Effect of ethanol on the DNA-polymerase activity in the liver subcellular fractions of adult and old rats

The effect of 5% ethanol on DNA polymerase activity in nuclei, mitochondria, microsomes and cytosol of intact and regenerating liver of adult and old rats has been studied. No changes in DNA polymerase activity were detected in subcellular fractions of adult rat liver. On the contrary, the increased activity of intact liver nuclei and decreased activity of regenerating liver microsomes was observed with ageing. These age-dependent peculiarities of DNA polymerase activity in response to 5% ethanol may be related to changes in the enzyme molecules or microenvironment associated with ageing.     

The activity of polynucleotide phosphorylase in polyribosomes of regenerating liver of adult rats, liver of newborn rats and in some reinoculated tumours]

Specific activity and level of polynucleotide phosphorylase (PNPase) in polyribosomes of regenerating liver of adult rats, liver of newborn rats and in malignant tumours of rat (sarcoma M-1 and hepatoma 27) were studied. 24 hours after partial hepatectomy the specific activity and level of PNPase in regenerating liver decreased 3--4 times in the fraction of polyribosomes, bound to the endoplasmic reticulum membranes, and remained at a constantly low level in the fraction of free polyribosomes. The PNPase activity also showed a sharp decrease in the fraction of membrane-bound polyribosomes from newborn rats liver and could not be detected either in free or in bound polyribosomes from sarcoma M-1 or hepatoma 27. The PNPase activity in the fraction of bound polyribosomes increased with a decrease in the rate of liver growth (regenerating liver and newborn rats liver), and reached the level normal for adult animals. Possible mechanisms of regulation of the PNPase activity in animal tissue were studied. It was found that a 2-fold administration of cyclic 3,5'-AMP to intact animals (5 mg per 100 g of body weight) with an interval of 8 hours, corresponding to the interval between two peaks of the increase in cyclic 3,5'-AMP concentration following partial hepatectomy, diminished the PNPase specific activity in polyribosomes by 30%. A factor, presumably of protein origin, which induced a release of PNPase from polyribosomes of normal rat liver but did not affect the activity of the liberated enzyme, was detected in the cell sap of sarcoma M-1 and hepatoma 27.     

Increased adenylosuccinase activity in hepatomas and kidney tumors.

Adenylosuccinase activity was measured in adult, neonatal and regenerating liver, in nine transplantable hepatomas of different growth rates, and also in kidney cortex and 2 transplantable kidney tumors in the rat. The activity was increased 1.4 to 2.2-fold in all the tumors, irrespective of the growth rate or the degree of histological differentiation of the neoplasms. Adenylosuccinase activity in the average liver cell of one-day-old rats was 21% of the adult value. In the rapidly-growing regenerating adult liver the enzyme activity did not change. It is concluded that the consistent elevation of adenylosuccinase activity in the tumors was linked with the neoplastic transformation and the increase was specific to neoplasia.     

Neoplastic transformation-linked alterations in adenylosuccinate synthetase activity.

Adenylosuccinate synthetase has been measured in rats in normal, differentiating, and regenerating liver, transplantable hepatomas of different growth rates, kidney cortex, and a transplantable kidney tumor. The activity was increased 1.6 to 3.7-fold in all the tumors. The activity showed no correlation with the degree of histological or biochemical differentiation of the tumors, nor with their growth rate. Adenylosuccinate synthetase activity in regenerating liver was unchanged and in neonatal liver it was much lower than in adult liver. It is concluded that the ubiquitous increase in the tumors of liver and kidney was linked with the neoplastic transformation.     

Cytological analysis of the intact and regenerating liver of different strains of rats]

Comparative cytological studies were conducted on control and regenerating liver of two strains (August and Cotton) of rats, 2/3 of the liver was resected; 5 or 6 animals were sacrificed at each of the following postoperative periods: in 30 hours, 3, 8, 42 and 120 days. The number of binuclear cells, the size of mononuclear hepatocytes and their nuclei, the mitotic activity, and ploidity of hepatocytes were determined. The intact and regenerating liver of the August rats differed from the intact and regenerating liver of the Cotton rats by a number of cytological indices, excluding the mitotic activity. A conclusion was drawn that the observed interstrain differences in the cytological indices providing regeneration of the liver after resection in the August and Cotton rats depended on the genotype of the given strain.     

The UDP-N-acetylglucosamine-asparagine-sequon N-acetyl-beta-D-glucosaminyltransferase activity in preparations of rough endoplasmic reticulum from regenerating rat liver.

The UDP-N-acetylglucosamine-asparagine-sequon N-acetylglucosaminyltransferase activity in preparations of rough endoplasmic reticulum from rat liver is less than 1% of that in preparations from rabbit liver. The activity of the enzyme is increased about 20-fold in preparations from regenerating rat liver within 48h after partial hepatectomy. A smaller, but still marked, increase (8-10-fold) occurs in preparations of rough endoplasmic reticulum from sham-operated rats.     

Increase in the number of atrial natriuretic hormone receptors in regenerating rat liver.

Forty-eight hours after partial (approximately 67%) hepatectomy the activity of the particulate guanylate cyclase was increased by 2-fold in the regenerating rat liver. This increase was not an artifact of membrane isolation procedures, and as determined by 125I-labeled Tyr-28 atrial natriuretic hormone-(1-28) ANF binding, was accompanied by a 2-fold increase in the number of ANF receptors. The Kd of the receptors in membranes of regenerating livers was not significantly different from the Kd of the receptors in livers of sham-operated rats. The linear synthetic descysteine analog of ANF, analog I, which binds only to the 66-kDa receptors, displaced approximately 40% of the specifically bound 125I-ANF in liver membranes from both hepatectomized and sham-operated (control) animals. Affinity cross-linking studies with 125I-ANF confirmed the increase in the 116-kDa ANF receptor in membranes of regenerating livers. In perfused livers derived from control and hepatectomized animals, the basal rates of cGMP production were not significantly different. However, atriopeptin II-stimulated cGMP production was twice as great in regenerating livers as compared with controls. These data demonstrate that the increase in particulate guanylate cyclase activity observed during liver regeneration is due to an increase in the 116-kDa ANF receptor-associated activity. Additionally, our data demonstrate that the regenerating rat liver may be a valuable model with which to study the role of the hepatic ANF receptor/particulate guanylate cyclase.     

A comparison by species, age and sex of cysteinesulfinate decarboxylase activity and taurine concentration in liver and brain of animals

Cysteinesulfinate decarboxylase activity and taurine concentration were determined in liver and brain of rats, mice, cats, guinea-pigs and sheep. Values were compared for male and female animals and in some cases measurements were also made in animals of different ages. Cysteinesulfinate decarboxylase activity and taurine concentration were also measured in liver and brain of male and female rat pups during the postnatal period. Hepatic cysteinesulfinate decarboxylase activity increased in both male and female rat pups during the postnatal period and then declined markedly in female rats so that activity in adult males was 16-fold that in adult females. Cysteinesulfinate decarboxylase activity in liver of 5- to 6-week old kittens was 73 times that observed in liver of 15-month old cats. Taurine level in liver of guinea-pigs was much lower than that in liver of any other species studied.     

Changes in polyamine-oxidizing capacity of peroxisomes under various physiological conditions in rats

Rat liver peroxisomal polyamine oxidase activity was determined under various physiological conditions by using the peroxidase method with phenol and 4-aminoantipyrine. N1-Acetylpolyamines such as N1-acetylspermine and N1-acetylspermidine were better substrates than the free polyamines. The polyamine oxidase activity in rat peroxisomes increased significantly when cell proliferation was high. The activity began to appear in fetal liver at the 16th approximately 18th day of pregnancy and peaked in neonatal liver on the first day (approx. 1.7-times higher than in adult liver). In regenerating rat liver, only polyamine oxidase activity among the peroxisomal enzymes tested was increased considerably 12 h after partial hepatectomy (approx. 2.8-fold over the control liver). Finally, the enzyme activity was significantly increased by administration of clofibrate, a peroxisome proliferator, which also causes hepatomegaly. In all cases, the increase in polyamine oxidase activity was not more than 3-fold. Since the level of polyamine oxidase activity in the normal liver is more than adequate in relation to the level of the substrates, the slight but significant increase under conditions of cell proliferation may have a role in modulating levels of polyamines in the proliferating liver tissue.     

Acceleration of rat liver phospholipid metabolism after long-term cadmium administration

Male Wistar rats, 6 weeks old, were allowed free access to water containing cadmium chloride at a concentration of 250 ppm as cadmium (Cd) for 6 and 12 months. The growth, as measured by body weight of Cd-treated rats, was significantly retarded. Electron microscopic studies revealed the appearance of small vacuoles in the cytoplasm, and involution of the rough endoplasmic reticulum (RER) in both the liver and whole kidney. When radioactive precursors of phospholipids, H3(32)PO4 and [1(3)-H]glycerol, were injected (ip) into cd-treated rats, the incorporation of 32P into phosphatidylcholine (PC) in the liver was increased 3.2- and 5.8-fold after 6- and 12-month Cd administration, respectively, and that of 3H into PC was also increased 2.3- and 2.2-fold after 6- and 12-month Cd administration, respectively. In the kidney, however, the incorporation rates of these radioactive precursors were little affected by long-term Cd administration. In the liver of rats treated with Cd for 6 and 12 months, the activity of CDP-choline:cholinephosphotransferase was increased by 20-30% over the control. It was shown that de novo synthesis of PC, which is a major constituent of biological membranes, was accelerated by long-term Cd administration in the liver but not in the kidney. These results suggest the possibility of regenerating the membranes in damaged hepatocytes after 6 and 12 months of Cd administration.     

Effects of liver regeneration on tRNA contents and aminoacyl-tRNA synthetase activities and sedimentation patterns.

The tRNA content and aminoacyl-tRNA synthetases of regenerating liver in the phase of rapid growth were compared with those of livers from both intact and sham-operated rats. At 48 h after hepatectomy, the amount of active tRNA (called 'total acceptor capacity') is significantly higher in regenerating liver than in control livers, owing to a general, possibly not uniform, increase in the various tRNA families, which suggests that it may contribute to the increased protein synthesis and to decreased protein degradation as well. The activities of most, but not of all, aminoacyl-tRNA synthetases in cell sap of regenerating liver tend to be greater than normal. Increased activity of histidyl-tRNA synthetase fits in with the possibility that the mechanisms that control the rate of protein degradation through aminoacylation of tRNAHis in cultured cells [Scornik (1983) J. Biol. Chem. 258, 882-886] also operate in the liver and play a role in regeneration. Sedimentation analysis of cell sap in sucrose density gradients shows a shift of prolyl-tRNA synthetase activity toward the high-Mr form in regenerating liver. This change might be related to the positive protein balance and to growth in vivo, since it is also observed in the anaplastic Yoshida ascites hepatoma AH 130.     

Intranuclear distribution of mouse liver nuclear DNA polymerase

Adult mouse liver nuclei and their subfractions corresponding to heterochromatin, nucleoli, membranes, and euchromatin were studied for DNA-polymerase activity. The intact nuclei and the two heavy nuclear fractions contained rather low activity while the two light fractions (membranes and euchromatin) had no activity at all. In the two heavy fractions, the activity was stimulated by β-mercaptoethanol and depressed by p-hydroxymercuribenzoate and by omission of one or more nucleotides. A nuclease activity, detected in the intact nuclei, may also be present in the nuclear subfractions. DNA-polymerase activity in the heavy fractions of mouse liver nuclei is discussed in relation to other published results.     

A characterization of gamma-glutamyltranspeptidase in normal, perinatal, premalignant and malignant rat liver

gamma-Glutamyltranspeptidase displays the following order of activity in tissues of the Fischer 344 rat: kidney much greater than small intestine much greater than cerebral cortex = testis greater than lung much greater than liver = heart. The activity of the hepatic enzyme in rats is: 4-fold higher in females than males; 4-fold higher in male Wistar, Sprague-Dawley and Zucker rats than male Fischer 344 rats; increased 10-fold in very old vs young male Fischer 344. The hepatic enzyme displays significant species variation: the mouse and rat liver enzymes are similar and low in activity, while duck, dog, pig and beef enzymes are 7, 13, 86 and 92-fold higher, respectively, in activity than the male Fischer rat liver enzyme. A liver plasma membrane isolation procedure has been devised which selects for the sinusoidal face of the liver parenchymal cell as assessed by marker enzyme analysis: for these plasma membranes the purification of gamma-glutamyltranspeptidase is 21.5 and the recovery is 42% indicating that this is the cellular and subcellular locus of the enzyme in rat liver. The characteristics of the liver plasma membrane from female rats are: pH optimum of 8.0; classical Michaelis-Menten kinetics; Km of 1.43 mM and Vmax of 33.3 nmol X mg-1 X min-1. In Fischer 344 rats, gamma-glutamyltranspeptidase activities are elevated over adult levels in perinatal liver: in fetal liver homogenates and plasma membranes the activities are increased 179 and 109-fold, respectively. The activity peaks just after birth and declines rapidly over the first 15 postnatal days. The activity of the liver enzyme in the male Fischer 344 rat exhibits a progressive increase throughout diethylnitrosamine-induced hepatocarcinogenesis: it is increased 7.8-fold in homogenates and 5.4-fold in plasma membranes at the early premalignant stage; 74-fold in homogenates and 31-fold in plasma membranes at the later hyperplastic nodular premalignant stage; and 174-fold in homogenates and 61-fold in plasma membranes at the hepatoma stage. The gradual drop in purification during hepatocarcinogenesis is associated with the appearance of the enzyme in the blood.     

Effect of propylthiouracil on liver regeneration in rats after partial hepatectomy.

The effect of propylthiouracil (PTU) on the growth activity of intact liver and liver regenerating after partial (65-70%) hepatectomy (PH) was studied in rats. PTU (Propycil, Kali-Chemie, FRG) was dissolved in drinking water (1 g PTU per litre) and this was given to the rats, as their sole source of fluids, three days before PH and then up to the end of the experiment. In rats given PTU, marked inhibition of liver DNA synthesis and the mitotic activity of hepatocytes was found after PH. This effect was potentiated to some extent by partial inanition of the experimental animals given PTU, as demonstrated in a paired feeding test in control rats. PTU inhibition of DNA synthesis in intact and regenerating liver also took effect in thyroidectomized rats, even with substitution (thyroid hormone) therapy. The experiments demonstrated that the effect of propylthiouracil on DNA synthesis in the liver is mediated primarily by way of its direct effect on the liver.     

The heme biosynthetic pathway in the regenerating rat liver. The relation between enzymes of heme synthesis and growth

Enzymes of heme synthesis, porphyrins and heme content of regenerating rat livers were examined. During the first three days of regeneration the weights of livers of one-third and two-third hepatectomized rats increased 1.5-fold and 2.7-fold and the activity of porphobilinogen deaminase increased 2-fold and 4-fold and was inversely correlated with ferrochelatase activity. delta-Aminolevulinic acid synthase and delta-aminolevulinic acid dehydratase activities were reduced. Concomitantly an increase in the concentration of porphyrins and a decrease in that of heme were observed. The changes in the biosynthetic pathway of heme during rapid growth of the liver are discussed.     

CFUs of the normal and regenerating liver in the sexually mature mouse

Functional properties of CFUs have been studied in intact and regenerating liver of mice. According to a number of properties (proliferative activity, character of colony growth) CFUs in the liver are similar to CFUs in the peripheral blood and, probably, make the same population. In the regenerating liver relative contents of CFUs 3-5 days after a partial resection is substantially increasing. Concentration of CFUs (endogenic) increases significantly also in a locally injured and regenerating lobe, comparing to the intact lobe that is in the same organ. The transplanted bone marrow CFUs prevail in number in the regenerating liver over the intact liver. It is concluded that increasing contents of CFUs in the regenerating liver depend mainly on its creased property to invade and/or to hold the extrahepatic CFUs.     

Characteristics of hepatocyte proliferation in the regenerating rat liver with hydroxyurea synchronization of the proliferative processes

5-week old rats underwent a partial hepatectomy (2/3 of the liver was removed). The operated rats were repeatedly injected hydroxyurea, within 9-24 or 12-24 hours following operation. Proliferation of hepatocytes and non-parenchymal cells in the regenerating liver was studied. Five or six injections of hydroxyurea increased the labelling index of hepatocytes up to 62.5% and induced proliferation of bile duct cells significantly.