Heavy metal chelator TPEN attenuates fura-2 fluorescence changes induced by cadmium, mercury and methylmercury.
Heavy metal chelator TPEN attenuates fura-2 fluorescence changes induced by cadmium, mercury and methylmercury.
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DOI：
10.1292/jvms.15-0620
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发表时间：
2016-06-01
期刊：
影响因子：
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通讯作者：
Shiraishi M
中科院分区：
文献类型：
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作者：
Ohkubo M;Miyamoto A;Shiraishi M
已知重金属刺激会诱导许多细胞类型中的钙 (Ca2+) 动员。负载 Ca2+ 指示剂 fura-2 的细胞中的重金属对细胞内 Ca2+ 浓度测量的干扰是一个持续存在的问题。在本研究中，我们利用特定的细胞渗透性重金属螯合剂TPEN分析了重金属对人SH-SY5Y神经母细胞瘤细胞中fura-2荧光比率的影响。氯化锰 (30–300 µM) 不会导致 fura-2 荧光比率发生显着变化。高浓度 (300 µM) 的醋酸铅导致 fura-2 荧光比率略有升高。相反，用氯化镉、氯化汞或甲基汞 (3-30 µM) 刺激会导致 fura-2 荧光比率明显升高，且呈剂量依赖性。在用 10 或 30 µM 氯化镉刺激的细胞中，添加 TPEN 将升高的 fura-2 荧光比率降低至基础水平。在用汞或甲基汞刺激的细胞中，TPEN 的添加显着降低了较低浓度 (10 µM) 汞或甲基汞引起的 fura-2 荧光比的升高，但不降低较高浓度 (30 µM) 引起的。使用 Ca2+ 通道阻滞剂（例如维拉帕米、2-APB 或氯化镧）进行预处理，会对 fura-2 荧光比产生不同的影响。我们的研究描述了几种重金属对二价阳离子动员的影响以及重金属对神经元细胞的毒性。
Stimulation with heavy metals is known to induce calcium (Ca2+) mobilization in many cell types. Interference with the measurement of intracellular Ca2+ concentration by the heavy metals in cells loaded with Ca2+ indicator fura-2 is an ongoing problem. In this study, we analyzed the effect of heavy metals on the fura-2 fluorescence ratio in human SH-SY5Y neuroblastoma cells by using TPEN, a specific cell-permeable heavy metal chelator. Manganese chloride (30–300 µM) did not cause significant changes in the fura-2 fluorescence ratio. A high concentration (300 µM) of lead acetate induced a slight elevation in the fura-2 fluorescence ratio. In contrast, stimulation with cadmium chloride, mercury chloride or MeHg (3–30 µM) elicited an apparent elevation of the fura-2 fluorescence ratio in a dose-dependent manner. In cells stimulated with 10 or 30 µM cadmium chloride, the addition of TPEN decreased the elevated fura-2 fluorescence ratio to basal levels. In cells stimulated with mercury or MeHg, the addition of TPEN significantly decreased the elevation of the fura-2 fluorescence ratio induced by lower concentrations (10 µM) of mercury or MeHg, but not by higher concentrations (30 µM). Pretreatment with Ca2+ channel blockers, such as verapamil, 2-APB or lanthanum chloride, resulted in different effects on the fura-2 fluorescence ratio. Our study provides a characterization of the effects of several heavy metals on the mobilization of divalent cations and the toxicity of heavy metals to neuronal cells.