Involvement of human glutathione S-transferase isoenzymes in the conjugation of cyclophosphamide metabolites with glutathione.

Alkylating agents can be detoxified by conjugation with glutathione (GSH). One of the physiological significances of this lies in the observation that cancer cells resistant to the cytotoxic effects of alkylating agents have higher levels of GSH and high glutathione S-transferase (GST) activity. However, little is known about the GSH-/GST-dependent biotransformation of alkylating agents, including cyclophosphamide. Cyclophosphamide becomes cytostatic after the enzymatic formation of 4-hydroxycyclophosphamide. The ultimate alkylating species formed from cyclophosphamide is phosphoramide mustard. In this paper we describe the involvement of purified human glutathione S-transferases isoenzymes GST A1-1, A2-2, M1a-1a, and P1-1 in the formation of two types of glutathionyl conjugates of cyclophosphamide, i.e., 4-glutathionylcyclophosphamide (4-GSCP) and monochloromonoglutathionylphosphoramide mustard. When 0.1 mM 4-hydroxycyclophosphamide and 1 mM GSH was incubated in the presence of 10 microM GST A1-1, A2-2, M1a-1a, and P1-1 the formation of 4-GSCP was 2-4-fold increased above the spontaneous level. Enzyme kinetic analysis demonstrated the lowest Km (0.35 mM) for GST A1-1. Km values for the other GST enzymes ranged from 1.0 to 1.9 mM. Glutathione S-transferase A1-1 (40 microM) also increased the conjugation of phosphoramide mustard and GSH (both 1 mM) 2-fold, while the other major human isoenzymes, A2-2, M1a-1a, and P1-1, did not influence the formation of monochloromonoglutathionylphosphoramide mustard. These results indicate that only one enzyme within the class of human GST alpha enzymes was able to catalyze the reaction of the aziridinium ion of phosphoramide mustard with glutathione. Thus increased levels of GST A1-1 in tumor cells can contribute to an enhanced detoxification of phosphoramide mustard and hence to the development of drug resistance. Since all of the human GSTs tested did catalyze the formation of 4-GSCP, the role of 4-GSCP either as a transport form of activated cyclophosphamide or as a detoxification product is discussed.