Graduate School of Medicine, Korea University, Sungbuk-Ku, Seoul, Republic of Korea.
Proteomics. 2010 May;10(9):1831-46. doi: 10.1002/pmic.200900674.
The effects of di(2-ethylhexyl) phthalate (DEHP) on proteins secreted by HepG2 cells were studied using a proteomic approach. HepG2 cells were exposed to various concentrations of DEHP (0, 2.5, 5, 10, 25, 50, 100, and 250 microM) for 24 or 48 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and comet assays were then conducted to determine the cytotoxicity and genotoxicity of DEHP, respectively. The MTT assay showed that 10 microM DEHP was the maximum concentration that did not cause cell death. In addition, the DNA damage in HepG2 cells exposed to DEHP was found to increase in a dose- and time-dependent fashion. Proteomic analysis using two different pI ranges (4-7 and 6-9) and large size 2-DE revealed the presence of 2776 protein spots. A total of 35 (19 up- and 16 down-regulated) proteins were identified as biomarkers of DEHP by ESI-MS/MS. Several differentiated protein groups were also found. Proteins involved in apoptosis, transportation, signaling, energy metabolism, and cell structure and motility were found to be up- or down-regulated. Among these, the identities of cystatin C, Rho GDP inhibitor, retinol binding protein 4, gelsolin, DEK protein, Raf kinase inhibitory protein, triose phosphate isomerase, cofilin-1, and haptoglobin-related protein were confirmed by Western blot assay. Therefore, these proteins could be used as potential biomarkers of DEHP and human disease associated with DEHP.
采用蛋白质组学方法研究了邻苯二甲酸二（2-乙基己基）酯（DEHP）对 HepG2 细胞分泌蛋白的影响。将 HepG2 细胞分别暴露于不同浓度的 DEHP（0、2.5、5、10、25、50、100 和 250 μM）24 或 48 h。然后进行 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）和彗星试验，分别测定 DEHP 的细胞毒性和遗传毒性。MTT 试验表明，10 μM DEHP 是不会引起细胞死亡的最大浓度。此外，还发现暴露于 DEHP 的 HepG2 细胞的 DNA 损伤呈剂量和时间依赖性增加。使用两种不同的等电点范围（4-7 和 6-9）和大型 2-DE 的蛋白质组学分析显示存在 2776 个蛋白斑点。通过 ESI-MS/MS 鉴定出 35 个（19 个上调和 16 个下调）蛋白作为 DEHP 的生物标志物。还发现了几个差异表达的蛋白质组。发现参与细胞凋亡、运输、信号转导、能量代谢以及细胞结构和运动的蛋白质上调或下调。其中，半胱氨酸蛋白酶抑制剂 C、Rho GDP 抑制剂、视黄醇结合蛋白 4、凝胶蛋白、DEK 蛋白、Raf 激酶抑制蛋白、磷酸丙糖异构酶、原肌球蛋白 1、副肌球蛋白和触珠蛋白相关蛋白的鉴定通过 Western blot 试验得到了证实。因此，这些蛋白质可以作为 DEHP 的潜在生物标志物以及与 DEHP 相关的人类疾病的标志物。
