Effect of acrolein and glutathione depleting agents on thioredoxin
Effect of acrolein and glutathione depleting agents on thioredoxin
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DOI：
10.1016/j.tox.2004.06.056
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发表时间：
2004-11-15
期刊：
影响因子：
4.5
通讯作者：
Kehrer, JP
中科院分区：
文献类型：
--
作者：
Yang, XM;Wu, XL;Kehrer, JP
丙烯醛是一种广泛存在的环境污染物，它与亲核试剂，特别是胞状硫醇反应迅速。除了谷胱甘肽（GSH），硫氧还蛋白（Trx）和硫氧还蛋白还原酶（TR）含有巯基，并可与亲电试剂反应。在本研究中，用5-25 μ M丙烯醛处理A549细胞30分钟以剂量依赖性方式丧失细胞Trx活性。超过90%的Trx活性在25 μ M或更高的浓度下丧失。与此相反，Trx蛋白质含量，如通过蛋白质印迹法评估的，在丙烯醛处理30分钟后没有立即改变。丙烯醛处理后4 h，Trx活性和蛋白水平均升高。然而，Trx活性在24小时仍低于对照水平。在暴露于丙烯醛的A549细胞中观察到与TR类似的剂量-反应关系。然而，在剂量小于或等于75 μ M丙烯醛后，TR活性迅速恢复，4小时达到正常水平。马来酸二乙酯（DEM），一种常见的但不是高度特异性的，用于消耗GSH的试剂，也使Trx失活。A549细胞暴露于1 mM DEM 2 h后，细胞GSH减少50%，Trx活性降低67%以上。较低的DEM剂量（0. 125 mM和0.25 mM）处理1 h对GSH无显著影响，但分别使Trx活性显著降低12%和23%。与丙烯醛暴露后立即类似，DEM不影响Trx蛋白水平。将Trx-1-GFP融合蛋白转染入A549细胞。当融合蛋白表达时，通过胰岛素还原测定，Trx组分是无活性的。总之，Trx和TR被丙烯醛灭活。此外，GSH消耗剂DEM使Trx失活比其消耗GSH更有效。Trx- 1 -GFP融合蛋白虽然易于表达，但似乎几乎没有活性或没有活性，这可能是因为Trx-1的小尺寸（12 kDa）受到较大GFP的影响。(C)2004爱思唯尔爱尔兰有限公司保留所有权利。
Acrolein is a widespread environmental pollutant that reacts rapidly with nucleophiles, especially cellular thiols. In addition to glutathione (GSH), thioredoxin (Trx) and thioredoxin reductase (TR) contain thiol groups and may react with electrophiles. In the present study, A549 cells treated with 5-25 muM acrolein for 30 min lost cellular Trx activity in a dose-dependent fashion. Over 90% of Trx activity was lost at concentrations of 25 muM or greater. In contrast, Trx protein content, as assessed by western blotting, was not altered immediately after the 30 min acrolein treatment. Both Trx activity and protein levels increased 4 h after the acrolein treatment. However, Trx activity remained below control levels at 24 h. A similar dose-response relationship was seen with TR in A549 cells exposed to acrolein. There was, however, a rapid recovery of TR activity such that it attained normal levels by 4 h after doses less than or equal to75 muM acrolein. Diethyl maleate (DEM), a common but not highly specific, agent used to deplete GSH, also inactivated Trx. A 2 h exposure of A549 cells to 1 mM DEM depleted cellular GSH by 50% and diminished Trx activity by over 67%. Lower DEM doses (0. 125 mM and 0.25 mM) for 1 h had no significant effect on GSH but significantly decreased Trx activity 12 and 23%, respectively. Similar to immediately after acrolein exposure, DEM did not affect Trx protein levels. A Trx-1-GFP fusion protein was transfected into A549 cells. While the fusion protein was expressed, the Trx component was inactive by the insulin reducing assay. In summary, Trx and TR are inactivated by acrolein. In addition, the GSH depleting agent DEM inactivates Trx somewhat more effectively than it depletes GSH. The Trx- 1 -GFP fusion protein, while readily expressed, appears to have little or no activity, perhaps because the small size of Trx-1 (12 kDa) is affected by the larger GFP. (C) 2004 Elsevier Ireland Ltd. All rights reserved.