INVOLVEMENT OF HUMAN GLUTATHIONE-S-TRANSFERASE ISOENZYMES IN THE CONJUGATION OF CYCLOPHOSPHAMIDE METABOLITES WITH GLUTATHIONE

Alkylating agents can be detoxified by conjugation with glutathione (G SH). One of the physiological significances of this lies in the observ ation that cancer cells resistant to the cytotoxic effects of alkylati ng agents have higher levels of GSH and high glutathione 5-transferase (GST) activity. However, little is known about the GSH-/GST-dependent biotransformation of alkylating agents, including cyclophosphamide. C yclophosphamide becomes cytostatic after the enzymatic formation of 4- hydroxycyelophosphamide. The ultimate alkylating species formed from c yclophosphamide is phosphoramide mustard. In this paper we describe th e 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 glut athionyl conjugates of cyclophosphamide, i.e., 4-glutathionylcyclophos phamide (4-GSCP) and monochloromonoglutathionylphosphoramide mustard. When 0.1 mM 4-hydroxycyclophosphamide and 1 mM GSH was incubated in th e presence of 10 mu M GST A1-1, A2-2, M1a-1a, and p1-1 the formation o f 4-GSCP was 24-fold increased above the spontaneous level. Enzyme kin etic analysis demonstrated the lowest K-m, (0.35 mM) for GST A1-1. K-m values for the other GST enzymes ranged from 1.0 to 1.9 mM. Glutathio ne S-transferase A1-1 (40 mu M) also increased the conjugation of phos phoramide mustard and GSH (both 1 mM) 2-fold, while the other major hu man isoenzymes, A2-2, M1a-1a, and P1-1, did not influence the formatio n of monochloromonoglutathionylphosphoramide mustard. These results in dicate that only one enzyme within the class of human GST Lu enzymes w as able to catalyze the reaction of the aziridinium ion of phosphorami de mustard with glutathione. Thus increased levels of GST A1-1 in tumo r 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 o f 4-GSCP either as a transport form of activated cyclophosphamide or a s a detoxification product is discussed.