Induction of megamitochondria by some chemicals inducing oxidative stress in primary cultured rat hepatocytes.

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 microg 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 microg 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.

Cycloheximide and 4-OH-TEMPO suppress chloramphenicol-induced apoptosis in RL-34 cells via the suppression of the formation of megamitochondria.

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.

Free radical-induced megamitochondria formation and apoptosis.

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 O2- 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.

Folliculo-stellate cells and intercellular communication within the rat anterior pituitary gland.

Folliculo-stellate (FS) cell are agranular and arranged around a follicle. They contain the S-100 protein and beta-adrenergic receptors. It has been suggested that they can act as stem cells, since they show mitotic figures, and could transform into granular or chromophilic cells according to the concept of a "cell renewal system." Cell-to-cell interactions among pituitary cells have been described, and recent progress with freeze-fracture electron microscopy has provided novel observations of the cell surface and gap junctions within the rat or teleost fish pituitary gland, or in cultured rat pituitary cells. In adult rats, the anterior pituitary was composed of lobules incompletely separated by a basement membrane. Follicles consisted exclusively of FS cells. Gap junctions were observed only between adjacent FS cells, in rare cases on the tips of their cytoplasmic processes. Thus, the FS cells, connected by gap junctions, made up a dense cellular network throughout the pituitary. Gap and tight junctions were absent on granular cells. Elongated follicles with columnar FS cells were observed in 10-day-old rats and were separated into smaller units. The number of gap junctions rapidly increased with age until 40-45 days of age. Few S-100 protein positive cells were observed on day 10, along the marginal cell layer and near the so-called postero-lateral wing. The frequency of positive cells increased with age and by day 40; numerous cells were observed throughout the anterior lobe. Gap junction number also varied with the stage of the estrous cycle, and frequency; during diestrus, they were half of that during proestrus or estrus. The number of gap junctions increased in late pregnancy and in lactating rats, probably due to changes in estrogen and progesterone. Hormone (LH-RH and testosterone) treated groups of rats showed accelerated development by almost 10 days, compared with controls. In castrated male rats, the ultrastructure of the pituitary remained immature even at 40 days of age, when the number of gap junctions was a quarter or less than the number in intact rats. Testosterone treatment restored the frequency of gap junctions to a normal level. We conclude that the appearance of gap junctions in the pituitary cells and maturation of the gland are dependent to a large degree upon gonadal steroids.

Immunohistochemical study of the post-natal development of the folliculo-stellate cells in the rat anterior pituitary gland.

We investigated the post-natal development of cell-to-cell communication within the rat anterior pituitary gland cells using immunohistochemistry of the S-100 protein. Tissues of animals from 10 to 60 days of age were analyzed. At 10 days of age, S-100 protein-containing cells were rarely observed. With age, the population of S-100 immunostained cells increased until day 40 when they were found to be quite numerous. No further changes were noted from day 40 through day 60. From our previous studies, we conclude that the cells which reacted with the S-100 antiserum were folliculo-stellate cells and their developmental pattern parallels that of the hypophyseal-gonadal axis.

Septate-like junctions in the normal male rat pituitary gland.

Septate-like junctions were observed in the rat anterior pituitary gland of the adult male solely between adjacent folliculo-stellate cells. Considering their location, it is presumed that their function is cellular adhesion and mechanical support for the hypophyseal follicles.

Sealing of the follicular lumen of the anterior pituitary gland of the male rat.

It is commonly accepted that follicular lumina of the adult rat anterior pituitary gland are tightly sealed by junctional complexes, especially tight junctions. In this report, we describe the presence of follicular lumina that are unsealed. Peroxidase (HRP) was used to study such structures and when injected through the femoral vein, was observed in association with a few follicular lumina, on their microvilli and around the cilia of folliculo-stellate cells. The existence of peroxidase-positive follicles clearly shows that follicles of the hypophysis are not always firmly sealed by tight junctions. The folliculo-stellate cells which faced the peroxidase-positive follicles displayed HRP deposits which were membrane bound within their cytoplasm. These findings suggest an absorptive function for the folliculo-stellate cells.

Intercellular communication within the rat anterior pituitary gland: VI. Development of gap junctions between folliculo-stellate cells under the influence of ovariectomy and sex steroids in the female rat.

BACKGROUND: Farquhar (1957) initially described the folliculo-stellate cells in the rat anterior pituitary gland and found them to be located in groups around follicles throughout the anterior lobe. Soji and his co-workers have published a series of reports concerning cell-to-cell communication in the male rat hypophysis involving folliculo-stellate cells as mediated through a gap junctional network and recorded a reduction in junctional number following castration that was reversed by the administration of testosterone (Soji and Herbert, 1990, Anat. Rec., 226:342-346; Soji et al., 1990, Anat. Rec., 226:337-341). METHODS: Animals were ovariectomized at 10 days of age and separated into three groups: (1) intact control, (2) ovariectomized controls, (3) ovariectomized and given either estradiol, testosterone, or progesterone. On days 10, 20, 30, 40, and 45, the pituitary gland from animals in each group was removed and processed for ultrastructural examination. RESULTS: Gap junctions in the intact control female rats initially appeared between adjacent folliculo-stellate cells on day 20. Their numbers linearly increased until the animals reached the age of 45 days. In contrast, there was a suppression in the number of gap junctions present in the ovariectomized groups and a marked enhancement of the junctions in each of the three steroid-treated groups. CONCLUSIONS: These findings support the observations made in the male rat in which it was found that the development of gap junctions in the anterior pituitary gland of the rat is dependent in part on the presence of sex steroid hormones.

Mechanism of the formation of megamitochondria induced by copper-chelating agents. II. Isolation and some properties of megamitochondria from the cuprizone-treated mouse liver.

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 megamitochondria 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.

Mechanism of the formation of megamitochondria induced by copper-chelating agents. I. On the formation process of megamitochondria in cuprizone-treated mouse liver.

Processes of the formation of cuprizone-induced megamitochondria in mouse liver have been studied in detail by electron microscopy. The earliest change observed was the presence of large intramitochondrial granules. The next stage was the formation of myelin figures by which mitochondria were apparently connected. The third stage was characterized by megamitochondria connected with each other by their outer membranes. Continuity of mitochondria were further examined by serial sections, and megamitochondria were proved to be connected to each other far more frequentlythan expected on one plane of section. A model for the mechanism of megamitochondrial formation is proposed based on electron microscopic evidences, involving the fusion of mitochondrial membranes. Possibility is also discussed that cuprizone-induced megamitochondria may fuse to one single branching mitochondrion.

Steroidogenesis in the zona glomerulosa of the adrenal coretx. I. Isolation and some properties of mitochondria from the zona glomerulosa of the bovine adrenal cortex.

Isolation procedures for mitochondria from the zona glomerulosa of the bovine adrenal cortex are described and the properties of the mitochondria thus prepared are compared with those isolated from the zona fasciculoreticularis. The cristal membranes of mitochondria in the zona glomerulosa in situ are tubular or tubulovesicular, whereas those of mitochondria in the zona fasciculoreticularis in situ are vesicular. When mitochondria are isolated from the former zone, they invariably showed the condensed configuration regardless of isolation media, whereas those isolated from the latter zone in an ST medium showed the orthodox configuration. When Ca2+ was added to mitochondria isolated either from the zona glomerulosa or the zona fasciculoreticularis in an STE medium in the condensed configuration, a transition from the condensed to the orthodox configuration tool place; the cristal membranes of mitochondria from the zona glomerulosa became tubular or tubulovesicular and those of mitochondria from the zona fasciculoreticularis became vesicular. Contaminations of mitrochondria of the zona glomerulosa with other cellular organelles were examined using various marker enzymes. There was no difference in cytochrome content between mitochondria of the two zones specified above. The coupling efficiency of mitochondria of the zona glomerulosa was found to be remarkably effected by temperature during the isolation procedures. Effects of various substrates, isolation media, and bovine serum albumin on the coupling efficiency of mitochondria of the zona glomerulosa are also described.

Mechanism of the formation of megamitochondria induced by copper-chelating agents. III. Formation and some biochemical properties of megamitochondria induced by diethyldithiocarbamate (DDC).

Sodium diethyldithiocarbamate (DDC), a copper-chelating agent, has induced megamitochondria in mouse hepatocytes simply by feeding the animal with a diet containing the noxious agent. Megamitochondria have been isolated from the liver, specified above, in a medium containing albumin. Phosphorylating capacities of such megamitochondria have revealed that they are tightly coupled. Biochemical properties of megamitochondria, such as cytochrome contents, activities of copper-containing enzymes. and contents of Cu2+, strongly suggest that copper-chelating action of the agent may not be related to the formation of megamitochondria just as in the case of cuprizone-induced megamitochondria. Moreover, contents of divalent metals such as Ca2+ and Mg2+ were drastically decreased in the mitochondrial preparation specified above. Similarities in biochemical aspects of DDC-induced megamitochondria to those of cuprizone-induced megamitochondria together with ultrastructural changes in the liver and clinical appearances of the mouse treated with the agent would strongly suggest that the mechanism of the formation of megamitochondria induced either by DDC or by cuprizone may be the same.

Stabilization of configurational states and enzyme activities in subcellular fractions after fixation with extremely low concentrations of glutaraldehyde.

The activities of various enzymes in some subcellular organelle fractions were examined after fixation in glutaraldehyde of various concentrations. A high speed centrifuge was used to shorten the fixation time. At the lowest concentration (0.01%) glutaraldehyde stabilized instable configurational states of mitochondria as revealed by electron microscopy. In addition, at this concentration, at least 70% of the original monoamine oxidase, ATPase and cytochrome oxidase activities were preserved. The activity of acid phosphatase, on the other hand, was enhanced in a lysosomal fraction when fixed with the aldehyde at higher concentrations, e.g. 0.1% and 1.0%. It is possible that the aldehyde at higher concentrations has the same effects on the lysosomal membrane as freeze-thawing. Glucose-6-phosphatase activity was well-preserved in a microsomal fraction fixed with 0.01% glutaraldehyde but was decreased drastically when the concentration of the aldehyde was greater than 0.05%.

Zonation of the adrenal cortex. I. Isolation of mitochondria from the zona glomerulosa of the bovine adrenal cortex.

Isolation procedures for mitochondria from the zona glomerulosa of the bovine adrenal cortex are described in comparison to those isolated from the zona fasciculo-reticularis. The cristal membranes of mitochondria in the zona glomerulosa in situ are tubular or tubulo-vesicular, whereas those of mitochondria in the zona fasciculo-reticularis in situ are vesicular. When mitochondria are isolated from the former zone, they invariably showed a condensed configuration regardless of isolation media, whereas those isolated from the later zone in a ST medium showed an orthodox configuration. When Ca2+ was added to mitochondria isolated form either zone in an STE medium in the condensed configuration, transition from the condensed to the orthodox configuration took place; the cristel membrane of mitochondria from the zona glomerulosa became tubular or tubulo-vesicular and those of mitochondria from the zona fasciculo-reticularis became vesicular. Purity of mitochondria thus obtained from the zona glomerulosa was examined by electron microscope and marker enzymes. Coupling efficiency of mitochondria was found to be remarkably affected by temperature during the isolation procedures and a choice of substrates.

Swelling of free-radical-induced megamitochondria causes apoptosis.

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.

Two types of the enlargement of mitochondria related to apoptosis: simple swelling and the formation of megamitochondria.

Our recent finding that free radical-induced formation of megamitochondria (MG) is followed by apoptosis has prompted us to investigate the correlation between the MG formation and the swelling of mitochondria which is considered to play a key role in early stages of apoptotic processes of the cell. Mitochondria of rat hepatocytes or RL-34 cells and those isolated from rat livers became enlarged up to three times in their diameters when they were exposed to a hypotonic medium. MG induced in the liver of rats placed on a 1% hydrazine-diet for 4-5 days or those induced in the liver of mice placed on a 2% chloramphenicol (CP)-diet for 9-10 days were endowed with a dense matrix whereas those fed with the toxic diets for longer periods of time became enlarged further and their matrix became extremely pale indicating that MG in the latter animals became swollen secondarily. The membrane potential, the content of cytochrome c and the rate of the generation of reactive oxygen species (ROS) of MG in the former animals were almost unchanged compared to those of mitochondria in control animals whereas those of MG in the latter animals became distinctly decreased. These results may suggest that free radical-induced MG possibly cause apoptosis via their secondary swelling.

Steroidogenesis in the zona glomerulosa of the adrenal cortex. II. Distribution of cytochrome P-450 in the zona glomerulosa of the bovine adrenal cortex.

Microsomes were obtained from the zona glomerulosa of the bovine adrenal cortex. Contamination of microsomes with other cellular organelles was examined using various marker enzymes and the electron microscope. Distribution of cytochrome P-450 in mitochondria and in microsomes was determined to be 0.73 and 0.32 nmol/mg protein, respectively. The CO difference spectrum was affected not only by the concentration of added deoxycholate but also by the incubation time after addition. Approximately 40-50% of cytochrome P-450 in the samples was converted to cytochrome P-420 within 20-30 sec of incubation with deoxycholate. The content of RNA, phospholipids, and cytochrome b5 in microsomes obtained from the zona glomerulosa is also evaluated in comparison to that in microsomes obtained from the zona fasciculoreticularis.

Zonation of the adrenal cortex. II. Effect of BSA on coupling efficiency of mitochondria isolated from the zona glomerulosa of the bovine adrenal cortex.

Effect of bovine serum albumin on coupling efficiency of mitochondria isolated from the zona glomerulosa of the bovine adrenal cortex in various media was examined polarographically and electron microscopically. Albumin restored the coupling efficiency of mitochondria isolated from the zona glomerulosa regardless of isolation media when succinate or malate was oxidizable substrate. Respiratory controls greater than 5 were obtained. Albumin, however, had no effect when glutamate, beta-hydroxybutylate and pyruvate were the oxidizable substrates. The conditions have been found under which mitochondria of the zona glomerulosa stay in the orthodox configuration and yet coupled.

Zonation of the adrenal cortex. III. Distribution of cytochrome P-450 in the zona glomerulosa of the bovine adrenal cortex.

A microsomal fraction was obtained from the zona glomerulosa of the bovine adrenal cortex. Glucose-6-phosphate activity of the fraction was found to be much lower than that of the liver. Contents of RNA and phospholipids, besides electron microscopic findings, of the fraction also indicate that it is rich in smooth-surfaced endoplasmic reticulum. Distribution of cytochrome P-450 in the zona glomerulosa was studied using various fractions including the microsomal fraction described above. The amount of cytochrome P-450 in mitochondria and that in microsomes were determined to be 0.73 and 0.32 nmoles/mg protein, respectively. The CO-difference spectrum was affected not only by the concentration of added deoxycholate but also by the incubation time after addition. Approximately 40-50% of cytochrome P-450 in the samples were converted to cytochrome P-420 within 20-30 seconds of incubation with deoxycholate.