Effects of pyrimidine salvage inhibitors on uracil incorporation of <i>Toxoplasma gondii</i>

, Youn, J H; , Nam, H W; , Kim, D J; , Choi, W Y

doi:1990.28.2.79

Korean J Parasito > Volume 28(2):1990 > Article

Original Article


Korean J Parasitol. 1990 Jun;28(2):79-84. English. Published online Mar 20, 1994. http://dx.doi.org/10.3347/kjp.1990.28.2.79
Copyright © 1990 by The Korean Society for Parasitology


Effects of pyrimidine salvage inhibitors on uracil incorporation of Toxoplasma gondii
J H Youn,H W Nam,D J Kim and W Y Choi
Department of Parasitology, Catholic University Medical College, Seoul 131-701, Korea.


Abstract
Metabolic inhibitors which act in the process of pyrimidine salvage influenced on the uracil incorporation into nucleic acids of Toxoplasma. Inhibitors of dihydrofolate reductase, pyrimethamine and methotrexate, and inhibitors of thymidylate synthase, fluoro-uridine, fluoro-dUMP and fluoro-uracil, diminished isotopic uracil uptake in dose-dependent manners. Azauridine which suppresses de novo pyrimidine biosynthesis did not affect the salvage even in a relatively high dose. These results suggested that the activation of uracil salvage should be closely related with the function of TMP biosynthetic enzymes. The pattern of thymidine uptake had no differences between control HL-60 cells and Toxoplasma infected cells, which did not reflect the specific proliferation of Toxoplasma. It can be exploited to characterize the effects of various compounds related with the proliferation of Toxoplasma, especially its DNA synthesis.

Figures


	Fig. 1 Inhibition of uracil incorporation by dihydrofolate reductase inhibitors in Toxoplasma gondii. Various concentrations of inhibitors, pyrimethamine (▪) and methotrexate (□ during the time of co-culture were treated. Effects of inhibitors were described as a percent ratio of isotopic uracil uptake to control which was not treated.


	Fig. 2 Effects of pyrimethamine (•,◦) and methotrexate (▪,▫) on thymidine incorporation in Toxoplasma-infected HL-60 cells (-) and HL-60 cells alone (…).


	Fig. 3 Inhibition of uracil incorporation by thymidylate synthase inhibitors in Toxoplasma gondii. Various concentrations of inhibitors, fluoro-uridine (-▪-), fluoro-dUMP (-▫-) and fluorouracil (-◦-) were treated. Effects of inhibitors were described as a percent ratio of isotopic uracil uptake to control which was not treated.


	Fig. 4 Effects of azauridine on uracil incorporation of Toxoplasma gondii (-•-) and HL-60 cells (-▪-).

Tables


	Table 1 Acute effects of inhibitors with the concentration which diminish the uracil incorporation to 50% of control group

References


1.	Bzik DJ, Li WB, Horii T, Inselburg J. Molecular cloning and sequence analysis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene. Proc Natl Acad Sci U S A 1987;84(23):8360–8364.

2.	Choi WY, Yoo JE, Nam HW, Oh CY, Kim SW, Katakura K, Kobayashi A. Toxoplasma antibodies by indirect latex agglutination tests in zoo animals. Korean J Parasitol 1987;25(1):13–23.

3.	Coderre JA, Beverley SM, Schimke RT, Santi DV. Overproduction of a bifunctional thymidylate synthetase-dihydrofolate reductase and DNA amplification in methotrexate-resistant Leishmania tropica. Proc Natl Acad Sci U S A 1983;80(8):2132–2136.

4.	Ferone R, Roland S. Dihydrofolate reductase: thymidylate synthase, a bifunctional polypeptide from Crithidia fasciculata. Proc Natl Acad Sci U S A 1980;77(10):5802–5806.

5.	Hammond DJ, Gutteridge WE, Opperdoes FR. A novel location for two enzymes of de novo pyrimidine biosynthesis in trypanosomes and Leishmania. FEBS Lett 1981;128(1):27–29.

6.	Hammond DJ, Gutteridge WE. UMP synthesis in the kinetoplastida. Biochim Biophys Acta 1982;718(1):1–10.

7.	Hammond DJ, Gutteridge WE. Purine and pyrimidine metabolism in the Trypanosomatidae. Mol Biochem Parasitol 1984;13(3):243–261.

8.	Kim KA, et al. J Catholic Med Coll 1989;42(2):407–415.

9.	Miller RL, Lindstead D. Purine and pyrimidine metabolizing activities in Trichomonas vaginalis extracts. Mol Biochem Parasitol 1983;7(1):41–51.

*10.*	Morgan K, et al. Parasitol 1974;60(2):364–367.

*11.*	North TW, Wyler DJ. DNA synthesis in promastigotes of Leishmania major and L. donovani. Mol Biochem Parasitol 1987;22(2-3):215–221.

*12.*	Pfefferkorn ER, Pfefferkorn LC. Toxoplasma gondii: characterization of a mutant resistant to 5-fluorodeoxyuridine. Exp Parasitol 1977;42(1):44–55.

*13.*	Pfefferkorn ER, Pfefferkorn LC. Specific labeling of intracellular Toxoplasma gondii with uracil. J Protozool 1977;24(3):449–453.

*14.*	Pfefferkorn ER. Toxoplasma gondii: the enzymic defect of a mutant resistant to 5-fluorodeoxyuridine. Exp Parasitol 1978;44(1):26–35.

*15.*	Schwartzman JD, Pfefferkorn ER. Toxoplasma gondii: purine synthesis and salvage in mutant host cells and parasites. Exp Parasitol 1982;53(1):77–86.

*16.*	Pfefferkorn ER, Eckel ME, McAdams E. Toxoplasma gondii: the biochemical basis of resistance to emimycin. Exp Parasitol 1989;69(2):129–139.

*17.*	Sheffield HG, Melton ML. Effect of pyrimethamine and sulfadiazine on the fine structure and multiplication of Toxoplasma gondii in cell cultures. J Parasitol 1975;61(4):704–712.

*18.*	Wang CC, et al. J Protozool 1975;24(4):564–568.

*19.*	Wang CC, Simashkevich PM. Purine metabolism in the protozoan parasite Eimeria tenella. Proc Natl Acad Sci U S A 1981;78(11):6618–6622.

*20.*	Wang CC, Verham R, Tzeng SF, Aldritt S, Cheng HW. Pyrimidine metabolism in Tritrichomonas foetus. Proc Natl Acad Sci U S A 1983;80(9):2564–2568.