclear change (condensation of chromatin) and the intactness of intracellular organelles, including mitochondria, based on light and electron microscopic observations. However, data have accumulated to demonstrate that the opening of megachannels of mitochondrial membranes, resulting in the swelling of the organelles, notably by Ca 2+ and free radicals, is the crucial step in the apoptotic processes of the cell. Application of fluorescent dyes to mitochondria, combined with flow cytometry, has made it possible to detect subtle changes in the structure and function of the organelles related to apoptosis. The present article overviews structural aspects of mitochondria related to apoptosis, including the free radical-induced formation of Megamitochondria.
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MECHANISM OF THE FORMATION OF Megamitochondria INDUCED BY COPPER‐CHELATING AGENTS
Pathology International, 2009Co-Authors: Takashi Wakabayashi, Masahisa Asano, Chieko KuronoAbstract:Megamitochondria have been isolated from the liver of the cuprizone-fed mouse with the aid of bovine serum albumin. Phosphorylating capacities of Megamitochondria, specified above, in terms of respiratory control ratios and ADP/O ratios have revealed that they are not uncoupled completely. Biochemical properties of megamito-chondria which are related to the metabolism of copper have shown that copper-chelating action of cuprizone may not be directly related to the formation of Megamitochondria in vivo. Namely, cytochrome contents, activities of cytochrome oxidase and monoamine oxidase and contents of copper of Megamitochondria were unchanged compared with those of the control. However, contents of divalent metals such as Ca++ and Mg++, especially that of the former, in Megamitochondria decreased significantly. It is suggested that cuprizone may alter Mg++/Ca++ ratios when it is administered in vivo, and that changes in the ratio might play a key role in the formation of Megamitochondria.
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swelling of free radical induced Megamitochondria causes apoptosis
Experimental and Molecular Pathology, 2000Co-Authors: Masaaki Teranishi, Chieko Kurono, Mariusz Karbowski, Tsuyoshi Soji, Jan H Spodonik, Takashi WakabayashiAbstract:Abstract 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.
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functional aspects of Megamitochondria isolated from hydrazine and ethanol treated rat livers
Pathology International, 2000Co-Authors: Takashi Wakabayashi, Chieko Kurono, Yuji Nishizawa, Jiro Usukura, Mariusz Karbowski, Masaaki Teranishi, Tsuyoshi SojiAbstract:: It is essential to analyze functions of Megamitochondria (MG) to elucidate the mechanism of the formation of MG induced under various pathological conditions. The MG fraction obtained by a routine isolation procedure for normal mitochondria always consists of a mixed population of mitochondria enlarged to various degrees and also normal-sized ones. The purpose of the present study is to answer the question of whether or not data obtained from the MG fraction consisting of such a heterogeneous population of mitochondria with respect to their sizes really reflect functions of MG. In the present study mitochondria were obtained from the livers of rats treated with a 1% hydrazine diet for 8 days and those given 32% ethanol in drinking water for up to 2 months using various isolation procedures. Results obtained are summarized as follows: (i) mitochondria enlarged to various degrees and normal-sized ones are sometimes connected with each other by a narrow stalk in the hepatocyte of hydrazine-treated animals, and such connections are maintained to some extent when mitochondria are isolated; and (ii) mitochondria obtained from experimental animals by a routine isolation procedure for mitochondria ((700-7000)gR2"') and those obtained by alternative isolation procedure yielding the heavy ((500-2000)gR2"') and light ((2000-7000)gR2"') fractions show some functional similarities: decreases in the content of cytochrome a + a3; decreases in oxygen consumptions and phosphorylating abilities; decreases in monoamine oxidase and cytochrome c oxidase activities; lowered membrane potential of mitochondria; decreases in the rate of the generation of reactive oxygen species. These results may suggest that mitochondria enlarged to various degrees and normal-sized ones are functionally similar to each other and that the MG fraction obtained by a routine isolation procedure for normal mitochondria can be applied to the study of the function of MG.
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cycloheximide and 4 oh tempo suppress chloramphenicol induced apoptosis in rl 34 cells via the suppression of the formation of Megamitochondria
Biochimica et Biophysica Acta, 1999Co-Authors: Mariusz Karbowski, Chieko Kurono, Michal Wozniak, Tsuyoshi Soji, Mariusz Ostrowski, Masaaki Teranishi, Takashi WakabayashiAbstract:Toxic effects of chloramphenicol, an antibiotic inhibitor of mitochondrial protein synthesis, on rat liver derived RL-34 cell line were completely blocked by a combined treatment with substances endowed with direct or indirect antioxidant properties. A stable, nitroxide free radical scavenger, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and a protein synthesis inhibitor, cycloheximide, suppressed in a similar manner the following manifestations of the chloramphenicol cytotoxicity: (1) Oxidative stress state as evidenced by FACS analysis of cells loaded with carboxy-dichlorodihydrofluorescein diacetate and Mito Tracker CMTH2MRos; (2) Megamitochondria formation detected by staining of mitochondria with MitoTracker CMXRos under a laser confocal microscopy and electron microscopy; (3) apoptotic changes of the cell detected by the phase contrast microscopy, DNA laddering analysis and cell cycle analysis. Since increases of ROS generation in chloramphenicol-treated cells were the first sign of the chloramphenicol toxicity, we assume that oxidative stress state is a mediator of above described alternations of RL-34 cells including MG formation. Pretreatment of cells with cycloheximide or 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, which is known to be localized into mitochondria, inhibited the Megamitochondria formation and succeeding apoptotic changes of the cell. Protective effects of cycloheximide, which enhances the expression of Bcl-2 protein, may further confirm our hypothesis that the Megamitochondria formation is a cellular response to an increased ROS generation and raise a possibility that antiapoptotic action of the drug is exerted via the protection of the mitochondria functions.
Mariusz Karbowski - One of the best experts on this subject based on the ideXlab platform.
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swelling of free radical induced Megamitochondria causes apoptosis
Experimental and Molecular Pathology, 2000Co-Authors: Masaaki Teranishi, Chieko Kurono, Mariusz Karbowski, Tsuyoshi Soji, Jan H Spodonik, Takashi WakabayashiAbstract:Abstract 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.
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functional aspects of Megamitochondria isolated from hydrazine and ethanol treated rat livers
Pathology International, 2000Co-Authors: Takashi Wakabayashi, Chieko Kurono, Yuji Nishizawa, Jiro Usukura, Mariusz Karbowski, Masaaki Teranishi, Tsuyoshi SojiAbstract:: It is essential to analyze functions of Megamitochondria (MG) to elucidate the mechanism of the formation of MG induced under various pathological conditions. The MG fraction obtained by a routine isolation procedure for normal mitochondria always consists of a mixed population of mitochondria enlarged to various degrees and also normal-sized ones. The purpose of the present study is to answer the question of whether or not data obtained from the MG fraction consisting of such a heterogeneous population of mitochondria with respect to their sizes really reflect functions of MG. In the present study mitochondria were obtained from the livers of rats treated with a 1% hydrazine diet for 8 days and those given 32% ethanol in drinking water for up to 2 months using various isolation procedures. Results obtained are summarized as follows: (i) mitochondria enlarged to various degrees and normal-sized ones are sometimes connected with each other by a narrow stalk in the hepatocyte of hydrazine-treated animals, and such connections are maintained to some extent when mitochondria are isolated; and (ii) mitochondria obtained from experimental animals by a routine isolation procedure for mitochondria ((700-7000)gR2"') and those obtained by alternative isolation procedure yielding the heavy ((500-2000)gR2"') and light ((2000-7000)gR2"') fractions show some functional similarities: decreases in the content of cytochrome a + a3; decreases in oxygen consumptions and phosphorylating abilities; decreases in monoamine oxidase and cytochrome c oxidase activities; lowered membrane potential of mitochondria; decreases in the rate of the generation of reactive oxygen species. These results may suggest that mitochondria enlarged to various degrees and normal-sized ones are functionally similar to each other and that the MG fraction obtained by a routine isolation procedure for normal mitochondria can be applied to the study of the function of MG.
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cycloheximide and 4 oh tempo suppress chloramphenicol induced apoptosis in rl 34 cells via the suppression of the formation of Megamitochondria
Biochimica et Biophysica Acta, 1999Co-Authors: Mariusz Karbowski, Chieko Kurono, Michal Wozniak, Tsuyoshi Soji, Mariusz Ostrowski, Masaaki Teranishi, Takashi WakabayashiAbstract:Toxic effects of chloramphenicol, an antibiotic inhibitor of mitochondrial protein synthesis, on rat liver derived RL-34 cell line were completely blocked by a combined treatment with substances endowed with direct or indirect antioxidant properties. A stable, nitroxide free radical scavenger, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and a protein synthesis inhibitor, cycloheximide, suppressed in a similar manner the following manifestations of the chloramphenicol cytotoxicity: (1) Oxidative stress state as evidenced by FACS analysis of cells loaded with carboxy-dichlorodihydrofluorescein diacetate and Mito Tracker CMTH2MRos; (2) Megamitochondria formation detected by staining of mitochondria with MitoTracker CMXRos under a laser confocal microscopy and electron microscopy; (3) apoptotic changes of the cell detected by the phase contrast microscopy, DNA laddering analysis and cell cycle analysis. Since increases of ROS generation in chloramphenicol-treated cells were the first sign of the chloramphenicol toxicity, we assume that oxidative stress state is a mediator of above described alternations of RL-34 cells including MG formation. Pretreatment of cells with cycloheximide or 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, which is known to be localized into mitochondria, inhibited the Megamitochondria formation and succeeding apoptotic changes of the cell. Protective effects of cycloheximide, which enhances the expression of Bcl-2 protein, may further confirm our hypothesis that the Megamitochondria formation is a cellular response to an increased ROS generation and raise a possibility that antiapoptotic action of the drug is exerted via the protection of the mitochondria functions.
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free radical induced Megamitochondria formation and apoptosis
Free Radical Biology and Medicine, 1999Co-Authors: Mariusz Karbowski, Chieko Kurono, Michal Wozniak, Yuji Nishizawa, Jiro Usukura, Tsuyoshi Soji, Mariusz Ostrowski, Masaaki Teranishi, Takashi WakabayashiAbstract: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, H 2 O 2 , 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-H 2 -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 G 1 phase, and apoptotic sub-G 1 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 H 2 O 2 and O 2 − 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.
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induction of Megamitochondria by some chemicals inducing oxidative stress in primary cultured rat hepatocytes
Biochimica et Biophysica Acta, 1997Co-Authors: Mariusz Karbowski, Chieko Kurono, Yuji Nishizawa, Yumiko Horie, Tsuyoshi Soji, Takashi WakabayashiAbstract:Abstract Effects of hydrazine, hydrogen peroxide and bromobenzene, inducers of free radicals, and those of erythromycin and cycloheximide, inhibitors of protein synthesis on structural changes of mitochondria in primary monolayer culture of rat hepatocytes were examined using laser confocal microscope and electron microscope. After 22 h of incubation of hepatocytes with 0.2 mM hydrogen peroxide or 10 μg ml−1 of erythromycin, mitochondria became extremely enlarged. Mitochondria of hepatocytes isolated from control rats became slightly to moderately enlarged in the presence of 2 mM hydrazine, while those of hepatocytes isolated from phenobarbital-pretreated animals became extremely enlarged in the presence of 2 mM hydrazine. Cycloheximide (0.5–10.0 μg ml−1) and bromobenzene (0.1–1.0 mM) failed to induce structural changes of mitochondria. The level of cytochrome P-450 in freshly prepared hepatocytes from phenobarbital-treated rats was 2.5 times higher than that from the control rats, and remained about three times higher than the latter after 22 h of incubation with 2 mM hydrazine. The level of malondialdehyde was invariably elevated when Megamitochondria were induced. These results may suggest that oxidative stress is intimately related to the mechanism of the formation of Megamitochondria and that the inhibition of cytoplasmic protein synthesis seems not to contribute the phenomenon. However, the detailed mechanism by which free radicals may induce Megamitochondria remains to be elucidated.
Yuji Nishizawa - One of the best experts on this subject based on the ideXlab platform.
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functional aspects of Megamitochondria isolated from hydrazine and ethanol treated rat livers
Pathology International, 2000Co-Authors: Takashi Wakabayashi, Chieko Kurono, Yuji Nishizawa, Jiro Usukura, Mariusz Karbowski, Masaaki Teranishi, Tsuyoshi SojiAbstract:: It is essential to analyze functions of Megamitochondria (MG) to elucidate the mechanism of the formation of MG induced under various pathological conditions. The MG fraction obtained by a routine isolation procedure for normal mitochondria always consists of a mixed population of mitochondria enlarged to various degrees and also normal-sized ones. The purpose of the present study is to answer the question of whether or not data obtained from the MG fraction consisting of such a heterogeneous population of mitochondria with respect to their sizes really reflect functions of MG. In the present study mitochondria were obtained from the livers of rats treated with a 1% hydrazine diet for 8 days and those given 32% ethanol in drinking water for up to 2 months using various isolation procedures. Results obtained are summarized as follows: (i) mitochondria enlarged to various degrees and normal-sized ones are sometimes connected with each other by a narrow stalk in the hepatocyte of hydrazine-treated animals, and such connections are maintained to some extent when mitochondria are isolated; and (ii) mitochondria obtained from experimental animals by a routine isolation procedure for mitochondria ((700-7000)gR2"') and those obtained by alternative isolation procedure yielding the heavy ((500-2000)gR2"') and light ((2000-7000)gR2"') fractions show some functional similarities: decreases in the content of cytochrome a + a3; decreases in oxygen consumptions and phosphorylating abilities; decreases in monoamine oxidase and cytochrome c oxidase activities; lowered membrane potential of mitochondria; decreases in the rate of the generation of reactive oxygen species. These results may suggest that mitochondria enlarged to various degrees and normal-sized ones are functionally similar to each other and that the MG fraction obtained by a routine isolation procedure for normal mitochondria can be applied to the study of the function of MG.
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free radical induced Megamitochondria formation and apoptosis
Free Radical Biology and Medicine, 1999Co-Authors: Mariusz Karbowski, Chieko Kurono, Michal Wozniak, Yuji Nishizawa, Jiro Usukura, Tsuyoshi Soji, Mariusz Ostrowski, Masaaki Teranishi, Takashi WakabayashiAbstract: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, H 2 O 2 , 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-H 2 -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 G 1 phase, and apoptotic sub-G 1 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 H 2 O 2 and O 2 − 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.
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induction of Megamitochondria by some chemicals inducing oxidative stress in primary cultured rat hepatocytes
Biochimica et Biophysica Acta, 1997Co-Authors: Mariusz Karbowski, Chieko Kurono, Yuji Nishizawa, Yumiko Horie, Tsuyoshi Soji, Takashi WakabayashiAbstract:Abstract Effects of hydrazine, hydrogen peroxide and bromobenzene, inducers of free radicals, and those of erythromycin and cycloheximide, inhibitors of protein synthesis on structural changes of mitochondria in primary monolayer culture of rat hepatocytes were examined using laser confocal microscope and electron microscope. After 22 h of incubation of hepatocytes with 0.2 mM hydrogen peroxide or 10 μg ml−1 of erythromycin, mitochondria became extremely enlarged. Mitochondria of hepatocytes isolated from control rats became slightly to moderately enlarged in the presence of 2 mM hydrazine, while those of hepatocytes isolated from phenobarbital-pretreated animals became extremely enlarged in the presence of 2 mM hydrazine. Cycloheximide (0.5–10.0 μg ml−1) and bromobenzene (0.1–1.0 mM) failed to induce structural changes of mitochondria. The level of cytochrome P-450 in freshly prepared hepatocytes from phenobarbital-treated rats was 2.5 times higher than that from the control rats, and remained about three times higher than the latter after 22 h of incubation with 2 mM hydrazine. The level of malondialdehyde was invariably elevated when Megamitochondria were induced. These results may suggest that oxidative stress is intimately related to the mechanism of the formation of Megamitochondria and that the inhibition of cytoplasmic protein synthesis seems not to contribute the phenomenon. However, the detailed mechanism by which free radicals may induce Megamitochondria remains to be elucidated.
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mechanism of the formation of Megamitochondria in the mouse liver induced by chloramphenicol
Toxicology Letters, 1996Co-Authors: Tatsuo Matsuhashi, Michal Wozniak, Yuji Nishizawa, Jiro Usukura, Takashi WakabayashiAbstract:Abstract Correlation between chloramphenicol-induced formation of Megamitochondria in the mouse liver and oxidative stress was studied by lipid peroxidation analysis and electron microscopic technique. Chloramphenicol suppressed increases in the body weight and liver weight of experimental animals and at the same time induced a remarkable increase in lipid peroxidation in the liver during the formation of Megamitochondria. A spin trapping agent, 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl, abolished all these changes induced by chloramphenicol. Namely, both the body weight and liver weight ofchloramphenicol-treated animals stayed at the same levels as those of the control, and the formation of Megamitochondria was completely suppressed. Allopurinol, a xanthine oxidase (EC 1.2.3.2) inhibitor, partly inhibited the changes induced by chloramphenicol, as described above. These results suggest that chloramphenicol-induced formation of Megamitochondria is not simply ascribed to the suppression of the dividing process of mitochondria due to lowered protein synthesis in mitochondria but is intimately related to oxidative stress. Furthermore, the results obtained with allopurinol may indicate that enhanced levels of lipid peroxidation observed in chloramphenicol-treated animals are partly due to enhanced rate of the degradation of purine nucleotides catalyzed by xanthine oxidase.
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suppression of the hydrazine induced formation of Megamitochondria in the rat liver by coenzyme q10
Toxicologic Pathology, 1995Co-Authors: Kayo Adachi, Jerzy Popinigis, Tatsuo Matsuhashi, Yuji Nishizawa, Jiro Usukura, Takashi WakabayashiAbstract:The effects of coenzyme Q10 (CoQ10) on the hydrazine-induced changes in the structure of mitochondria and those in antioxidant systems of the liver were investigated using rats as experimental animals. Animals were placed on a powdered diet containing 1.0% hydrazine for 7-8 days in the presence or absence of the combined treatment with CoQ10. Results obtained were as follows: (a) treatment of animals with CoQ10 prevented the hydrazine-induced formation of Megamitochondria in the liver; (b) changes observed in the liver of the hydrazine-treated animals in comparison to the control were increases in the contents of α-tocopherol and CoQ analogs, increases in the levels of lipid peroxidation, decreases in the level of reduced glutathione with increases in that of oxidized glutathione, and increases in the ratio of unsaturated to saturated fatty acids in phospholipid domains of mitochondrial membranes; and (c) administration of CoQ10 to hydrazine-treated animals suppressed enhanced lipid peroxidation and impro...
Jiro Usukura - One of the best experts on this subject based on the ideXlab platform.
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functional aspects of Megamitochondria isolated from hydrazine and ethanol treated rat livers
Pathology International, 2000Co-Authors: Takashi Wakabayashi, Chieko Kurono, Yuji Nishizawa, Jiro Usukura, Mariusz Karbowski, Masaaki Teranishi, Tsuyoshi SojiAbstract:: It is essential to analyze functions of Megamitochondria (MG) to elucidate the mechanism of the formation of MG induced under various pathological conditions. The MG fraction obtained by a routine isolation procedure for normal mitochondria always consists of a mixed population of mitochondria enlarged to various degrees and also normal-sized ones. The purpose of the present study is to answer the question of whether or not data obtained from the MG fraction consisting of such a heterogeneous population of mitochondria with respect to their sizes really reflect functions of MG. In the present study mitochondria were obtained from the livers of rats treated with a 1% hydrazine diet for 8 days and those given 32% ethanol in drinking water for up to 2 months using various isolation procedures. Results obtained are summarized as follows: (i) mitochondria enlarged to various degrees and normal-sized ones are sometimes connected with each other by a narrow stalk in the hepatocyte of hydrazine-treated animals, and such connections are maintained to some extent when mitochondria are isolated; and (ii) mitochondria obtained from experimental animals by a routine isolation procedure for mitochondria ((700-7000)gR2"') and those obtained by alternative isolation procedure yielding the heavy ((500-2000)gR2"') and light ((2000-7000)gR2"') fractions show some functional similarities: decreases in the content of cytochrome a + a3; decreases in oxygen consumptions and phosphorylating abilities; decreases in monoamine oxidase and cytochrome c oxidase activities; lowered membrane potential of mitochondria; decreases in the rate of the generation of reactive oxygen species. These results may suggest that mitochondria enlarged to various degrees and normal-sized ones are functionally similar to each other and that the MG fraction obtained by a routine isolation procedure for normal mitochondria can be applied to the study of the function of MG.
-
free radical induced Megamitochondria formation and apoptosis
Free Radical Biology and Medicine, 1999Co-Authors: Mariusz Karbowski, Chieko Kurono, Michal Wozniak, Yuji Nishizawa, Jiro Usukura, Tsuyoshi Soji, Mariusz Ostrowski, Masaaki Teranishi, Takashi WakabayashiAbstract: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, H 2 O 2 , 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-H 2 -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 G 1 phase, and apoptotic sub-G 1 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 H 2 O 2 and O 2 − 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.
-
mechanism of the formation of Megamitochondria in the mouse liver induced by chloramphenicol
Toxicology Letters, 1996Co-Authors: Tatsuo Matsuhashi, Michal Wozniak, Yuji Nishizawa, Jiro Usukura, Takashi WakabayashiAbstract:Abstract Correlation between chloramphenicol-induced formation of Megamitochondria in the mouse liver and oxidative stress was studied by lipid peroxidation analysis and electron microscopic technique. Chloramphenicol suppressed increases in the body weight and liver weight of experimental animals and at the same time induced a remarkable increase in lipid peroxidation in the liver during the formation of Megamitochondria. A spin trapping agent, 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl, abolished all these changes induced by chloramphenicol. Namely, both the body weight and liver weight ofchloramphenicol-treated animals stayed at the same levels as those of the control, and the formation of Megamitochondria was completely suppressed. Allopurinol, a xanthine oxidase (EC 1.2.3.2) inhibitor, partly inhibited the changes induced by chloramphenicol, as described above. These results suggest that chloramphenicol-induced formation of Megamitochondria is not simply ascribed to the suppression of the dividing process of mitochondria due to lowered protein synthesis in mitochondria but is intimately related to oxidative stress. Furthermore, the results obtained with allopurinol may indicate that enhanced levels of lipid peroxidation observed in chloramphenicol-treated animals are partly due to enhanced rate of the degradation of purine nucleotides catalyzed by xanthine oxidase.
-
suppression of the hydrazine induced formation of Megamitochondria in the rat liver by coenzyme q10
Toxicologic Pathology, 1995Co-Authors: Kayo Adachi, Jerzy Popinigis, Tatsuo Matsuhashi, Yuji Nishizawa, Jiro Usukura, Takashi WakabayashiAbstract:The effects of coenzyme Q10 (CoQ10) on the hydrazine-induced changes in the structure of mitochondria and those in antioxidant systems of the liver were investigated using rats as experimental animals. Animals were placed on a powdered diet containing 1.0% hydrazine for 7-8 days in the presence or absence of the combined treatment with CoQ10. Results obtained were as follows: (a) treatment of animals with CoQ10 prevented the hydrazine-induced formation of Megamitochondria in the liver; (b) changes observed in the liver of the hydrazine-treated animals in comparison to the control were increases in the contents of α-tocopherol and CoQ analogs, increases in the levels of lipid peroxidation, decreases in the level of reduced glutathione with increases in that of oxidized glutathione, and increases in the ratio of unsaturated to saturated fatty acids in phospholipid domains of mitochondrial membranes; and (c) administration of CoQ10 to hydrazine-treated animals suppressed enhanced lipid peroxidation and impro...
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suppression of the hydrazine induced formation of Megamitochondria in the rat liver by α tocopherol
Experimental and Molecular Pathology, 1994Co-Authors: Jerzy Antosiewicz, Yuji Nishizawa, Jiro Usukura, Takashi WakabayashiAbstract:Abstract The effects of α-tocopherol on the hydrazine-induced changes in the structure of mitochondria and those in the enzymatic and nonenzymatic antioxidant systems of the liver were investigated using rats as experimental animals. Animals were divided into four groups: animals of the first group were placed on a powdered diet containing 1.0% hydrazine for 7 days; those of the second and third groups received a control diet and α-tocopherol (ip, daily, 700 mg/kg body wt). On the fourth day and thereafter for up to 7 days, the control diet was replaced by a 1.0% hydrazine diet for the animals of the second group; those of the fourth group served as the control. After 10 days (in the case of animals of the first group, 7 days), animals were sacrificed. Results obtained were as follows: (1) Treatment of animals with α-tocopherol partly prevented the hydrazine-induced formation of Megamitochondria in the liver. (2) Treatment of animals with hydrazine induced remarkable increases in the contents of α-tocopherol in mitochondria (4.8 times), microsome (1.4 times), and homogenate (2.9 times) of the liver compared with those of the control. Treatment of animals with α-tocopherol did not raise the concentration of α-tocopherol in mitochondria of the liver. The highest concentration of α-tocopherol in motochondria of the liver was obtained in animals given hydrazine plus α-tocopherol (7.2 times higher than the control). (3) The amount of lipid-soluble fluorophores as an indicator of nonenzymatic oxidative stress was remarkably increased in mitochondria, microsome, and homogenate of the liver of hydrazine-treated animals. (4) Among enzymes protecting the cell from the oxidative stress activities of superoxide dismutase and glutathione peroxidase were almost the same among four groups of animals, while the activity of catalase was decreased distinctly in hydrazine-treated animals. Administration of α-tocopherol to these animals did not improve its activity. The present study has clearly demonstrated that α-tocopherol, a typical scavenger for free radicals, prevents the hydrazine-induced formation of Megamitochondria in the liver. However, we failed to correlate free radicals to biochemical and physicochemical changes of mitochondrial membranes induced by hydrazine. Previously, we have demonstrated that a ratio of unsaturated to saturated fatty acids in phospholipid domains of mitochondrial membranes increases with hydrazine treatment. Since increases in the ratio specified above are a key event in the membrane fusion process we are now studying how the ratio is modified by hydrazine focussing on desaturase activity in the liver, and results will be reported soon.
Chieko Kurono - One of the best experts on this subject based on the ideXlab platform.
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MECHANISM OF THE FORMATION OF Megamitochondria INDUCED BY COPPER‐CHELATING AGENTS
Pathology International, 2009Co-Authors: Takashi Wakabayashi, Masahisa Asano, Chieko KuronoAbstract:Megamitochondria have been isolated from the liver of the cuprizone-fed mouse with the aid of bovine serum albumin. Phosphorylating capacities of Megamitochondria, specified above, in terms of respiratory control ratios and ADP/O ratios have revealed that they are not uncoupled completely. Biochemical properties of megamito-chondria which are related to the metabolism of copper have shown that copper-chelating action of cuprizone may not be directly related to the formation of Megamitochondria in vivo. Namely, cytochrome contents, activities of cytochrome oxidase and monoamine oxidase and contents of copper of Megamitochondria were unchanged compared with those of the control. However, contents of divalent metals such as Ca++ and Mg++, especially that of the former, in Megamitochondria decreased significantly. It is suggested that cuprizone may alter Mg++/Ca++ ratios when it is administered in vivo, and that changes in the ratio might play a key role in the formation of Megamitochondria.
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swelling of free radical induced Megamitochondria causes apoptosis
Experimental and Molecular Pathology, 2000Co-Authors: Masaaki Teranishi, Chieko Kurono, Mariusz Karbowski, Tsuyoshi Soji, Jan H Spodonik, Takashi WakabayashiAbstract:Abstract 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.
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functional aspects of Megamitochondria isolated from hydrazine and ethanol treated rat livers
Pathology International, 2000Co-Authors: Takashi Wakabayashi, Chieko Kurono, Yuji Nishizawa, Jiro Usukura, Mariusz Karbowski, Masaaki Teranishi, Tsuyoshi SojiAbstract:: It is essential to analyze functions of Megamitochondria (MG) to elucidate the mechanism of the formation of MG induced under various pathological conditions. The MG fraction obtained by a routine isolation procedure for normal mitochondria always consists of a mixed population of mitochondria enlarged to various degrees and also normal-sized ones. The purpose of the present study is to answer the question of whether or not data obtained from the MG fraction consisting of such a heterogeneous population of mitochondria with respect to their sizes really reflect functions of MG. In the present study mitochondria were obtained from the livers of rats treated with a 1% hydrazine diet for 8 days and those given 32% ethanol in drinking water for up to 2 months using various isolation procedures. Results obtained are summarized as follows: (i) mitochondria enlarged to various degrees and normal-sized ones are sometimes connected with each other by a narrow stalk in the hepatocyte of hydrazine-treated animals, and such connections are maintained to some extent when mitochondria are isolated; and (ii) mitochondria obtained from experimental animals by a routine isolation procedure for mitochondria ((700-7000)gR2"') and those obtained by alternative isolation procedure yielding the heavy ((500-2000)gR2"') and light ((2000-7000)gR2"') fractions show some functional similarities: decreases in the content of cytochrome a + a3; decreases in oxygen consumptions and phosphorylating abilities; decreases in monoamine oxidase and cytochrome c oxidase activities; lowered membrane potential of mitochondria; decreases in the rate of the generation of reactive oxygen species. These results may suggest that mitochondria enlarged to various degrees and normal-sized ones are functionally similar to each other and that the MG fraction obtained by a routine isolation procedure for normal mitochondria can be applied to the study of the function of MG.
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cycloheximide and 4 oh tempo suppress chloramphenicol induced apoptosis in rl 34 cells via the suppression of the formation of Megamitochondria
Biochimica et Biophysica Acta, 1999Co-Authors: Mariusz Karbowski, Chieko Kurono, Michal Wozniak, Tsuyoshi Soji, Mariusz Ostrowski, Masaaki Teranishi, Takashi WakabayashiAbstract:Toxic effects of chloramphenicol, an antibiotic inhibitor of mitochondrial protein synthesis, on rat liver derived RL-34 cell line were completely blocked by a combined treatment with substances endowed with direct or indirect antioxidant properties. A stable, nitroxide free radical scavenger, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and a protein synthesis inhibitor, cycloheximide, suppressed in a similar manner the following manifestations of the chloramphenicol cytotoxicity: (1) Oxidative stress state as evidenced by FACS analysis of cells loaded with carboxy-dichlorodihydrofluorescein diacetate and Mito Tracker CMTH2MRos; (2) Megamitochondria formation detected by staining of mitochondria with MitoTracker CMXRos under a laser confocal microscopy and electron microscopy; (3) apoptotic changes of the cell detected by the phase contrast microscopy, DNA laddering analysis and cell cycle analysis. Since increases of ROS generation in chloramphenicol-treated cells were the first sign of the chloramphenicol toxicity, we assume that oxidative stress state is a mediator of above described alternations of RL-34 cells including MG formation. Pretreatment of cells with cycloheximide or 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, which is known to be localized into mitochondria, inhibited the Megamitochondria formation and succeeding apoptotic changes of the cell. Protective effects of cycloheximide, which enhances the expression of Bcl-2 protein, may further confirm our hypothesis that the Megamitochondria formation is a cellular response to an increased ROS generation and raise a possibility that antiapoptotic action of the drug is exerted via the protection of the mitochondria functions.
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free radical induced Megamitochondria formation and apoptosis
Free Radical Biology and Medicine, 1999Co-Authors: Mariusz Karbowski, Chieko Kurono, Michal Wozniak, Yuji Nishizawa, Jiro Usukura, Tsuyoshi Soji, Mariusz Ostrowski, Masaaki Teranishi, Takashi WakabayashiAbstract: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, H 2 O 2 , 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-H 2 -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 G 1 phase, and apoptotic sub-G 1 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 H 2 O 2 and O 2 − 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.