Nucleoside-mediated mitigation of 5-fluorouracil-induced toxicity in synchronized murine erythroleukemic cells.
5-Fluorouracil (5-FU) is a chemotherapeutic agent known to retard embryonic growth and induce cleft palate and limb deformities. The predominant mechanism underlying its toxic action is thought to be inhibition of thymidylate synthetase (TS), and hence thymidine triphosphate (dTTP) synthesis, resulting in alteration of the balance of deoxynucleotide (dNTP) pools and disruption of DNA synthesis. Indeed, previously we demonstrated retarded cell-cycle progression concurrent with a 60% decrease in TS activity in rat whole embryos following maternal exposure to 40 mg/kg 5-FU on Gestational Day 14 and in the murine erythroleukemic cell (MELC) suspension culture following exposure to 5-25 microM 5-FU for 2 hr. In the study described herein, we used high-performance liquid chromatography (HPLC) to demonstrate in both of these model systems that 5-FU exposure results in similar patterns of dNTP perturbations: a prolonged decrease in dTTP and dGTP levels and an increase in dCTP and dATP. In addition, we used centrifugal elutriation to synchronize MELC in the phases of the cell cycle (G0/G1 and early S) most sensitive to 5-FU to investigate the ability of nucleoside supplementation to mitigate 5-FU-induced toxicity. Our data indicate that following a 2-hr exposure to 5-25 microM 5-FU, supplementation with 1-10 microM thymidine (TdR) for 24 hr partially reverses 5-FU-induced toxicity as evidenced by increased cellular proliferation and cell-cycle progression and amelioration of 5-FU-induced perturbations of protein synthesis and cellular membrane permeability compared to unsupplemented 5-FU-exposed cells. However, TdR concentrations >/=100 microM inhibited growth or were cytotoxic. In comparison, supplementation with 10 microM-10 mM of deoxycytidine (CdR) was not toxic, but effected a dose-dependent recovery from 5-FU-induced toxicity. At 1-100 microM, neither deoxyadenosine nor deoxyguanosine supplementation reduced 5-FU-induced toxicity; at higher concentrations, both purine nucleotides inhibited cell growth. Although these results support the hypothesis that 5-FU disrupts the MELC cell cycle by depleting dTTP (a perturbation that is reversible by TdR supplementation), they also indicate that CdR supplementation offers an additional recovery pathway.
Elstein
K H
KH
National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, USA. elstein.kenneth@epamail.epa.gov
Mole
M L
ML
Setzer
R W
RW
Zucker
R M
RM
Kavlock
R J
RJ
Rogers
J M
JM
Lau
C
C
D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000633:toxicity*; D002453:Cell Cycle / Q000187:drug effects; D003841:Deoxycytidine / Q000494:pharmacology*; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D004622:Embryo, Mammalian / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D005472:Fluorouracil / Q000633:toxicity*; D004915:Leukemia, Erythroblastic, Acute / Q000473:pathology*; D051379:Mice; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D013936:Thymidine / Q000494:pharmacology*; D013942:Thymine Nucleotides / Q000378:metabolism
D000964:Antimetabolites, Antineoplastic; D003848:Deoxyguanine Nucleotides; D013942:Thymine Nucleotides; D003841:Deoxycytidine; C029603:deoxyguanosine triphosphate; C024157:thymidine 5'-triphosphate; D005472:Fluorouracil; D013936:Thymidine