Effects of extracellular purines on cytotoxicity of methotrexate

Nucleoside and base modulation of the cytotoxicity of nucleic acid and folate antimetabolite drugs has been widely discussed. Many investigators have observed reduced toxicity due to circumvention of drug-induced inhibition of de novo purine and pyrimidine synthesis. However, exogenous purine nucleosides and bases may also enhance the cytotoxicity of even moderate concentrations of antifolate drugs (MTX and PTX) which inhibit dihydrofolate reductase. In this study, the effects of nucleosides in the medium on the cytotoxicity and deoxyribonucleoside triphosphate pools after brief exposure of cultured cells to methotrexate have been studied in cultured L1210 murine leukaemia cells.

Cell viability was determined by trypan blue exclusion assay. Colony formation was assessed by microtitration cloning assay. The deoxyribonucleotides were measured by a modification of the DNA polymerase assay. Purines were extracted with trioctylamine and 1,1,2-trichlorotrifluoroethane buffer and concentrations of purine bases were determined by HPLC.

Subculture of drug-treated cells in fresh medium containing 10% FCS led to greater toxicity than sub culture in 'conditioned' medium, i.e. fresh medium in which logarithmically growing cells had been cultured for 24 h before separation. Cells resuspended in fresh medium had increased dATP and sustained inhibition of dTTP levels, while cells subcultured in 'conditioned' medium had no elevation of dATP. Hypoxanthine concentration determined by HPLC in 'conditioned' medium was 0.9 microM compared to 6.7 microM in fresh medium. Resuspension of drug-treated cells in conditioned medium supplemented with 10 or 100 microM HX enhanced cytotoxicity and increased the dATP levels.

These results add further evidence that purines present in normal culture conditions are important determinants of methotrexate cytotoxicity. Elevation of dATP levels after methotrexate treatment is an important modulator of cytotoxicity.