Unimpaired induction of drug-metabolizing enzymes in hepatocytes isolated from rats with micronodular cirrhosis

To test further the competence of the cirrhotic liver to metabolize xenobiotics, hepatocytes were isolated from control and CCl4-induced cirrhotic male or female rats. Histologically micronodular cirrhosis was present in all CCl4-treated rats, while control rats had normal livers. Portal perfusion pressure and intrahepatic collagen content were also significantly increased by CCl4 administration. In male rats, no significant differences in levels of circulating transaminases nor in alkaline phosphatase was observed between cirrhotic and control rats, while CCl4-treated females had slightly higher than normal serum transaminase levels at the time of the studies. Hepatocytic cytochrome P-450 and basal xenobiotic biotransformation were unaffected by micronodular cirrhosis in both genders; calculation of the aminopyrine and 7-ethoxycoumarin intrinsic clearances (Cli) revealed, however, a slightly decreased transformation potential in hepatocytes obtained from cirrhotic females, a phenomenon not observed in cirrhotic male rats. It is speculated that the observed reduction in Cli may have been independent of cirrhosis per se, owing to the perduring cytotoxic effect of CCl4 as evidenced by the higher than normal level of transaminases in female rats. Finally, male rats were subjected to in vivo administration of phenobarbital or 3-methylcholanthrene; both compounds led to significant induction of the mixed-function oxidase system, which was similar in magnitude and in selectivity in control and cirrhotic rats as illustrated by calculation of the Michaelis-Menten kinetic parameters for aniline p-hydroxylation, aminopyrine-N-demethylation, 7-ethoxycoumarin-O-deethylation, and p-nitrophenol UDP-glucuronyl transferase. We conclude that in well-established but compensated and hepatolysis-free micronodular cirrhosis, hepatocytes are fully able to transform xenobiotics and to respond normally and selectively to inducers of drug metabolism.     

Electron microscopy autoradiographic investigation of DNA synthesis in a nucleolus

A compensatory increase in the number of the DNA synthezising hepatocytes was induced in mice by the CCl4 injection. Some variants of the distribution of the labeled DNA were detected in the nucleus after a short-term (20-30 min) labeling of the hepatocytes by thymidine-3H. In one of the variants the label was localized almost exclusively in the body of the nucleolus. Some features of the replication of intranucleolar DNA were noted: a rather poor labeling at this time of all the other nucleus structures including nucleolush eterochromatin, synchronism of DNA synthesis in different nucleoli of one nucleus; the decrease in electron density of the body of the nucleolus and a high label concentration above it.     

Effect of "vilon" on cirrhotically changed rat liver. Liver regeneration, and status of glycogen-forming function of hepatocytes

Effects of a dipeptide preparation "Vilon" on rehabilitation of functional activity of hepatocytes and regeneration of the cirrhotically altered rat liver were studied. The liver cirrhosis was produced by poisoning of rats for 4 months with carbon tetrachloride (CCl4). On the end of the poisoning with CCl4, one group of animals was not submitted to any further actions, whereas animals of the other group were injected "Vilon" (1.7 micrograms/kg) daily for 5 days. On smears of isolated hepatocytes, contents of total glycogen (TG), and its labile and stable fractions (LF and SF) were determined in addition to cell ploidy levels and the total protein content. In liver homogenates, activities of glucose-6-phosphatase (G6P), glycogen synthase (GS), and glycogen phosphorylase (GP) were measured. In 2 weeks after the drug application, G6P activity being reduced in cirrhosis 1.2 times, elevated under effect of "Vilon". In non-treated rats the contents of TG and its fractions and of G6P activity remained at the level characteristic of the cirrhotic liver prior to "Vilon" administration. In both groups of rats, GP and GS activities in the cirrhotically altered liver did not differ from their control values throughout the entire experiment. "Vilon" has been shown to exert a weak stimulating effect on regeneration of the cirrotically altered rat liver: in hepatocytes of the second group of rats the total protein content and ploidy levels were higher than those in the first group by 4.7 and 11.5%, respectively.     

RNA synthesis in the circular nucleoli of hepatocytes]

By analysis of serial sections it has been revealed that the so-called ring-like nucleoli of hepatocytes consist of a cavity with an amorphous contents surrounded by fibrillar and granular material. Such nucleoli are sometimes encountered in normal animals; the number of ring-like nucleoli increases considerably in chronic pathological process caused by repeated CCl4 injections. The capacity of RNA synthesis in the ring-like nucleoli was revealed by means of electron-microscopic autoradiography.     

The hepatocytes of the brown trout (<Emphasis Type="Italic">Salmo trutta</Emphasis> f. <Emphasis Type="Italic">fario</Emphasis>): a stereological study of their number and size during the breeding cycle

A firm knowledge of the normal structure is crucial for evaluating pathological processes and morphofunctional correlations. Stereological liver structure characterization had its debut for mammals in the 1960s, but only in the 1980s did it start to be used in fishes. Using stereology, our aim was to verify the hypothesis that in parallel with the well-known annual seasonal changes in the liver–body ratio of brown trout, hepatocytes would vary their number and/or size, and that gender differences likely exist. Three-year-old specimens were used. Five animals per gender were examined in May (endogenous vitellogenesis), September (exogenous vitellogenesis), and February (spawning season end). The liver was fixed by perfusion, and its total volume estimated. Systematically sampled material was embedded in epoxy or in metachrylate resins. Stereology was executed on light and electron microscopy images. Unbiased design-based techniques were applied, using physical disectors and differential point counting. Target parameters were the relative (per unit volume) and total number of hepatocytes, the mean cell and nuclear volumes, and the total volumes of hepatocytes and their nuclei. Data support that in both genders the number of hepatocytes and the volume of its nucleus change along the breeding cycle. The cell number increased from endogenous to exogenous vitellogenesis (accompanying relative liver size gains), later followed by a decline in the cell number, still detectable after the spawning season. The total liver volumes of the cell and nucleus also increased from May to September in females, despite that the mean hepatocyte nuclear volume showed a minimum in September. No statistical changes in the mean cell volume were detected, regardless of the tendency for lower mean values in September. Changes were more marked in females and showed a higher correlation with the gonad weight. It was firstly suggested that numerical (rather than cell size) changes govern the shifts of the relative liver weight seen during the brown trout annual breeding cycle, and eventually of other fishes. We hypothesized that there are seasonal cycles of hepatocyte mitosis (from after spawning to exogenous vitellogenesis) and of apoptosis (at spawning). These cycles would be regulated by sex steroids, being more striking in females.     

Glycogen synthesizing function of hepatocytes in rats with liver cirrhosis after treatment with chorionic gonadotropin]

Using rat liver hepatocytes, methods of cytofluorimetry (Kudryavtseva et al., 1974) and biochemistry were applied to comparative studies of the total glycogen content, including its labile (LF) and stable (SF) fractions, and activities of glucose-6-phosphatase, glycogen phosphorylase and glycogensynthetase in these. The liver hepatocytes were examined in norm, and under conditions of CCl4 poisoning of rats, both 6 months after a chronic poisoning, and 1, 3 and 6 months following poisoning cessation. All the experimentally poisoned rats were divided into two conventional groups: rats of one group received, apart from poisoning, a complex treatment with chorionic gonadotropin (CG); the other group rats received, no treatment. The material used for examination was obtained from serial functional biopsies of each experimental animal. It has been shown that under cirrhosis the content of the total glycogen in hepatocytes increased by 3 times, and that of its SF even by 9.7 times. The treatment with CG for 1 month resulted in its reducing to the norm, and 3 to 6 months treatments normalized contents of both the glycogen fractions. In the group of non-treated rats no similar changes were registered. Besides, in the cirrotic rats the activity of glucose-6-phosphatase was shown to increase by 4 times. After CG treatment it was seen to decrease by 3 times. Thus, CG may be regarded as an optimum and more effective agent for restoring abnormalities in cirrotic liver, compared to some other stimulating factors, such as hepatectomy (Kudryavtseva et al., 1996) or rich-carbohydrate diet (Kudryavtseva et al., 1998).     

Cyclic nucleotides levels in the liver of rats treated with CCl4.

Treatment of rats with two different doses of CCl4 (respectively 2.5 and 0.5 ml/kg body wt. intragastrically) is followed by a rapid increase in the cAMP content of the liver. With 0.5 ml of CCl4, the increase occurs as early as 30 min after poisoning, namely about 4-5 h before the onset of triglyceride accumulation in the liver. The maximum increase has been at 6 h after administration of the hepatotoxin. In both experimental conditions, normal values are recovered only after 36-48 h. cGMP level appears unmodified during the whole observation period. Therefore the ratio cGMP/CAMP decreases consistently. The ATP level decreases significantly between 2 and 12 h. The increase in liver triglycerides level after CCl4 can be also a consequence of an impairment of microtubule function, leading to a decreased release of lipoprotein micelles from hepatocytes into the blood stream.     

Morphometry of hepatocyte mitochondrial apparatus in normal and cirrhotic rat liver

Morphometric electron microscopy study of the hepatocyte mitochondrial apparatus and morphofunctional analysis of the degree of pathological alterations were carried out on the liver of rats with CCL4-cirrhosis (experimental group). Chronic poisoning of rats with CCL4 for 6 months led to a 4.2-fold increase in proportion of connective tissue and to a decrease in the number of hepatocytes in the liver by 21.8 %. Dry mass and ploidy of hepatocytes in the cirrhotis liver rose as compared with norm by 20.6 and 9.3%, respectively. Activities of alanine and aspartate aminotransferases in blood of rats of experimental group exceeded normal ones 2.0 and 1.4 times, respectively. Concentration of total bilirubin in blood of the cirrhotic animals increased 1.7 times, while concentration of total protein decreased by 22%. Concentration of diene conjugates in the liver of rats of experimental group increased 2.1 times as compared with normal one, while the level of malonic dialdehyde - by 34%. Activities of superoxide dismutase and catalase in the cirrhotic liver were lower than in the normal liver were lower than in the normal liver by 16 and 23 %, respectively. Morphometry of the hepatocyte mitochondrial apparatus has shown that in spite of an increase in the voluminous density of mitochondria in hepatocytes of the cirrhotic liver (by 28 %), concentration of internal mitochondrial membranes in the cells was reduced almost 1.5 times, while the total length of internal membrane in a single mitochondrion was reduced about twice as compared with norm. Thus, despite compensation of the partial loss of hepatocytes because of their polyploidization and hypertrophy, the specific synthetic activity of cells in the case of cirrhosis is decreased due to deterioration of the antioxidant system and electron transport chain of the mitochondrial apparatus.     

Synthesis and localization of alpha fetoprotein in liver regeneration in mice]

Typical mature hepatocytes constituting not over several per cent of the total amount of preserved hepatocytes served as the principal site of the alpha-fetoprotein (AFP) localization in the liver of mice regenerating after the CCl4 poisoning or partial hepatectomy. Morphologically they failed to differ from the principal mass of hepatocytes and retained an antigen of the bile capillaries on the surface. A change id to the dynamics of the AFP level in the animal serum. Apparently in regeneration of the mouse liver the principal AFP production was realized by mature hepatocytes.     

Effect of a food ration with a low-protein content on the processes of liver parenchyma recovery in rats after tetrachloromethane damage

Male Wistar rats weighing 190-200 g were fed a low protein diet. Atrophy of cytoplasm was observed after a 3-week malnutrition. In early periods of liver regeneration (6 hr after CCl4 poisoning), an increase in DNA synthesis was accompanied by an increase in summary concentration of organelle membranes. Though their ploidy was higher than in the control, volume of hepatocytes and summary area of organelles surface membrane was smaller than in standard diet. These data show a reduced capacity for realization of genetic program during the regeneration period in protein-deficient rats.     

Dynamics of acid phosphatase activity in the liver in the process of involution of cirrhosis

Changes in the total activity of acid phosphatase in the liver as well as changes in the enzyme activity in hepatocytes and connective tissue cells of fibrosis layers were investigated, using quantitative histochemical method, in the process of mouse cirrhosis involution. After discontinuation of CCl4 injection, the animals with cirrhosis were divided into two groups. In the first group the resection of the left lobe of the liver was performed. The animals of the second group were not subject to operation. The results demonstrate that there is a close correlation between lysosomal hydrolase activity of hepatocytes and connective tissue cells of the liver and collagen resorption during cirrhosis involution. The most intensive lysis of collagen takes place within the first three weeks of cirrhosis involution in both experimental groups. Partial resection in cirrhosis has no significant effect on the changes and level of total activity of lysosomal hydrolase enzymes in the liver during cirrhosis involution.     

beta-Hexosaminidase activity in the acute phase of CCl4 poisoning in the rat

beta-Hexosaminidase (Hex) activity has been shown to be increased in the sera of patients with chronic liver diseases as well as in rats with CCl4-induced liver cirrhosis. In this study, serum and liver Hex activity was determined in rats during the acute phase of CCl4 poisoning, a widely used animal model of acute necrotic liver damage. The results showed a statistically significant decrease of Hex activity in the sera of rats 36 h after CCl4 poisoning (5.84 +/- 2.90 U/l), as compared to controls (11.58 +/- 1.35 U/l; p less than 0.001). No significant change was observed in liver tissue of CCl4-treated animals and controls. A significant correlation between the decrease in Hex and the increase in serum aspartate aminotransferase in serum was found. The results are consistent with the hypothesis that this lysosomal enzyme could be released by non-parenchymal liver cells, such as activated macrophages; its increased activity could be the expression of macrophage activation, as demonstrated in patients with chronic liver diseases.     

Short-term effects of carbon tetrachloride on the lipoprotein secretion in isolated rat hepatocytes

Short-term exposure of isolated rat hepatocytes in suspension to a low dose of CCl4 (20 micrograms/ml) leads within minutes to characteristic structural alterations. The earliest reaction is a disappearance of the microvilli border 5 min after starting the incubation. After 10 min the number of Golgi VLDL is decreased by about 80% and reaches zero after 20 min. The reduction in Golgi VLDL is associated with a decrease in the volume density of the Golgi complexes by about 50% compared with controls and by a marked elevation of intracytoplasmic and intralysosomal lipid deposits after 20 min incubation. Concomitantly with these alterations the total number of VLDL particles within single and multiple particle secretory vesicles located along the cell periphery decreases by about 50% 10 min after CCl4 exposure. This is followed 10 min later by a significant increase of about 20% compared with the corresponding controls. The elevation in the total number of VLDL is combined with an increase in the number of the multiple particle secretory vesicles. The particle content per vesicle, however; is significantly lower compared with controls. No reaction is detectable in the mitochondria, whereas the amount of RER appears to be decreased and that of the SER increased. The incubation of 14C-sodium palmitate prelabeled hepatocytes in the presence of CCl4 leads to a significantly higher content of labeled lipids in the total Golgi fraction and in the cytosol 20 min after CCl4 administration, whereas considerably less labeled lipids are secreted into the incubation medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

不同生殖期鳜肝脏超微结构变化的观察

应用透射电镜对生殖季节与非生殖季节鳜肝脏超微结构的变化进行了观察。鳜肝细胞含有单个卵圆形的核,核仁清楚;细胞质内含有粗面内质网、线粒体、糖原颗粒和脂滴等细胞器和内含物。胆小管由相邻的数个肝细胞质膜凹陷围成,而肝血窦则由内皮细胞的胞质构成。还发现了贮脂细胞、枯否氏细胞和成纤维细胞。胆小管腔和窦周隙内浸润许多由肝细胞发出的微绒毛结构。鳜肝细胞的超微结构在产卵前后呈现明显变化：产卵前的肝细胞内富含线粒体、糖原颗粒和脂滴,粗面内质网发达;而产卵后的肝细胞内核仁发生迁移,部分细胞核囊泡化,糖原颗粒和脂滴排空,少数肝细胞具双核结构。非生殖期多数肝细胞核含有双核仁结构,胞质内溶酶体数量增多。     

Studies on satellite nucleoli in rat hepatocytes and Novikoff hepatoma cells

Summary The present study was undertaken to provide information on the presence and frequency of satellite nucleoli in cells with increased nucleolar biosynthetic activity. The number of hepatocytes containing satellite nucleoli was analyzed in rat liver, regenerating liver 18 h after partial hepatectomy and in Novikoff hepatoma ascites cells. In comparison with hepatocytes of normal liver, the number of both stimulated hepatocytes and malignant hepatoma cells containing satellite nucleoli was significantly reduced. The results also indicated that whereas most satellite nucleoli contain protein C23, a smaller percentage contain protein B23.     

Simultaneous induction of galectin-3 phosphorylated on tyrosine residue, p21(WAF1/Cip1/Sdi1), and the proliferating cell nuclear antigen at a distinctive period of repair of hepatocytes injured by CCl4

The period of repair of hepatocytes injured by CCl4 and signaling proteins intrinsic to this period were examined. A 30 kDa polypeptide detected by immunoblot analysis using anti-phosphotyrosine antibody in livers from rats 48 to 72 h after administration of a single dose of CCl4 was identified as galectin-3 induced in cytoplasm of periportal hepatocytes and phosphorylated on tyrosine residue(s). Simultaneously, these hepatocytes induced p21(WAF1/Cip1/Sdi1) in the nucleus and the proliferating cell nuclear antigen in both the nucleus and the cytoplasm, suggesting that hepatocytes during this distinctive period are quiescent and repair cellular damage. Trabecular architecture of hepatocytes with the proliferating cell nuclear antigen only in the nucleus was found at 96 h. These findings indicate that galectin-3 is a novel member of signaling proteins downstream of tyrosine kinase, and suggest that it plays roles in supporting repair or survival of the injured hepatocytes rather than their proliferation that is likely to be initiated later than 72 h.     

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.     

3-dimensional organization of the nucleolus and the nucleolus-organizing regions in differentiated cells. I. Ring-shaped nucleoli in the endotheliocytes, smooth-muscle cells and fibroblasts in the mouse

The method of ultra-thin serial sections was used to study the three-dimensional structure and to perform the quantitative analysis of ring-shaped nucleoli of kidney and liver endotheliocytes, smooth muscle cells of kidney arterioles and fibroblasts of mice. Spatial models of ring-shaped nucleoli with one fibrillar centre are given. For the quantitative analysis the following parameters were measured: the number and volumes of nucleoli, fibrillar centers, RNP-containing structures, the vacuolar system and the RNP-index (the latter is a ratio of RNP-part and fibrillar center volumes). Nucleoli of the same type of cells, occasionally in the same nucleus, were found to differ sharply in their fibrillar center shape. Differences in the mean volume values of nucleoli, fibrillar centers and the RNP-part between some cell populations are sufficiently well pronounced. Within the same population ring-shaped nucleoli have, as a rule, specific volume values of nucleoli, RNP and fibrillar centers. The comparison of quantitative data obtained on different cell types showed that the mean RNP-index values were the most stable parameter. The structural relation between fibrillar centers, intra- and perinucleolar chromatin and lacunar region is shown. The structural organization of intranucleolar chromatin and rRNA in the nucleolar body and in fibrillar centers is discussed.