Dirven H A, Dictus E L, Broeders N L, van Ommen B, van Bladeren P J
TNO Nutrition and Food Research Institute, Division of Toxicology, Zeist, The Netherlands.
Cancer Res. 1995 Apr 15;55(8):1701-6.
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 (t1/2 = 3300 min). In the presence of glutathione, the rate of disappearance of thiotepa increased greatly (t1/2 = 282 min). Both monoglutathionyl thiotepa and diglutathionyl thiotepa conjugates were identified by 31P NMR and mass spectrometry. Addition of GST A1-1 (alpha) to an incubation of thiotepa and GSH further increased the rate of disappearance of thiotepa (t1/2 = 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 microM 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. Kms for the GST A1-1 (alpha) and P1-1 (pi) 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.
非酶促和谷胱甘肽S-转移酶（GST）催化的谷胱甘肽（GSH）结合反应被认为是烷基化细胞抑制药物在细胞内失活的一种机制。在本研究中，我们描述了对三功能烷基化剂噻替派（三（1-氮丙啶基）硫化膦）的谷胱甘肽依赖性生物转化的研究。31P NMR研究表明，噻替派在pH 7.4的0.07 M磷酸盐缓冲液中稳定（t1/2 = 3300分钟）。在谷胱甘肽存在下，噻替派的消失速率大大增加（t1/2 = 282分钟）。通过31P NMR和质谱鉴定了单谷胱甘肽基噻替派和双谷胱甘肽基噻替派共轭物。将GST A1-1（α）添加到噻替派和GSH的孵育体系中，进一步提高了噻替派的消失速率（t1/2 = 100分钟），并提高了单谷胱甘肽基噻替派的形成速率。双谷胱甘肽基噻替派的形成速率未改变，表明双谷胱甘肽基噻替派的形成不受GST A1-1催化。通过HPLC进一步研究了纯化的人GST对单谷胱甘肽基噻替派形成的作用。在含有0.2 mM噻替派、1 mM GSH和40 μM GST的孵育体系中，GST A1-1和P1-1使单谷胱甘肽基共轭物的形成比非酶促形成增加了30 - 35倍，而GST A2-2和M1a-1a并未催化该共轭物的形成速率。GST A1-1（α）和P1-1（π）催化形成单谷胱甘肽基噻替派的Km值在5 - 7 mM范围内。由于肿瘤中的pH可能低于正常细胞，因此还研究了GST P1-1催化形成单谷胱甘肽基噻替派的pH依赖性。在所有测试的pH值（范围为5.5 - 8.5）下，均观察到GST P1-1和A1-1对单谷胱甘肽基共轭物形成有显著的催化作用。还研究了GST对替派（三（1-氮丙啶基）氧化膦）单谷胱甘肽基共轭物形成的作用，替派是噻替派形成的主要代谢产物。GST A1-1和P1-1均可使谷胱甘肽共轭物的形成比自发水平增加37 - 46倍，而GST M1a-1a和A2-2同样未提高该共轭物的形成速率。这些研究结果表明，噻替派/替派中的氮丙啶部分是GST A1-1和P1-1的底物。因此，GST催化的噻替派谷胱甘肽结合反应可能是对噻替派产生耐药性的一个重要因素。
