Aims

We had previously reported that addition of putrescine to the culture medium was reported to reduce methylmercury toxicity in C17.2 neural stem cells. Here, we have examined the inhibition of methylmercury-induced cytotoxicity by putrescine using ODC1-overexpressing C17.2 cells.

Materials and methods

We established stable ODC1-overexpressing C17.2 cells and evaluated methylmercury-induced apoptosis by examining the TUNEL assay and cleaved caspase-3 levels. Mitochondria-mediated apoptosis was also evaluated by reduction of mitochondrial membrane potential and recruitment of Bax and Bak to the mitochondria.

Key findings

ODC is encoded by ODC1 gene, and putrescine levels in ODC1-overexpressing cells were significantly higher than in control cells. Overexpression of ODC1 and addition of putrescine to the culture medium suppressed DNA fragmentation and caspase-3 activation, which are observed when apoptosis is induced by methylmercury. Moreover, mitochondrial dysfunction and reactive oxygen species (ROS) generation, caused by methylmercury, were also inhibited by the overexpression of ODC1 and putrescine; pretreatment with ODC inhibitor, however, promoted both ROS generation and apoptosis by methylmercury. Finally, we found that Bax and Bak, the apoptosis-promoting factors, to be increased in mitochondria, following methylmercury treatment, and the same was inhibited by overexpression of ODC1. These results suggest that overexpression of ODC1 may prevent mitochondria-mediated apoptosis by methylmercury via increase of putrescine levels.

Significance

Our findings provide important clues to clarify mechanisms involved in the defense against methylmercury toxicity and suggest novel biological functions of putrescine.

中文翻译：

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由于ODC1过表达而增加的腐胺水平可防止甲基汞诱导的线粒体功能障碍相关凋亡。

目的

我们以前曾报道过，在培养基中添加腐胺会降低C17.2神经干细胞中甲基汞的毒性。在这里，我们研究了使用过表达ODC1的C17.2细胞对腐胺对甲基汞诱导的细胞毒性的抑制作用。

材料和方法

我们建立了稳定的ODC1过表达的C17.2细胞，并通过检查TUNEL分析和裂解的caspase-3水平评估了甲基汞诱导的细胞凋亡。线粒体介导的细胞凋亡还通过降低线粒体膜电位以及将Bax和Bak募集到线粒体来评估。

主要发现

ODC由编码ODC1基因，并在腐胺水平ODC1 -overexpressing细胞显著比对照细胞高。ODC1的过表达和向培养基中添加腐胺抑制了DNA片段化和caspase-3活化，这在甲基汞诱导细胞凋亡时观察到。此外，ODC1的过表达也抑制了由甲基汞引起的线粒体功能障碍和活性氧（ROS）的产生。和腐胺; 但是，使用ODC抑制剂进行预处理可以通过甲基汞促进ROS的产生和细胞凋亡。最后，我们发现在甲基汞处理后，线粒体中的凋亡促进因子Bax和Bak会增加，而ODC1的过表达抑制了线粒体中的凋亡。这些结果表明，ODC1的过表达可能通过增加腐胺水平来阻止甲基汞介导的线粒体介导的凋亡。

意义

我们的发现提供了重要的线索，以阐明参与防御甲基汞毒性的机制，并提出腐胺的新型生物学功能。