Muylle Frederik, Robbens Johan, De Coen Wim, Timmermans Jean-Pierre, Blust Ronny
Department of Biology, Laboratory for Ecophysiology, Biochemistry and Toxicology, University of Antwerp, Belgium.
Comp Biochem Physiol C Toxicol Pharmacol. 2006 Jun;143(2):242-51. doi: 10.1016/j.cbpc.2006.02.008. Epub 2006 Feb 28.
The cDNA of a zinc transporter-1 (ZnT-1) gene was cloned from an established cell line derived from common carp (Cyprinus carpio) using rapid amplification of cDNA ends (RACE). Using real-time quantitative PCR, we showed that both zinc (Zn) and cadmium (Cd) transiently upregulate ZnT-1 mRNA to comparable levels. The loosely bound cellular Zn pool, as estimated using the Zn-specific probe FluoZin-3, was increased threefold after exposure to 250 microM ZnCl(2). Correspondingly, the ZnT-1 mRNA level at 24 h was induced about fivefold, reflecting the need for more zinc export capacity. Total cellular zinc levels were not different from the controls after 72 h of exposure to 10, 50, or 250 microM ZnCl(2). A loss of total cellular Zn but little labile zinc changes were observed with up to 25 microM Cd. At 72 h, the total Zn was partially restored to the control levels, only 1 microM Cd allowed for a full recovery. Downregulation of ZnT-1 mRNA and partial loss of loosely bound Zn were observed with 50 microM Cd. Our results clearly show that although Zn and Cd can both regulate Zn export in EPC cells, the effects on the cellular Zn pools are quite different.
利用cDNA末端快速扩增技术（RACE），从鲤科鲤属鲤鱼建立的细胞系中克隆出锌转运蛋白1（ZnT-1）基因的cDNA。通过实时定量PCR，我们发现锌（Zn）和镉（Cd）均可瞬时上调ZnT-1 mRNA至相当水平。使用锌特异性探针FluoZin-3估算，暴露于250微摩尔氯化锌（ZnCl₂）后，松散结合的细胞内锌池增加了三倍。相应地，24小时时ZnT-1 mRNA水平诱导增加约五倍，反映出需要更多的锌输出能力。暴露于10、50或250微摩尔氯化锌（ZnCl₂）72小时后，细胞内总锌水平与对照组无差异。暴露于高达25微摩尔镉时，细胞内总锌减少，但不稳定锌变化不大。72小时时，总锌部分恢复至对照水平，仅1微摩尔镉可实现完全恢复。观察到50微摩尔镉可使ZnT-1 mRNA下调，松散结合的锌部分丧失。我们的结果清楚地表明，虽然锌和镉均可调节EPC细胞中的锌输出，但对细胞内锌池的影响差异很大。
