Abstract
Development of resistance of cancer cells against cyclophosphamide (CP) is probably associated with an increased conjugation with glutathione. 31P 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°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 62 × 10 −3 min −1, and was 54 × 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.
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