The Reversal of Methotrexate Cytotoxicity to Mouse Bone Marrow Cells by Leucovorin and Nucleosides

The cytotoxic effect of methotrexate (MTX) for mouse bone marrow cells has been studied by in vitro culture of the granulocyte precursor cell (CFU-C) in a medium containing dialyzed fetal calf serum and dialyzed L-cell supernatant. The formation of 50-cell colonies was inhibited to 50% of control by 10-8 m MTX. Further increases in MTX concentration rapidly abolished colony formation by CFU-C. The potential of leucovorin and nucleosides to rescue the CFU-C from MTX toxicity was studied. Toxicity of 10-7 m MTX was completely reversed by equimolar concentrations of leucovorin, but with higher MTX concentrations, relatively more leucovorin was required. While 10-5 m MTX was rescued by 10-3 m leucovorin, rescue of the toxic effect of 10-4 m MTX by 10-3 m leucovorin was not observed. In contrast to the rescue by leucovorin, toxicity of all MTX concentrations up to 10-4 m was completely prevented by 10-5 m thymidine with 10-5 m adenosine, inosine, or hypoxanthine. Single nucleosides or thymidine with guanosine were ineffective, as were lower concentrations (≤ 10-6 m) of the effective combinations. Thus, while leucovorin reversed the MTX toxicity to CFU-C competitively, rescue by nucleosides was noncompetitive. The significance and possible usefulness of these findings for chemotherapeutic protocols are discussed.