Htike Nang T T, Maekawa Fumihiko, Soutome Haruka, Sano Kazuhiro, Maejima Sho, Aung Kyaw H, Tokuda Masaaki, Tsukahara Shinji
Area of Regulatory Biology, Division of Life Science, Graduate School of Science and Engineering, Saitama University Saitama, Japan.
Center for Health and Environmental Risk Research, National Institute for Environmental Studies Tsukuba, Japan.
Front Neurosci. 2016 Jun 29;10:297. doi: 10.3389/fnins.2016.00297. eCollection 2016.
There is serious concern about arsenic in the natural environment, which exhibits neurotoxicity and increases the risk of neurodevelopmental disorders. Adverse effects of arsenic have been demonstrated in neurons, but it is not fully understood how arsenic affects other cell types in the brain. In the current study, we examined whether sodium arsenite (NaAsO2) affects the cell cycle, viability, and apoptosis of in vitro-cultured astrocytes isolated from the cerebral cortex of mice. Cultured astrocytes from transgenic mice expressing fluorescent ubiquitination-based cell cycle indicator (Fucci) were subjected to live imaging analysis to assess the effects of NaAsO2 (0, 1, 2, and 4 μM) on the cell cycle and number of cells. Fucci was designed to express monomeric Kusabira Orange2 (mKO2) fused with the ubiquitylation domain of hCdt1, a marker of G1 phase, and monomeric Azami Green (mAG) fused with the ubiquitylation domain of hGem, a marker of S, G2, and M phases. NaAsO2 concentration-dependently decreased the peak levels of the mAG/mKO2 emission ratio when the ratio had reached a peak in astrocytes without NaAsO2 exposure, which was due to attenuating the increase in the mAG-expressing cell number. In contrast, the mAG/mKO2 emission ratio and number of mAG-expressing cells were concentration-dependently increased by NaAsO2 before their peak levels, indicating unscheduled S phase entry. We further examined the fate of cells forced to enter S phase by NaAsO2. We found that most of these cells died up to the end of live imaging. In addition, quantification of the copy number of the glial fibrillary acidic protein gene expressed specifically in astrocytes revealed a concentration-dependent decrease caused by NaAsO2. However, NaAsO2 did not increase the amount of nucleosomes generated from DNA fragmentation and failed to alter the gene expression of molecules relevant to unscheduled S phase entry-coupled apoptosis (p21, p53, E2F1, E2F4, and Gm36566). These findings suggest that NaAsO2 adversely affects the cell cycle and viability of astrocytes by inducing unscheduled S phase entry coupled with cell death that may be caused by mechanisms other than apoptosis.
人们对自然环境中的砷深感担忧，砷具有神经毒性并会增加神经发育障碍的风险。砷对神经元的不良影响已得到证实，但砷如何影响大脑中的其他细胞类型尚不完全清楚。在本研究中，我们检测了亚砷酸钠（NaAsO₂）是否会影响从小鼠大脑皮质分离的体外培养星形胶质细胞的细胞周期、活力和凋亡。对表达基于荧光泛素化的细胞周期指示剂（Fucci）的转基因小鼠的培养星形胶质细胞进行实时成像分析，以评估NaAsO₂（0、1、2和4 μM）对细胞周期和细胞数量的影响。Fucci被设计为表达与hCdt1的泛素化结构域融合的单体库萨比拉橙2（mKO2），hCdt1是G1期的标志物，以及与hGem的泛素化结构域融合的单体淡绿荧光蛋白（mAG），hGem是S、G2和M期的标志物。当mAG/mKO2发射率在未暴露于NaAsO₂的星形胶质细胞中达到峰值时，NaAsO₂浓度依赖性地降低了该发射率的峰值水平，这是由于表达mAG的细胞数量增加减弱所致。相反，在mAG/mKO2发射率和表达mAG的细胞数量达到峰值之前，NaAsO₂使其浓度依赖性增加，表明出现了非计划的S期进入。我们进一步研究了被NaAsO₂迫使进入S期的细胞的命运。我们发现，在实时成像结束时，这些细胞中的大多数都死亡了。此外，对星形胶质细胞中特异性表达的胶质纤维酸性蛋白基因拷贝数的定量分析显示，NaAsO₂导致其浓度依赖性降低。然而，NaAsO₂并没有增加由DNA片段化产生的核小体数量，也没有改变与非计划S期进入相关的凋亡相关分子（p21、p53、E2F1、E2F4和Gm36566）的基因表达。这些发现表明，NaAsO₂通过诱导非计划的S期进入以及可能由凋亡以外的机制引起的细胞死亡，对星形胶质细胞的细胞周期和活力产生不利影响。
