Selection and characterization of 5-fluoroorotate-resistant Plasmodium falciparum.
Previous studies have shown that 100 nM 5-fluoroorotate (5-FO) is sufficient to block the in vitro proliferation of Plasmodium falciparum without causing toxicity to mammalian cells. In anticipation of potential drug resistance, a study was undertaken to identify P. falciparum cells that would proliferate in the presence of 5-FO. About 3 x 10(6) UV-irradiated as well as nonirradiated parasites were subjected to a one-step selection with 100 nM 5-FO both in the absence and in the presence of preformed pyrimidines (uracil, uridine, thymine, and thymidine). The P. falciparum cells that emerged after 3 weeks were cloned, and the 90% inhibitory concentration of 5-FO for the cloned cells was found to be 100- to 400-fold greater than that for the parent cell line. Two clones that were further characterized retained resistance to 5-FO even after prolonged propagation in culture without drug pressure. Since the mutants were not cross-resistant to 5-fluorouracil or to dihydrofolate reductase inhibitors, it was unlikely that alteration of thymidylate synthase or overproduction of the bifunctional dihydrofolate reductase-thymidylate synthase was responsible for 5-FO resistance. Similarly, resistance was not due to the expression of a pyrimidine salvage pathway since the cells were not pyrimidine auxotrophs, they did not show increased utilization of pyrimidine nucleosides, and they did not show increased susceptibility to 5-fluoropyrimidine nucleosides. When the selection experiments were repeated, without mutagenesis, in the presence of 10(-7) M 5-FO with fewer than 10(6) parasites or in the presence of more than 10(-7) M 5-FO with more than 10(8) parasites, viable mutants could not be recovered from the cultures. The implications of these findings for the in vivo use of 5-FO for malaria chemotherapy are discussed.
Rathod
P K
PK
Department of Biology, Catholic University of America, Washington, DC 20064.
Khosla
M
M
Gassis
S
S
Young
R D
RD
Lutz
C
C
D000818:Animals; D004351:Drug Resistance; D005472:Fluorouracil / Q000494:pharmacology; D009963:Orotic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000528:radiation effects; D011743:Pyrimidines / Q000378:metabolism; D014466:Ultraviolet Rays
D011743:Pyrimidines; D009963:Orotic Acid; C001242:5-fluoroorotic acid; D005472:Fluorouracil