THE INTERACTION OF GLUTATHIONE WITH 4-HYDROXYCYCLOPHOSPHAMIDE AND PHOSPHORAMIDE MUSTARD, STUDIED BY P-31 NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY

Development of resistance of cancer cells against cyclophosphamide (CP) is probably associated with an increased conjugation with glutathione. P-31 NMR spectroscopy was used to monitor the time courses for the chemical conjugation with glutathione of the CP metabolites 4-hydroxycyclophosphamide (4-OHCP) and phosphoramide mustard (PM) at 24 degrees C. PM incubated with a 10-fold molar excess of glutathione showed a disappearance of the PM signal (t(1/2)= 112 min), accompanied by an increase of two signals, attributed to the intermediate PM monoglutathione conjugate and the PM diglutathione conjugate. After 680 min, only a signal assigned to the PM diglutathione conjugate was found. This conjugate was relatively stable. The formation of the PM diglutathione conjugate was confirmed with fast atom bombardment mass spectrometry (FAB-MS). The rate constant for the disappearance of the PM signal in incubations with glutathione was 6.2 x 10(-3) min(-1), and was 5.4 x 10(-3) min(-1) in incubations without glutathione, indicating that the rate-limiting step in both reactions is the formation of aziridinium ions. When 4-OHCP was incubated with a 10-fold molar excess of glutathione, six signals were found which were not present in spectra of incubations without glutathione. In addition to the signals assigned to the mono- and diglutathionyl conjugates of PM, four signals were found of which the pattern of formation in time was identical. These four signals correspond to the four stereoisomers of 4-glutathionylcyclophosphamide (4-GSCP). The formation of 4-GSCP was confirmed with FAB-MS. Within 120 min after the start of the reaction no free 4-OHCP or aldophosphamide signals were found in the spectra. Free PM was detected in all spectra indicating that degradation of 4-GSCP gives rise to PM, the ultimate cytotoxic metabolite of CP. 4-GSCP therefore appears an important pool of phosphoramide mustard, which in turn can be deactivated by glutathione.