Moffatt P, Plaa G L, Denizeau F
Département de Pharmacologie, Faculté de Médecine, Université de Montréal, Québec, Canada.
Toxicol Appl Pharmacol. 1996 Jan;136(1):200-7. doi: 10.1006/taap.1996.0025.
Metallothionein (MT) is a small cysteine-rich metal-binding protein involved in Zn and Cu homeostasis as well as in heavy metal detoxication. It is also believed that when MT is overexpressed, it can confer resistance against alkylating agents. However, the mechanisms involved are still poorly understood. The purpose of the present work was to investigate whether metal treatment, which induces MT synthesis, could protect isolated rat hepatocytes against the cytotoxic effects of the alkylating agents methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Exposure to 12.5 microM ZnSO4 for 18 hr raised MT levels approximately 15-fold (as measured by the 109Cd-heme assay). When these cells were exposed to increasing concentrations of MNNG, a significant reduction in cell death (as measured by lactate dehydrogenase leakage into extracellular medium) was observed (LC50 = 468 +/- 20 microM vs 362 +/- 13 microM for control cells). On the other hand, Zn pretreatment was not accompanied by resistance against MMS toxicity. In addition, the synthesis of graded amounts of MT, achieved by incubation with various concentrations of Zn or Cu, led to a high correlation between MT levels and the extent of hepatocyte survival. Cd (another MT inducer) failed to protect hepatocytes from MNNG cytotoxicity. Time-course studies also revealed a good correlation between the onset of MT induction by Zn (> 3 hr) and that of protection against MNNG (> 3 hr). The stability of MT in the presence of MNNG was studied by incubating 109Cd-labeled MT with MNNG and by analyzing the mixture using Sephadex G-75 Chromatography. Direct interaction of MNNG with rabbit liver (Cd,Zn)-MT was demonstrated by the release of 109Cd bound to MT. Similar results were obtained with 109Cd-exposed hepatocytes, 109Cd being redistributed from MT to high-molecular-weight proteins after incubation with MNNG. None of the metals used to induce MT modulated glutathione (GSH) because it remained at control levels after 18 hr. However, within 15 min of incubation, MNNG had completely depleted GSH in both control and Zn-pretreated hepatocytes equally. This was followed by a marked decline in MT levels. Taken together, these results suggest that Zn- and Cu-induced tolerance against killing by MNNG appears to be related to the accumulation of MT. The mechanism of protection might reside in the antioxidant properties of MT and on its ability to scavenge electrophilic species.
金属硫蛋白（MT）是一种富含半胱氨酸的小分子金属结合蛋白，参与锌和铜的体内平衡以及重金属解毒。也有人认为，当MT过表达时，它可以赋予细胞对烷化剂的抗性。然而，其中涉及的机制仍知之甚少。本研究的目的是探讨诱导MT合成的金属处理是否能保护分离的大鼠肝细胞免受烷化剂甲磺酸甲酯（MMS）和N-甲基-N'-硝基-N-亚硝基胍（MNNG）的细胞毒性作用。用12.5 microM硫酸锌处理18小时可使MT水平提高约15倍（通过109Cd-血红素测定法测量）。当这些细胞暴露于浓度不断增加的MNNG时，观察到细胞死亡显著减少（通过乳酸脱氢酶泄漏到细胞外培养基中来测量）（LC50 = 468 +/- 20 microM，而对照细胞为362 +/- 13 microM）。另一方面，锌预处理并未使细胞对MMS毒性产生抗性。此外，通过与不同浓度的锌或铜孵育来合成不同量的MT，导致MT水平与肝细胞存活程度之间具有高度相关性。镉（另一种MT诱导剂）未能保护肝细胞免受MNNG的细胞毒性作用。时间进程研究还揭示了锌诱导MT（> 3小时）的开始与对MNNG的保护（> 3小时）之间具有良好的相关性。通过将109Cd标记的MT与MNNG孵育并使用Sephadex G-75柱色谱分析混合物，研究了MNNG存在下MT的稳定性。MNNG与兔肝（镉，锌）-MT的直接相互作用通过与MT结合的109Cd的释放得到证实。用109Cd处理的肝细胞也得到了类似的结果，与MNNG孵育后，109Cd从MT重新分布到高分子量蛋白质中。用于诱导MT的金属均未调节谷胱甘肽（GSH），因为18小时后其仍保持在对照水平。然而，孵育15分钟内，MNNG使对照和锌预处理的肝细胞中的GSH均完全耗尽。随后MT水平显著下降。综上所述，这些结果表明锌和铜诱导的对MNNG杀伤的耐受性似乎与MT的积累有关。保护机制可能在于MT的抗氧化特性及其清除亲电物质的能力。
