Effects of methotrexate on RNA and purine synthesis of astrocytes in primary culture.

There is increasing evidence to indicate that astrocytes are primary targets for methotrexate (MTX) neurotoxicity. However, the mechanism by which MTX exerts its deleterious effect on astroglial cells is not known. Methotrexate acts by inhibiting dihydrofolate reductase and in other cell systems has been reported to inhibit thymidylate synthesis, purine synthesis or both. To determine the mechanism involved in MTX-induced toxicity to the nervous system, RNA synthesis was studied in two week-old primary astrocyte cultures by measuring [3H]Uridine (Urd) incorporation 24 hours after exposure to varying concentrations of MTX. De novo purine synthesis was also studied by measuring incorporation of [14C]glycine and [14C]formate in cultured astrocytes. The radioactivity level of incorporated Urd in culture decreased to 48%, 53% and 43% after exposure to 1, 10 and 100 microM MTX. Total [14C]glycine incorporation was not affected while incorporation of [14C]formate was almost completely inhibited by MTX. The MTX-induced inhibition of [3H]Urd incorporation was not reversed by concomitant addition of exogenous purine bases (1 and 10 microM adenine, guanine and hypoxanthine) or nucleosides (1 and 10 microM adenosine, guanosine and inosine) to the MTX-treated cultures. On the other hand, addition of formyl-tetrahydrofolate reversed the MTX-induced reduction in [3H]Urd incorporation, indicating that the RNA inhibition was due to depletion of folate-dependent substrates for purine synthesis. Our results provide evidence that inhibition of purine and RNA synthesis may be the underlying mechanism involved in MTX-induced injury to the astrocytes, and may be important in the pathogenesis of MTX encephalopathy.