XI1 - 4 310

UPTP,RE OF NUCLEIC ACID PRECURSORS IN ?AT BRAIN CORTEX SLICES IN VITRO

C.S. Kjeldsen, Department of Neurology, Hvidovre Hospital, Copenhagen, Denmark.

Purine and pyrimidine nucleotide synthesis in brain is based on the use of simple metabolites (de novo synthesis) or precursors which are supplied to the brain from extraneural tissues (sal- vage pathway). The nucleotide metabolism in brain has to be con- sidered in the evaluation of drugs for brain cancer chemothera- py, since many of these drugs exert their action on the nucleo- tide and nucleic acid metabolism in cancer cells. Biochemical tests for prediction of chemotherapeutic sensitivity in treat- ment of cancer have mostly consisted in enzyme studies or 2 vivo studies on whole animals. The present study suggests that brain cortex slices form a suitable test system in which the mechanisms of antimetabolite action on nucleotide and nucleic acid metabolism can be studied on intact brain cells without the influence of the blood-brain barrier. The incorporation of nucleic acid precursors into the nucleotide pool in brain cells was investigated by measuring the uptake and metabolism of 14-C-labelled purine base and nucleoside in rat brain cortex slices in vitro. Adult male Wistar rats were deca- pitated without anesthesia. One slice of 0.5 mm thickness was cut fgom the lateral surface of each hemisphere and incubated at 37 C in 4 ml of bicarbonate Ringer solution aerated with 0 +CO ( 9 5 : 5 ) . The slices were preincubated for 40 min., with u6- labglled base or nucleoside present in the medium for the last 10 min. before addition of 14-C-labelled hypoxanthine, guanine, adenine or adenosine. The slices were exposed to the label for 1, 2.5, 5, 10 or 30 min. The uptake of isotope was measured by scintillation counting, and the metabolism of the labelled com- pounds by the tissue was evaluated by thin layer chromatography. Results are expressed as space, l / l O O mg tissue. Values ex- ceeding 85 , i ~ l / l O O mg (corresponLng to tissue water) indicate concentrative uptake or metabolic transformation. Table 1. shows the time course of 14-C-labelled purine base or nucleoside uptake by the brain cortex slices. Adenine is rapid- ly taken up by the tissue, and since almost no ,free adenine (difference between total uptake and nucleotide fraction) is present in the tissue, the phosphorylation of adenine to AMP must be responsible for this rapid uptake. Uptake and metabo- lism of hypoxanthine and guanine are slower than the adenine uptake, and free hypoxanthine or guanine is present extra- as well as intracellularly in concentrations equal to the the ex- ternal medium concentration after 30 rain. exposure to isotope. Hypoxanthine and guanine are phosphorylated to nucleotides by the same enzyme, but the nucleotide formation from hypoxanthine is much more rapid than the guanine phosphorylation. The nucleo- side adenosine is converted to AMP by adenosine kinase, and the rate of the reaction seemsnot to be SO fast as the phosphoryla- tion of adenine. The inhibitory action of tvupurine analogues, mercaptopurine (MP) and mercaptopurine riboside (MPR) on base and nucleoside membrane transport and phosphorylation was also tested. The up- take and metabolism of adenine and adenosine were not influenced

3 1 1

Table 1. Total uptake and metabolism of 14-C-labelled purines, expressed as space,~1/100 mg wet weight, of rat brain cortex slices exposed to the isotope for 1-30 minutes. Medium concen- trations of unlabelled hypoxanthine, guanine, adenine or ade- nosine, 2 uM; of mercaptopurine ( M P ) , 1 mM; of mercaptopurine riboside (MPR), 5 mM. Results are means 2 S.D. of 5-17 experi- ments.

14-C-adenine, total uptake Nucleotide fraction

14-C-adenosine, total uptake Nucleotide fraction

14-C-hypoxanthine, total uptake

Inhibition by MP,% Inhibition by MPR,%

Nucleotide fraction

Inhibition by MP,% Inhibition by MPR,%

14-C-guanine, total uptake Nucleotide synthesis

1

50.9 515.0 36.7 26.0

30.6 28.9 18.3 21.4

34.9 25.3

5.9 22.8

28.7 26.3 9.2

23.5

Time of exposure to

2.5

100.8 +17.0 90.9 22.6

60.1 26.4 45.3 21.4

49.3 216.4

6.1 21.8

37.3

2.7 27.5

20.9

5

176.0 272.2 158.0 +9.0

112.9 +19.3 83.2 28.2

59.9 +26.4

-

9.5 24.3

55.0 +6.9 15.5 +2.3

isotopes , min 10 30

409.1 259. I 377.6 24.5

198.8 251.4 144.7 212.1

117.6 204.6 226.7 256.7

24.6% 40.1% 22.8% 29.3%

35.9 118.3 +14.2 218.8

62.2% 89.8% 64.1% 77.3%

70.9 101.0

8.9 19.2 22.2 27.0

211.2 +9.1

by the drugs (results not presented). Howeger, the presence of 1 mM mercaptopurine in the medium inhibited the phosphorylation of hypoxanthine 89.8% (30 min. value). The total uptake was on- ly inhibited 40.1%, indicating that the membrane transport was not influenced. The use of the riboside even at a concentration of 5 mM seems to be no advance over the use of the base, since the inhibition of the hypoxanthine phosphorylation by the two drugs is equal. These results show that mercaptopurine acts as a hypoxanthine analogue in brain, but tell nothing about the exact mechanism of its action. Mercaptopurine may compete with hypoxanthine in the phosphorylation reaction, or it may actbya feed-back mechanism after being converted to mercaptopurine monophosphate. The results also show that mercaptopurine would be a poor anticancer drug in the therapy of brain cancer. It has been shown that mercaptopurine and perhaps the.riboside penetrate the blood-brain barrier, but the metabolic effects of the two drugs are small at pharmacological concentrations, and only become maximal at toxic concentrations.