药学学报, 2020, 55(2): 265-271
王梦梦, 张旭霞, 尹俊, 任湘祎, 李欣悦, 陈红红. 基于竞争ELISA法的铀促排螯合剂体外筛选模型的建立[J]. 药学学报, 2020, 55(2): 265-271.
WANG Meng-meng, ZHANG Xu-xia, YIN Jun, REN Xiang-yi, LI Xin-yue, CHEN Hong-hong. Establishment of an in vitro screening model for uranium decorporation chelators based on the competitive ELISA method[J]. Acta Pharmaceutica Sinica, 2020, 55(2): 265-271.

王梦梦, 张旭霞, 尹俊, 任湘祎, 李欣悦, 陈红红
复旦大学上海医学院放射医学研究所, 上海200032
血液中的铀[U(Ⅵ)]与去铁转铁蛋白(apotransferrin,apo-Tf)形成稳定的络合物在U(Ⅵ)进入细胞致细胞毒性中发挥重要作用。本研究通过探讨螯合剂与apo-Tf竞争结合U(Ⅵ)的作用,建立了一种基于酶联免疫吸附实验(enzyme linkedimmunosorbent assay,ELISA)的U(Ⅵ)促排螯合剂体外筛选新模型,通过方法学研究确定了该检测方法的最佳包被抗原apo-Tf、Tf抗体、二抗稀释比例和U(Ⅵ)处理的浓度,验证了该方法的稳定性和重现性,并采用该模型考察了4种螯合剂邻苯二酚-3,6二甲撑亚氨基四乙酸(CBMIDA)、Tiron、DTPA-CaNa3、DTPA-ZnNa3与apo-Tf竞争结合U(Ⅵ)的能力,其强弱顺序为:CBMIDA ≈ Tiron > apo-Tf > DTPA-CaNa3 ≈ DTPA-ZnNa3;采用动物实验观察以上螯合剂的排U(Ⅵ)效果发现,CBMIDA和Tiron立即给药能显著提高U(Ⅵ)内污染小鼠24 h尿U(Ⅵ)排出、明显降低肾脏和股骨U(Ⅵ)蓄积;而DTPA-CaNa3和DTPA-ZnNa3则无明显促排效果,与竞争ELISA法的检测结果相一致。动物实验符合复旦大学药学院动物伦理委员会规程。以上结果表明,该方法快速、简便、重现性好,能够用于U(Ⅵ)促排螯合剂的快速筛选。
关键词:          螯合剂      去铁转铁蛋白      竞争酶联免疫吸附实验      筛选方法     
Establishment of an in vitro screening model for uranium decorporation chelators based on the competitive ELISA method
WANG Meng-meng, ZHANG Xu-xia, YIN Jun, REN Xiang-yi, LI Xin-yue, CHEN Hong-hong
Institute of Radiation Medicine, Shanghai Medical College, Fudan University, Shanghai 200032, China
Uranium[U(Ⅵ)] in the blood is known to form stable complexes with apotransferrin (apo-Tf), which plays an important role in mediating the cytotoxicity induced by U(Ⅵ) transported to cells. The present study aimed to establish an new in vitro screening model of U(Ⅵ) decorporation agents through exploring the capability of chelating agents competing with U(Ⅵ) binding to apo-transferrin based on enzyme-linked immunosorbent assay (ELISA). The optimal concentrations of apo-Tf coated antigen, Tf antibody, secondary antibody and U(Ⅵ) treatment were achieved and the stability and reproducibility of this method were validated by methodology study. Using this model, the ability of four chelating agents to mobilize the U(Ⅵ) binding to apo-Tf was evaluated, and the rank of competitiveness was catechol-3,6-bis(methyleiminodiacetic acid) (CBMIDA) ≈ Tiron > apo-Tf > DTPA-CaNa3 ≈ DTPA-ZnNa3. The efficacy of these chelating agents in removal of U(Ⅵ) was tested by animal experiments. The results showed that immediate administration of CBMIDA or Tiron after injection of U(Ⅵ) in mice significantly promoted urinary U(Ⅵ) excretion and reduced U(Ⅵ) accumulation in kidneys and femurs, while DTPA-CaNa3 and DTPA-ZnNa3 have no obvious effects as compared to U(Ⅵ)-exposed mice alone, which was consistent with the results of competitive ELISA method. The animal experiments conform to the rules of the Animal Research Ethics Committee of School of Pharmacy of Fudan University. These results show that the new proposed method is rapid, simple and convenient with good reproducibility and has the potential to be used for in vitro screening of U(Ⅵ) decorporation agents.
Key words:    uranium    chelating agent    apotransferrin    competitive enzyme linked immunosorbent assay    screening method   
收稿日期: 2019-08-27
DOI: 10.16438/j.0513-4870.2019-0687
基金项目: 国家自然科学基金资助项目(81972971).
通讯作者: 陈红红,Tel/Fax:86-21-64437130,E-mail:hhchen@shmu.edu.cn
Email: hhchen@shmu.edu.cn
PDF(531KB) Free
王梦梦  在本刊中的所有文章
张旭霞  在本刊中的所有文章
尹俊  在本刊中的所有文章
任湘祎  在本刊中的所有文章
李欣悦  在本刊中的所有文章
陈红红  在本刊中的所有文章

[1] Faa A, Gerosa C, Fanni D, et al. Depleted uranium and human health[J]. Curr Med Chem, 2018, 25:49-64.
[2] Craft E, Abu-Qare A, Flaherty M, et al. Depleted and natural uranium:chemistry and toxicological effects[J]. J Toxicol Environ Health Part B, 2004, 7:297-317.
[3] Bleise A, Danesi PR, Burkart W. Properties, use and health effects of depleted uranium (DU):a general overview[J]. J Environ Radioact, 2003, 64:93-112.
[4] Wang XM, Ji GX, Shi C, et al. Structural and thermodynamic stability of uranyl-deferiprone complexes and the removal efficacy of U(Ⅵ) at the cellular level[J]. Dalton Trans, 2018, 47:8764-8770.
[5] Averseng O, Hagège A, Taran F, et al. Surface plasmon resonance for rapid screening of uranyl affine proteins[J]. Anal Chem, 2010, 82:9797-9802.
[6] El Hage Chahine J, Hémadi M, Ha-Duong N. Uptake and release of metal ions by transferrin and interaction with receptor 1[J]. Biochim Biophys Acta, 2012, 1820:334-347.
[7] Deblonde GJ, Sturzbecher-Hoehne M, Mason AB, et al. Receptor recognition of transferrin bound to lanthanides and actinides:a discriminating step in cellular acquisition of f-block metals[J]. Metallomics, 2013, 5:619-626.
[8] Malard V, Gaillard J, Bérenguer F, et al. Urine proteomic profiling of uranium nephrotoxicity[J]. Biochim Biophys Acta, 2009, 1794:882-891.
[9] Shaki F, Hosseini M, Ghazi-Khansari M, et al. Toxicity of depleted uranium on isolated rat kidney mitochondria[J]. Biochim Biophys Acta, 2012, 1820:1940-1950.
[10] Pierrefite-Carle V, Santucci-Darmanin S, Breuil V, et al. Effect of natural uranium on the UMR-106 osteoblastic cell line:impairment of the autophagic process as an underlying mechanism of uranium toxicity[J]. Arch Toxicol, 2017, 91:1903-1914.
[11] Wagner SE, Burch JB, Bottai M, et al. Ground water uranium and cancer incidence in South Carolina[J]. Cancer Cause Control, 2011, 22:41-50.
[12] Hemadi M, Ha-Duong N, El Hage Chahine J. Can uranium be transported by the iron-acquisition pathway? Ur uptake by transferrin[J]. J Phys Chem B, 2011, 115:4206-4215.
[13] Domingo JL, Ortega A, Llobet JM, et al. Effectiveness of chelation therapy with time after acute uranium intoxication[J]. Fundam Appl Toxicol, 1990, 14:88-95.
[14] Fukuda S, Ikeda M, Nakamura M, et al. Efficacy of oral and intraperitoneal administration of CBMIDA for removing uranium in rats after parenteral injections of depleted uranium[J]. Radiat Prot Dosim, 2009, 133:12-19.
[15] Fukuda S, Ikeda M, Nakamura M, et al. Effects of pH on DU intake and removal by CBMIDA and EHBP[J]. Health Phys, 2007, 92:10-14.
[16] Zhang XX, Bao YZ, Wang MM, et al. Competitive combination with uranium (Ⅵ) between chelating agent and metallothionein[J]. Chin J Radiol Health (中国辐射卫生), 2019, 28:12-16.
[17] Hao YH, Huang JW, Liu C, et al. Differential protein expression in metallothionein protection from depleted uranium-induced nephrotoxicity[J]. Sci Rep, 2016, 6:38942.
[18] Guo M, Lu XW, Ran XY, et al. Molecular transport mechanism of pefloxacin mesylate binding with transferrin[J]. Acta Pharm Sin (药学学报), 2012, 47:1503-1510.
[19] Wang M, Ding WJ, Wang DQ. Binding mechanism of uranyl to transferrin implicated by density functional theory study[J]. RSC Adv, 2017, 7:3667-3675.
[20] Benavides-Garcia MG, Balasubramanian K. Structural insights into the binding of uranyl with human serum protein apotransferrin structure and spectra of protein-uranyl interactions[J]. Chem Res Toxicol, 2009, 22:1613-1621.
[21] Gkouvatsos K, Papanikolaou G, Pantopoulos K. Regulation of iron transport and the role of transferrin[J]. Biochim Biophys Acta, 2012, 1820:188-202.
[22] Montavon G, Apostolidis C, Bruchertseifer F, et al. Spectroscopic study of the interaction of U(Ⅵ) with transferrin and albumin for speciation of U(Ⅵ) under blood serum conditions[J]. J Inorg Biochem, 2009, 103:1609-1616.
[23] Sutton M, Burastero SR. Uranium(Ⅵ) solubility and speciation in simulated elemental human biological fluids[J]. Chem Res Toxicol, 2004, 17:1468-1480.
[24] Carrière M, Thiebault C, Milgram S, et al. Citrate does not change uranium chemical speciation in cell culture medium but increases its toxicity and accumulation in NRK-52E cells[J]. Chem Res Toxicol, 2006, 19:1637-1642.
[25] Mirto H, Barrouillet MP, Henge-Napoli MH, et al. Influence of uranium(Ⅵ) speciation for the evaluation of in vitro uranium cytotoxicity on LLC-PK1 cells[J]. Hum Exp Toxicol, 1999, 18:180-187.
[26] Lan LA, Wu SY, Yu SD, et al. Fast and direct determination of catechol-3,6-bis(methyleiminodiacetic acid) prototype in beagle dog plasma using liquid chromatography tandem mass spectrometry:a simplified and high throughput in-vivo method for the metal chelator[J]. J Chromatogr A, 2019, 1596:84-95.
[27] Bao YZ, Wang D, Hu YX, et al. Efficacy of chelator CBMIDA-CaNa2 for the removal of uranium and protection against uranium-induced cell damage in human renal proximal tubular cells[J]. Health Phys, 2013, 105:31-38.
1.张晓菲 李 超 赵长琦 刘丽宏.水溶性印迹壳聚糖对贫铀致细胞毒性的解毒作用[J]. 药学学报, 2011,46(5): 513-520
2.暴一众 王 丹 胡昱兴 徐爱红 孙梅贞 陈红红.螯合剂BPCBG对铀的促排效果和防护铀致人肾小管上皮细胞损伤的作用[J]. 药学学报, 2011,46(11): 1308-1313