Biosynthesis of cytidine nucleotides in rat liver after administration of D-galactosamine

Chem.-BioL Interactions, 35 (1981) 217--228 © Elsevier/North-Holland Scientific Publishers Ltd.

217

BIOSYNTHESIS O F CYTIDINE NUCLEOTIDES IN R A T L I V E R A F T E R ADMINISTRATION O F D-GALACTOSAMINE

J. SEIFERT and E. BUCHAR Institute o f Pharmacology, Czechoslovak Academy o f Sciences Albertov 4, 128 O0 Prague 2 (Czechoslovakia)

(Received July 20th, 1980) (Revision received December 23rd, 1980) (Accepted January 6th, 1981)

SUMMARY Following the administration o f D-galactosamine the utilization of [2-14C]orotic acid for the synthesis of the cytidine components o f the acidsoluble extract and liver R N A cytosine is markedly decreased. The depression of the specific activity of the cytidine components takes place after application o f low doses of the drug which do n o t interfere with the specific activity of the uridine components of the acid-soluble extract or of liver R N A uracil. Simultaneously the administration of [U-14C]cytidine paralleled by its enhanced liver uptake. The total a m o u n t of uridine as well as cytidine components of the acid-soluble extract following the administration of D-galactosamine increases; however, the molar ratio of both pyrimidines does not change. The alterations of the cytidine metabolism after the administration of the drug are accompanied b y the increased level of microsomal c y t o c h r o m e P-450.

INTRODUCTION The administration of D-galactosamine to rats results in the impairment of the hepatocyte function; the morphological changes in cells resemble those during viral hepatitis in man [1--4]. Of the biochemical consequences of the treatment with this substance the most remarkable is the inhibition of liver R N A synthesis. The mechanism underlying this change is the depletion of cell UTP since this substrate of R N A polymerase reaction is being taken up for the synthesis o f UDP-hexosamines [ 5--8]. UTP is also a precursor of CTP synthesis. This terminal product of the synthesis of pyrimidine ribonucleotides is also an immediate precursor in the synthesis of R N A and of cytidine cofactors o f phospholipids biosynthesis. Simultaneously, it gives rise to CDP in the cells; the reduction o f its sugar moiety leads to dCDP from which dCTP and dTTP, t w o precursors o f DNA synthesis, are formed.

218 The synthesis of CTP is enhanced in tissues with elevated DNA synthesis [9] as well as in tumors; the increase can be correlated with the t u m o r growth rate of various hepatomes [10]. In addition CTP is a substmte from which cyclic CMP is formed. This cyclic pyrimidine m o n o p h o s p h a t e seems to be related more closely to cell proliferation than the metabolism of cyclic AMP and cylic GMP [11,12]. This paper describes changes in the metabolism o f cytidine components in rat liver where the concentration of cellular UTP is reduced b y D-galactosamine. MATERIALS AND METHODS Male rats (Wistar strain) weighing 150--170 g were used. The pelleted diet (produced by Velaz, Prague) was composed of the following ingredients: wheat (60%), dried fat milk (11%), casein (15%), wheat germs (4%), soy beans (2%), dried lucerne flour (4%), dried yeast (0.6%), CaCO3 (1.6%), Plastin (0.8%) and Konvit (1%). The latter t w o ingredients represent an additional source of minerals and vitamins. The diet can be classified as low in fat (5.5%). ( F o o d and water were given ad libitum). D-Galactosamine (Serva Feinbiochemica, 200 mg/kg of b o d y wt.) was dissolved in 0.9% NaC1 and injected intraperitoneally in a volume of 0.3 ml. [2-14C]Orotic acid (spec. act., 47 /~Ci//~mol) and [U-14C]cytidine (spec. act., 290 /~Ci/~mol) were product of the Institute for Research, Production and Uses of Radioisotopes, Prague. For the in vivo application the radioisotopes were diluted with 0.9% NaC1 and injected intraperitoneaUy in a volume of 0.3 mh Each experimental group consisted of 3 animals; for each determination at least 2 groups of animals were used. The determination of the concentration and specific activity of the acidsoluble pyrimidine components of the liver, the specific activity of R N A uracil and cytosine was assayed as previously described [13]. C y t o c h r o m e P-450 was assayed according to Omura and Sato [14]. The difference spectrum was recorded in Unicam model 1800 spectrophotometer. The quantity of protein in the mierosomal suspension was determined according to Lowry et al. [15] using bovine serum albumin as a standard. In experiments with labeled cytidine the radioactivity values present in the extracellular space of the liver were subtracted from the total values of specific activity determined in the acid-soluble extracts. The correction was based on the assumption that the liver contains a b o u t 17% (w/w) of extracellular fluid whose radioactivity is approximately the same as the radioactivity of blood plasma. The radioactivity of blood plasma was measured in Instagel Packard (0.2 ml of blood plasma + 3.0 ml of Instagel). The results were expressed as means + S.E. The term of uptake denotes the appearance of radioactivity from an exogenous labeled substrate in the cell regardless of its transport mechanism or regardless of its metabolic conversion.

219 RESULTS

After the administration of D-galactosamine to rats (200 mg/kg), the total quantity of uridine and cytidine components of the acid-soluble extract of the liver is increased during the interval of 2--6 h. The molar ratio of the two pyrimidine components remains essentially unchanged (Fig. I). The utilization of [2-14C]orotic acid for the synthesis of the uridine components of the acid-soluble extract is slightly decreased 2--4 h after the administration of D-galactosamine; startingfrom the 6th hour the values are again increased. W h e n the values are recalculated in terms of the increased content of uridine components in 1 g of tissue weight (total specific activity), the values after the administration of D-galactosamine are increased during all the time intervals investigated. The specific activities of the cytidine components of the acid-soluble extract are markedly depressed 2--6 h after the administration of the drug; the depression is marked even after recalculation of the values for the increase of the total quantity of the cytidine components in 1 g of wet tissue weight (Fig. 2A). The changes in the specific activity of R N A uracil are decreased 2---6 h after the administration of the drug. The curve of the specific activity of R N A cytosine is analogous to the curve of the specific activity of the cytidine components of the acid-soluble extract (Fig. 2B).

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225 The total quantity of both uridine and cytidine components of the acidsoluble extract increases as a function of the quantity of D-galactosamine applied. The molar ratio of both pyrimidines remains constant (Table I). The utilization of [2-~4C]oro*Ac acid for the synthesis of the uridine components of the acid-soluble extract decreases starting from a dose of 200 mg/ kg of the drug. After recalculation of the values for the higher concentration of the uridine components in 1 g of liver after the administration of D-galactosamine (total specific activity) the values are higher in all cases. A decrease of the specific activity of the cytidine components can be observed even after the administration of 25 mg/kg of body weight of the drug. The depression of the specific activity is maximal starting from doses of 100 mg/kg. When the values are recalculated for the increased content of the cytidine components of the acid-soluble extract of hepatocytes (total specific activity), the radioactivity values of the cytidine components remains in all cases lower than in the control group (Fig. 3A). A similar course show the pyrimidine components of RNA (Fig. 3B). The [U-~4C]cytidine uptake by the liver is highest 2--6 h after the administration of D-galactosamine and starts to decrease later (Fig. 4A). The utilization of labeled cytidine for the synthesis of RNA cytosine increases 4--6 h after the administration of D-galactosamine, then decreases. The specific activity of RNA uracil is increased starting from the 6th hour after the administration of the drug (Fig. 4B). The [U-Z4C]cytidine uptake markedly increases starting from a dose of 50 mg/kg, body weight D-galactosamine. The uptake remains essentially unaltered over a dose range of 100--400 mg/kg (Fig. 5A). The utilization of labeled cytidine for the synthesis of RNA cytosine significantly decreases starting from a dose of 100 mg/kg of D-galactosamine and reflects the inhibition of RNA synthesis. Analogous changes undergoes also the specific activity of RNA uracil (Fig. 5B). Starving for 24 h increases the utilization of [2-~4C]orotic acid for the synthesis of both the uridine components of the acid-soluble extract and of T A B L E III CYTOCHROME P-450 CONTENT OF LIVER MICROSOMES D - G a l a c t o s a m i n e ( 2 0 0 m g ] k g b o d y w t . ) was i n j e c t e d i n t r a p e r i t o n e a l l y , n = n u m b e r o f d e t e r m i n a t i o n s ; f r o m g r o u p o f 2 animals. Control

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226

RNA uracil. The utilization of this precursor for the synthesis of the cytidine is decreased. The administration of D-galactosamine has the same effect on starving rats as on animals fed ad libitum (Table II). Starting from the 2nd hour after the administration of the drug the level of microsomal cytochrome P-450 is increased (Table III). The addition of D-galactosamine in concentrations of 1.0 and 10 mM to the measuring cell does not affect the values of the difference spectrum of cytochrome P-450. DISCUSSION The administration of D-galactosamine to rats inhibits the synthesis of RNA, proteins, glycoproteins, phospholipids and glycogen in the liver [4--6,8,16]. The inhibition of both rRNA [6] and mRNA [7] synthesis is a result of the depletion of UTP whose concentration in the liver drops below the Kin-range of RNA polymerase [ 5]. It was shown in studies with labeled guanosine that RNA biosynthesis in vivo is decreased by 90% [8] after the administration of D-galactosamine at the dose level of 200 mg/ kg of body weight. The degree of inhibition of RNA synthesis, measured in terms of [2-14C]orotic acid utilization for the synthesis of uridine nucleotides, is less marked. The relative mild decrease of the specific activity of RNA uracil may be explained by the fact, that after the administration of the drug the pool of UTP is reduced [5]; thus if the labelled orotic acid is injected the specific activity of UTP might be higher. The utilization of [2-14C]orotic acid for the synthesis of cytidine components of the acid-soluble extract and RNA cytosine is decreased by several orders more than the utilization for the synthesis of uridine components. The depression of the de novo synthesis of cytidine components of the liver can be caused not only by a decrease of the concentration of cellular UTP below the Km-range for CTP synthetase. The inhibition of the cytidine nucleotide synthesis is not paralleled by a depression of the CTP concentration in the cells. Whereas the UTP concentration drops after the administration of D-galactosamine 13 times the level of CTP is increased twice; the GTP and ATP concentration is not influenced very markedly [5]. The paradox increase of CTP concentration can be accounted for by increased transport from circulation and by its phosphorylation [4,17]. Higher concentrations of CTP can affect also the activity of CTP synthetase [18], the phosphorylation of cytidine and uridine [19] and also the nucleolar RNA synthesis [20]. The cumulation of CTP in the liver [5,17] after the administration of the drug can be caused by its depressed utilization for RNA synthesis under the condition of inhibition of the synthesis, by its decrease degradation, or even by changes in its utilization for the synthesis of the cytidine cofactors of phospholipids synthesis, whose synthesis is likewise inhibited by D-galactosamine [16,21]. It has been observed, that the initial reaction of phospholipid metabolism in brain tissue is inhibited by higher concentrations of cytidine nucleoside polyphosphates, mainly by CTP [22].

227 The experiments with labeled cytidine have shown that the transport of preformed cytidine is markedly increased even after the administration of low D-galactosamine doses. Depending on the time of contact with the drug the increase of the uptake of preformed cytidine is inversely proportional to the decreased utilization of labeled orotic acid for the synthesis of the cytidine components of the acid-soluble extract. The decrease of the utilization of labeled orotic acid for the synthesis of the cytidine components and the increased uptake of the cytidine by the liver after the administration of D-galactosamine are identical with the changes which occur in the liver after the administration of lipid-soluble xenobiotics inducing the mixed-function oxygenases of liver microsomes [23--26]. It is an interesting fact that the changes in the metabolism of the cytidine components of the liver after the administration o f D-galactosamine are also paralleled by a higher cytochrome P-450 level of the microsomes. The rise of the cytochrome P-450 level after the administration of the drugs occurs with simultaneous inhibition of both r R N A [6] and m R N A [7] synthesis. This fact together with the early cytochrome P-450 increases after the administration of D-galactosamine shows that the increase of this cytochrome can be governed by a mechanism other than the classical inductory mechanism. REFERENCES 1 D. Keppler, R. Lesh, W. Reutter and K. Decke~ Experimental hepatitis induced by D-galactosamine, Exp. Mol. Pathol., 9 (1968) 279. 2 R.S. Koff, G. Gordon and S.M. Sabesin, D-Galactosamine hepatitis.I. Hepatocellular injury and fatty liver following a single dose, Proc. Soc. Exp. Biol. Med., 137 (1971) 696. 3 A. Medline, F. Schaffner and H. Popper, Ultrastructural features in galactosamineinduced hepatitis, Exp. Mol. Pathol., 12 (1970) 201. 4 C. Schuchhardt, K. Felgenhauer, T.W. Wagner, H. Enzan and R. Lesch, Adaptive changes of rat liver cells induced by repeated intraperitoneal injection of D-galactosamine. I. Light microscopic, autoradiographic and biochemical studies of parenchyreal alteration, D N A and R N A synthesis, Virchows Arch. B. Cell Pathol., 26 (1977) 43. 5 D.O.R. Keppler, J. Pausch and K. Decker, Selective uridine triphosphate deficiency induced by D-galactosamine in liver and reversed by pyrimidine nucleotide precursors. Effect on ribonucleic acid synthesis,J. Biol. Chem., 249 (1974) 211. 6 J. Herzog and J. Farber, Fibrillar nucleolar remnants do not contain macromolecular precursors of ribosomal R N A . Demonstration by the effects of D-galactosamine, Exp. Cell. Res., 93 (1975) 505. 7 R.D. Reynolds and W. Reutter, Inhibition of induction of rat liver tyrosine aminotransferase by D-galactosamine, J. Biol. Chem., 248 (1973) 1562. 8 H. Shinozuka, J.L. Farber, Y. Konishi and T. Anukarahanonta, D-Galactosamine and acute livercell injury, Fed. Proc., 32 (1973) 1516. 9 D.D. Genchev, Activity of cytidine triphosphate synthetase in normal and neoplastic tissues, Experientia, 29 (1973) 789. 10 J.C. Williams, H. Kizaki, G. Weber and H.P. Morris, Increased C T P synthetase activity in cancer cells,Nature (Lond)., 271 (1978) 71. 11 A. Bloch, Cytidine 3t-5r-monophosphate (cyclic CMP). I. Isolation from extracts of leukemic L-1210 cells,Biochem. Biophys. Res. Commun., 58 (1974) 652.

228 12 D.M. Helfman, N.L. Brackett and J.F. Kuo, Depression of cytidine 3'-5'-cyclic monophosphate phosphodiesterase activity in developing tissues of guinea pigs, Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 4422. 13 J. Seifert and E. Buchar, Increased transport of [U-~4C]cytidine into rat liver after administration of ~-hexachlorocyclohexane, Naunyn-Schmiedeberg's Arch. Pharmacol., 303 (1978) 101. 14 T. Omura and R. Sato, The carbon monoxide-binding pigment of liver microsomes, J. Biol. Chem., 239 (1964) 2370. 15 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265. 16 K. Decker and D. Keppler, Galactosamine induced liver injury, in: H. Popper and F. Schafner (Eds). Progress in Liver Diseases, Vol. IV, Grune & Stratton, New York, 1972, p. 183. 17 H. Henninger, A. Holstege, B. Hermann, T. Anukarahanonta and D.O.R. Keppler, Depletion of cytidine triphosphate as a consequence of cellular uridine triphosphate deficiency, FEBS Lett., 103 (1979) 165. 18 R.P. McPortland and W. Weinfeld, Cooperative effects of CTP on calf liver CTP synthetase, J. Biol. Chem., 254 (1979) 11394. 19 E.P. Anderson and R.W. Brockman, Feedback inhibition of uridine kinase by cytidine triphosphate and uridine triphosphate, Biochem. Biophys. Acta, 91 (1964) 380. 20 Y. Nagamine, D. Mizuno and S. Natori, Inhibitory effects of nucleoside triphosphates on nucleolar RNA synthesis, J. Biochem., 85 (1979) 839. 21 W. Bachmann, E. Harms, B. Hassels, H. Henninger and W. Reuter, Studies of rat liver plasma membrane. Altered protein and phospholipid metabolism after injection of D-Galactosamine, Biochem. J., 166 (1977) 455. 22 F. Possmayer, CDP-choline reversal of CMP and CTP inhibition of phosphatidic acid synthesis by rat brain preparations, Biochem. Biophys. Res. Commun., 61 (1974) 1415. 23 J. Seifert and J. V~cha, Inhibition of [6-~4C]orotic acid incorporation into the cytosine moiety of the ribonucleic acid of rat liver cytoplasmic ribosomes after phenobarbital administration, Mol. Pharmacol., 9 (1973) 259. 24 J. Seifert and J. V~cha, Microsomal inducers of drug-metabolizing enzymes suppress cytidine nucleotide biosynthesis in rat liver, Arch. Biochem. Biophys., 167 (1975) 366. 25 J. Seifert, J. V~cha, E. Buchar and M. Seifertov~, The molecular biology of drug induction: the biosynthesis of pyrimidine nucleotides and nucleic acids after the administration of inducers, in: R.W. Estabrook and E. Lindenlaub (Eds.), The Induction of Drug Metabolism, F.K. Schattauer Verlag, Stuttgart-New York, 1979, p. 81. 26 J. Seifert, J. V~cha and M. Seifertov~, Liver growth, biosynthesis of cytidine nucleotides and level of cytochrome P-450 in rat liver after administration of ~-hexachlorocyclohexane, Chem.-Biol. Interact., 20 (1978) 227.