Modulation of 5-fluorouracil metabolism by thymidine. In vivo and in vitro studies on RNA-directed effects in rat liver and hepatoma.

The effects of thymidine (TdR) co-administration on the cytotoxicity and incorporation of 5-fluorouracil (5-FU) into RNA of various tissues was studied in rats bearing an ascites hepatoma (AH 130). The role of pyrimidine degradation in determining the modulating effects of TdR on the formation of FU-RNA was studied in hepatocytes and AH 130 cells in vitro. TdR (500 mg/kg) potentiated the antitumour effect of 5-FU (150 mg/kg) and also increased host toxicity as judged by changes in body weight. TdR given alone did not significantly affect tumour growth and body weight gain. Examination of the effect of TdR on the incorporation of 5-FU into RNA revealed a differential modulation of RNA-directed toxicity in different tissues. Incorporation of 5-FU into RNA in tumour and bone marrow was increased 2- and 4-fold, respectively. In spleen and kidney the incorporation increased by approximately 50%, but the values did not reach statistical significance. In contrast, the incorporation into RNA of liver and intestinal mucosa was decreased to ca 35% of the control. TdR at concentrations of 40 microM-40 mM progressively inhibited the degradation of 5-FU and decreased the incorporation of 5-FU into RNA of hepatocytes in vitro. In AH 130 cells in vitro TdR did not significantly influence the metabolism of 5-FU and the incorporation into RNA. These results demonstrate that the enhanced incorporation of 5-FU into tumour RNA in vivo after pretreatment with TdR is related not to local effects on the tumour cells but rather to an increased bioavailability of the drug. Although co-administration of TdR did not selectively enhance the antitumour effect of 5-FU, a differential toxicity in host tissues was indicated by the modulated incorporation of 5-FU into RNA.

Effect of N-phosphonacetyl-L-aspartate and D-glucosamine on the incorporation of 5-fluorouridine into normal tissues and an adenocarcinoma in the rat.

Cytostatic treatment of liver metastases from colorectal cancer has been of limited value. Higher drug levels in the target substances of the tumor may improve the results. It was the aim of this investigation to examine the effect of PALA (N-phosphonacetyl-L-aspartate) and D-glucosamine on the level of uracil nucleotides in the liver and in an N-methyl-N-nitrosoguanidine-induced adenocarcinoma of the colon transplanted to the liver of rats, and on the incorporation of 3H-FUrd into the acid-soluble fraction, the RNA and the DNA of the tumor and of several normal tissues. Combined treatment with PALA and D-glucosamine reduced the UTP pool in the liver and the tumor. D-glucosamine alone increased UDP-N-acetyl-hexosamine in liver tissue. Pretreatment with PALA and D-glucosamine increased incorporation of 3H-FUrd into RNA of the liver and kidney, and into the DNA fraction of the liver, but had no effect on 3H-FUrd incorporation in the tumor.

Metabolism of uridine and determination of liver ribonucleic acid synthesis in developing and adult mice.

The metabolism of [5-3H]uridine and the incorporation of the precursor into liver RNA was studied in developing (13-day-old) and adult (45-day-old) mice. Different time-courses of labelling and increased amounts of labelled catabolic products of uridine were found in liver and blood of developing mice compared with adult animals. This is suggested to be a consequence of enlarged metabolite pools resulting from a lower total amount of uracil-degrading enzymes in the developing mice. The labelling of the uracil nucleotides was decreased in the developing liver. However, in spite of a lower specific radioactivity of UTP, the RNA-specific radioactivity of developing liver was increased compared with adult liver. Also the labelling of liver RNA with [6-14C]orotic acid was found to be increased in developing mice, thus indicating a higher rate of RNA synthesis in these animals. A more pronounced difference in liver RNA labelling between the developing and the adult mice obtained with the use of [14C]orotic acid than with [3H]uridine may suggest that the de novo pathway, relative to the salvage pathways, is more important in developing than in adult liver.

Incorporation of orotic acid into nucleotides and RNA in mouse organs during 60 minutes.

Mouse kidney and liver were found to increase their levels of radioactivity above that of serum from 2 to 60 min after administration of [6-14C]orotic acid. In spleen, thymus and brain, the radioactivity level reached a maximum soon after the injection and then decreased, as did that in serum. Sixty minutes after the injection, 44% of the administered isotope dose was found in the kidneys, 22% in the liver and 0.75% in the spleen. The 14C activity in liver UTP increased rapidly and then remained constant for 60 min. The ratio between the activities in uridine phosphates and UDP-sugars was 3:4 from 10- 60 min after injection. In the liver and kidneys, the RNA 14C activities at 60 min after injection were 15% of the activity in their acid-soluble fractions. Intraperitoneal administration was found to be preferable to intravenous administration for studies on nucleotides and RNA in mouse liver, due to the delayed incorporation of the [14C]orotic acid activity into the nucleotide pool.

Serum and liver radioactivity levels in mice after intraperitoneal and subcutaneous injection of [14C]orotic acid.

[14C]Orotic acid was rapidly distributed in blood after both i.p. and s.c. injection but was not completely absorbed from the peritoneal cavity until 20 min after injection. S.c. injection should be an acceptable alternative to i.p. injection although the incorporation into the liver acid soluble- and RNA-fractions was somewhat delayed after the s.c. injection.

Utilization of labelled uridine, cytidine and orotic acid for determination of ribonucleic acid synthesis in mouse liver.

[3H]uridine and [3H]orotic acid were equally utilized for labelling of RNA in mouse liver. Incorporation of [3H]cytidine was 2-3 times as high as that of [3H]-labelled uridine or orotic acid. These results differ from findings in rat liver, where both cytidine and orotic acid are better utilized for RNA labelling than is uridine. The ratio between liver RNA [3H]-activity and volatile [3H]-activity was 2, 3 and 13, respectively, at 300 min after injection of labelled uridine, orotic acid and cytidine, indicating an efficient chanelling of cytidine into liver anabolic pathways.

RNA synthesis in regenerating mouse liver evaluated by incorporation of [methyl-14C]methionine and by determination of RNA polymerase activity.

The synthesis of RNA during mouse liver regeneration was studied by two different methods at 24 and 48 h after partial hepatectomy. Total chromatin-bound RNA polymerase activity showed an increase of 32% at 24 h after partial hepatectomy. At 48 h a slight increase in total activity was also observed in regenerating liver, but the difference was not significant. The increase in total RNA polymerase activity was due to a rise in RNA polymerase I plus III activity. This enzyme activity was increased at both 24 and 48 h. The increase was 57% at 24 h and 51% at 48 h. When [methyl-14C]methionine was used for labelling of methyl groups in rRNA, there was an increased specific radioactivity of this class of RNA at both 24 h and 48 h. The increases were 263 and 103% at 24 and 48 h respectively. Thus both methods revealed an increased synthesis of rRNA during mouse liver regeneration. The results are discussed in relation to previous results from this laboratory [Yngner, Carlberg, Lewan & Engelbrecht (1979) Hoppe-Seyler's Z. Physiol. Chem. 360, 1069-1074; Yngner, Engelbrecht, Lewan & Annerfeldt (1979) Biochem. J. 178, 1-8; Yngner, Bengtsson, Carlberg, Engelbrecht & Wieslander (1980) Exp. Cell. Biol. 48, 393-403], which have shown that the incorporation of orotic acid or uridine into RNA is not increased in mouse liver regenerating after partial hepatectomy.

Increased urinary polyamine excretion during liver regeneration.

Tumor growth is a process associated with both cell proliferation and cell death. The increase in polyamine excretion observed in cancer patients may be partly due to leakage of polyamines from proliferating cells, which all contain an elevated polyamine level. However, the increased polyamine excretion may also be due to a release of polyamines from dead or damaged cells. To determine if actively proliferating cells release polyamines, the urinary polyamine excretion was measured during a proliferative event associated with minimal cell necrosis. Rats subjected to partial hepatectomy were used as an experimental model. Their 24-hr urines were collected during 6 consecutive days following the operation. Rat liver regeneration is characterized by a proliferation wave with a maximum 24 hr after the operation. The 24-hr urinary putrescine excretion reached a maximum 2 days after the operation and then decreased. The 24-hr urinary spermidine excretion increased during the second day following operation and remained essentially unchanged during the rest of the experimental period. Although there is an apparent correlation between elevated urinary polyamine excretion and the proliferative activity, concurrent permeability changes and necrotic events may contribute to the increase in polyamine excretion.

Interrelations between uptake of [14C]orotic acid and labeling of UTP and RNA in rat and mouse liver after partial hepatectomy.

The uptake and utilization of [6-14C]orotic acid for UTP and RNA synthesis were studied in rat and mouse liver at 24 h after partial hepatectomy. Rat liver concentrated radioactivity relative to blood several-fold better than did mouse liver after both sham-operation and partial hepatectomy. The results showed that in mouse liver, contrary to rat liver, the orotic acid uptake was not increased after the partial hepatectomy. In rat liver, the precursor uptake and the labeling of UTP increased by about 75% whereas the specific radioactivity of RNA increased 2 to 3-fold after the operation, thus indicating an increased RNA synthesis. Mouse liver showed no increased [14C]orotic acid uptake or labeling of UTP or RNA at 24 h after partial hepatectomy.

Anabolism versus catabolism of [5-3H]uridine and its relationship to ribonucleic acid labelling in mouse liver after partial hepatectomy.

The balance between anabolism and catabolism of [5-(3)H]uridine was studied in the mouse after partial hepatectomy. Labelling of RNA and UDP-glucose was determined and evaluated in relation to changes in the specific radioactivity of UTP. The amounts of labelled catabolic products of uridine were increased several-fold in liver and blood after partial hepatectomy. The specific radioactivity of RNA decreased to about 60% of the control value at 6h and was in the same range as that of control liver at 24h after operation. Decreased labelling of RNA and UDP-glucose was attributable to decreased specific radioactivity of UTP. No changes in the size of the UTP pool or in the balance between uridine anabolism and catabolism were found that could explain the decreased specific radioactivity of UTP. Rather, the alterations in the labelling of this metabolite induced by the partial hepatectomy may be related to decreased phosphorylating capacity in the liver cells and/or dilution of the labelled precursor in an expanded uridine pool. The enhanced amounts of uridine catabolic products in liver and blood were probably a consequence of accumulation and altered incorporation of the metabolites from the blood into the liver cells. Despite the increased amounts of labelled catabolic products and the decreased labelling of RNA, the results reported here actually suggest decreased uridine catabolism and slightly increased RNA synthesis in mouse liver after partial hepatectomy. The results stress the importance of proper controls in determination of nucleic acid synthesis and in metabolic studies by use of labelled precursors.

Urinary polyamine excretion as related to cell death and cell proliferation induced by carbon tetrachloride intoxication.

This study addresses the question whether urinary polyamine excretion is related to cell death or cell proliferation. CCl4 intoxication of the rat was used as the experimental model. Treatment with CCl4, a hepatotoxic haloalkane, produces an initial phase of liver cell death succeeded by a regenerative phase of growth, during which the liver is restored. The highest rate of putrescine (and spermidine) excretion occurred during the first 24 hr of CCl4 intoxication and coincided with the period of maximum liver damage. During subsequent liver regeneration the rate of excretion of both polyamines decreased.

Effects of administration routes on the uptake of uridine and 5-fluorouridine into an adenocarcinoma transplanted to rat liver.

In a model of secondary liver cancer in Wistar rats, the effect of different administration routes on the uptake of 3H-uridine into tumor and several normal tissues was studied. The rats were inoculated with a tumor cell suspension in the central liver lobe. Ten days later, they were distributed into four groups with a catheter placed in the gastroduodenal artery, the portal vein, one of the femoral veins, or in the peritoneal cavity. 3H-uridine was injected 46 h later and after an additional 90 minutes the animals were anesthetised and pieces of liver tumor and normal tissues were removed and frozen. The incorporation into the acid-soluble fraction and RNA was analyzed. In a separate experiment, 3H-fluorouridine was administered by the gastroduodenal artery and a comparison was made with the uptake of 3H-uridine. A significantly higher amount of uridine was incorporated into the tumor by the arterial route. The intraportal and intraperitoneal routes were comparable, while a somewhat higher incorporation was found by the systemic route. The consequences of using the different routes upon the incorporation into RNA of tumor and dose-limiting organs are demonstrated. With the aid of this experimental model, it is possible to evaluate further the effect by different manipulations on different drugs regarding the administration route.

Possible significance of changes in the energy metabolism for the release of liver lactate dehydrogenase and for the uptake and incorporation of [14C]-orotic acid into liver ribonucleic acid after partial hepatectomy.

The mouse liver revealed no increased incorporation of [14C]-orotic acid into either the total acid-soluble fraction, the uridine triphosphate or the RNA at 6 and 24 h after partial hepatectomy. In regenerating mouse and rat liver, the concentration of adenosine triphosphate was decreased 15-20% at 6 h, but was in the same range as that of the controls at 24 h. The adenosine monophosphate concentration of mouse liver increased 4-fold and 2-fold at these times after partial hepatectomy, respectively. The results indicate no direct relationship between the energy metabolism and the uptake and incorporation of orotic acid into RNA of regenerating liver. The activity of mouse plasma lactate dehydrogenase 5 (LDH 5) was increased 12-fold at 6 h and 5-fold at 24 h after partial hepatectomy. In rat, the LDH 5 activity was increased 2-fold at 6 h but was not different from that of the controls at 24 h. An increased leakage of LDH 5, possibly related to the decreased energy content of the liver, was thus revealed by the partially hepatectomized mice.