Department of Neurochemistry, Mossakowski Medical Research Centre, Polish Academy of Sciences, Pawinskiego 5, 02-106, Warsaw, Poland.
Department of Neurochemistry, Mossakowski Medical Research Centre, Polish Academy of Sciences, Pawinskiego 5, 02-106, Warsaw, Poland.
Neurotoxicology. 2017 Dec;63:126-136. doi: 10.1016/j.neuro.2017.09.014. Epub 2017 Sep 29.
In the present study, primary cultures of rat cerebellar granule cells (CGC) and the RT Profiler PCR array were used to examine the effect of acutely applied brominated flame retardant tetrabromobisphenol A (TBBPA) on the expression of 84 genes related to the main modes of programmed cell death. CGC, at the 7th day of culture, were exposed to 10 or 25μM TBBPA for 30min. Then, 3, 6, and 24h later, the viability of the cells was examined by the staining with propidium iodide (PI) or using the calcein/ethidium homodimer (CA/ET) live/dead kit, and RNA was extracted for the evaluation of gene expression by RT-PCR. At 3, 6 and 24h after the treatment, the number of viable neurons decreased, according to the PI staining method, to 75%, 58% and 41%, respectively, and with the CA/ET method to 65%, 58% and 28%, respectively. In CGC analyzed 3h after the treatment with 25μM TBBPA or 6h after 10μM TBBPA, the only change in the gene expression was a reduction in the expression of Tnf, which is associated with autophagy and may activate some pro-apoptotic proteins. Six hours after 25μM TBBPA, only 2 genes were over-expressed, a pro-apoptotic Tnfrsf10b and Irgm, which is related to autophagy, and the genes that were suppressed included the anti-apoptotic gene Xiap, the necrosis-related Commd4, pro-apoptotic Abl1, 5 genes involved in autophagy (App, Atg3, Mapk8, Pten, and Snca) and 2 genes that participate in two metabolic pathways: Atp6v1g2 (pro-apoptotic and necrosis) and Tnf (pro-apoptotic, autophagy). Autophagy-related Snca and Tnf remained under-expressed 24h after treatment with 25μM TBBPA, which was accompanied by the over-expression of the pro-apoptotic Casp6, the anti-apoptotic Birc3, 2 genes related to autophagy (Htt and Irgm) and 2 genes (Fas and Tp53) that are involved in both apoptosis (pro-apoptotic) and autophagy. These results show a complex pattern of TBBPA-evoked changes in the expression of the genes involved in the programmed neuronal death, indicating no induction of programmed necrosis, an early suppression of the autophagy and anti-apoptotic genes, followed by a delayed activation of genes associated with apoptosis.
在本研究中，我们使用原代培养的大鼠小脑颗粒细胞（CGC）和 RT Profiler PCR 阵列来研究急性应用溴化阻燃剂四溴双酚 A（TBBPA）对与细胞程序性死亡主要方式相关的 84 个基因表达的影响。CGC 在培养的第 7 天，用 10 或 25μM 的 TBBPA 处理 30min。然后，在处理后 3、6 和 24h，用碘化丙啶（PI）染色或使用 calcein/ethidium homodimer（CA/ET）活/死试剂盒检测细胞活力，并提取 RNA 用于通过 RT-PCR 评估基因表达。PI 染色法显示，处理后 3、6 和 24h，神经元存活率分别下降至 75%、58%和 41%，CA/ET 法显示，神经元存活率分别下降至 65%、58%和 28%。在 TBBPA 处理 25μM 3h 或 10μM 6h 后分析 CGC 的基因表达，唯一的变化是 TNF 基因表达下调，这与自噬有关，可能激活一些促凋亡蛋白。TBBPA 处理 25μM 6h 后，只有 2 个基因过表达，一个是促凋亡的 TNFRSF10B，另一个是与自噬有关的 Irgm，受抑制的基因包括抗凋亡基因 XIAP、坏死相关的 Commd4、促凋亡基因 Abl1、5 个参与自噬的基因（APP、ATG3、MAPK8、PTEN 和 SNCA）和 2 个参与两条代谢途径的基因：ATP6V1G2（促凋亡和坏死）和 TNF（促凋亡、自噬）。TBBPA 处理 25μM 24h 后，自噬相关的 SNCA 和 TNF 仍表达下调，同时促凋亡基因 Casp6、抗凋亡基因 Birc3、2 个与自噬相关的基因（Htt 和 Irgm）和 2 个参与凋亡（促凋亡）和自噬的基因（Fas 和 TP53）过表达。这些结果显示 TBBPA 诱导的与程序性神经元死亡相关基因表达的变化呈现复杂模式，表明未诱导程序性坏死，早期抑制自噬和抗凋亡基因，随后延迟激活与凋亡相关的基因。
