Folate Depletion Induced by Methotrexate Affects Methionine Synthase Activity and Its Susceptibility to Inactivation by Nitrous Oxide TORUNN FISKERSTRAND, PER MAGNE UELAND and HELGA REFSUM

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 mM) was associated with a doseand 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. MTX is an antifolate drug that inhibits dihydrofolate reductase, the enzyme responsible for the regeneration of tetrahydrofolate from dihydrofolate. This effect is associated with depletion of cellular reduced folates, and thereby inhibition of numerous folate-dependent processes. Impeding of the thymidylate synthase and enzymes involved in purine biosynthesis impairs DNA synthesis, which probably explains the cytotoxicity of MTX (Bertino, 1993). Met synthase (5-methyltetrahydrofolate-homocysteine methyltransferase, EC 2.1.1.13) catalyzes a folate-dependent reaction, in which 5-methyltetrahydrofolate (5-methyl-THF) functions as methyldonor, thereby converting Hcy to Met. Cobalamin (Cbl) serves as cofactor in this reaction. Because Met synthase operates at the point of convergence of folate, Cbl and sulfur amino acids (Finkelstein, 1990), impaired function of this enzyme may have secondary effects on many cellular processes, including S-adenosylmethionine-dependent methylation reactions and polyamine synthesis. The anesthetic agent nitrous oxide is the only drug which has been reported to directly inhibit Met synthase, and it probably oxidizes enzyme-bound cob(I)alamin formed during the catalytic cycle (Banerjee and Matthews, 1990). Notably, this effect of nitrous oxide may account for its diverse biological effects, including the megaloblastic changes in human bone marrow (Nunn, 1987; Amess et al., 1978), antileukemic effect reported in patients (Ikeda et al., 1989; Eastwood et al., 1963) and experimental animals (Ermens et al., 1989b; Abels et al., 1990) and altered metabolism and plasma level of Hcy and related sulfur compounds (Nunn, 1987; Ermens et al., 1991a; Christensen et al., 1994). The side effects reported for nitrous oxide (Nunn, 1987) point to significant biological consequences of Met synthase inhibition. Depletion of 5-methyl-THF by MTX (Bunni 1988, Ermens 1991b, Baram 1987) suggests that some effect of this antifolate drug may be related to inhibition of Met synthase. Furthermore, because the action of both nitrous oxide and MTX converges on 5-methyl-THF metabolism, one may expect a dynamic interaction between these two drugs (Ueland et al., 1986b; Goldhirsch et al., 1987; Ermens et al., 1991b). The aim of our work was to investigate changes of the Met synthase system secondary to the folate depletion induced by MTX, and to compare these changes to those caused by niReceived for publication October 4, 1996. 1 This work was supported by grants from the Norwegian Cancer Society and the Norwegian Research Council. ABBREVIATIONS: MTX, methotrexate; Met, methionine; THF, tetrahydrofolate; Hcy, homocysteine; Cbl, cobalamin; Thd, thymidine; Hx, hypoxanthine; DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphate-buffered saline; HPLC, high-performance liquid chromatography. 0022-3565/97/2823-1305$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 282, No. 3 Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 282:1305–1311, 1997