Derepression of hepatocyte proliferation with changes in the functional state of the reticuloendothelial system

Experiments on rats and mice were performed to study the effects of different substances modifying RES functions on hepatocyte proliferation. It was shown that as early as 24 hours after Kupffer cell (KC) overloading with colloidal iron particles the number of hepatocytes in mitosis increased. The mitotic rate increased by 32 h and decreased between 48 and 72 h following iron injection. Forty-eight h after injection of latex particles the hepatocyte mitotic peak could be identified. Twenty-four and 48 h after zymozan injection DNA synthesis in sinusoidal liver cells correspondingly increased. Hepatocytes in mitosis appeared 5 days later, reaching the peak value after 9 days followed by a decrease 12 days after zymozan injection. The depression of the hepatocyte mitotic rate was also observed 9 days after BCG and 15 days after prodigiozan injection. The data are suggestive of the importance of KC as potential inducers of hepatocyte proliferation.     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on stimulated DNA synthesis in the liver and kidney of the rat

The administration of the toxic agent 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) to rats at 0, 24 or 72 h before 70% partial hepatectomy had no significant effect on DNA synthesis, measured by [³H]thymidine incorporation 24 h after the operation, in the remaining liver lobes. However, dioxin depressed DNA synthesis in the folate-stimulated rat kidney when given from 0–9 days before the folic acid. A similar effect was observed using inorganic lead to stimulate kidney mitosis. This effect does not appear to be due to direct interaction of dioxin with DNA, inhibition of the protein synthesis required for DNA synthesis, alteration of the time course of events in the mitotic cycle of the kidney or the depression of food intake of the rats caused by dosage with dioxin. Experiments with methylcholanthrene suggest that levels of drug-metabolising enzymes in the liver or kidney might possibly have an indirect effect on folate-stimulated kidney mitosis.     

DNA synthesis and mitotic activity in cells of regenerating rat liver following x-irradiation in stages GO and Gl]

Effect of X-rays on DNA synthesis and mitotic activity of regenerating liver cells has been studied. The irradiation was performed at a dose of 630 rad before hepatectomy and 2.5 and 6 hours after the stimulation of liver. With the stimulated liver being irradiated, the number of cells synthetizing DNA and entering into mitosis was seen reduced almost twice, whereas DNA synthesis and entering into mitosis were delayed, resp., by 4 and 6 hours. Irradiation of liver before the stimulation brings about a delay in DNA synthesis and in start of mitosis by 2 and 4 hours, resp., without reducing the numbers of cells capable to synthesize DNA and to enter mitosis.     

Effect of prolactin on DNA methylation in the liver and kidney of rat

Prolactin is an important growth modulatory hormone in fetal and adult tissues. It stimulates DNA synthesis and enzymatic markers of the G1 phase of cell cycle in rat liver and other tissues. In this study the effects of prolactin on 5-methyl cytosine content in liver and kidney of rats was studied using HPLC. Prolactin treatment caused hypomethylation of DNA in the liver and kidney of immature rats at 48 h after treatment and the effect remained even at 72 h. Prolactin also caused hypomethylation of DNA in the kidney and liver of adult rats at 48 h after treatment. These results indicate that prolactin probably regulates DNA methylation in the liver and kidney of immature and adult rats.     

Chronobiology of the proliferative events related to angiogenesis in mice liver regeneration after partial hepatectomy

In liver regeneration the formation of new capillary blood vessels is a fundamental requirement for cellular proliferation. Vascular endothelial growth factor (VEGF) is involved in the events of angiogenesis, the mRNA of which is expressed in both hepatocytes and non-parenchymal cells. In this experimental design we try to establish if during liver regeneration in mouse, the expression of VEGF is produced before or after the hepatocytes proliferation. C3H/S adult male mice were divided in three groups in order to study: VEGF expression; S-phase index (SI); and mitotic activity (MA) of hepatocytes. The results that were analyzed by ANOVA, show that VEGF expression starts to increase 26 h after PH with a peak at 28 h. Furthermore, the DNA synthesis (DNAs) reaches maximal level 42 h after pH, meanwhile the MA of the hepatocytes shows an increase 8h after the DNAs peak. In conclusion, it could be argued that the chronobiology of the events related to liver regeneration in mice started with a release of VEGF by the hepatocytes, followed by its DNAs and mitosis.     

Dynamic pathways of selenium metabolism and excretion in mice under different selenium nutritional statuses

The selenoprotein, cellular glutathione peroxidase (cGPx), has an important role in protecting organisms from oxidative damage through reducing levels of harmful peroxides. The liver and kidney in particular, have important roles in selenium (Se) metabolism and Se is excreted predominantly in urine and feces. In order to characterize the dynamics of these pathways we have measured the time-dependent changes in the quantities of hepatic, renal, urinary, and fecal Se species in mice fed Se-adequate and Se-deficient diets after injection of (82)Se-enriched selenite. Exogenous (82)Se was transformed to cGPx in both the liver and kidney within 1 h after injection and the synthesis of cGPx decreased 1 to 6 h and continued at a constant level from 6 to 72 h after injection. The total amount of Se associated with cGPx in mice fed Se-deficient diets was found to be less than in mice fed Se-adequate diets. This finding indicated that cGPx synthesis was suppressed under Se-deficient conditions and did not recover with selenite injection. Excess Se was associated with selenosugar in liver and transported to the kidney within 1 h after injection, and then excreted in urine and feces within 6 h after injection. Any excess amount of Se was excreted mainly as a selenosugar in urine.     

DNA synthesis and mitotic activity in short-term cultures of hepatocytes isolated from regenerating rat liver

Cell suspensions were prepared from normal and regenerating liver of adult rats by perfusion with a calcium-chelating agent (EGTA), collagenase and hyaluronidase, and the cells were incubated in culture medium. In cultures prepared from regenerating liver at 20 h after partial hepatectomy, 23 ± 4% of parenchymal cells initially incorporated [³H]TdR. This incorporation was shown to reflect semiconservative DNA replication. At least some parenchymal cells were able to complete their DNA synthesis and to progress through G2 and mitosis. Numbers of hepatocytes in mitosis increased up to 12 h of culture. On the other hand, no entry of hepatocytes into the S period was detectable in cultures prepared from normal or regenerating liver.     

Urethane inhibition of DNA synthesis in the hepatocytes of the regenerating liver in mice

Hepatocarcinogen urethane (ethyl carbamate) inhibits DNA synthesis in the regenerating mice liver when administered at the peak of stimulated proliferation--46 hours after partial hepatectomy. The inhibition is temporary and reversible. The maximum inhibition of 3H-thymidine incorporation in the cells is observed 12 hours after urethane administration, and the effect is removed following 20 hours after administration. Another effect of urethane consists in the lengthening of the period of DNA synthesis by 1.38 times, as estimated by the Quastler-Sherman method, though it does not affect the length of G2-period or mitosis. Possible mechanisms of the effect of urethane on the initiation of DNA synthesis and on the rate of DNA replication are discussed.     

Recovery kinetics of epidermal cell proliferation after two intraperitoneal injections of epidermal extracts (chalone)

The cell kinetic effect of two intraperitoneal (IP) injections of 5 mg of crude lyophilized skin extracts given 12 h apart was assessed during the recovery period (5 to 52 h after last injection) by measuring epidermal labelling indices and the specific activity after tritiated thymidine (3HTdR) injection, and by determining the cell cycle phase distribution by flow cytometry. The skin extracts produced an epidermal chalone effect and inhibited both DNA synthesis and mitosis. A slow recovery took place from 5 to between 22 and 36 h after the last chalone injection. During this period the cell flux into DNA-synthesis and mitosis slowly recovered, but the exits were blocked and cells accumulated in the respective phases. The fluxes out opened up at the S phase about 22 h, and at the M phase about 30 h after the second chalone injection. A secondary inhibitory effect was observed at about 40 h, followed by a subsequent recovery to normal at 52 h. The similarity between the recovery kinetics after chalone and that observed after hydroxyurea (HU) is emphasised.     

Initiation of DNA synthesis in the liver and other tissues of adult mice by a growth factor (EACF) isolated from acellular fluid of the Ehrlich ascites carcinoma

The mitogenic effect of a new growth factor that we recently isolated from the acellular ascitic fluid of the Ehrlich ascites carcinoma grown in vivo was examined. We have called this factor EACF (Ehrlich ascites carcinoma factor). EACF caused initiation of DNA synthesis in the liver, submandibular gland, exorbital lacrimal gland and epithelium of the tongue of adult mice after i.p. injection at a protein concentration of 3 micrograms per 25 g of body weight. For all tissues examined, except the tongue, EACF initiated DNA synthesis at about 48 to 60 h after injection, with the maximum effect at approx. 85 h, and the stimulatory effect lasting approx. 60 h. The initiation of DNA synthesis in liver, which is normally characterized by only an occasional cell passing through the S phase, by EACF is of particular interest. The initiation of DNA synthesis in the liver was not prevented by hypophysectomy. Evidence also indicates that a similar heat-labile growth promoting factor(s) is present in calf serum.     

Mitogenic effects of certain cathepsins and calciferin on the intact liver in vivo

Calciferin, a new parathyroid hormone stimulating the release of cathepsins D and L (but not B) from isolated lysosomes, or the release of cathepsin D from erythrocytes or ghosts in vitro, elevated free cathepsin D in the blood, and at the same time stimulated DNA synthesis in the intact liver when it was injected into mice. Both calciferin and free cathepsin D in the blood (rats) were elevated concomitantly soon after 70% hepatectomy, reaching a peak around 5 hr. The cathepsin D-elevation was almost proportional to fractional hepatectomies. Cathepsin L (but not B), when injected intraperitoneally into mice, stimulated DNA synthesis and mitosis in the intact liver much like cathepsin D, the effect of which was reported earlier. In contrast to the mitogenic effects of calciferin or cathepsins (D and L) in vivo, only cathepsin L (but not cathepsin D or calciferin) in low concentrations appeared to stimulate DNA synthesis in the cultured liver cells, and also stimulated adenylate cyclase of isolated liver plasma membranes in vitro. Dibutyryl-cyclic AMP in concentrations lower than 10(-5) M also stimulated DNA synthesis in cultured liver cells.     

Retinoic acid repressed the expression of c-fos and c-jun and induced apoptosis in regenerating rat liver after partial hepatectomy.

Retinoic acid (RA), which was injected within 4 h after partial hepatectomy (PH), inhibited DNA synthesis in regenerating liver. The inhibition was accompanied by apoptosis, evidenced by in situ end labeling and gel electrophoresis of DNA fragmentation. Characteristic DNA fragmentation was obvious at 4 h and reached a maximum at 8 h after injection. Northern blot analysis revealed that RA repressed the expression of c-fos and c-jun at 15 and 30 min with the up-regulation of retinoic acid receptor gamma (RARgamma) and RARbeta at 2 h after PH. The transglutaminase II mRNA level and activity were increased by RA injection at 4 h and 8 h after PH, respectively. The mRNA levels of thymidylate synthase and thymidine kinase, which are rate determining enzymes of DNA synthesis, decreased in RA injected rats. No change was seen in the expression of p53 and p21WAF1/CIP1 which have been suggested to participate in the apoptosis process. These results suggest that RA exerts the antiproliferative activity only on the early stage of liver regeneration accompanied by the repression of c-fos and c-jun expression and induction of apoptosis.     

Course of liver regeneration after partial hepatectomy in rats treated with dialysates of intact organs

A number of growth phenomena observed in vitro have shown that cells, at high densities, produce and release substances which, when they have reached a given concentration, arrest further growth. In vivo, these possibilities can be studied on the model of rapid regeneration of the rat liver after 65-70% partial hepatectomy (PH). We evaluated the course of liver regeneration after PH in animals treated with dialysates (DIA) of intact rat tissues. In addition to kidney and lymph node DIA, we were particularly interested in the effect of liver and spleen DIA. The experiments were carried out on male rats weighing 210-240 g. The relevant DIA was administered 24 h prior to PH; the controls were given physiological saline. The animals were killed just before PH and 24, 48, 30 and 72 h and 14 days after. DIA obtained from intact liver tissue inhibited the regeneration process induced by PH and its effect persisted 48 h after PH. Compared with the controls and with the rats given kidney DIA, DNA synthesis in the liver 24 h after PH was reduced to 77%. After spleen DIA, several (still hypothetical) factors probably acted together synergically (factors belonging to the immune system--RES--and spleen-produced factors capable of promoting proliferation of the hepatocytes--the "portal blood factor"). We arrived at this conclusion from an evaluation of liver DNA synthesis 24 h after 24h after PH, when synthesis was altogether markedly raised, but attained far higher values after the administration of spleen DIA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of a single dose of dimethylnitrosamine on biosynthesis of nucleic acid and protein in rat liver and kidney

1. Administration of a single dose of dimethylnitrosamine to rats temporarily fed on a protein-deficient diet causes a high incidence of kidney tumours. The effect of such a dose of dimethylnitrosamine (40mg/kg body wt.) on metabolism of nucleic acids and protein in rat liver and kidneys was examined during the week immediately after administration. 2. Incorporation of [(14)C]leucine and [(14)C]orotate into hepatic macromolecules was inhibited within 5h of injection of dimethylnitrosamine, and did not recover for at least 5 days. Interpretation of these results is complicated by the concomitant extensive hepatic necrosis. 3. Renal RNA synthesis was assayed by incorporation of [(14)C]orotate in vivo and measurement of DNA-dependent RNA polymerase activity in vitro. Both systems indicate biphasic inhibition; minimal activity was recorded 9h and 3 days after treatment. Changes in incorporation of [(14)C]leucine into renal protein were similar but less marked. 4. Sucrose-density-gradient analysis of renal cytoplasmic RNA indicated increased synthesis of rRNA 24h after injection of the nitrosamine. The rate of loss of radioactivity from kidney ribosomes pre-labelled with [(14)C]orotate was not modified by dimethylnitrosamine. 5. Dimethylnitrosamine increased incorporation of [(3)H]-thymidine into renal DNA. The three distinct periods of stimulated synthesis observed are discussed, with particular reference to recently published morphological studies of the sequential development of kidney tumours induced by dimethylnitrosamine in protein-depleted rats.     

Autoradiographic study of the rate of DNA synthesis in the duct epithelial cells of the epididymis and of the brain subependymal zone in the rat following whole-body x-ray irradiation

The experimental data have been analyzed on the labeled cell distribution related to the grain count over the nucleus in autographs of histological sections (5 mcm) made of the rat brain subependymal zone and of epididymis duct epithelium at different time after 3H-thymidine injection and X-irradiation in the dose of 300 cGr. These results served some additional grounds to the recently established conclusion that a repeated successive decrease in the rate of DNA synthesis is occurring in the cell system starting from stem cell (level I) to semistem cell (levels II-VII) (Gracheva, 1982 r). 5 days after irradiation, at the peak of reparative proliferation, the cell reproduction was intensified, these cells having normally both middle and high levels of DNA synthesis. This process is running of the background of the inhibition of reproduction of cells with the inherent low level of DNA synthesis which are to start differentiation after mitosis. All this makes for the increase in the mean grain count over the nuclei, without changes of the inherent rates of DNA synthesis in the successive generations of the stem cells.     

Modulation of the mitotic action of ethylene dibromide

Refeeding rats treated with a single high dose of ethylene dibromide (1,2-dibromoethane, EDB) induced liver DNA synthesis. The peak of DNA synthesis, as measured by [methyl-3H]thymidine incorporation was attained after 24 h in refed rats and at 48 h in fasted ones. Fasting enhances the EDB action leading to liver cell necrosis, as shown by elevation of serum enzymes' activities, glutamic pyruvic transaminase (GPT) and sorbital dehydrogenase (SDH). A low dose of EDB administered during 2 and 3 weeks slightly enhanced the liver DNA synthesis and elevated the activity of serum enzymes. Phenobarbitone (PB) treatment of rats together with low dose of EDB during 2 weeks prevented the enzyme activity elevation and attenuated the DNA synthesis. Diethyldithiocarbamate (DDC) pretreatment potentiated the DNA synthesis in fed rats after both a small dose of EDB for 2 weeks and after a single high-dose treatment. In DDC pretreated rats, the high single dose of EDB caused biochemical perturbations in serum and liver representative of liver cell necrosis; changes in serum enzymes' activities also were noticed as early as 2 h after EDB toxication. The possible function of modulators on the mitogenic or the necrogenic action of EDB is discussed.     

The effect of vinyl chloride monomer,chloroethylene oxide and chloracetaldehyde on DNA synthesis in regenerating rat liver

Vinyl chloride monomer used in the manufacture of polyvinyl chloride is a chemical of increasing industrial importance but has recently been incriminated as a carcinogen, producing a mutagenic effect after being metabolized to active metabolites. The initial effect of vinyl chloride monomer and two of its presumed metabolites, chloracetaldehyde and chloroethylene oxide, on DNA synthesis was investigated in vivo in regenerating rat liver. The established control curve for the DNA synthesis rate after partial hepatectomy demonstrated two waves of synthetic activity at 21 and 30 h. Vinyl chloride, injected intravenously immediately on completion of the operation, depressed the first wave of DNA synthesis by 49.6%. The second peak of DNA synthetic activity was similar to that of the control. Chloracetaldehyde and chloroethylene oxide both produced similar effects on the first wave of DNA synthesis after partial hepatectomy, inhibiting the DNA synthesis rate by approx. 50%. After a regenerating period of 27 h, however, they produced very different effects, chloroethylene oxide raising the control DNA synthesis rate at 30 h by 49% while chloracetaldehyde tended to desynchronize the well-defined second peak of the control. The test compounds have been compared to literature reports of the inhibitory effects of various carcinogens on DNA synthesis.     

The control of nucleic acid and nicotinamide nucleotide synthesis in regenerating rat liver

1. The effect of injecting nicotinamide on the incorporation of [(14)C]orotate into the hepatic nucleic acids of rats after partial hepatectomy was investigated. 2. At 3h after partial hepatectomy the rapid incorporation of [(14)C]orotate into RNA, and at 20h after partial hepatectomy the incorporation of [(14)C]orotate into both RNA and DNA, were inhibited in a dose-dependent fashion by the previous injection of nicotinamide. 3. The injection of nicotinamide at various times before the injection of [(14)C]orotate at 20h after partial hepatectomy revealed an inhibition of the incorporation of orotate into RNA and DNA which was non-linear with respect to the duration of nicotinamide pretreatment. 4. The induction of a hepatic ATP depletion by ethionine demonstrated that the synthesis of hepatic NAD and NADP in partially hepatectomized rats was more susceptible to an ATP deficiency than in control rats. 5. The total hepatic activity of ribose phosphate pyrophosphokinase (EC 2.7.6.1) was assayed at various times after partial hepatectomy and found to be only marginally greater than the maximum rate of hepatic NAD synthesis induced in vivo by nicotinamide injection between 12 and 24h after partial hepatectomy. 6. It is suggested that a competition exists between NAD synthesis and purine and pyrimidine nucleotide synthesis for available ATP and particularly 5-phosphoribosyl 1-pyrophosphate. In regenerating liver the competition is normally in favour of the synthesis of nucleic acid precursors, at the expense of NAD synthesis. This situation may be reversed by the injection of nicotinamide with a subsequent inhibition of nucleic acid synthesis.