Laboratory of Clinical Pharmaceutics, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan.
Laboratory of Bioorganic & Natural Products Chemistry, Kobe Pharmaceutical University, 4-19-1, Motoyama-kita, Higashinada, Kobe 658-8558, Japan.
Metallomics. 2020 Jun 24;12(6):868-875. doi: 10.1039/d0mt00015a.
Copper (Cu) is an essential trace element and acts as a redox cofactor for many enzymes; however, excess Cu is toxic to cells. Hydrogen sulfide (H2S) is a well-known toxic gaseous molecule, but it has various biological effects such as neuromodulation and vasodilation. H2S was recently demonstrated to be involved in the detoxification of heavy metals, including zinc and cadmium, suggesting that H2S helps to maintain the homeostasis of heavy metals in cells. However, it is unclear how H2S impacts cellular Cu dynamics. In this study, we examined the effects of H2S on Cu cytotoxicity. Human neuroblastoma SH-SY5Y cells were exposed to CuSO4 in the presence of the H2S donor NaHS. CuSO4 alone slightly induced cell injury, whereas the combination of CuSO4 and NaHS (Cu/NaHS) increased Cu cytotoxicity. The Cu chelator bathocuproinedisulfonic acid mitigated Cu/NaHS-induced cytotoxicity. Compared with CuSO4 alone, Cu/NaHS markedly promoted ROS generation, mitochondrial dysfunction, and a decrease in ATP production. In addition, reporter assay using the metal responsive element (MRE)-driven reporter plasmid revealed that Cu/NaHS augmented Cu-dependent MRE activation. The amount of intracellular Cu was significantly higher in cells treated with Cu/NaHS than in those treated with CuSO4 alone. Moreover, Cu/NaHS markedly suppressed the level of the Cu exporter ATP7A, but not ATP7B, protein, whereas the combination did not affect that of the Cu importer CTR1 protein. Taken together, we conclude that the marked decrease in the ATP7A protein level by Cu/NaHS promotes intracellular Cu accumulation and leads to increased Cu cytotoxicity.
铜（Cu）是一种必需的微量元素，作为许多酶的氧化还原辅助因子；然而，过量的铜对细胞是有毒的。硫化氢（H2S）是一种众所周知的有毒气态分子，但它具有多种生物学效应，如神经调制和血管扩张。最近的研究表明，H2S 参与了包括锌和镉在内的重金属的解毒，表明 H2S 有助于维持细胞内重金属的内稳态。然而，H2S 如何影响细胞内的 Cu 动力学尚不清楚。在本研究中，我们研究了 H2S 对 Cu 细胞毒性的影响。人类神经母细胞瘤 SH-SY5Y 细胞在 H2S 供体 NaHS 的存在下暴露于 CuSO4。CuSO4 单独作用时轻微诱导细胞损伤，而 CuSO4 和 NaHS 的组合（Cu/NaHS）增加了 Cu 细胞毒性。Cu 螯合剂 bathocuproinedisulfonic acid 减轻了 Cu/NaHS 诱导的细胞毒性。与单独的 CuSO4 相比，Cu/NaHS 显著促进了 ROS 的产生、线粒体功能障碍和 ATP 生成的减少。此外，使用金属反应元件（MRE）驱动的报告质粒进行的报告基因检测显示，Cu/NaHS 增强了 Cu 依赖性 MRE 激活。与单独用 CuSO4 处理的细胞相比，用 Cu/NaHS 处理的细胞中细胞内 Cu 的量明显更高。此外，Cu/NaHS 显著抑制了 Cu 外排蛋白 ATP7A 的水平，但不影响 Cu 输入蛋白 CTR1 的水平，而这种组合对 Cu 输入蛋白 CTR1 的水平没有影响。总之，我们的结论是，Cu/NaHS 显著降低 ATP7A 蛋白水平促进了细胞内 Cu 的积累，并导致 Cu 细胞毒性增加。
