CORRELATED MORPHOMETRIC AND BIOCHEMICAL STUDIES ON THE LIVER CELL : II. Effects of Phenobarbital on Rat Hepatocytes

The changes occurring in rat hepatocytes during a 5 day period of treatment with phenobarbital were determined by morphometric and biochemical methods, particular attention being paid to the endoplasmic reticulum. The hepatocytic cytoplasm played an overwhelming part in the liver hypertrophy, while the hepatocytic nuclei contributed to only a moderate extent. The endoplasmic reticulum accounted for more than half of the increase in cytoplasmic volume. The increase in the volume and number of hepatocytic nuclei in the course of phenobarbital treatment was associated with changes in the ploidy pattern. Until the 2nd day of treatment both the rough-surfaced endoplasmic reticulum (RER) and the smooth-surfaced endoplasmic reticulum (SER) participated in the increase in volume and surface of the whole endoplasmic reticulum (ER). Subsequently, the values for RER fell again to control levels, whereas those for SER continued to increase, with the result that by the 5th day of treatment the SER constituted the dominant cytoplasmic element. The specific volume of mitochondria and microbodies (peroxisomes) remained constant throughout the duration of the experiment, while that of the dense bodies increased. The specific number of mitochondria and microbodies displayed a significant increase, associated with a decrease in their mean volume. The phenobarbital-induced increase in the phospholipid and cytochrome P-450 content of the microsomes, as well as in the activities of microsomal reduced nicotinamide-adenine dinucleotide phosphate-cytochrome c reductase and N-demethylase, was correlated with the morphometric data on the endoplasmic reticulum.     

NEW OBSERVATIONS ON MICROBODIES : A Cytochemical Study on CPIB-Treated Rat Liver

The liver of male rats has been studied after CPIB stimulation by using the peroxidase reaction for localizing catalase in hepatic cells. CPIB administration leads to an increase in the number of microbodies, and it is suggested that one mechanism by which microbody proliferation occurs is a process of fragmentation or budding from preexisting microbodies. Reaction product was observed not only within the microbody matrix, but outside the limiting membrane of the microbody and in association with ribosomes of adjacent rough endoplasmic reticulum. This localization of reaction product is interpreted as evidence that catalase after synthesis on rough endoplasmic reticulum may accumulate near microbodies and may be transferred directly into these organelles without traversing the cisternae of the endoplasmic reticulum or Golgi apparatus.     

Isolation and characterization of organelles from soybean suspension cultures

Whole homogenates from cells of Glycine max grown in suspension culture were centrifuged on linear sucrose gradients. Assays for marker enzymes showed that distinct peaks enriched in particular organelles were separated as follows: endoplasmic reticulum (density 1.10 g/cm³, NADH-cytochrome-c reductase), Golgi membranes (density 1.12 g/cm³, inosine diphosphatase), mitochondria (density 1.18—1.19 g/cm³, fumarase, cytochrome oxidase) and microbodies (density 1.21—1.23 g/cm³, catalase). In cells which had ceased to grow (stationary phase) only a single symmetrical catalase peak at density 1.23 g/cm³ was observed on the sucrose gradient. During the phase of cell division and expansion a minor particulate catalase component of lighter density was present; its possible significance is discussed.     

Effect of clofibrate application on morphology and enzyme content of liver peroxisomes.

Male albino rats (Sprague Dawley) were fed for 2-6 weeks on a diet containing 0.75% clofibrate. Liver cell fractions obtained from these animals were assayed for peroxisomal enzymes. In the cell homogenate the catalase activity was doubled, whereas the activity of urate oxidase was found to be only slightly depressed. The activity of carnitine acetyltransferase increased several times. In liver peroxisomes purified by isopycnic gradient centrifugation the specific activity of urate oxidase decreased appreciably showing that peroxisomes formed under the proliferative influence of clofibrate are not only modified with respect to their morphological characteristics but also to their enzymic equipment. This is also obvious from the changes in peroxisomal carnitine acetyltransferase activity which was enhanced by clofibrate to more than the fivefold amount. In purified mitochondria this enzyme was even more active: clofibrate advances both, the peroxisomal and the mitochondrial moiety of carnitine acetyltransferase. Morphological and cytochemical studies showed an increase in the number of microbodies and as compared to the controls microbodies were lying in groups more frequently. Small particles located closely adjacent to "normal" sized peroxisomes were found particularly after short feeding periods. While the number of coreless microbodies increased studies gave no clear evidence for an increase in marked shape irregularities of the peroxisomes.     

Isolation of organelles from carrot cell suspension cultures

Summary Organelles isolated from carrot cell suspension cultures by density gradient centrifugation and identified by their specific marker enzymes were found at the following mean densities on the sucrose gradient: microbodies 1.25 g/cm³ (catalase), mitochondria 1.18 (fumarase), endoplasmic reticulum 1.09 g/cm³ (NADH-cytochrome c reductase). Further enzyme assays were done for characterization of microbodies from carrot cultures.This work was supported by the Deutsche Forschungsgemeinschaft.     

CYTOCHEMICAL AND DEVELOPMENTAL CHANGES IN MICROBODIES (GLYOXYSOMES) AND RELATED ORGANELLES OF CASTOR BEAN ENDOSPERM

Structural changes in endosperm cells of germinating castor beans were examined and complemented with a cytochemical analysis of staining with diaminobenzidine (DAB). Deposition of oxidized DAB occurred only in microbodies due to the presence of catalase, and in cell walls associated with peroxidase activity. Seedling development paralleled the disappearance of spherosomes (lipid bodies) and matrix of aleurone grains in endosperm cells. 6 to 7 days after germination, a cross-section through the endosperm contained cells in all stages of development and senescence beginning at the seed coat and progressing inward to the cotyledons. Part of this aging process involved vacuole formation by fusion of aleurone grain membranes. This coincided with an increase in microbodies (glyoxsomes), mitochondria, plastids with an elaborate tubular network, and the formation of a new protein body referred to as a dilated cisterna, which is structurally and biochemically distinct from microbodies although both apparently develop from rough endoplasmic reticulum (ER). In vacuolate cells microbodies are the most numerous organelle and are intimately associated with spherosomes and dilated cisternae. This phenomenon is discussed in relation to the biochemical activities of these organelles. Turnover of microbodies involves sequestration into autophagic vacuoles as intact organelles which still retain catalase activity. Crystalloids present in microbodies develop by condensation of matrix protein and are the principal site of catalase formerly in the matrix.     

Stereological study of endoplasmic reticulum, Golgi complex and secretory granules in the B-cells of normal and alloxan-treated mice.

Different B-cell organelles (lamellar and vesicular endoplasmic reticulum, Golgi complex, whole secretory granules and secretory granule cores) were studied stereologically in pancreatic islets from control mice and mice killed 10 or 60 min following alloxan injection. Ten min following alloxan a significant decrease was observed in the volume, surface and numerical densities of whole secretory granules and their cores, and a significant increase was found in the volume and surface densities of vesicular endoplasmic reticulum. At the 60 min observation time, a significant decrease was seen in the volume density of lamellar endoplasmic reticulum and Golgi complex, and in the volume, surface and numerical densities of whole secretory granules and their cores, and a significant increase was observed in the volume and surface densities of vesicular endoplasmic reticulum, and in the mean values for volume and surface of whole secretory granules and their cores. The stereological data indicate swelling of endoplasmic reticulum, decreased Golgi area, and decreased number and total volume and surface of secretory granules during the first hour after alloxan administration to mice. The observations may be consistent with inhibited insulin synthesis.     

BALB/C小鼠肝细胞结构的年龄变化——光镜与电镜的计量形态学研究

作者研究了1月、6月和12月三个不同年龄组的BALB/c小鼠的肝细胞结构。利用计量形态学方法在光镜与电镜两个水平上对肝细胞整体及重要细胞器的年龄变化进行定量分圻,发现在所观察的结构参数中大部分随年龄而发生动力学变化。文中讨论了某些变化的可能意义。     

Lysosomal involvement in the removal of clofibrate-induced rat liver peroxisomes. A biochemical and morphological analysis

Peroxisomal proliferators induce in rodents hepatic hyperplasia and hypertrophy; the significant increase in the peroxisomal population is accompanied by specific and reversible induction of some peroxisomal enzymes. In suckling rats born from clofibrate-treated mothers, a massive removal of proliferated organelles occurs within 3 days of recovery. In the present paper we examined the early stages of the recovery period in liver of male rats treated with clofibrate for 5 days. The lysosomal involvement in the removal of drug-induced peroxisomes was investigated under physiological conditions, ie in the absence of inhibitors of the autophagic process. Biochemical results indicate that peroxisomal β-oxidation, but not catalase activity, returns to the control values within the examined period. Total acid phosphatase activity is not affected by clofibrate treatment, but following fractionation on a linear density gradient the lysosomal marker enzyme activity is shifted towards lower density values, particularly at day 1 and 2 of recovery. This class of organelles possibly represents lysosomes involved in active autophagic processes. Acid phosphatase cytochemistry shows an increase of lysosome number at day 1 of recovery. Combination of acid phosphatase cytochemistry either with catalase cytochemistry or with catalase immunogold labelling allows to reveal organelles containing both marker enzymes. These results strongly support the involvement of autophagic processes in the removal of proliferated peroxisomes.     

Structural and functional changes after the administration of tetrachlormethane in the liver of rats fed on diets with different protein contents

Morphological and biochemical changes characterizing the degree of liver damage and the development of liver repair were studied in rats fed 21 days on a low protein diet (LPD), a standard diet (SLD) and a high protein diet (HPD) and then given a single i.p. injection of tetrachlormethane (CCl4) in a dose of 0.75 ml/kg body weight. The HPD was found to increase sensitivity to CCl4, but it also promoted the liver repair process, as seen from the increment in liver DNA synthesis and the total DNA content of the liver, increased ploidy of the hepatocytes and growth of the size of their nuclei and of the hepatocytes themselves. An increase in the total surface area of the membranes of the granular endoplasmic reticulum and the inner and outer membrane of the mitochondria, but a decrease in the surface area of the membranes of the smooth endoplasmic reticulum, were also observed after the administration of CCl4. The LPD raised liver resistance to CCl4, but the development of liver repair activity differed from the process after the SLD and HPD, since polyploidy of the hepatocytes (especially the growth of octaploid cells) predominated and there was also an increase in the number of binuclear hepatocytes. Cell hypertrophy was expressed less in rats fed on the LPD than in animals given the HPD. As far as liver repair was concerned, the HPD showed no explicit advantages over the SLD.     

Decrease of hepatic catalase level by treatment with diallyl sulfide and garlic homogenates in rats and mice

Diallyl sulfide (DAS) is a flavor compound derived from garlic and is active in the inhibition of chemically induced cytotoxicity and carcinogenicity in animal models. This study was conducted to examine the effects of the treatment of DAS and garlic homogenates on the activities of catalase, glutathione peroxidase, and superoxide dismutase. Male Sprague-Dawley rats were treated with DAS i.g. at daily doses of 50 or 200 mg/kg for 8 days, causing the hepatic catalase activity to decrease by 55 and 95%, respectively. Such a decrease in hepatic catalase activity was also observed when the DAS treatment was extended to 29 days. Western blot analysis showed that the DAS treatments resulted in corresponding decreases in the liver catalase protein level. No significant change in the catalase activity in the kidney, lung, and brain was observed with the treatments, but a slight decrease in heart catalase activity was observed. These treatments did not cause significant changes in superoxide dismutase and glutathione peroxidase activities in these tissues. Treatment with DAS at a daily dose of 200 mg/kg for 1-7 days resulted in a gradual decrease in the liver catalase activity to 5% of the control level, but it did not decrease the erythrocyte catalase activity. Treatment of rats with fresh garlic homogenates (2 or 4 g/kg, i.g., daily for 7 days) caused a 35% decrease in liver catalase activity. A/J mice treated with DAS and garlic homogenates also showed a decrease in the liver catalase activity. Diallyl sulfone (DASO2), a DAS metabolite, however, did not effectively decrease catalase activity in mice. The catalase activity was not inhibited by either DAS or DASO2 in vitro. The present results demonstrate that treatment with DAS and garlic homogenates decrease the hepatic catalase level in rats and mice.     

Effect of rehydration of rat liver tissue after water deprivation.

Male albino rats were deprived of water for 6 days, then they were allowed to drink tap water ad libitim. The structure of the liver was examined by light and electron microscopy, and the protein and dry matter contents, oxygen consumption and glucose-6-phosphatase activity of the liver were determined after rehydration. At 10 minutes, the mitochondria showed signs of division and a peculiar transformation of the cristae. At 60 minutes, the membranes of the rough endoplasmic reticulum were found to have proliferated. At 12 hours, the smooth-surfaced membranes showed hypertrophy and the bile canaliculi were distended. At 24 hours all rehydration induced organelle alterations were declining. The biochemical findings agreed well with the fine structural changes and both were indicative of an enchanced functional capacity of liver cells during rehydration.     

Ascorbyl free radical and dehydroascorbate formation in rat liver endoplasmic reticulum

The mechanism of ascorbate oxidation was studied in rat liver microsomes. A continuous consumption of the added ascorbate was observed, which was accompanied with a prompt appearance of ascorbyl free radical and dehydroascorbate. Microsomes sustained steady-state level of ascorbyl free radical and dehydroascorbate till ascorbate was present in the medium. Ascorbyl free radical formation was diminished when microsomes had been pretreated with heat or trypsine. It was also decreased by addition of quercetin, econazole or metal chelators, including the copper specific neocuproine. Enzymatic (superoxide dismutase, catalase) and nonenzymatic (dimethyl sulfoxide, mannitol) antioxidants did not modify the microsomal production of ascorbyl free radical. Investigation of the subcellular distribution of ascorbate oxidation showed that the microsomal fraction of liver had the highest activity. The decrease of ascorbate oxidation after protease treatment and the negligible increase upon permeabilization of microsomal vesicles showed that a membrane protein is responsible for the activity, which is exposed to the outer surface of the endoplasmic reticulum. The results indicate the presence of a primary enzymatic ascorbate oxidation in rat liver endoplasmic reticulum which is able to generate dehydroascorbate, an important source of the oxidizing environment in the endoplasmic reticulum.     

ELECTRON MICROSCOPIC RADIOAUTOGRAPHIC DETECTION OF SITES OF PROTEIN SYNTHESIS AND MIGRATION IN LIVER

The sites of synthesis of proteins and their subsequent migration in rat liver have been studied during a 75 min period after labeling of liver-slice proteins by exposure to leucine-H³ for 2 min. Incorporation of the label into protein began after 1 min and was maximal by 4 min. Electron microscopic radioautography showed that synthesis of proteins in hepatocytes occurs mainly on ribosomes, particularly those in rough endoplasmic reticulum and, to some extent, in nuclei and mitochondria. Most of the newly formed proteins leave the endoplasmic reticulum in the course of 40 min, and concurrently labeled proteins appear in Golgi bodies, smooth membranes, microbodies, and lysosomes. A likely pathway for the secretion of some or all plasma proteins is from typical rough endoplasmic reticulum to a zone of reticulum which is partially coated with ribosomes, to the Golgi apparatus, and thence to the cell periphery. The formation of protein by reticuloendothelial cells was measured and found to be about 5% of the total protein formed by the liver.     

Morphometric studies on lipoprotein particles in developing rat liver and their corticosteroid-induced changes during the late gestational period

Summary Changes in lipoprotein particles in hepatocytes of the fetal rat liver have been studied morphometrically from days 15–21 of gestation. On all these days, distinct lipoprotein particles are found within the cisternae of the RER, Golgi complexes and Golgi-derived secretory vesicles. Their mean diameter is 30–31 nm. The number of Golgi complexes per hepatocyte, the lipoprotein particle number per Golgi complex and the volume density of the latter remain unchanged within the developmental period examined. The volume density of lipid droplets, however, shows a significant decrease during this time.Following corticosteroid treatment, the mean diameter of lipoprotein particles, the number of lipoprotein particles per Golgi complex, the volume density of the Golgi complex, and that of the lipid droplets increase significantly within the examined period, whereas the number of Golgi complexes per hepatocyte is reduced. These data support the view that triglyceride production in the fetal liver is directly or indirectly stimulated by corticosteroids administered to the pregnant rat, thus giving rise to larger amounts of hepatic lipoproteins and lipids.Abbreviations LP lipoprotein - VLDL very low density lipoproteins (d< 1.006 g/ml) - RER rough endoplasmic reticulum - ER endoplasmic reticulum     

Effect of citrate and aminotriazole on matrical plates induced in hepatic microbodies.

Feeding of acetylsalicylic acid (ASA), clofibrate and dimethrin induces formation of matrical plates in heptaic microbodies. Administration of sodium citrate prevents formation of matrical plates in rats fed ASA, but not in rats treated with clofibrate or dimethrin. Propionate has a similar effect on rats fed ASA but not those fed clofibrate. Feeding of aminotriazole prevents formation of matrical plates in rats fed ASA without inhibiting the proliferation of microbodies. Sodium citrate and aminotriazole and feeding decrease also the activity of hepatic catalase in rats fed ASA.     

PEROXIDASE ACTIVITY IN RAT LIVER MICROBODIES AFTER AMINO-TRIAZOLE INHIBITION

The in vivo effects of 3-amino-1,2,4-triazole (AT) on the fine structure of microbodies in hepatic cells of male rats has been studied by the peroxidase-staining technique. Within 1 hr of intraperitoneal injection AT abolishes microbody peroxidase-staining, and the return of staining coincides temporally with the known pattern of return of catalase activity following AT inhibition; this is further evidence that the peroxidase staining of microbodies is due to catalase activity. Peroxidase staining reappears in the microbody matrix without evidence of either massive degradation or rapid proliferation of the organelles. Furthermore, during the period of return of activity, ribosomal staining occurs adjacent to microbodies whose matrix shows little or no peroxidase staining. These observations are interpreted as evidence that (a) catalase is capable of entering preexisting microbodies without traversing the cisternae of the rough endoplasmic reticulum or the Golgi apparatus, and that (b) the ribosomal staining is probably not cytochemical diffusion artifact and may represent a localized site of synthesis or activation of catalase.     

The endoplasmic reticulum in regenerating liver cells

Summary Light-microscopic analysis of mouse liver homogenates six days after partial hepatectomy, showed a higher percentage of nuclei with adherent cytoplasm than homogenates from normal liver. This observation was true for animals with either a slow or rapid recovery of body weight after the operation. The phenomenon was not a function of the changes in the proportions of parenchymal and non-parenchymal tissue in the regenerating liver.Electron-microscopic analysis of random samples from normal and regenerating livers indicated an increase in the perinuclear rough endoplasmic reticulum, and a displacefment of the glycogen depots within the regenerating cells six days after partial hepatectomy.The marked resistance towards homogenization, shown by the cytoplasm of the regenerating cells, may have been due to the observed increase of perinuclear membranes. However, qualitative changes of the cell membranes and a general decrease of proteolytic activity connected with liver regeneration may also have contributed.     

Ultrastructural, immunocytochemical and morphometric characterization of liver peroxisomes in gray mullet, Mugil cephalus

Peroxisomes of the hepatocytes of gray mullets, Mugil cephalus, were characterized cytochemically and immunocytochemically using antibodies against the peroxisomal proteins catalase and palmitoyl-coenzyme A (CoA) oxidase. In addition, morphometric parameters of peroxisomes were investigated depending on the hepatic zonation, the age of the animals and the sampling season. Mullet liver peroxisomes were reactive for diaminobenzidine, but presented a marked heterogeneity in staining intensity. Most of the peroxisomes were spherical or oval in shape, although irregular forms were also observed. Their size was heterogeneous, with profile diameters ranging from 0.2 to 3 microm. Peroxisomes tended to occur in clusters, usually near the mitochondria and lipid droplets. They also showed a very close topographical relationship to the smooth endoplasmic reticulum. Mullet liver peroxisomes did not contain cores or nucleoids as rodent liver peroxisomes, but internal substructures were observed in the matrix, consisting of small tubules about 60 nm in diameter and larger semicircles 120 nm in diameter. The volume density of peroxisomes was higher in periportal hepatocytes of mullets sampled in summer than in pericentral hepatocytes, indicating that mullet peroxisomes vary depending on physiological and environmental conditions. By immunoblotting, the mammalian antibodies cross-react with the corresponding proteins in whole homogenates of mullet liver. Paraffin sections immunostained with the antibodies against catalase and palmitoyl-CoA oxidase showed a positive reaction corresponding to peroxisomes localized in the hepatocyte cytoplasm. In agreement, the ultrastructural study revealed that catalase and palmitoyl-CoA oxidase are exclusively localized in the peroxisomal matrix in fish hepatocytes, showing a dense gold labeling. The presence of the peroxisomal beta-oxidation enzyme palmitoyl-CoA oxidase in peroxisomes indicated that these organelles play a key role in the lipid metabolism of fish liver.     

Fine structural and cytochemical analysis of phragmosomes (microbodies) during cytokinesis inAllium root tip cells

Summary The ontogeny and distribution of phragmosomes (microbodies) during cytokinesis inAllium sativum root tip cells have been studied and complemented with a cytochemical analysis of reactivity with diaminobenzidine (DAB). Incubation in different DAB media revealed the presence of catalase but not peroxidase in these organelles, identifying them as a type of microbody associated with the forming cell plate. Only vacuoles, segments of endoplasmic reticulum and portions of the mature walls stained positively with DAB for peroxidase activity. Microbodies begin to appear in the region of the future cell plate as cells enter late anaphase. They exhibit a moderately electron-opaque anucleoid matrix and are continuous with segments of endoplasmic reticulum (ER). Certain observations have led us to consider that certain aspects of plate formation inAllium require the participation of microbodies: (a) their pronounced numerical increase at the onset of plate formation, (b) their intimate association with regions of the plate where vesicle fusion is in progress, and (c) their rapid numerical decline following vesicular fusion and concomitant cell plate formation. The characteristic spatial association observed between microbodies and the plate-forming vesicles may well reflect their mutual involvement in the metabolism of carbohydrates comprising the middle lamella, being coordinated by metabolic activities in the cytosol, mitochondria and dictyosomes.This study was supported in part by NIH training grant HD 174 to Dr.Hewson Swift and the Marquette University Committee on Research Grants 5641 and 5532.