Qian Yongchang, Venkatraj Jijayanagaram, Barhoumi Rola, Pal Ranadip, Datta Aniruddha, Wild James R, Tiffany-Castiglioni Evelyn
Department of Integrative Biosciences, Texas A&M University, College Station Center for Environmental and Rural Health, College Station, TX 77843-4458, USA.
Toxicol Appl Pharmacol. 2007 Mar;219(2-3):162-71. doi: 10.1016/j.taap.2006.11.030. Epub 2006 Dec 6.
The objective of this study was to evaluate the comparative non-cholinergic neurotoxic effects of paraoxon, which is acutely neurotoxic, and diisopropyl fluorophosphate (DFP), which induces OPIDN, in the human neuroblastoma SY5Y and the human astrocytoma cell line CCF-STTG1. SY5Y cells have been studied extensively as a model for OP-induced neurotoxicity, but CCF cells have not previously been studied. We conducted a preliminary human gene array assay of OP-treated SY5Y cells in order to assess at the gene level whether these cells can distinguish between OP compounds that do and do not cause OPIDN. Paraoxon and DFP induced dramatically different profiles of gene expression. Two genes were upregulated and 13 downregulated by at least 2-fold in paraoxon-treated cells. In contrast, one gene was upregulated by DFP and none was downregulated at the 2-fold threshold. This finding is consistent with current and previous observations that SY5Y cells can distinguish between OPs that do or do not induce OPIDN. We also examined gene array results for possible novel target proteins or metabolic pathways for OP neurotoxicity. Protein levels of glucose regulated protein 78 (GRP78) revealed that paraoxon exposure at 3 microM for 24 h significantly reduced GRP78 levels by 30% in neuroblastoma cells, whereas DFP treatment had no effect. In comparison with SY5Y neuroblastoma cells, paraoxon and DFP (3 microM for 24 h) each significantly increased GRP78 levels by 23-24% in CCF astrocytoma cells. As we have previously evaluated intracellular changes in Ca(2+) levels in SY5Y cells, we investigated the effects of paraoxon and DFP on cellular Ca(2+) homeostasis in CCF by studying cytosolic and mitochondrial basal calcium levels. A significant decrease in the ratio of mitochondrial to cytosolic Ca(2+) fluorescence was detected in CCF cultures treated for either 1 or 3 days with 1, 3, 10, or 30 microM paraoxon. In contrast, treatment with DFP for 1 day had no significant effect on the ratio of mitochondrial to cytosolic Ca(2+) fluorescence; after 3 days treatment, only 30 microM decreased the ratio. These results are consistent with the finding that paraoxon induced a greater decrease than did DFP of intracellular esterase activity in CCF cells. The changes seen in the ratio of mitochondrial to cytosolic Ca(2+) represent a good indicator of the degree of injury induced by each chemical tested. This work further develops in vitro models that distinguish between compounds that cause OPIDN and those that induce acute neurotoxicity only. The study also exposes additional OP-induced toxicities that may be obscured in vivo.
本研究的目的是评估对氧磷（急性神经毒性）和二异丙基氟磷酸酯（DFP，可诱发有机磷中毒性神经病，OPIDN）在人神经母细胞瘤SY5Y细胞和人星形细胞瘤细胞系CCF-STTG1中的非胆碱能神经毒性作用。SY5Y细胞作为有机磷（OP）诱导神经毒性的模型已得到广泛研究，但CCF细胞此前尚未被研究过。我们对经OP处理的SY5Y细胞进行了初步的人类基因芯片分析，以便在基因水平评估这些细胞能否区分导致和不导致OPIDN的OP化合物。对氧磷和DFP诱导出截然不同的基因表达谱。在经对氧磷处理的细胞中，有两个基因上调，13个基因下调至少2倍。相比之下，DFP仅上调了一个基因，在2倍阈值时无基因下调。这一发现与目前及以往观察结果一致，即SY5Y细胞能够区分诱导或不诱导OPIDN的OP。我们还检查了基因芯片结果，以寻找可能的OP神经毒性新靶蛋白或代谢途径。葡萄糖调节蛋白78（GRP78）的蛋白水平显示，在神经母细胞瘤细胞中，3 microM对氧磷处理24小时可使GRP78水平显著降低30%，而DFP处理则无此作用。与SY5Y神经母细胞瘤细胞相比，对氧磷和DFP（3 microM处理24小时）均可使CCF星形细胞瘤细胞中的GRP78水平显著升高23 - 24%。由于我们之前已评估过SY5Y细胞内Ca(2+)水平的变化，因此通过研究胞质和线粒体基础钙水平，我们研究了对氧磷和DFP对CCF细胞中细胞Ca(2+)稳态的影响。在用1、3、10或30 microM对氧磷处理1天或3天的CCF培养物中，检测到线粒体与胞质Ca(2+)荧光比值显著降低。相比之下，用DFP处理1天对线粒体与胞质Ca(2+)荧光比值无显著影响；处理3天后，只有30 microM降低了该比值。这些结果与对氧磷比DFP诱导CCF细胞内酯酶活性更大程度降低的发现一致。线粒体与胞质Ca(2+)比值的变化是所测试的每种化学物质所致损伤程度的良好指标。这项工作进一步建立了区分导致OPIDN的化合物和仅诱导急性神经毒性的化合物的体外模型。该研究还揭示了一些在体内可能被掩盖的OP诱导的其他毒性。
