[Dynamics of pro- and macroglycogen content in hepatocytes of normal and cirrhotic rat liver at different stages of glycogenesis].

The content and structure of glycogen in hepatocytes of normal and cirrhotic rat liver has been studied at definite time intervals after the administration of glucose to starving animals. In the study, an original cytofluorimetric method for detection and quatification of proglycogen (PG) and macroglycogen (MG) content in isolated hepatocytes was applied. This method is based on using Schiff reagents with different spectral characteristics. It has been determined that the content MG content in the hepatocytes of control rats increases in 10 min after initiation of glycogenesis by 52% (P < 0.01). MG content in the cells of cirrhotic liver increased only after 20 min (43%, P < 0.05) after glucose administration to starving animals. The coefficient of correlation between MG content and the total glycogen content in the hepatocytes at different stages of glycogenesis ranged from 0.90 to 0.99 (P < 0.001) in both groups of rats. Increase in PG content in hepatocytes of control rats appeared within 10-30 and 45-70 min. In the case of cirrhosis PG content increased only 60 min after the start of glycogenesis, but after 120 min it was 1.5 times higher than the control values (P < 0.001). The correlation coefficient between the PG and the total glycogen content in rat liver cells averaged 0.86 (P < 0.001) and 0.77 (P < 0.001) in control and experimental groups, respectively. Thus, the change in total glycogen content in hepatocytes of normal and cirrhotic liver is associated mainly with the level of MG. In normal cells, contribution of PG is most significant in the early glycogenesis (10-30 min), and in the cirrhotic liver--in the later stages.

[Rat heat structural and functional characteristics and gas exchange parameters after experimental myocardial infarction].

Rat heart structural and functional changes and gas exchange parameters were investigated in six months after experimental myocardial infarction. Left ventricular end-systolic and end-diastolic dimensions in rats with chronic heart failure were 78 and 30% higher than in control respectively. Left ventricle cavity volume in systole and diastole were 5 and 2 times increased respectively. Left ventricular cavity stretching was accompanied by thinning of the interventricular septum. Left ventricular structural changes leads to its functional deterioration. Left ventricular contraction fraction was reduced by 60%, and the ejection fraction--by 52% in comparison with control. Gas exchange investigation revealed that in six month after myocardial infarction oxygen consumption of operated rats was increased by 30% and production of carbon dioxide by more than 40%. Respiratory quotient, which reflects the nature of oxidized substrates, in rats with myocardial infarction was amounted to 0.85, indicating significant increase in the contribution of carbohydrates as an energy substrate for myocardial metabolism.

[DNA image-fluorimetry of individual human chromosomes].

Mucrofluorimetric method for the determination of DNA content in individual human chromosomes has been developed. The method is based on a preliminary identification of chromosomes with Hoechst 33258, followed by staining of the chromosomes with Feulgen reaction using Schiffs reagent type ethidium bromide-SO2, then measuring the fluorescence intensity of the chromosomes using an image analyzer. The method allows to determine the DNA content of individual chromosomes with accuracy up to 4.5 fg. DNA content of individual human chromosomes, their p-and q-arms as well as homologous chromosomes were measured using the developed method. It has been shown that the DNA content in the chromosomes of normal human karyotype is unstable. Fluctuations in the DNA content in some chromosomes can vary 35-40 fg.

[Cellular mechanisms of regeneration of rats' liver after experimental myocardial infarction].

Morphological changes and regeneration activity of the rats' liver after an experimental myocardial infarction (MI), caused by a permanent left coronary artery occlusion, were investigated. It has been shown that in 6 months after MI there were considerable changes of the rats' liver circulatory system: the quantity of vessels per unit of area increased by 118%, thickness of their walls by 19%, and the average square of vessels lumens by 159%. The percentage of connective tissue in 6 months after MI increased more than in one and a half time in comparison with control. Inflammatory and necrotic changes in rats' liver remained for 6 months after MI. The liver injury caused by MI led to activation of regeneration processes in its parenchyma. In 6 months after MI, the number of 4c- hepatocytes decreased by 12% in comparison with control, and the number of 4c x 2- and 8c-hepatocytes increased by 45 and 71%, respectively. The mean level of hepatocytes ploidy increased in 6 months after MI by 11%. The dry mass of rats' hepatocytes increased in 6 months after MI by 19% in comparison with control. Thus, liver regeneration after MI is more due to hepatocytes hypertrophy than to their polyploidization.

[Analysis of glycogen structure in rat hepatocytes using cytochemical and FRET methods].

Using cytochemical and FRET (Forster, Resonance Energy Transfer) methods, the glycogen structure in rat hepatocytes was investigated during fasting and at different time intervals after per os glucose administration to animals. Hepatocytes on slides were stained with fluorescent PAS-reaction. Staining the slides with ethidium bromide-SO2 (EtBr-SO2) for 40 min revealed a labile glycogen fraction (LE), and the subsequent staining the same samples with auramine-SO2 (Au-SO2) for 50 min showed a stable glycogen fraction (SF) in the cells. The total glycogen content (LF and SF) in the hepatocytes at different stages of refeeding was determined by means of cytofluorimetry, and then efficiency of FRET was measured in the same cells. Registration of FRET in several areas of the cells was carried out on a laser scanning confocal microscope Leica TCS SP5 with application of FRET AB (Acceptor Photobleaching) procedure. In this procedure, auramine served as a donor (D) and ethidium bromide was an acceptor (A). It was shown that the efficiency of FRET varied from 10 to 14 % during refeeding, while the glycogen structure had a marked influence on the value of this parameter. FRET efficiency was shown to correlate with the ratio A/D in the cells of hungry rats and at the early stages after glucose administration to animals, which reflected the degree of filling of the external tiers of glycogen molecules of glucose residues. At later stages, this correlation was either less pronounced or absent. It was found that the FRET efficiency can vary by 3-4 times at the same value of A/D. Since the probability of energy transfer from D to A is proportional to 1/R6, where R is a distance between D and A, such variations of the FRET efficiency indicate that the glycogen molecules possess a labile structure in which the chain of glucose residues can deviate from its axis by a distance of about half their diameter.

[Regeneration of the liver of Chinese hamster Cricetulus griseus].

Using cytofluorimetry and interferometry, hepatocyte DNA, dry mass and distribution of hepatocyte ploidy classes were measured in hamsters Cricetulus griseus in 1 month after partial hepatoctomy. Ploidy of normal liver hepatocyte was 2.35 +/- 0.03 (mean +/- SD) c. Modal ploidy class was presented by mononuclear hepatocytes with diploid nuclei (82.4 +/- 1.3 %). Hepatocyte dry mass was 605.2 +/- 4.8 pg. One month after partial hepatectomy the distribution of ploidy classes and dry mass of hepatocyte did not change. A similar hepatectomy in mice resulted in significant polyploidization of liver parenchyma: the middle level of hepatocyte ploidy increased by 32% and mononuclear octaploid cells, the number of which increased 5-fold, composed modal ploidy class in place of 4cx2-hepatocytes predominated in control mice. The number of 8cx2-hepatocytes in the liver of mice creased by more than 5-fold. Thus, in contrast with mice, in hamsters Cricetulus griseus an increase in the liver mass followed partial hepatectomy depended completely on hepatocyte proliferation.

[Interrelation between the glycogen content in hepatocytes and their size in normal and cirrhotic rat liver].

The dependence between the size of hepatocytes and glycogen content in them was investigated in normal and cirrhotic liver from fasting rats as well as from rats in 10 and 60 minutes after per os glucose administration. The cytophotometric method used allows glycogen and DNA contents determining in the same cell and its dry mass as well. It has been shown that the dry mass and the glycogen content in hepatocytes from normal and cirrhotic liver are proportional to genes dose. Normal liver hepatocytes of various ploidy classes show distinct correlation between the cell size and the glycogen content. No similar dependence is observed in hepatocytes population from the cirrhotic liver, apparently, because the liver lobule structure is disturbed, and heterogeneity of the hepatocytes microenvironment condition is increased.

[Mechanisms of hepatocyte multinucleation in rats exposed to N-nitrosodimethylamine].

Mechanisms of hepatocyte multinucleation were investigated in rats exposed to N-nitrosodimethylamine (NDMA).Using immunohistochemical reaction to y-tubulin it was established that the number of cells containing three and more centrosomes increased in 48 h after NDMA injection. It was shown that formation of extra-centrosomes in hepatocytes was enhanced by oxidative stress induced by cytochromes P450 superfamily in the course of NDMA metabolism. NDMA administration led to a sharp increase in cytochrome P450 content in the liver, especially in 24 and 48 h (3.3 and 2.8 times respectively) after NDMA injection. Extensive staining of cytoplasm in the centrolobular hepatocytes was revealed by immunohistochemical reaction to cytochrome P450 2E1 in 24 and 48 h after the NDMA injection. Malone dialdehyde (the derivative of lipid peroxidation) was shown to increase 1.1-2.0 times, whereas catalase activity as of the antioxidative agent reduced to 1.1-1.3 times in that time. In 72-120 h after NDMA treatment, the number of cells with three or more centrosomes, the intensity of cytoplasmic staining, cytochrome P450 and malone dialdehyde contents in the liver were shown to decrease, whereas catalase activity increased. In 48 h after treatment, binucleated hepatocytes with various 3H-thymidine distribution in nuclei appeared in NDMA-treated cell populations evidencing of asynchronous DNA synthesis. Immunohistochemical reaction against Ki-67 proliferation marker revealed asynchronous nuclear proliferation activity in binucleated cells spreading not only to S-phase, but also to other phases of cell cycle, and namely G1, G2 and M. Thus, main mechanisms of hepatocyte multinucleation under NDMA exposure are accounted for hyperamplification of centrosomes as a consequence of oxidative stress and for asynchronous DNA synthesis in the nuclei of binucleate hepatocyte followed by asynchronous acytokinetic mitosis.

[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.

[New protective effect of simvastatin in rats with experimental steatohepatitis].

The intragastric introduction of carbon tetrachloride (CCl4) (0.2 ml/kg in 50% oil suspension, twice a week) and ethyl alcohol (5% solution ad libitum as the only available drink) in rats over a period of four weeks results in the development of inflammation, fibrosis, and fatty dystrophy in the liver. Such a fast formation of liver damage is obviously caused by potentiating effect of alcohol in combination with CCl4. Simultaneous injection of simvastatin (1 mg/kg, intragastrically) in rats with ethanol--CCl4 hepatitis decreased fatty dystrophy and produced certain anticytolytic and anticholestatic effects without potentiation of microsomal oxidation system damage by hepatotoxins. In addition, simvastatin shows hypolipidemic activity, which is manifested primarily by a decrease in the general holesterol level in the blood serum.

[Microfluorimetric analysis of dynamics of genomic changes of Chinese hamster fibroblasts CHL V-79 RJK with multidrug resistance].

Results of karyological analysis of cells CHL V-79 RJK selected for resistance to ethidium bromide (EB) causing multidrug resistance (MDR) (line Vebr-5) were compared with the data of microfluorimetric determination of DNA content in individual chromosomes of the karyotype. The analysis was performed at the 11th and 88th passages. Karyotyping of Vebr-5 has shown the presence of an additional genetic material (ADM) in the form of homogenously or differentially stained regions (HSRs and DSRs, respectively) in two chromosomes (Z1 and Z6, loci 1 p29-31 and 1q26, respectively). HSRs in Z6, in the region of localization of the wild type of gene mdr, had unstable length and structure characteristic of morphological markers of amplification of genes of the family mdr. During long cultivation of Vebr-5 in the presence of EB (88 passages), the instability of HSRs in Z6 increased. Results of microfluorimetric analysis of Vebr-5 at the 11th passage have shown an increase in the DNA content not only in chromosomes Z1 and Z6 marked by HSRs, but also in three chromosomes (Z5, Z12 and Z13) that have no visual morphological changes. The corresponding analysis at the 88th passage has also revealed non-random changes in the DNA content in four more chromosomes: an increase in Z14, while a decrease in chromosomes 8, Z7, and Z9. A decrease of the DNA content in chromosomes is considered to be a result of a partial loss of genetic material, while its increase is a result of its translocation and (or) amplification. Coefficient of variation of the DNA content changes for large chromosomes amounted to about 9%. while for small chromosomes it is about 26%, which indicates that small chromosomes have greater potential for instability than the large ones. The data obtained not only confirm, but also enlarge the concept of directions and character of destabilization of the cell genetic apparatus in the process of neoplastic transformation due to the MDR acquisition by cells.

[Cellular mechanisms of cirrhotic rat liver regeneration. II. Proliferation, polyploidization and hypertrophy after partial hepatectomy].

Using cytofluorimetry and absorptional cytophotometry, hepatocyte DNA and total protein contents were measured in intact and cirrhotic rats in 1, 3 and 6 months after partial hepatectomy (PH). It has been found that within one month of intact rat liver regeneration the level of hepatocyte ploidy rised by 25% to remain elevated for the next 6 months. This was due mainly to reducing the number of cells with diploid nuclei (2c 2-fold, 2c x 2 - 6.6-fold) and to rising the number of octaploid hepatocytes. In cirrhotic animals the ploidy level in hepatocytes increased in 3 months after PH, and decreased by 15% in 6 months. The number of hepatocytes with diploid nuclei (2c and 2c x 2) increased within 3-6 months in both control and cirrhotic rats. The protein content per diploid hepatocyte rised by 30% within 3-6 months of liver regeneration after PH. Special calculations have shown that within 3 months after PH the increase in the liver mass of control and cirrhotic rats was due completely to hepatocyte DNA synthesis, i. e. proliferation and polyploidization. Within the next 3 months of liver regeneration after PH, the contribution of polyploidization to liver mass increase was negative because of depolyploidization of liver parenchyma cell population. At this time hypertrophy was the main process determining the liver mass increase.

[Kinetic features of a white rat hepatocyte population under hormonal disbalance before and after partial hepatectomy].

Bilateral adrenalectomy, followed in 4 days by a partial hepatectomy, was performed using white rats weighing as much as 120-140 g. Under hormonal disbalance caused by bilateral adrenalectomy, the number of polyploid (4c, 4c x 2, and 8c) hepatocytes significantly increased, compared to that in non-operated control rats. Six hours after a partial hepatectomy, the share of highly ploid hepatocytes falls, being accompanied by a 9-fold increase in mitotic index. It is supposed that under hormonal disbalance condition, a partial hepatectomy may induce "early" mitoses in hepatocytes blocked in G2-phase of the cell cycle.

[The influence of hepatoprotector 2-ethylthiobenzimidazole hydrobromide (bemithyl) on the content of glycogen in cirrhotic rat liver hepatocytes located in various microenvironments]. / Vliianie gepatoprotektora 2-étiltiobenzimidazola gidrobromida (bemitila) na soderzhaniie glikogena v gepatotsitakh tsirroticheski izmenennii pecheni, nakhodiashchikhsia v razlichnykh usloviiakh mikrookruzheniia.

Using absorption and fluorescent cytophotometry methods, glycogen contents were studied in hepatocytes located in liver lobules and in hepatocytes, which make the general population of these cells in normal and cirrhotic rat liver. In cirrhosis, the content of glycogen in hepatocytes located in lobules obviously rises in comparison with the norm, but to a lesser degree, than in hepatocytes making the general population of these cells in cirrhotic liver. The content of glycogen in hepatocytes, located in lobules of pathologically changed liver in bemithyl treated rats, did not differ from the norm. At the same time, the glycogen content in hepatocytes, representing the general population of these cells in cirrhotically altered bemithyl injected rat liver, remained higher than in the norm. The data obtained indicate that distinctions in particular cell microinvironment, obviously present in cirrhotic liver, render essential influence on hepatocyte functional activity.

[Liver pathology in patients with primary hemochromatosis--homozygous carriers of C282Y mutation ]. / Patologiia pecheni u bol'nykh pervichnym gemokhromatozom--gomozigotnykh nositelei C282y-mutatsii.

Liver pathology was studied in 3 patients with primary chemochromatosis. In two cases so-called iron free foci with signs of hepatocytes with feature of dysplasia were found. Many siderosomes were found ultrastructurally in the cytoplasma of hepatocytes. Histological markers of virus infection were absent in a patient with positive serum HbsAg and HCV-Ab. Alcohol did not produce typical histological changes. In this case grave liver reticuloendothelial hemosiderosis typical for secondary hemochromatosis and overloading with iron of spleen pulp according to MR imaging were observed.

[Dynamics of glycogen content in the normal and cirrhotic liver following glucose administration to starving rats]. / Dinamika soderzhaniia glikogena v normal'noi i tsirroticheskaia izmenennoi pecheni posle vvedeniia gliukozy golodnym krysam.

Using biochemical, cytofluorimetric and television cytophotometric methods, glycogen contents were studied in normal and cirrhotic rat liver at various intervals after glucose administration to fasting animals. The obtained data indicate that after a 48 h fasting glycogen contents in normal and cirrhotic liver are equally poor. A marked rise of glycogen content in cirrhotic liver was observed only 20-30 min after glucose administration to rats. It has been established that at all intervals after glucose administration to rats hepatocytes of the portal lobule zone, both in normal and in cirrhotic liver, accumulate more glycogen than those of the central zone. Again, the intensity of glycogen accumulation in cirrhotically altered liver is significantly lower than in normal liver, due, presumably, to a lower rate of glycogen synthesis in pathologically changed liver.

[Features of the effect of bemethyl on glycogen metabolism in hepatocytes of pathological changed human liver]. / Osobennosti vliianiia bemitila na metabolizm glikogena v gepatotsitakh patologicheski izmenennoi pecheni cheloveka.

Effect of actoprotector bemithyl (2-ethylthiobenzimidazole hydrobromide) on glycogen metabolism in hepatocytes of patients with chronic hepatitis and liver cirrhosis was investigated. Using cytofluorimetric method, the content of glycogen and its fractions in isolated hepatocytes was measured. The treatment with bemithyl resulted in a decrease in glycogen levels in hepatocytes, and in a marked restoration of fractional glycogen composition as compared to the basic therapy. Besides, it was established that the degree of glycogen decrease in cells of patients with chronic hepatitis depended on the increase of glucose-6-phosphatase activity (r = 0.75, P < 0.05), and on the levels of glycogen in hepatocytes prior to bemitil treatment (r = = 0.87, P < 0.01). Positive changes in glycogen metabolism after bemithyl treatment are pronounced in patients with chronic hepatitis. These positive alterations take place simultaneously with the conservation of basic structural disturbances in the liver parenchyma. However, even in this case, the indices of glycogen metabolism do not reach the normal levels.

[Effect of bemythyl on carbohydrate metabolism in cirrhotic rat liver]. / Issledovanie vliianiia bemitila na uglevodnyi obmen tsirroticheski izmenennoi pecheni krys.

Effect of actoprotector bemitil (2-ethylthiobenzimidazole hydrobromide) on glycogen content and activities of glycogen synthase, glycogen phosphorylase, and glucose-6-phosphatase was studied in cirrhotically altered rat liver. The contents of glycogen and its fraction were determined a cytofluorimetrically (Kudryavtseva et al., 1974). In cirrhosis, the total glycogen content in hepatocytes increases by nearly 3 times, while the amount of a stable fraction of glycogen rises by 7.5 times. Glucose-6-phosphatase activity fell to the level of 25% compare to the norm. Activities of glycogen synthase and glycogen phosphorylase in the cirrhotic liver did not differ from the norm. In cirrhotically altered liver, bemitil produced a decrease in the total glycogen content due to a decrease in glycogen synthase activity in an increase in glucose-6-phosphatase and glycogen phosphorylase activities. The above results suggest a favorable effect of bemitil on cirrhotic liver.

[Effect of partial hepatectomy on the glycogen level in hepatocytes in the portal and central lobule zones in cirrhotically-changed rat liver]. / Vliianie chastichnoi gepatéktomii na urovni glikogena v gepatotsitakh portal'noi i tsentral'noi zon dol'ki tsirroticheski izmenennoi pecheni krys.

Using cytophotometric method, the content of glycogen was studied in hepatocytes of the portal and central zones of a liver lobule in norm, in cirrhosis, and 1, 3, and 6 months after a partial hepatectomy of the normal and cirrhotic rat liver. As we showed earlier, glycogen content in cirrhotic liver hepatocytes rose 2-3-fold, along with obvious impairment of glycogen metabolic heterogeneity in these. In cirrhotic liver glycogen dominates in the central zone, whereas in norm more glycogen is observed in the portal one. The objective of this study was to find out to what degree a partial hepatectomy of cirrhotic liver may promote recovery of the metabolic glycogen heterogeneity in hepatocytes. Glycogen was determined in hepatocytes, using a quantitative variant of PAS-reaction on sections of the material obtained from serial supravital punctate liver biopsies. Glycogen amount in hepatocytes of different liver lobule zones was determined by an image analyzer technique that allows to bring together the cytophotometric analysis of the substance with its localization in a particular liver lobule. Results of these studies have shown that a partial hepatectomy of cirrhotic liver promotes restoration of the hepatocyte metabolic heterogeneity in the liver lobule.

[Effect of "vilon" on cirrhotically changed rat liver. Liver regeneration, and status of glycogen-forming function of hepatocytes]. / Vliianie preparata "vilon" na tsirroticheski izmenennuiu pecheni krys. Regeneratsiia pecheni i sostoianie glikogenoobrazovatel'noi funktsii gepatotsitov.

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.