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Nonenzymatic and glutathione S-transferase (GST) catalyzed glutathione (GSH) conjugation has been postulated as a mechanism by which alkylating cytostatic drugs can be inactivated intracellularly. In this study, we describe studies on the glutathione-dependent biotransformation of thiotepa (tris(1-aziridinyl)phosphine sulfide), a trifunctional alkylating agent. 31P NMR studies showed that thiotepa is stable in 0.07 M phosphate buffer, pH 7.4 (t( 1/2 ) = 3300 min). In the presence of glutathione, the rate of disappearance of thiotepa increased greatly (t( 1/2 ) = 282 min). Both monoglutathionyl thiotepa and diglutathionyl thiotepa conjugates were identified by 31P NMR and mass spectrometry. Addition of GST A1-1 (α) to an incubation of thiotepa and GSH further increased the rate of disappearance of thiotepa (t( 1/2 ) = 100 min) and increased the rate of formation of monoglutathionyl thiotepa. The rate of formation of diglutathionyl thiotepa was not altered, suggesting that the formation of diglutathionyl thiotepa is not catalyzed by GST A1-1. The role of purified human GST on the formation of monoglutathionyl thiotepa was further studied by HPLC. In incubations with 0.2 mM thiotepa, 1 mM GSH, and 40 μM GST, both GST A1-1 and P1-1 enhanced the formation of the monoglutathionyl conjugate 30-35-fold above the nonenzymatic formation, while GST A2-2 and M1a-1a did not catalyze the rate of formation of this conjugate. K(m)s for the GST A1-1 (α) and P1-1 (π) catalyzed formation of monoglutathionyl thiotepa were in the 5-7 mM range. Since the pH in tumors might be lower than in normal cells, the pH dependency of the GST P1-1 catalyzed formation of monoglutathionyl thiotepa was also studied. At all pHs tested (range, 5.5-8.5), a marked catalytic effect of both GST P1-1 and A1-1 on the formation of monoglutathionyl conjugates was noted. The role of GST on the formation of monoglutathionyl conjugates of tepa (tris(1-aziridinyl)phosphine oxide), the major metabolite formed from thiotepa, was also studied. Both GST A1-1 and P1-1 could enhance the formation of the glutathione conjugate 37-46-fold above the spontaneous levels, while GST M1a-1a and A2-2 again did not increase the rate of formation of this conjugate. The results of these studies show that the aziridine moieties in thiotepa/tepa are substrates for both GST A1-1 and P1- 1. Thus, GST catalyzed glutathione conjugation of thiotepa might be an important factor in the development of drug resistance towards thiotepa.

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 μM 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 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. Glutathione S-transferase A1-1 (40 μM) 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 α- 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.

Contains fulltext : mmubn000001_160089344.pdf (Publisher’s version ) (Open Access) Promotores : J. Noordhoek, P. Henderson en F. Jongeneelen 131 p.