Effect of Pb, Cu and Zn on development and Wnt/ß-catenin signaling pathway genes expression of Ctenopharyngodon idella.

Pollution of the aquatic environment with heavy metals is a serious environmental problem, since they accumulate in aquatic organisms and can affect their development and worsen their condition. According to the scheme of Fig. 1 zinc (Zn), copper (Cu) or lead (Pb) were studied when exposed to concentrations of: Zn (0.01; 0.1; 1 mg/L), Cu (0.001; 0.01; 0.1 mg/L), Pb (0.006; 0.06; 0.6 mg/L) for 144 h after fertilization (hpf) on the grass carp (Ctenopharyngodon idella), one of the important commercial fish species of Kazakhstan, the activity of superoxide dismutase (SOD) and the expression of genes of the Wnt/ß-catenin signaling pathway involved in development. All metals significantly reduced survival, hatching rate, and changed biometric parameters and heart rate of cupid larvae. In addition, these metals (mainly Pb and Cu) inhibited superoxide dismutase (SOD) activity and mRNA transcription of genes encoding genes of the Wnt/ß-catenin signaling pathway. These results showed that Pb, Cu and Zn not only affect the survival and development of fish at an early stage of life, but also cause oxidative stress and prevent fish detoxification.

[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.

[Mutagenic effect of the rocket fuel component asymmetric dimethylhydrazine on rats of various ages].

Mutagenic effect of asymmetric dimethylhydrazine (ADMH) on rats of different age groups upon acute and subacute treatment and protective effect of a Limonium gmelinii preparation. Genotoxic effect of ADMH depending on the dose and duration of treatment was established. The phytopreparation lacked mutagenicity and toxicity and had a protective effect in combination with the xenobiotic.

[Cellular mechanisms of postnatal growth of rat liver in during chronic exposure of cadmium sulfate and strontium chloride]. / Kletochnye mekhanizmy postnatal'nogo rosta pecheni krys pri khronicheskom vozdeistvii sul'fata kadmiia i khlorida strontsiia.

A cytophotometric investigation was performed to study the ploidy level and total protein content in hepatocytes of rats of different ages (1, 7, 14, 21, 30, 90, 180, 365 days), both intact and chronically treated with cadmium sulfate or strontium chloride. It was established that during the first month of postnatal ontogenesis, compositions of liver parenchyma cell population of intact and treated rats did not differ. Compared to control animals, the process of cell polyploidization in the liver of rats treated with heavy metal salts of 30-90 days proceeded slower, especially in Cd(2+)-treated rats. Within 180-365 days the cell polyploidization in the treated animals increased. The proportion of (4c x 2)-hepatocytes in 1 year old Cd(2+)- or Sr(2+)-treated rats increased, resp., by 2.7 and 1.5 times, and that of 8c hepatocytes was higher by 3.9 and 1.5 times than in the control, the average ploidy level rising by 20 and 5%. respectively. It was established that until 90 days the rate of protein accumulation in liver cells of intoxicated rats was slower than in intact animals. Thus, the average protein content per diploid hepatocyte in Cd(2+)- or Sr(2+)-treated 30 day old rats was lower by 20 and 16%, respectively, compared to control animals. The protein content increased in liver cells of Cd(2+)- or Sr(2+)-intoxicated rats following 90 and 180 days, respectively, and this process was exclusively associated with cell polyploidization. During the first 3 weeks after birth, no significant difference was observed in the extent of involvement of cell proliferation, polyploidization and hypertrophy in the growth of liver in intact and intoxicated animals. At this period the liver was growing due completely to cell proliferation and hypertrophy. During 21-30 days the contribution of cell proliferation to the liver growth of intact rats was not significant (29%), whereas it remained at higher level (50%) in the treated animals. In 30-90 days after birth, the involvement of proliferation process to the liver growth of intoxicated rats decreased to 25-28%, while in intact animals it increased up to 37%. At this period the cell polyploidization plays an essential role in the growth of liver in both intact and intoxicated animals to reach in average 37-46%. The contribution of polyploidization and hypertrophy to the liver growth of Cd(2+)-treated rats within 30-90 days was obviously higher than in Sr(2+)-treated animals. Both at the late (3-12 months) and at the early (1-21 days) stages of experiments, the pattern of correlation of different cell components in the growing liver of intact and intoxicated rats differed only a little.

[Effect of cadmium sulfate and strontium chloride on the glycogen content in hepatocytes of rats of various ages]. / Vliianie sul'fata kadmiia i khlorida strontsiia na soderzhanie glikogena v gepatotsitakh krys raznogo vozrasta.

Cytophotometry and image analysis being used, hepatocyte glycogen contents were measured in periportal and pericentral zones of liver lobules at different stages (1, 7, 14, 21, 30, 90, 180 and 365 days) of postnatal development of both intact rats and rats exposed to chronic CdSO4 (1 mg/kg body weight) and SrSO4 (6.5 mg/kg body weight) intoxication. The glycogen content in hepatocytes of intact rats increased continuously in the course of development being most highest at the initial stage of development. The glycogen content ratio in cells of portal and central zones of liver lobules varied during ontogenesis. The maximum value of this ratio is reached on the 21st day after the rat birth, dropping sharply at later age to reach its minimum in adults. Intoxication of rats by Cd2+ and Sr2+ within 1-90 days interval reduced hepatocyte glycogen levels, compared to normal liver. The prolongation of rat treatment with heavy metals for 90-365 days led to glycogen accumulation in hepatocytes. Rat intoxication with heavy metals for 1 year brought about the increase in both glycogen content per cell and glycogen concentration. Cd2+ treatment for 30-90 days resulted in glycogen accumulation inhibition in both the investigated zones of liver lobules. Thereafter an increased glycogen accumulation took place in hepatocytes of the portal and central liver lobules. Following Cd2+ treatment, the value of the ratios of glycogen levels in the portal and central liver lobules was lower than in the normal liver on all stages of the postnatal rat development. The lowest value (< 1.0) of this ratio was reached in the cirrhotic liver. Distinct from Cd2+ treatment of rats, the treatment with Sr2+ does not lead to significant changes in glycogen levels in cells of different zones of liver lobules. Nevertheless certain destructive changes in glycogen-forming function of hepatocytes after Sr2+ treatment are apparent. This is suggested from the lower glycogen levels in the portal and central zones of liver lobules on 30-180 days interval compared to the normal liver. Besides, the values of ratios in glycogen levels in the portal and central zones of liver lobules in 14 and 21 days old rats was noticeably lower than in the intact rats of the same age.

[An assessment of the relative contribution of the processes of cellular proliferation, polyploidization and hypertrophy to the increase in liver mass at various stages in the postnatal development of rats]. / Otsenka otnositel'nogo vklada protsessov proliferatsii, poliploidizatsii i gipertrofii kletok v uvelichenie massy pecheni na raznykh stadiiakh postnatal'nogo razvitiia krys.

Changes in isolated hepatocyte dry mass, its ploidy and liver mass at different stages of the rat postnatal ontogeny were investigated. The determination of these processes and special calculation made it possible to estimate quantitatively a relative contribution of cell proliferation, polyploidization and hypertrophy, not associated with DNA synthesis to the increase in the liver mass at different stages of the rat development. During the first week after the rat's birth, its liver growth is provided by 61 and 39% with hepatocyte proliferation and hypertrophy, respectively. Between the 14th and the 21st days of development, when considerable functional changes occur in the rat liver, the contributions of proliferative and polyploidization processes, and of cell hypertrophy into the liver mass increasing are roughly identical. Later on, the contribution of cell hypertrophy into the liver growth is noticeably reducing to reach within 1-2 months only 1%. On this developmental stage the liver mass increment by 2/3 is provided due to cell proliferation and by 1/3--to its polyploidization. As a whole, the accelerated growth of the rat liver from the birth to sex puberty is described as follows: the contribution of processes of proliferation and polyploidization, and of cell hypertrophy correspond to 28, 30 and 42%, respectively; afterwards, the liver growth being retarded. Within the period from 2 to 6 months, the liver mass increase is provided mainly (up to 76%) by cell proliferation, the shares of polyploidization and cell hypertrophy being 8 and 16%, respectively.

[Protein content in rat hepatocytes of varying degrees of ploidy during the period of postnatal development]. / Soderzhanie belka v gepatotsitakh raznoi stepeni ploidnosti u krys v postnatal'nyi period razvitiia.

Protein contents in individual mononucleate and binucleate hepatocytes of different ploidy have been measured by a method that involves the following procedures: cell staining with naphthol yellow, protein content measurement, the Feulgen reaction (the fluorescent variant), and DNA content measurement in the same cells. During the postnatal development, protein contents in hepatocytes of different ploidy was shown to be unchanged up to the 14th day, then, within the next 2 months it increased by 50-60% to remain at the same level. At all the stages of development, regardless of the variation of protein contents in the liver of individual animals, the ratio of protein contents in diploid binucleate hepatocytes to that in diploid mononucleate cells makes 2.0.

[Glycogen content in the DNA-synthesizing and nonsynthesizing hepatocytes of rats of different ages]. / Soderzhanie glikogena v sinteziruiushchikh i ne sinteziruiushchikh DNK gepatotsitakh krys raznogo vozrasta.

The glycogen content in DNA synthesizing and non-synthesizing hepatocytes has been conducted cytochemically during the rat postnatal development. Within all the periods of investigation, the average glycogen content in DNA synthesizing hepatocytes (phase S) are shown to be lower than that in hepatocytes being in phases G0 and G1. The absolute value of reduction of the glycogen content with diploid mononucleate hepatocytes in S-phase is almost the same for rats of all the age groups examined. The absolute value of reduction of the glycogen content in diploid binucleate hapatocytes in S-phase is twice as much as that in diploid mononucleate hepatocytes being at the same stage of the cell cycle. Regardless of the postnatal development stage and of the total glycogen content in the liver, the glycogen content in DNA non-synthesizing hepatocytes of different ploidy is in conformity with the genome number.

[Glycogen concentration in DNA-synthesizing and non-DNA-synthesizing hepatocytes of week-old rats]. / Soderzhanie glikogena v sinteziruiushchikh i ne sinteziruiushchikh DNK gepatotsitakh nedel'nykh krysiat.

Using the combined cytochemistry, that allows to determine glycogen and DNA contents, and to localize 3H-thymidine label in one and the same cell, glycogen content was measured in both 3H-TdR-marked and 3H-TdR-non-marked hepatocytes of week-old rats. The vast majority of hepatocytes in a liver population of such rats are mononuclear 3H-TdR-non-marked cells with a 2C DNA content, which corresponds to the diploid chromosome set. The ratio of binuclear hepatocytes with diploid nuclei is in average 0.6%. At one injection of 3H-TdR, 6.6% of hepatocytes are marked in average which are almost exclusively mononuclear cells with 2c to 4c DNA content. In binuclear hepatocytes with two diploid nuclei (2c X 2), the label was seen only in single instances. The average glycogen content in S-phase hepatocytes is aproximately one third of that in hepatocytes that did not start DNA synthesis (G1 and G2 phases). The glycogen content in hepatocytes is seen reducing during S-phase. The glycogen content in G2-phase hepatocytes does not differ from that of hepatocytes before they start DNA synthesis. The availability of glycogen is not necessary for the beginning of S-phase: DNA synthesis in hepatocytes of week-old rats can start and proceed irrespective of the presence of glycogen in these. The glycogen content in binuclear hepatocytes with diploid nuclei that did not start DNA-synthesis is twice as much as in mononuclear hepatocytes before they started DNA synthesis.

[Cytofluorimetric study of the content of glycogen and its fractions in rat liver cells in the course of the 1st week of postnatal ontogeny]. / Tsitofluorimetricheskoe issledovanie soderzhanie glikogena i ego fraktsii v kletkakh pecheni krys v techenie 1-i ned postnatal'nogo ontogeneza.

A cytofluorometric study of the total glycogen and its fractions in rat liver cells using the fluorescent PAS reaction was made during 1--7 days of the postnatal development. It was established that glycogen content was small on the first two days of development. The glycogen content increases only on the third day after birth. The glycogen of the rat liver cells during a first week of the postnatal development is different from that detected in adult liver cells in two aspects: in 3 day old hepatocytes soluble and stable glycogen fractions are equal, while in adult rat liver cells the former makes 80--90%; during the first week of the postnatal development, the stable fraction of rat liver cell is more labile, while in the adult rat liver the soluble fraction of glycogen is more labiles.

[Cytofluorimetric study of the glycogen content in hepatocytes of varying ploidy in adult rats]. / Tsitofluorimetricheskoe issledovanie soderzhaniia glikogena v gepatotsitakh razlichnoi ploidnosti u vzroslykh krys.

A combined method, that allows measuring glycogen and DNA contents in one of the same cell, was applied for quantitative determination of these in mono- and binucleate hepatocytes with different ploidy obtained from adult rats. The mean glycogen content was shown to increase proportionally to the genome number within the changes of the hepatocyte ploidy from 2 to 8c.

[Method of determining the concentration of glycogen, DNA, 3H-thymidine label and dry weight in one and the same cell]. / Metod opredeleniia v odnoi i toi zhe kletke soderzhaniia glikogena, DNK, 3H-timidinovoi metki i sukhogo vesa.

A method is proposed for determination of glycogen, DNA, 3H-thymidine incorporation and dry weight in the same cell, the technique being based on successive discovery and measuring of each of these indices. Cells are obtained from animals, previously injected with 3H-thymidine, to be charted on preparation, made pictures and measured in square units. Then on preparations embedded into glycerine or vaseline oil, the optical path difference of rays for the nucleus and cytoplasm of selected cells is measured with the interferencial microscope. This is followed by the fluorescent PAS reaction and the content of glycogen is registered microfluorimetrically in the same cells. Preparations after that are treated with a freshly prepared water solution of 0.025% borohydride sodium, stained with the routine or fluorescent Feulgen reaction, and DNA content is determined in the same cells in which glycogen and delta delta were previously measured. The stained nuclei are photographed, their areas are measured and the dry weight of the nucleus and cytoplasm of marked cells is calculated from the values of the nuclear areas and of delta delta. Eventually the preparations are covered by emulsion and exposed, and 3H-thymidine-containing nuclei are determined, the index of marked nuclei and the marking intensity over the nucleus are calculated. As a result, a precise and reliable determination of glycogen, DNA, dry weight and 3H- or 14C-thymidine incorporation is made in either of the marked cell.

[Conditions for preserving glycogen in smears of isolated cells. II. Cytofluorimetric study of the effect of fixation on the glycogen content of cells]. / Issledovanie uslovii sokhraneniia glikogena na mazkakh izolirovannykh kletok. II. Tsitofluorimetricheskoe issledovanie vliianiia fiksatsii na soderzhanie glikogena v kletkakh.

The influence of fixation on the intensity and specificity of the fluorescence of PAS-reaction (F-PAS) in the rat's liver cells was examined. 1.4 types of fixatives, routinely used in polysacchride cyto- and histochemistry, were tried: 100% metanol, 100% and 80% ethanol, acetone, 10% neutral formalin, buffered neutral formalin, mixtures of various fluids: ethanol : acetone (1 : 1), ethanol : formalin (9 : 1), ethanol : acetic acid (3 : 1), formaline : ethanol : acetic acid (1.0 : 8.5 : 0.5), fixatives of Carnoy, Bouin, Rossman and Shabadash. The cell fluorescence intensity after F-PAS reaction with Schiff's reagent auramine--SO2 and the cell autofluorescence were measured cytofluorometrically. It was shown that all the fixatives, besides Bouin's and Shabadash's fluids provide rather good preservation of the cell glycogen. Results obtained from the cytofluorometry of F-PAS reaction, autofluorescence and from the morphological studies of F-Pas stained cells, suggested that the best fixatives were 100% metanol and 100% ethanol.

[Conditions for glycogen retention in smears of isolated cells. I. Cytofluorimetric analysis of glycogen content in cells obtained by different methods of isolation]. / Issledovanie uslovii sokhraneniia glikogena na mazkakh izolirovannykh kletok. I. Tsitofluorimetricheskii analiz soderzhaniia glikogena v kletkakh, poluchennykh razlichnymi metodami.

A cytofluorimetrical study was made of glycogen content on smears of isolated rat liver cells, obtained by perfusion of different solutions: 1/15 M phosphate buffer, the Locke solution, only the Locke solution plus sodium citrate, a calcium-free Locke solution, 0.25 M sucrose, physiological saline, Versene. No loss of glycogen occurred during any perfusion procedure, however, it took place during the smear preparation. The main cell injury is observed due to mechanical factors. An additional treatment of liver, after perfusion, by homogenization and centrigugation at mild conditions decreases the cell glycogen content by 30%. The least cell injury and the best glycogen retention was achieved when phosphate buffers were employed for cell isolation.

[A microfluorimetric study of glycogen fractions in rat liver under different conditions of liver perfusion and animal nutrition]. / Mikrofluorimetricheskoe issledovanie fraktsii glikogena v kletkakh pecheni krya pri razlichnykh usloviiakh perfuzii pecheni i pitaniia zhivotnykh

A study was made of the amount of a labile and a stable glycogen fractions in the rat liver cells under various feeding regimes and different durations of liver perfusion. The amount of the glycogen fraction revealed after a 40 minutes' treatment in a Schiff type reagent--Auramine--SO2 was found most chaneable at hunger, at feeding with carbohydrate rich food and at liver perfusion. This fraction is removed from the cells after the treatment with cold trichloroacetic acid (TCA-fraction). The amount of the glycogen fraction revealed after a more prolonged treatment of cells (90 minutes) in Auramine--SO2 and extracted only with hot KOH (KOH fraction), is relatively stable. According to the cytochemical evidence, the TCA and KOH fraction contents in the rat liver cells reach 80--85 and 15--20%, resp. The cytochemical evidence provided obtained with the fluorescence PAS-reaction permits to consider as identic the glycogen fractions revealed with biochemical methods.