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 glutathion e. P-31 NMR spectroscopy was used to monitor the time courses for the chemical conjugation with glutathione of the CP metabolites 4-hydroxyc yclophosphamide (4-OHCP) and phosphoramide mustard (PM) at 24 degrees C. PM incubated with a 10-fold molar excess of glutathione showed a di sappearance of the PM signal (t(1/2)= 112 min), accompanied by an incr ease of two signals, attributed to the intermediate PM monoglutathione conjugate and the PM diglutathione conjugate. After 680 min, only a s ignal assigned to the PM diglutathione conjugate was found. This conju gate was relatively stable. The formation of the PM diglutathione conj ugate was confirmed with fast atom bombardment mass spectrometry (FAB- MS). The rate constant for the disappearance of the PM signal in incub ations 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 incubation s without glutathione. In addition to the signals assigned to the mono - and diglutathionyl conjugates of PM, four signals were found of whic h the pattern of formation in time was identical. These four signals c orrespond to the four stereoisomers of 4-glutathionylcyclophosphamide (4-GSCP). The formation of 4-GSCP was confirmed with FAB-MS. Within 12 0 min after the start of the reaction no free 4-OHCP or aldophosphamid e signals were found in the spectra. Free PM was detected in all spect ra indicating that degradation of 4-GSCP gives rise to PM, the ultimat e cytotoxic metabolite of CP. 4-GSCP therefore appears an important po ol of phosphoramide mustard, which in turn can be deactivated by gluta thione.