Exposure to nitrous oxide interferes selectively with the coenzyme function of vitamin B12 and causes inactivation of methionine synthetase, with subsequent impairment of folate metabolism and reduction of cellular proliferation. In a rat leukemia model (BNML) we investigated the combined administration of nitrous oxide, inactivating vitamin B12, and methotrexate (MTX), a folate antagonist inhibiting the enzyme dihydrofolate reductase. Through different mechanisms, both agents decrease the availability of tetrahydrofolate, and subsequently of other reduced folates, with increased impairment of folate-dependent synthesis of thymidylate. Effects on leukemic growth and on hematological values in rats demonstrated enhancement of the therapeutic effect of MTX by exposure to nitrous oxide. With several treatment schedules, the results of combined treatment were seen to be better than additive when compared with the effects of single agents. In particular, pretreatment of leukemic rats with nitrous oxide for 3 days before administration of MTX appeared effective. With higher doses of MTX, concomitant exposure to nitrous oxide even resulted in toxic effects. These findings were in accordance with the results of some metabolic studies performed in leukemic rats.(ABSTRACT TRUNCATED AT 250 WORDS)
Myelo-cytotoxicity of extended nitrous oxide (N2O) inhalation was described almost forty years ago and then incidentally applied already with temporary success for suppressing leukemia. In 1948 the accompanying megaloblastic maturation arrest was explained by inactivation of the methylcobalamin coenzyme and subsequent folate deficiency. We studied the anti-leukemic effect of N2O on a transplantable acute leukemia in B(rown) N(orway) rats. Progression of this B,N,M(yelocytic)L(eukemia) was measured as spleen and liver weights, and leukemic blood cell counts. The deoxyuridine (dU)-suppression test provided in vitro indication of the functional folate activity of leukemic cells. Breathing of N2O-oxygen considerably reduced but did not eradicate, BNML-proliferation. Addition of anti-metabolites, interfering with some enzyme in the folate metabolism beyond the methylcobalamin co-enzyme dependent methionine synthase step, acted at least synergistically. The anti-leukemic effect of cycloleucine, which reduces S-adenosyl-methionine synthesis by inactivation of methionine adenosyltransferase, was moderate but became much stronger with N2O inhalation. Methotrexate, a potent anti-leukemic agent by inhibiting tetrahydrofolate (THF) generation through inactivation of di-HF reductase, became highly anti-BNML, even in low dosage when combined with or preceded by N2O. 5-Fluorouracil, which inhibits methylene-THF dependent thymidilate synthase, itself was surprisingly anti-BNML, but also became much more potent with previous or concomitant N2O exposure. Preliminary dU-suppression test results with human acute leukemia cells, exposed to N2O and/or folate antagonists in vitro, correlated well with the in vivo BNML-experiments. Combining the anticobalamin activity of N2O with an anti-folate therefore seems to be a promising chemotherapeutic approach.
Summary Nitrous oxide (N20) inactivates the vitamin B12-dependent enzyme methionine synthetase with subsequent impairment of folate metabolism and a reduction of cellular proliferation. Indications exist that this effect is antagonized by S-adenosylmethionine (SAM), and it was investigated whether combination with an inhibitor of SAM synthesis, cycloleucine, would result in increased inhibition of growth in rat leukaemia model (BNML). Leukaemic growth was compared in untreated rats, in rats treated with either nitrous oxide/oxygen (1:1) or cycloleucine (50mg kg-1 i.p.), and in rats receiving both agents. Combined treatment resulted in the strongest reduction of leukaemic infiltration in spleen and liver, and this reduction often was more than the added effects of single treatments. Peripheral leukocyte counts were also lowest after combined treatment. The deoxyuridine suppression test, measuring folate-dependent de novo synthesis of thymidine, was more severely disturbed with combined treatment. Levels of vitamin B12 in plasma were reduced in rats receiving N20, but an increase in plasma folate occurred in all treated rats. These results indicate that a reduction of SAM synthesis by cycloleucine can increase the disturbance of folate metabolism that is caused by nitrous oxide, with a potentiation of the effects on leukaemic growth.
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