Ultrastructure of hepatocellular neoplasms in aflatoxin B1 (AFB1)-initiated rainbow trout (Oncorhynchus mykiss).

The fine structure of hepatocellular neoplasms from aflatoxin B1 (AFB1)-initiated rainbow trout was studied by transmission electron microscopy. Large, usually uniform hepatic nuclei, large nucleoli, abundant, dilated rough-surfaced endoplasmic reticulum, and reduced glycogen storage were common findings in both hepatocellular adenomas and hepatocellular carcinomas. In addition, the presence of poorly developed microvilli in the space of Disse and in bile canaliculi, the occurrence of few or no bile preductule cells and a striking increase in the size and number of intercellular spaces characterized hepatocellular carcinomas. The three latter characteristics of hepatocellular carcinomas suggest loss of inter-relationships between hepatocytes and the microvascular system (sinusoids), between hepatocytes and the biliary system, and between individual hepatocytes, respectively. With respect to these parameters, adenomas were more similar to normal liver than to carcinomas.     

Cavernous structures composed of hepatocytes in experimentally induced hepatocellular carcinoma

Cavernous structures composed of hepatocytes were observed for the first time in trabecular-patterned hepatocellular carcinomas by light, scanning and transmission electron microscopy. The carcinomas were induced by a single dose of diethylnitrosamine combined with subsequent administrations of 2-acetylaminofluorene and partial hepatectomy. The cavernous structures had numerous cavities walled by flat hepatocytes. In the cavities, dendritic hepatocytes, connected with each other by their long and slender processes, formed a network and were attached to the fenestrated sinusoidal endothelium via intercellular spaces between the flat hepatocytes. Dendritic hepatocytes had smooth surfaces, prominent nucleoli, decreased glycogen, and well organized cisternae. Dilated bile canaliculi were sometimes seen among flat and dendritic cells. Dendritic cells showed more morphological dearrangement than flat cells. Blood cells were observed in the cavities. Kupffer cells were not seen in the sinusoids among the cavernous structures. The cavernous structures in the hepatocellular carcinomas resembled the hepatocyte arrangement in fetal liver in which intercellular spaces of hepatocytes are extremely widened.     

CAVERNOUS STRUCTURES COMPOSED OF HEPATOCYTES IN EXPERIMENTALLY INDUCED HEPATOCELLULAR CARCINOMA

Cavernous structures composed of hepatocytes were observed for the first time in trabecular-patterned hepatocellular carcinomas by light, scanning and transmission electron microscopy. The carcinomas were induced by a single dose of diethylnitrosamine combined with subsequent administrations of 2-acetylaminofiuorene and partial hepatectomy. The cavernous structures had numerous cavities walled by flat hepatocytes. In the cavities, dendritic hepatocytes, connected with each other by their long and slender processes, formed a network and were attached to the fenestrated sinusoidal endothelium via intercellular spaces between the flat hepatocytes. Dendritic hepatocytes had smooth surfaces, prominent nucleoli, decreased glycogen, and well organized cisternae. Dilated bile canaliculi were sometimes seen among flat and dendritic cells. Dendritic cells showed more morphological dearrangement than flat cells. Blood cells were observed in the cavities. Kupffer cells were not seen in the sinusoids among the cavernous structures. The cavernous structures in the hepatocellular carcinomas resembled the hepatocyte arrangement in fetal liver in which intercellular spaces of hepatocytes are extremely widened.     

大鼠肝再生过程中毛细胆管增生的形态计量研究

Morphometric analysis of the liver during regeneration after partial hepatectomy showed that the mean area and number of bile canaliculi increased, while the volume density of microvilli related to the lumina of the canaliculi decreased. These changes were observed 12 hours after the operation, which gradually returned to normal on the third day after operation. It indicated that the decrease of volume density of microvilli was the result of enlargement of bile canaliculi, which was different from the decrease of microvilli due to passive widening of the canaliculi such as in cholestasis from loss of microvilli. All these phenomena suggest that enlargement of the area of bile canaliculi during liver regeneration may be due to the increase of membranous protein synthesis. Moreover, the increase of the number of bile canaliculi suggests that the proliferation of hepatocytes is associated with formation of new branches of bile canaliculi.     

Electron microscopic study on the injurious effects of chlorpromazine on rat liver cells.

The authors examined the effect of chlorpromazine administration on rat liver cells. The early alterations were limited to the pericanalicular region, but the dilatation of bile canaliculi and the destruction of canalicular microvilli, both characteristic of rats with cholestasis were not observed. It is suggested that beside the Golgi apparatus, the smooth surfaced endoplasmic reticulum, the plasma membranes of liver cells also have an important role in the formation of vesicles and vacuoles in the pericanalicular region. The progressive proliferation of the smooth surfaced endoplasmic reticulum is thought to be related to an increased overburdening of the biotransformation system of liver cells, which is the result of chronic drug administration. In the last period of the experiment there was a decrease in the quantity of rough surfaced endoplasmic reticulum and increased fatty infiltration, mitochondrial alterations in some liver cells and simultaneously numerous regenerating liver cells were observed. All these alterations are attributed by the authors to the direct liver injuring effect of chlorpromazine.     

The ultrastructure of primary hepatocellular cancer in man

Summary The authors carried out an electronmicroscopical study of material obtained by needle biopsy from 12 human hepatocellular carcinomas. Pleomorphism of the cell nuclei and the occurrence in great numbers of cytoplasmic infoldings into the nuclei were striking features. The nucleoli were enlarged and nucleoli of a peculiar target-like form were observed. The endoplasmic reticulum had a vesicularized, fragmented appearance and the membranes were degranulated. The frequent occurrence of fingerprint-like formations of endoplasmic reticulum were also observed. The cristae of the mitochondria were diminished and other degenerative changes were also regularly present. In one case peculiar electron-dense inclusions were seen in the mitochondria. The amount of glycogen granules in the tumour cells seemed to be considerably diminished. No significantly great changes were seen in the Golgi complex. Bile canaliculi as well as desmosomes were regularly found: usually as many as five or six participated in the formation of the canaliculi. Changes were seen also in the microvilli of the biliary and vascular poles.The relation of the tumour cells to the sinusoids proved in many respects to be similar to that in normal liver. It was a frequent finding, however, that the tumour cells formed plates consisting of several cell rows; furthermore, that a basement membrane had developed around the sinusoids, which thus became capillarized, while the sinusoid endothelium became multilayered and the Disse space filled by a fibrin-like substance.The authors compare the alterations observed in human hepatocellular carcinomas with the ultrastructural alterations found in other pathological conditions of the liver and in experimental liver tumours. Based on this comparison they offer conclusions concerning the functional cytopathological significance of the various alterations on the one hand, and the possible pathogenesis of hepatic tumours on the other.     

Ultrastructural studies of the hepatocytes after chronic exposure to low levels of halothane.

Adult rats were exposed to 10 ppm or 500 ppm halothane 8 hr/day and 5 days/wk for 8 wk or 4 wk, respectively. In the liver from animals which were exposed to 10 ppm of halothane, the rough endoplasmic reticulum in some hepatocytes accumulated a floccular, electron-dense material which gave the hepatocytes a dense and dark appearance. Increase in the matrical density and C-shaped transformation were observed in the mitochondria of some hepatocytes. In addition to these findings, areas of focal cytoplasmic degradation, dilatation of the bile canaliculi, peribiliary accumulation of lysosomes, and extensive dilatation of the smooth endoplasmic reticulum to form large cytoplasmic vacuoles were also observed in the hepatocytes of animals which had been exposed to 500 ppm halothane. Toxic potential of halothane upon chronic exposure is suggested.     

Histopathology of the liver and kidney during malaria: relation to malaria-induced dyslipoproteinemia

A kinetic study concerning histologic and cytologic alterations during Plasmodium chabaudi infection of Swiss mice has been carried out. In liver, a reversible focal and non ischemic necrosis and a vascular congestion were observed together with an accumulation of malarial pigment. The endoplasmic reticulum cisternae and Golgi saccules of hepatocytes were highly distended. Hepatocyte microvilli in biliary canaliculi and in Disse' spaces were markedly less developed and less numerous than in normal liver. Intracytoplasmic lipid globules were found in large amount in hepatocytes before the peak of parasitaemia. Their number and size gradually diminished thereafter. Hepatocytic mitochondria showed important unspecific modifications probably in relation, at last partly, to the tissue anoxia. Some hepatocytic changes (intracytoplasmic lipid globules, enlargement of endoplasmic reticulum cisternae and Golgi saccules) were consistent with an increased synthesis of lipoproteins (VLDL). The kidney showed only minor histological and ultrastructural changes. However haemosiderin was observed in proximal tubules and in their bordering cells. The deposit of immune complex reported previously do not appear associated with tissular or cellular important alterations.     

Expression of NCAM in activated portal fibroblasts during regeneration of the rat liver after partial hepatectomy

In the portal tract of the regenerating liver after partial hepatectomy, vascular and bile ductular remodeling takes place in response to the portal hyperdynamic state and parenchymal hyperplasia. In order to reveal phenotypical changes in the portal fibroblasts, we immunohistochemically investigated neural cell adhesion molecules (NCAM) and alpha smooth muscle actin (alphaSMA) expression and the ultrastructural changes in them during liver regeneration. In the control rat liver, portal fibroblasts were negative for both NCAM and alphaSMA. They became positive for both markers two days after partial hepatectomy, increased in staining intensity, reached a maximum at three to four days, then decreased, being still clearly positive at 14 days. Under an electron microscope, portal fibroblasts from the regenerating liver had larger amounts of cytoplasm and rough endoplasmic reticulum than those from the control liver; thus they might be activated. Additionally, periportal hepatic stellate cells in the regenerating liver were activated with alphaSMA, but without NCAM. The present study has demonstrated that portal fibroblasts express NCAM and alphaSMA in the regenerating liver after partial hepatectomy via transformation into myofibroblasts following reconstruction of the portal tracts.     

Characterizations of and interactions between bile ductule cells and hepatocytes in early stages of rat hepatocarcinogenesis induced by ethionine.

Numerous hepatic cell lineage pathways have been proposed for the development of hepatocarcinogensis induced by chemical carcinogens in rats. The roles of bile ductule cells and hepatocytes in the development of carcinogenesis were investigated using light and electron microscopic procedures to detect differences in morphology and in the phenotypic expression of antigens that are associated with each cell type. In early stages of hepatocarcinogenesis (4-10 weeks after initiation of feeding of a choline-deficient ethionine containing diet), both bile ductulelike (BDL) cells and hepatocytes were seen in mitosis. At the light microscope level, BDL cells showed intense cytoplasmic pyronin (RNA) staining and were positive for the antigens defined by monoclonal antibody 270.38 (bile ductule cells and "oval" cell marker) and glutathione-S-transferase (Yp isoform), whereas hepatocytes were positive for the antigens defined by monoclonal antibodies 270.26 and 258.26 (liver parenchymal cell markers), catalase activity (peroxisome marker) and adenosine triphospatase activity (bile canalicular marker). The authors frequently encountered BDL cells and hepatocytes in close proximity. Ultrastructural examination showed extensive plasma membrane appositions between a subset of BDL cells and hepatocytes. Desmosome structures, tight junctions, microvilli interdigitations and ATPase-positive bile canalicularlike structures were present along the contiguous plasma membrane domains of BDL cells and hepatocytes. Many of the BDL cells attached to hepatocytes were also attached to other BDL cells that had retained a basal lamina. In many cases, BDL cells connected to both hepatocytes and other BDL cells were no longer completely surrounded by basal lamina and had acquired a dual polarity as a consequence of their sharing apical and lateral membrane domains with both BDL cells and hepatocytes. BDL cells showed increased numbers of microperoxisomes (catalase positive organelles) and numerous free ribosomes. Hepatocytes showed a prominent development of the smooth endoplasmic reticulum, a feature prominent in hepatocytes within hyperplastic nodules. Since BDL cells and hepatocytes proliferate and BDL cells and hepatocytes develop intercellular junction sites, the authors propose that both cell types in early stages of carcinogenesis have the capacity to enter the cell lineage pathway leading to the development of hepatocarcinoma. Furthermore, the finding that BDL cells and hepatocytes form multiple attachment sites at the level of the plasma membrane, suggests the possibility that at some stage convergence of separate hepatic cell pathways may occur.     

KIF20A mRNA and its product MKlp2 are increased during hepatocyte proliferation and hepatocarcinogenesis

Mitotic kinesin-like protein 2 (MKlp2), a microtubule-associated motor, is required during mitosis exit for the final step of cytokinesis. It also contributes to retrograde vesicular trafficking from the Golgi apparatus to the endoplasmic reticulum in interphase. The KIF20A gene encoding MKlp2 is controlled by the E2F-retinoblastoma protein-p16 pathway, and its widely expressed mRNA is found in fetal and proliferating adult tissues. The expression pattern and function of MKlp2 in the adult liver, however, have not been investigated. We report herein that MKlp2 transiently accumulates in vivo during mouse liver regeneration after partial hepatectomy and is strongly overexpressed in preneoplastic and neoplastic mouse liver. In vitro in mitogen-stimulated primary hepatocytes, MKlp2 accumulated in the nucleus during the G2 phase of the cell cycle coincident with the mitotic kinase Aurora B. Human hepatoma cell lines exhibited high levels of MKlp2; however, it was undetectable in normal human hepatocytes. RNAi-mediated MKlp2 knockdown in hepatoma cells induced polyploidization consistent with its essential function in promoting cytokinesis and inhibited cell proliferation without inducing apoptosis. KIF20A mRNA was strongly accumulated in a large series of human hepatocellular carcinomas, with the highest expression observed in tumors with genomic instability. Accumulation of MKlp2 in normal proliferating, preneoplastic, and transformed hepatocytes suggests that MKlp2 contributes to both normal and pathologic hepatocyte proliferation and is linked to tumor aggressiveness in human hepatocellular carcinomas.     

Immunohistochemical location of prothymosin alpha in regenerating human hepatocytes and hepatocellular carcinomas

In the present paper we analysed the presence of prothymosin alpha (ProT) in human liver. In normal liver, ProT immunostaining was found in the nuclei of bile duct cells, but not in the hepatocytes. In contrast an intense immunoreactivity was observed in regenerative hepatocytes of chronic hepatitis, cirrhosis and in hepatocellular carcinomas. In all cases the immunostaining was restricted to the nuclei, but the nucleoli were always negative. Similar results were obtained for proliferating cell nuclear antigen. These findings confirm that ProT is related to cell proliferation and provides a new immunohistochemical proliferation marker for routinely processed samples.     

Ultrastructure of liver tumors induced in F344 rats by methapyrilene

Liver tumors induced in F344 rats by methapyrilene were studied by electron microscopy. The tumor cells constituting hepatocellular carcinomas showed a pronounced increase in the number of mitochondria and conformational changes of these organelles while the content of lipid, glycogen and smooth endoplasmic reticulum was greatly reduced. The cholangiocarcinomas consisted of bile duct epithelia at varying stages of squamous metaplasia.     

Early effects of 1,1-dichloroethylene on canalicular and plasma membranes: ultrastructure and stereology

The pathogenesis of 1,1-dichloroethylene (1,1-DCE)-induced hepatotoxicity was investigated in fasted male rats by identifying the earliest morphological alterations in organelles. In situ perfusion-fixed liver tissue was examined by light and electron microscopy at 1, 2, or 3 hr after oral administration of 25, 50, and 100 mg 1,1-DCE/kg in mineral oil. The earliest morphological alterations, which occurred within 1 to 2 hr after 1,1-DCE administration, were dilation of bile canaliculi with an increase in the number of microvilli or membrane fragments in canaliculi and the formation of canalicular diverticuli in centrolobular hepatocytes. Subsequently, microvilli on sinusoidal surfaces were disrupted or lost. Membrane whorls were frequently found in bile canaliculi, the space of Disse, and between the lateral membranes of hepatocytes at early times. As injury progressed, centrolobular hepatocytes retracted from endothelial cells and sinusoidal plasma membranes invaginated to form cytoplasmic vacuoles. Stereological analysis of centrolobular hepatocytes at the 25 mg/kg dose showed a significant increase in canalicular volume density by 3 hr and no detectable alteration in mitochondrial volume density. These results indicate that changes in canalicular shape and microvilli configuration are the earliest morphological alterations following 1-DCE ingestion.     

Ultrastructural changes in hepatocytes,sinusoidal endothelial cells and macrophages in hypothermic preservation of the rat liver with University of Wisconsin solution

To identify subtle changes which might lead to liver failure after liver transplantation, rat livers stored at 4° C in University of Wisconsin solution for 8, 16, 24, and 32 h were examined by transmission electron microscopy, scanning electron microscopy, cellular matrix maceration and freeze fracture for ultrastructural analysis. Endothelial cells exhibited aggregation of intramembrane particles (IMPs) at 8 h and produced tiny blebs accompanied by marked development of pits. As deterioration advanced, endothelial cells exposed the perisinusoidal faces of hepatocytes directly to the lumen with destruction of sieve plates. They then degraded with loss of IMPs. Macrophages followed a similar deterioration process to endothelial cells. Membranes of hepatocytes did not demonstrate aggregations of IMPs for 32 h. Rough endoplasmic reticulum (rER) lost ribosomes and smooth ER (sER) increased in amount and dilated in an irregular form. Autophagosomes appeared in the cytoplasm, engulfed cytoplasmic matrix containing intracellular organelles and became autophagic vacuoles. At 32 h bile canaliculi were filled with detached vesicles. This may be one of the causes of preservation related bile duct complications after liver transplantation.     

阻塞性黄疸大鼠毛细胆管壁的酶活性变化

The enzymic activities of hepatocytes, especially the bile canaliculi, of rats with experimental obstructive jaundice were studied by using electron microscopic cytochemistry. Common hepatic duct was ligated in rats, and liver tissue was taken from these animals 4 days after the operation for comparison with that of normal rats. The main results of this experiment were as follows: (1) Lumens of bile canaliculi were obviously enlarged. The microvilli became shortened and thickened, or even disappeared. The exoplasm was thickened as well. (2) The activity of Na(+)-K(+)-ATPase, G-6-Pase, Ca(++)-ATPase and NDPase in the wall of bile canaliculi was obviously reduced. (3) The activity of cytochrome oxidase in hepatocytes was also obviously reduced. The significance of the changes in these enzymic activity is discussed.     

Electron microscopic observation of hepatitis B virus budding from hepatocytes into bile canaliculi

In electron microscopic observation of a liver biopsy obtained from a hepatitis B surface antigen-positive patient, noncoated core particles were occasionally seen budding into the hepatocytic cisterni and many Dane particles were found in the pericanalicular vesicles of hepatocytes. Noncoated core particles were also localized in clusters within the bleb of microvilli. There were some core particles being protruded from microvilli into the lumen of bile canaliculi by budding. These findings suggest hepatitis B virus being released from the hepatocyte to the bile canaliculi by two different modes; through vesicle by reversed phagocytosis and from the microvilli by budding.     

Catalase-negative peroxisomes: transient appearance in rat hepatocytes during liver regeneration after partial hepatectomy.

Using light microscopy enzyme cytochemistry to localize catalase activity in peroxisomes, a population of peroxisome-negative hepatocytes was detected in livers of rats during liver regeneration induced by two-thirds partial hepatectomy. However, examination by electron microscopy revealed that this population of hepatocytes contained peroxisomes with a delimiting membrane and a nucleoid, but no cytochemically demonstrable catalase activity within their matrix. Regenerating livers 6, 18, 24, 36, 48 and 72 hours, and 1 week after partial hepatectomy showed hepatocytes without catalase activity. However, their numbers varied, with the most numerous appearing at 24 hours after partial hepatectomy. Mitosis of catalase-negative hepatocytes were seen along with mitosis of hepatocytes containing the normal complement of catalase-positive peroxisomes. The catalase-negative hepatocytes did not show evidence of apoptosis or necrotic cell death. Lysosomal acid phosphatase activity and bile canalicular ATPase activity were present in hepatocytes with catalase-negative peroxisomes. Another population of hepatocytes with a small number of catalase-positive peroxisomes appeared and were more numerous at 36 hours after partial hepatectomy; ultrastructurally, these hepatocytes contained both catalase-negative peroxisomes, which appeared to undergo dissolution, and catalase-positive peroxisomes, which were smaller in size. After complete restoration of the liver, all hepatocytes displayed essentially uniform numbers of catalase-positive peroxisomes. These studies indicated that during liver regeneration there is a transient loss of catalase in peroxisomes of some hepatocytes. These cells proliferate and with time acquire new catalase-positive peroxisomes. The observations are discussed in relation to peroxisome biogenesis, hepatocellular carcinogenesis, and oxidative stress during liver regeneration.     

Phenotypic characterization of hepatic proliferation. Antigenic expression by proliferating epithelial cells in fetal liver, massive hepatic necrosis, and nodular transformation of the liver

Different forms of hepatocellular proliferation are seen in fetal livers, massive hepatic necrosis, and nodular transformation (nodular regenerative hyperplasia) of the liver. In an attempt to characterize the proliferating cells in these conditions, we studied the expression of several antigens by immunohistochemical methods. Alpha-fetoprotein (AFP), alpha 1-antitrypsin (AAT), a hepatocellular export protein, carcinoembryonic antigen (CEA), a marker of bile duct epithelial cells, and hepatitis B virus antigens (HBsAg, HBcAg), were localized by the peroxidase-antiperoxidase method in 11 fetal livers, 10 cases of nodular transformation, and 7 cases of massive hepatic necrosis. AFP was the most prevalent antigen in fetal hepatocytes. Many hyperplastic hepatocytes in nodular transformation contained AAT, but not oncofetal antigens, supporting the differentiated hepatocellular nature of these cells. A similar staining pattern was seen in two-cell-thick plates of hepatocytes in massive hepatic necrosis. In contrast, the ductlike structures at the periphery of necrotic lobules contained both AAT and CEA, suggesting that these cells exhibit features of hepatocytes and bile duct epithelial cells. Therefore, the appropriate term for these regenerating cells appears to be "ductular" or "biliary hepatocytes".     

D-galactosamine hepatotoxicity. 3. Normoactive smooth endoplasmic reticulum and modification by phenobarbital

The administration of a single dose of d-galactosamine-HCL (375 mg/kg ip) to female rats produces severe hepatocellular necrosis. Ultrastructural studies disclose a variety of nuclear and cytoplasmic changes including focal and diffuse hyperplasia of the smooth endoplasmic reticulum. The functional capacity of the hyperplastic smooth endoplasmic reticulum was investigated by several criteria. Pentobarbital sleeping time determined 6, 16, and 24 hr after d-galactosamine injection was similar to that observed in saline-treated control animals. Hepatic microsomal protein concentration, cytochrome P-450 content, and NADPH cytochrome c-reductase activity measured at 20 hr were similar in d-galactosamine-treated and control animals. Thus, using these modalities, the hyperplastic smooth endoplasmic reticulum induced by d-galactosamine was not associated with an altered microsomal drug metabolizing activity.When phenobarbital was administered in a dose of 80 mg/kg for 4 days prior to d-galactosamine, the histological evidence of hepatocellular injury and lipid accumulation was less severe than in animals receiving d-galactosamine alone. The mechanism of the modification of hepatotoxicity and fatty liver by phenobarbital remains to be determined.