Folate Depletion Induced by Methotrexate Affects Methionine Synthase Activity and Its Susceptibility to Inactivation by Nitrous Oxide1

Abstract

We compared the effects of methotrexate (MTX) and nitrous oxide on the methionine (Met) synthase system in two variants of a human glioma cell line. The cells were protected from cytotoxic effect of MTX by adding thymidine and hypoxanthine to the cell culture medium. MTX (0–1 μM) was associated with a dose- and time-dependent reduction in 5-methyltetrahydrofolate (5-methyl-THF) in both cell lines. Already after 3 hr of exposure, 5-methyl-THF was reduced by 50% and after additional 48 hr, the level was undetectable. In addition to reduction in folate level, homocysteine (Hcy) remethylation in intact cells was markedly inhibited as judged by an increased export of Hcy from the cells, and Met synthase activity in cell extracts and level of cellular methylcobalamin (CH₃Cbl) declined. MTX reduced Hcy remethylation and CH₃Cbl level more efficiently than nitrous oxide. In both cell variants, the inactivation of Met synthase by nitrous oxide was almost completely prevented in cells pre-exposed to MTX. This indicates that there is no catalytic turnover in cells exposed to MTX, and emphasizes the importance of the sequence of administration for synergistic effect of this drug combination. In conclusion, our data show that MTX through depletion of 5-methyl-THF reduces both the Met synthase activity and the cellular CH₃Cbl level. Moreover, the effect of MTX on the Hcy remethylation is more pronounced than the inhibition caused by nitrous oxide. These observations should be taken into account in studies on MTX pharmacodynamics.