A. Čihák

Prolongation of the lag period preceding the enhancement of thymidine and thymidylate kinase activity in regenerating rat liver by 5-azacytidine

Abstract The administration of 5-azacytidine to rats 1 hr after partial hepatectomy leads to a complete inhibition of thymidine and thymidylate kinase in 24 hr-regenerating liver and to a prolongation of the lag period preceding their increased activity. While 5-azacytidine does not affect DNA synthesis in the liver of sham-operated animals, complete inhibition of its formation in 24 hr-regenerating liver was observed. Inhibition of liver protein synthesis following 5-azacytidine has a different time course and is in agreement with the degradation of liver polyribosomes. Maximal polyribosome degradation and the greatest inhibition of protein synthesis in regenerating liver occur 2–8 hr after 5-azacytidine administration. Decreased thymidine and thymidylate kinase activity in relation to the restored proteosynthetic capacity of the liver after 5-azacytidine administration is discussed.

Enhanced uridine kinase and RNA synthesis in regenerating rat liver after 5-azacytidine administration

Abstract The administration of 5-azacytidine to partially hepatectomized rats results in the increase of uridine kinase activity in cell-free liver extracts 24–72 hr after drug administration. At the same time the activity of uridine phosphorylase and of uridine 5′-nucleotidase is decreased, while uridinemonophosphate kinase and uridine 5′-triphosphatase are not affected. The repeated administration of 5-azacytidine leads to a further enhancement of uridine kinase which is 6- to 8-fold higher in 96-hr regenerating livers than in untreated controls. Simultaneously the enhanced incorporation of uridine into liver ribonucleic acids was observed. The metabolic alterations occurring in the liver at later phases after 5-azacytidine in vivo administration are discussed.

Transformation of 5-aza-2′-deoxycytidine-3H and its incorporation in different systems of rapidly proliferating cells

Abstract 5-Aza-2′-deoxycytidine-3H undergoes in Ehrlich ascitic cells phosphorylation and deamination resulting in the formation of new metabolites. In the acid-soluble pool of tetrahydrouridine-treated cells the level of deaminated analogue and of 5-azauracil formed by its phosphorolytic cleavage was substantially decreased. Both in vivo and in cell suspension in vitro 5-aza-2′-deoxycytidine-3H is incorporated into nucleic acids. In AKR mice with lymphatic leukemia the drug is preferentially incorporated into lymphatic tissues. While the blast infiltration of the liver results in a 3–4 fold increase of the uptake of the analogue the incorporation of thymidine-3H is enhanced 12–17 times to compare with the normal livers. Cytosine arabinoside depresses the incorporation of thymidine-3H as well as 5-aza-2′-deoxycytidine-3H. In regenerating rat livers thymidine, deoxycytidine but not 5-aza-2′-deoxycytidine are utilized with a greater efficiency than in the stationary liver. 5-Aza-2′-deoxycytidine is thus incorporated into rapidly proliferating cells while its uptake into lymphatic system is preferential.

Dual effect of 5-azacytidine on the synthesis of liver ribonucleic acids: Lack of the relationship between metabolic transformation of orotic acid in vitro and its incorporation in vivo

Abstract Administration of 5-azacytidine to rats results in inhibition of orotidylic acid decarboxylase in cell-free extracts of liver. Maximal effects were observed 2–8 hr after the administration of the analogue, and enzyme activity returned to control levels 30–36 hr later. The utilization of orotic acid for the synthesis of liver ribonucleic acids was inhibited only at 5–6 hr after drug. At subsequent times its incorporation was markedly enhanced, with maximal increases (400–450 per cent) being noted 18–24 hr after 5-azacytidine. Chromatographic separation of liver RNA isolated 2 and 24 hr after 5-azacytidine on a methylated albumin kieselguhr column did not indicate preferential inhibition or stimulation of the synthesis of individual types of RNA.

Stimulatory effect of cycloheximide and related glutarimide antibiotics on liver uridine kinase

Cycloheximide inhibits protein synthesis in a variety of mammalian cells, including hepatocytes, Lcells, and reticulocytes [l] . The drug prevents the transfer of amino acids from aminoacyl-tRNA to the growing polypeptide chain [2] and evidence has been obtained showing that cycloheximide inhibits both peptide initiation and extension by an effect on the donor site on ribosomes [3]. After the administration of cycloheximide the formation of ribosomes and the synthesis of nuclear 45 S RNA in rat liver are also decreased as a result of the reduced protein synthesis [4]. The mechanism of cycloheximide action is influenced by adrenal secretion [S] ; in adrenalectomized rats no inhibition of amino acid incorporation into liver proteins has been observed [6]. Besides inhibiting protein synthesis cycloheximide increases tyrosine aminotransferase activity [7, 81. It has been concluded that degradation as well as synthesis of the enzyme must be blocked in the cycloheximide-treated animals [9]. In the present report evidence is presented showing that another liver enzyme, uridine kinase, is influenced by the administration of cycloheximide and related glutarimide antibiotics.

Metabolic alterations of liver regeneration. XV. Cadmium-mediated depression of thymidine and thymidylate kinase induction in rats

Cadmium administered shortly before or after partial hepatectomy blocks in a dose-dependent manner the increase of thymidine and thymidylate kinase activities in regenerating rat livers. The effect of cadmium can be partially antagonized by simultaneous zinc administration. The intraperitoneal injection of cadmium is more effective than its subcutaneous administration. While there are in vitro differences in the sensitivity of thymidine kinase and thymidylate kinase towards Cd2+-, Zn2%- and Cu2+-ions, both enzymes are equally depressed following their in vivo administration. Cadmium displays the highest inhibitory activity and resembles in this respect beryllium [1].

Pb-precipitated protein fraction from calf brain containing highly active uridine kinase with different molecular properties

The de novo pyrimidine synthesis as well as pyrimidine nucleotides synthesized from preformed bases by the salvage pathway [l] can serve as a source of pyrimidines. Hogans et al. [2] observed recently that uridine was far superior to erotic acid in labelling RNA in rat brain. Among the tissues studied the preference for uridine over erotic acid for RNA synthesis was unique to neural tissue. Even though the concept that the biogenesis of pyrimidines for RNA synthesis in the whole animal occurs primarily through the de novo synthesis is generally accepted, the results suggest that rat brain utilizes preformed pyrimidines to a much greater extent than it utilizes the de novo pathway to supply its requirements for pyrimidine nucleotides. Uridine kinase is a representative and key enzyme of the salvage pathway [3]. The enzyme has been shown in chick embryo to be of greater importance at later stage of development and organogenesis than during early stages of development [4]. A similar observation was made during the study of a developmental pattern of uridine kinase and enzymes of the de novo pyiimidine biosynthetic pathway in developing rat cerebellum [5]. It was suggested that while the de novo synthesis provides the major source of pyrimidine nucleotides required for RNA synthesis in developing rat brain, mature brain obtains its pyrimidine nucleotides primarily from utilization of preformed pyrimidine bases and nucleotides. In this study an attempt to characterize and partially purify uridine kinase from calf brain is described. Simultaneously an effect of Pb” ions, resulting in an irreversible precipitation of the enzyme without affecting seriously its activity is presented.

Biotransformation and some metabolic effects of 5-(4-aminophenyl)-cytosine.

Abstract 5-(4-Aminophenyl)-cytosine possesses, according to results of clinical trials, good prophylactic activity against influenza A 2 . This contribution presents a survey of transformations of the compound in the organism and of its distribution in the tissues of a variety of animals. In the urine of mice, after the administration of 5-(4-arnmophenyl)-cytosine several metabolites were found, viz.: 5-(4-acetamidophenyl)-cytosine, 5-(3-hydroxy-4-aminophenyl)-cytosine, 5-(3-hydroxy-4-aeetamidophenyl)-cytosine, and traces of 2-hydroxy-4-acetainido-5-(4-acetamidophenyl)-pyrimidine and 5-(3-hydroxy-4-aminophenyl)-cytosine- O -glucuronide. A considerable portion of unchanged 5-(4-aminophenyl)-cytosine, however, was rapidly excreted in the urine and the faeces. When 5-(4-aminophenyl)-cytosine was administered to mice, a two-fold increase of incorporation of orotic-6- 14 C acid, administered simultaneously, was observed. Other 5-arylpyrimidines enhanced the incorporation of orotic-6- 14 C acid which wes utilized for the synthesis of liver ribonucleic acid as well. The incorporation of adenine-8- 14 C or thymidine-7- 14 C was, however, not influenced under identical conditions. While studying the site of inhibitory action it was further observed that the presence of 5-(4-aminophenyl)-cytosine and some other 5-arylpyrimidines was accompanied by a decreased activity of succinate dehydrogenase. The inhibition of succinate dehydrogenase in a cell-free rat liver extract by 5-(4-aminophenyl)-cytosine is of competitive character, the ratio of the Michaelis aed inhibition constants being 1.84.

Transformation of 5-aza-2′-[3H] deoxycytidine in Escherichia coli

5-Aza-2’deoxycytidine has preferential affinity for the lymphatic system [ 1,2] and displays pronounced growth-inhibitory action against various forms of experimental neoplasias [3,4] . Although 20-times less active in Escherichia coli B than 5-azacytidine the deoxy-aza analogue exhibits still remarkable antibacterial effects which can be completely removed by natural pyrimidine precursors [5] . The 5azapyrimidine deoxyribonucleoside is an efficient donor of the deoxyribosyl group promoting in E. coli the deoxyriboside-dependent incorporation of thymine [6]. However, the deamination to 5-aza2’deoxyuridine seems to be necessary for the utilization of the deoxyribose moiety of 5-aza-2’-deoxycytidine since its uptake was lost in bacterial mutants deficient in cytidine deaminase [7] where 5-aza-2’deoxycytidine had practically no inhibitory effect. It was proposed that the analogue enters the cells of E. coli via deamination followed by the phosphorolytic cleavage of the glycosidic bond [7] . The deamination of 5-aza-2’deoxycytidine takes place also in mice and results in the formation of 5-azauracil which has been isolated from the urine of drug-treated animals [2]. Recently the phosphorylation of 5-aza-2’-deoxycytidine to higher 5’-phosphates in parallel with the incorporation of the fraudulent deoxyribonucleotide into DNA was described in different systems of eukaryotic cells [4,8] . The aim of the present study was to follow the metabolic conversion and incorporation of 5-aza-2’-deoxycytidine in E. coli using tritium-labelled drug of high specific radioactivity.