Print Email Facebook Twitter The role of human glutathione S-transferase isoenzymes in the formation of glutathione conjugates of the alkylating cytostatic drug thiotepa Title The role of human glutathione S-transferase isoenzymes in the formation of glutathione conjugates of the alkylating cytostatic drug thiotepa Author Dirven, H.A.A.M. Dictus, E.L.J.T. Broeders, N.L.H.L. van Ommen, B. van Bladeren, P.J. Instituut CIVO-Toxicologie en Voeding TNO Publication year 1995 Abstract 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. Subject NutritionGlutathioneGlutathione reductaseTepaThiotepaConformational transitionDrug conformationDrug conjugationDrug protein bindingDrug resistanceEnzyme activityPriority journalChromatography, High Pressure LiquidFemaleGlutathioneGlutathione TransferaseHumanIsoenzymesKineticsLiverMagnetic Resonance SpectroscopyPlacentaPregnancySupport, Non-U.S. Gov'tThiotepa To reference this document use: http://resolver.tudelft.nl/uuid:48238acc-95cc-42bf-9ee9-7c0de05e6aa0 TNO identifier 232885 ISSN 0008-5472 Source Cancer Research, 55 (8), 1701-1706 Document type article Files To receive the publication files, please send an e-mail request to TNO Library.