Sequential changes in ornithine decarboxylase thymidine kinase, and other enzyme activities in regenerating liver in rats on controlled feeding schedules.

The changes in activity of five enzymes including ornithine decarboxylase (ODC), tyrosine aminotransferase (TAT), thymidine kinase (TK), ornithine aminotransferase (OAT) and serine dehydratase (SDH) in the early stage of the regenerating rat liver have been studied under a controlled feeding and lighting schedule. The first three enzyme activities were stimulated sequentially by partial hepatectomy. The earliest response was observed in ODC activity. A significant increase in this enzyme activity was observed at 2 hrs and the maximal level was at 4 hrs after the operation. TAT began to increase at 4 hrs and the maximal level was at 8 hrs. The TK activity was induced at about 24 hrs and the highest value was at 48 hrs after partial hepatectomy.A significant decrease in OAT activity was observed at 24 hrs after the operation and subsequently. Although a decrease in SDH activity was also observed this decrease did not seem to correlate directly with the regeneration process, since a lowered level of the enzyme activity was also found in the sham operated group.     

Alpha-fetoprotein and serum hormone levels following liver intoxication with carbon tetrachloride

Rats were submitted to carbon tetrachloride intoxication at two different doses. Serum level of estradiol, progesterone, cortisol and thyroxin were measured by radioimmunoassays and correlated with the histological evidences of liver regeneration and serum alpha-fetoprotein levels. A two fold increase of progesterone was observed 48hrs after CCl₄ administration. Cortisol levels were moderately increased at both doses of CCl₄. Despite the five fold increase of alpha-fetoprotein (which is the major estradiol binding protein in these sera) no changes in estradiol levels were observed. Thyroxin levels showed a two fold increase after 72hrs. This result contrasts with the drop of this hormone after partial hepatectomy which has been previously published. These experiments show that a new hormonal imbalance (directly or indirectly due to the toxic) is involved both in liver regeneration and alpha-feto-protein synthesis.     

Effect of treatment in vivo with N,N-dimethylnitrosamine or methyl methanesulphonate on the cytoplasmic DNA polymerase of regenerating rat liver.

A 10-16 fold increase in rat liver cytoplasmic DNA polymerase (DNA polymerase-alpha) was observed by 24 hrs after two thirds partial hepatectomy. Injection of either N,N-dimethylnitrosamine (DMN) or methyl methanesulphonate (MMS) At 6 or 12 hrs after partial hepatectomy completely inhibited this increased production of polymerase, but when given at 20 hours they had less effect. Neither compound altered the molecular size distribution of the enzyme. These data indicate that the lowered levels of DNA polymerase-alpha could play a major role in the reduction in DNA synthesis which occurs after carcinogen treatment.     

Synthesis of chromatin phospholipids

The presence of a phospholipid fraction associated with chromatin has been demonstrated by biochemical technique in rat hepatocytes. The composition of this fraction determined by chromatography with respect to that of the nuclei is characterized by low content of phosphatidylserine and high content in phosphatidylethanolamine. Also the synthesis and turnover studied after injection of [32P]O4(2-) show a different behaviour: the peak of activity is after 6 hrs in nuclei and microsomes, whereas in chromatin it occurs after 9 hrs. A second peak is evident after 24 hrs in chromatin and microsome phospholipids. Differences have been also shown by analyzing the single phospholipid radioactivity in time. The behaviour of chromatin phospholipids has also been studied during DNA premitotic synthesis in regenerating liver. It has been shown that there is no difference in synthesis in relation to that of DNA in nuclear phospholipids, whereas the specific activity of chromatin phospholipids begins to increase twelve hours after hepatectomy and continues throughout the period of the first mitotic wave, thus bringing to a summation with the beginning of the second wave. The role of this phospholipid fraction in relation to DNA synthesis and gene expression is discussed.     

Content, pattern and metabolic processing of rat-liver gangliosides during liver regeneration

During rat liver regeneration, the ganglioside content and distribution undergo significant changes after partial hepatectomy; total liver gangliosides increase remarkably till the 4th day after surgery, thereafter progressively decreasing to reach the values of sham-operated controls at the 12th day. The qualitative pattern is characterized by the 95% relative increase of GD1a at the 4th day and the 40% relative decrease of GD1b. In order to investigate the processes of ganglioside penetration into cells, degradation and biosynthesis, radiolabelled GM1 ([Sph-3H] GM1) was administered. One day after hepatectomy the liver uptake and metabolism of exogenous ganglioside were significantly reduced. Three days post-surgery these parameters were restored to control values; however an increased radioactivity incorporation was found in GD1a, thus suggesting an enhancement of its biosynthesis around the 4th day. The data reported here suggest that in the first two days after partial hepatectomy, the ganglioside degradation is reduced with a consequent increase of ganglioside content; later on the catabolic routes normalize and some biosynthetic processes leading to GD1a are enhanced. GD1a seems to be a marker of a peculiar transition phase of liver regeneration.     

Impaired hepatic regeneration in metallothionein-I/II knockout mice after partial hepatectomy

Although the translocation of metallothionein (MT) from cytoplasm to nucleus has been demonstrated in liver during times of high requirement for zinc (fetal development and the neonatal period), the role of MT in cellular growth is not well understood. In this study, a potential role of MT in liver regeneration was investigated in wild type (WT) and MT-I and MT-II gene knockout (MT-null) mice after 35% partial hepatectomy (PH) or sham laparotomy. Hepatic MT levels and proliferation index were measured at 0, 5, 15, 24, 36, 48, and 60 hrs after PH and 48 hrs after sham laparotomy (control). MT levels were increased in WT mice (peak at 24 hrs after PH) and declined to normal levels by 60 hrs after PH. Immunohistochemical staining for MT in WT mice indicated the presence of MT in both nucleus and cytoplasm of hepatocytes at 24 hrs after PH, whereas MT was present mainly in the cytoplasm at 36-60 hrs after PH and 48 hrs after sham laparotomy. Hepatic proliferation index in both WT and MT-null mice, as determined by argyrophilic nucleolar organizing region staining and proliferating cell nuclear antigen immunohistochemical staining, reached a peak at 48 hrs and declined by 60 hrs after PH. Cell proliferation was significantly less in MT-null mice as compared to WT mice during liver regeneration after PH. These results suggest that MT may play a positive role in hepatic regeneration after PH.     

Increase in nuclear aluminum concentration during hepatic regeneration: study by analytical ionic microscopy

20 hrs. after partial hepatectomy, a significant increase in the aluminum concentration of liver cell nuclei has been measured by analytical ion microscopy. These results indicate a possible role of aluminium in an in vivo induced proliferative response.     

Influence of subtotal hepatectomy on peroxisomes and peroxisomal enzymes of rat liver and isolated liver cell fractions.

The activities of peroxisomal enzymes of rat liver were followed 1 to 10 days after subtotal (60-70%) hepatectomy in homogenates prepared from regenerating livers and in cell fractions isolated from them. Catalase activity was found to be depressed in the total liver homogenate (H) as well as in the mitochondrial (M) and soluble (S) fractions, while it did not change appreciably in the microsomal (Mc) and lysosomal (L) fractions. Alpha-hydroxyacid oxidase behaved in a similar fashion. In contrast to these enzymes, urate oxidase activity remained unchanged in H, whereas it was decreased in M and increased in L and Mc during the first 5 days after operation. These results agree well with the assumption that microbody proliferation is initiated by the fragmentation of large peroxisomes. The different relations of peroxisomal enzyme activities during regeneration time are discussed with respect to the possible existence of various kinds of peroxisomes with different enzyme equipments and with different turnover rates. Biochemical examinations ions were paralleled to morphological and histochemical studies. An early increase in number of peroxisomes was found to occur during the first day after partial hepatectomy, which is accompanied by decrease in particle size. During the first mitotic wave (24-36 hrs post op.) the number of peroxisomes per cell was reduced to about the half. After this time number and size of the particles began to increase. Positive staining of ribosomes was frequently observed in the vicinity of peroxisomes after the application of the cytochemical catalase reaction (alkaline diaminobenzidine medium). This phenomenon is interpreted to represent rather a diffusion artifact than the cytochemical identification of newly synthesized catalase.     

Behaviour of tyrosine amino transferase and convertase during the first hours after hepatectomy in rats

The activity of rat liver tyrosine amino transferase (TAT) increases after hepatectomy with a first prominent peak at 8 h and a second peak at 18 h. This change in activity is probably due to de novo enzyme synthesis since it is prevented by actinomycin-D (AMD). In the same period an increase of the lysosomal converting enzyme (convertase) which catalyses the in vitro transition of TAT from form I to form III, has been observed; this is not accompanied by changes of other lysosomal enzymes, such as acid phosphatase and cathepsin L. The activity of convertase is equal to that of the controls (sham operated animals) 2 h after hepatectomy, increases three times at 5 h, maintains the same value at 8 h and then decreases slowly to control level after 24 h. The correlation between the activity changes of the two enzymes strongly suggests a physiological role of convertase in TAT turnover.     

Changes in UsnRNA biosynthesis during rat liver regeneration

Partial hepatectomy (P.H.) induces a partially synchronized growth response of liver under normal regulation of growth. In this phase changes in cellular morphology, radial distribution pattern of cells and other biological as well as major biochemical changes are well documented [24]. Here, we have shown that the cellular content of UsnRNAs altered during this proliferative phase as well. The level of spliceosomal UsnRNAs (U1, U2, U4–U6) gradually decreased by 30–50% upto 48 hrs of P.H. followed by gradual increase to reach the normal level within one month of P.H. The U3 snRNA level on the other hand, was nearly equal to that in normal liver at 48 hrs of P.H. but in 24 and 72 hrs of P.H. its level was high (4 fold) in contrast to that in other UsnRNAs. Thus, it is clear from our data that the level of all the six UsnRNAs decreased during 48 hrs of P.H. compared to that after first 24 hrs. This has been correlated in the kinetics of UsnRNAs' synthesis (in terms of labelling) in isolated hepatocytes, where the rate of labelling of all the six UsnRNAs increased 20–30% in 24 hrs regenerating hepatocytes (R.H.) followed by sharp decrease by 30–50% within next 24 hrs, compared to that in the normal hepatocytes. But from 72 hrs onwards in R.H. the rate of labelling of all the six UsnRNAs again increased by 30–50% (compared to that in normal hepatocytes) followed by decrease of their labelling-rate to reach the normal level in R.H. within one month of P.H. Thus, it may be concluded that the changes in UsnRNAs' level during the proliferative phase of liver regeneration may be either due to the alteration in the rate of synthesis (in terms of labelling) or along with it differential turn over rate; this phenomenon may have some consequences with the regenerative process of liver.This paper was published in Molecular and Cellular Biochemistry131:67–73, 1994. Kluwer Academic Publishers regret the publication of the only partly corrected version.     

Polypeptide synthetase activity of chromatin from eukaryotic cells]

It is shown that the polypeptide synthetase activity (PS-activity) of chromatin from rat liver is increased 9--21 hrs after partial hepatectomy. Among 9 amino acids studied alanine, methionine, lysine, tyrosine and arginine are not incorporated into the system in question. The highest rate of polymerization is observed in case of glutamic acid. The rate of glutamine, asparagine and glycine incorporation is 7--8 times slower. The PS-activity of chromatin is enhanced by chromatin preincubation with NAD (but not with its analogs). The activation is prevented by thymidine and nicotinamide. Storage of chromatin for 18 hrs at 2--4 degrees C results in a complete loss of PS-activity. Ability of "old" chromatin to incorporate of amino acids may be restored by its preincubation with NAD. Storage of chromatin in the presence of 5 mM cAMP does not decrease the PS-activity. It is assumed that in the system described poly-ADP ribose is an energy source for amino acid activation.     

Electron microscope study of chromatin in hepatocyte nuclei during the first hours after partial hepatectomy. V. Changes in the relative area of condensed chromatin and the density of chromatin fibril packing in the ultrathin sections

The degree of chromatin condensation was studied on ultrathin cell sections of guinea pig hepatocytes during the prereplicative period after partial hepatectomy. Three time points were chosen for analysis namely 2,5, 5 and 9 hrs after operation since they show marked increasing (2.5 hrs), decreasing (5 hrs) and repeated increasing (9 hrs) of the amount of ethidium bromide binding to chromatin. The degree of chromatin condensation was determined by measuring the area occupied by condensed chromatin and also by measuring the number of chromatin fibrils per a certain length. The condensed chromatin with varying localization in the nucleus were studied separately. The changes of nucleoplasmic chromatin were most pronounced: at 2.5 and 9 hrs after operation the decrease of the relative area and of the density of chromatin fibrils package was observed; these parameters were near to control at 5 hrs after operation. In general the changes in nucleoplasmic chromatin were correlated with the changes of the activity of the chromatin in the whole nucleus. The decondensation of the perimembranous chromatin was manifested in the decrease of its area and was expressed only at 9 hrs after operation. The perinucleolar chromatin was found to show the gradual decondensation which was manifested mainly by the decrease of its relative area. Thus the condensed chromatin seems to be a labile structure which undergoes essential changes in the process of the exit of the hepatocytes from G0-stage of the cell cycle, during the prereplicative period.     

DNA hypomethylation in ethionine-induced rat preneoplastic hepatocyte nodules

DNA from hepatocyte nodules induced in rats with dietary DL-ethionine and from the surrounding non-nodular liver contained less 5-methyldeoxycytidine per deoxycytidine when compared with that from normal adult liver. The degree of apparent hypomethylation, 37% in nodules and 20% in the surrounding liver, decreased somewhat (29% and 16% respectively) at 2 weeks after terminating the exposure to ethionine. Nodules and surrounding liver, like normal liver, responded to partial hepatectomy with a decrease in the 5-methyldeoxycytidine level at 24 hrs and a return to the level at the time of partial hepatectomy by 38 hrs. These findings indicate the need for careful control of cell proliferation in comparing the levels of a post-replicative DNA modification, methylation, in proliferating and non-proliferating cell populations. These findings also suggest that a portion of the hypomethylation in preneoplastic nodules may be due to a bona fide decrease in the level of cytosine methylation in the parental strand of DNA. This hypomethylation could be one basis for the altered gene expression in hepatocyte nodules, possible precursors for liver cancer.     

Nucleotide pool and the proliferative process: an NMR study of the liver regeneration

The metabolic changes occurring during liver regeneration after partial hepatectomy have been followed by examination of the perchloric extracts by means of NMR spectroscopy. Proton spectra show an abrupt decrease of glycogen, glucose and nucleotides, which are essentially ribonucleotides, in the first hours after hepatectomy. Nucleotides begin to store up again in the third day after hepatectomy, while glucose and glycogen storage builds up from the second day. 31P data evidence a sharp drop of phosphomonoesters, i.e. monophosphosugars (including AMP) and phosphocholine, and phosphodiesters, i.e. GPC and GPE, soon after hepatectomy and a recovery of the initial levels approximately at the 60th hour and a further increase of PDE at later times. The NMR findings are in agreement with the biochemical knowledge of the course of liver regeneration.     

Ploidy class-dependent metabolic changes in rat hepatocytes after partial hepatectomy

Glucose-6-phosphate dehydrogenase (G6PDH), succinate dehydrogenase (SDH) activity and the single-stranded RNA (ssRNA) content of isolated hepatocytes of different ploidy classes from adult male rats have been studied after partial hepatectomy using quantitative cytochemical means. The SDH activity and ssRNA content in all classes of hepatocytes are decreased during the first hours after operation followed by an increase above control values. The increase of both SDH activity and ssRNA content is significant only in the mononuclear diploid (MD) cells but not in the hepatocytes of higher ploidy classes and is related with the mitotic wave at 32 h after hepatectomy. After the mitotic wave, the values quickly return to normal levels. The G6PDH activity does not show any significant change in hepatocytes other than MD cells. In MD cells the G6PDH activity is elevated on a highly significant level up to a maximum value of 3.5 times the control value at 48 h after operation. The G6PDH activity in MD cells is returned to normal values within 14 days after operation. It is concluded that: 1. The MD cells show a distinct metabolic behaviour due to their function as stem cells of liver parenchyma and retain at least some of their fetal characteristics. 2. G6PDH activity is not a transformation-linked discriminant for neoplastic metabolism.     

Action of x-ray irradiation on the nuclear envelope of rat liver cells]

X-irradiation of isolated nuclear envelopes (NE) has revealed their high radiosensitivity, while irradiation of isolated intact nuclei in vitro, in the doses up to 5000 r 18--20 hours after partial hepatectomy, produced no morphological changes in NE. The damaging effect of irradiation on both nuclei and mitochondria (Mt) was revealed only with a decrease in cytochrome-c-oxidase (CO) activity in parallel with an increase in the radiation dose. One hour after the whole body irradiation of rats in the beginning of S-period, the damaging effect was recorded in both NE and Mt at the doses of 50 and 150 t, and was enhanced with the increase of irradiation dose. Morphological changes were observed mostly in the outer nuclear membrane, which lost its distinct outline and disappeared from some nuclear regions. Lethal radiation doses produced a decrease in the number of pore complexes (PC) with their evident segregation from the membranes. After irradiation in a dose of 1200 r, only the residue or "ghosts" of the PCs remained. After irradiation in doses up to 400 r, the CO-activity recovered during the first hour in Mt and during first two hours in the nuclei.     

Time dependence of the proton relaxation times of regenerating rat liver

The proton longitudinal relaxation time of regenerating rat liver has been found to increase during the first 24 hours after hepatectomy and to drop back to normal in the following hours. The decreased relaxation rate may be related to the increase of the water mobility due to the expansion of the intercellular spaces during the massive proliferation of the first day, or to the increased cell hydration which is known to occur during active cell proliferation. Equations have been derived for the proliferation process, and the competing inhibition process, active from the 24th hour, which can quantitatively account for the proton relaxation behaviour.     

Participation of the intracellular enzymes in the control of the mutation process

Summary The influence of repair and replication on the frequency of spontaneous chromosome aberrations and of those induced by gamma-irradiation is reported.Using the technique of labelling DNA with radioactive ³H-thymidine and measuring the radioactivity of DNA isolated from embryos, the time of initiation and the duration of DNA synthesis in barley seeds was studied after the soaking of the seeds had begun. The average duration of each phase of the first DNA synthesis cycle in soaking barley seeds was found to be as follows: pre-DNA synthesis stage, 10–11 hrs; DNA synthesis stage, 8 hrs. After gamma-irradiation, the intensity of DNA synthesis decreased and the beginning of DNA synthesis was delayed.It was found that the inhibition of repair by caffeine led to an increase in the frequency of both spontaneous and induced chromosome aberrations. Caffeine enhanced several times the frequency of chromosome and chromatid aberrations at the time of the maximal activity of repair enzymes. During DNA replication, caffeine had a lower effect on the realization of premutational lesions.An inhibitor of DNA replication — hydroxyurea — had no influence on the frequency of spontaneous chromosome aberrations during the replication period, whereas after gamma-irradiation, hydroxyurea enhanced the frequency of aberrations mainly at the stage of DNA replication.The relatively small mutagenic action of both agents (caffeine and hydroxyurea) was observed during all stages of the cell cycle of germinating barley seeds.     

Transcriptional and translational control of the biphasic increase in ornithine decarboxylase activity in liver

The requirements for protein and RNA synthesis for each of the two increases in liver ornithine decarboxylase activity after the injection of unoperated rats with a solution containing glucagon and after 70% hepatectomy were studied with cycloheximide and actinomycin D. Protein synthesis is required for both increases whereas RNA formation is essential for the first elevation only. The second increase appears to be dependent upon RNA that is made during the period of the first rise in activity.The two rises in the decarboxylase activity may be caused by different stimuli. After the injection of the mixture with glucagon, the first elevation is accompanied by increases in hepatic tyrosine aminotransferase activity and in the rate of transport into liver cells of the model amino acid, α-aminoisobutyrate. Neither an increase in the aminotransferase activity nor in amino acid uptake occurs, however, during the period of the second elevation in the decarboxylase activity.     

Topographic distribution of dividing hepatocytes in the regenerating liver lobule in the period of maximum mitotic activity

Topographic distribution of dividing hepatocytes was studied in the liver lobule after hepatectomy performed at 10-11.30 a. m. The studies were conducted 20-32 h after surgery during the first increase in mitotic activity. It has been established that dividing hepatocytes appear simultaneously in all lobule areas, i. e. that all the hepatocytes are capable of responding to regenerating stimuli. However, further behaviour of dividing hepatocytes becomes different. It is concluded that hepatocytes from outer and intermediate lobule areas possess a more pronounced proliferative potential during peak mitotic activity after partial hepatectomy.