Division of Life Science, Graduate School of Science and Engineering, Saitama University.
J Toxicol Sci. 2014 Apr;39(2):217-29. doi: 10.2131/jts.39.217.
A widely-used plasticizer di(2-ethylhexyl) phthalate (DEHP) is known to induce apoptosis in neurons, although the mechanisms responsible for DEHP-induced apoptosis is not well explored yet. We recently showed that exposure to DEHP increases the expression of hemeoxygenase (HO)-1, an oxidative stress related enzyme, in the mice brain. In this study, we investigated whether HO-1 is involved in DEHP-induced apoptosis using a mouse neuroblastoma cell line Neuro-2a, which forcibly express SCAT3, a fluorescent indicator of caspase-3 activity. The doses of DEHP at 1, 10 or 100 µM were used in the present study to mimic the level of human exposure to DEHP. Live image analysis of SCAT3-expressing Neuro-2a cells revealed that caspase-3 activity in the cells was significantly increased by DEHP at 100 µM but not 1 or 10 µM. We measured HO-1 mRNA level in Neuro-2a cells exposed to DEHP and found significant increase in HO-1 mRNA level by DEHP at 100 µM but not 1 or 10 µM. Live image analysis of SCAT3-expresisng Neuro-2a cells was further performed to determine the effects of HO-1 siRNA in DEHP-induced apoptosis via caspase-3 activation. We found that knockdown of HO-1 gene nullifies the effects of DEHP to activate caspase-3. These results suggest that HO-1 is involved in DEHP-induced apoptosis. Moreover, this study demonstrates that high-dose DEHP exposure induces caspase-3-dependent apoptosis, which is at least partially mediated by the up-regulation of HO-1 gene, in Neuro-2a cells.
一种广泛使用的增塑剂邻苯二甲酸二（2-乙基己基）酯（DEHP）已知可诱导神经元凋亡，尽管 DEHP 诱导凋亡的机制尚未得到充分探索。我们最近表明，暴露于 DEHP 会增加小鼠大脑中血红素加氧酶（HO）-1的表达，HO-1 是一种与氧化应激相关的酶。在这项研究中，我们使用一种表达 SCAT3 的小鼠神经母细胞瘤细胞系 Neuro-2a 来研究 HO-1 是否参与 DEHP 诱导的凋亡，SCAT3 是一种 caspase-3 活性的荧光指示剂。本研究使用 1、10 或 100µM 的 DEHP 剂量来模拟人类接触 DEHP 的水平。SCAT3 表达的 Neuro-2a 细胞的实时图像分析显示，细胞中的 caspase-3 活性在 100µM 的 DEHP 作用下显著增加，但在 1 或 10µM 的 DEHP 作用下没有增加。我们测量了暴露于 DEHP 的 Neuro-2a 细胞中的 HO-1 mRNA 水平，发现 100µM 的 DEHP 显著增加了 HO-1 mRNA 水平，但 1 或 10µM 的 DEHP 没有增加。进一步对 SCAT3 表达的 Neuro-2a 细胞进行实时图像分析，以确定 HO-1 siRNA 在通过 caspase-3 激活的 DEHP 诱导凋亡中的作用。我们发现，HO-1 基因的敲低消除了 DEHP 激活 caspase-3 的作用。这些结果表明 HO-1 参与了 DEHP 诱导的凋亡。此外，这项研究表明，高剂量 DEHP 暴露通过 caspase-3 依赖性凋亡诱导，至少部分是通过 HO-1 基因的上调介导的，在 Neuro-2a 细胞中。
