Guo T L, Miller M A, Shapiro I M, Shenker B J
Department of Pathology, University of Pennsylvania School of Dental Medicine, Philadelphia, Pennsylvania, 19104, USA.
Toxicol Appl Pharmacol. 1998 Dec;153(2):250-7. doi: 10.1006/taap.1998.8549.
The major objective of our study was to define the mechanism by which mercuric chloride (HgCl2) induces human T-cell death. Human peripheral blood T-cells were exposed to 0-40 microm HgCl2 and then analyzed for biochemical and molecular features of T-cell apoptosis. HgCl2-treated cells exhibited increased Hoechst 33258 fluorescence while maintaining their ability to exclude the vital stain 7-aminoactinomycin D. To further evaluate cell death and distinguish between apoptosis and necrosis, translocation of phosphatidylserine to the outer layer of the plasma membrane (annexin V binding), DNA fragmentation (TUNEL assay), and cleavage of poly (ADP-ribose) polymerase (PARP) were assessed. In the presence of 20-40 microm HgCl2, T-cells exhibited increased annexin V binding (28%) and DNA fragmentation (31%). HgCl2-dependent PARP cleavage was also observed by Western blot analysis. Because degradative changes associated with apoptosis are often preceded by disruption of mitochondrial function, HgCl2-treated cells were assessed for disruption of the mitochondrial transmembrane potential (DeltaPsim) and development of the mitochondrial permeability transition state. Using DiOC6(3), we demonstrated that HgCl2 exposure resulted in a decrease in the DeltaPsim. Because a decline in DeltaPsim can disturb the intracellular pH (pHi), we used the fluorescent probe, SNARF-1, to assess intracellular acidification. Treatment of T-cells with HgCl2 resulted in reduced pHi from 7.0 to 6.7. Concomitant with these observations, the fluorescent probe, hydroethidine, was utilized to demonstrate that uncoupled mitochondrial electron transport resulted in increased reactive oxygen species (ROS) generation. Interestingly, in spite of these alterations to mitochondrial function, translocation of cytochrome c to the cytosol was not detected; this correlated with enhanced bcl-2 levels in HgCl2-treated cells. In conclusion, HgCl2 exposure results in oxidative stress and activation of death signaling pathways leading to apoptosis. Collectively, our studies indicate that individual mercurial species are capable of inducing T-cell death by activating specific apoptotic cascades.
我们研究的主要目的是确定氯化汞（HgCl2）诱导人T细胞死亡的机制。将人外周血T细胞暴露于0 - 40微摩尔的HgCl2中，然后分析T细胞凋亡的生化和分子特征。经HgCl2处理的细胞Hoechst 33258荧光增强，同时保持其排斥活体染料7-氨基放线菌素D的能力。为了进一步评估细胞死亡并区分凋亡和坏死，我们评估了磷脂酰丝氨酸向质膜外层的易位（膜联蛋白V结合）、DNA片段化（TUNEL检测）以及聚（ADP - 核糖）聚合酶（PARP）的裂解。在存在20 - 40微摩尔HgCl2的情况下，T细胞表现出膜联蛋白V结合增加（28%）和DNA片段化增加（31%）。通过蛋白质印迹分析也观察到了HgCl2依赖性的PARP裂解。由于与凋亡相关的降解变化通常先于线粒体功能的破坏，因此对经HgCl2处理的细胞进行了线粒体跨膜电位（ΔΨm）破坏和线粒体通透性转变状态发展的评估。使用DiOC6(3)，我们证明HgCl2暴露导致ΔΨm降低。由于ΔΨm的下降会干扰细胞内pH（pHi），我们使用荧光探针SNARF - 1来评估细胞内酸化。用HgCl2处理T细胞导致pHi从7.0降至6.7。与这些观察结果一致，荧光探针氢乙锭被用于证明解偶联的线粒体电子传递导致活性氧（ROS）生成增加。有趣的是，尽管线粒体功能发生了这些改变，但未检测到细胞色素c向细胞质的易位；这与经HgCl2处理的细胞中bcl - 2水平升高相关。总之，HgCl2暴露导致氧化应激和死亡信号通路的激活，从而导致凋亡。总体而言，我们的研究表明，单个汞物种能够通过激活特定的凋亡级联反应诱导T细胞死亡。
