螯合剂与金属硫蛋白对铀的竞争结合作用
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摘要
目的通过探讨螯合剂与金属硫蛋白(MT)竞争结合铀(U)(VI)的作用,建立铀促排螯合剂体外筛选的新方法。方法采用竞争ELISA法,96孔酶标板经MT抗原包被、U(VI)或Zn~(2+)预处理、螯合剂(CBMIDA-CaNa_2、BPCBG和DTPA-CaNa_3)处理、MT抗体免疫结合、HRP偶联二抗结合及OPD显色反应后,在波长490 nm处检测吸光度值以评价螯合剂与MT竞争结合U(VI)、Zn~(2+)的能力。结果本反应体系的吸光度值随MT质量浓度和MT抗体浓度增加而增加,其中最佳MT抗原包被浓度和MT抗体浓度均为2μg/mL。与Zn~(2+)的作用相似,U(VI)亦可使反应体系的吸光度值随其浓度增加而降低,其中最佳U(VI)和Zn~(2+)浓度均为300μg/mL。加入CBMIDA-CaNa_2和BPCBG则能明显提高U(VI)处理体系的吸光度值,CBMIDA-CaNa_2的作用强于BPCBG,而DTPA-CaNa_3则无明显作用;各螯合剂与MT竞争结合U(VI)能力的强弱顺序与以往细胞和动物U(VI)促排实验的结果相吻合。这3种螯合剂对Zn~(2+)处理体系的吸光度值均无明显影响。结论 CBMIDA-CaNa_2和BPCBG能有效竞争结合于MT上的U(VI),DTPA-CaNa_3无明显作用,竞争ELISA法可作为体外筛选U(VI)促排螯合剂的方法,具有简便、快速和高通量的优势。
Objective To establish a new method for the in vitro screening of U(Ⅵ) decorporation agents by exploring the competitive combination with uranium(Ⅵ) between chelating agent and metallothionein(MT). Methods Competitive enzyme-linked immunosorbent assay(ELISA) was developed to detect the competitive ability of chelating agents to mobilize the U(Ⅵ) and Zn~(2+) binding to MT. MT antigen was coated on the surface of well of 96-well plates and then pretreated with U(Ⅵ) or Zn~(2+) and then individually treated with chelating agents of CBMIDA-CaNa_2, BPCBG and DTPA-CaNa_3 followed by reacted with MT antibody and the secondary antibody linked with horseradish peroxidase(HRP) enzyme. The absorption values were detected by spectrophotometry at 490 nm after the chromogenic reaction of OPD. Results The absorption values of this testing system increased with higher concentrations of coating MT and MT antibody, and the optimal concentration of coating MT and MT antibody were both 2 μg/mL. Similar to the effects of Zn~(2+), U(Ⅵ) treatment also reduced the absorption values of testing system, with the increase of U(Ⅵ) and Zn~(2+) concentration, and the optimal concentration of U(Ⅵ) and Zn~(2+) were both 300 μg/mL. Treatment with CBMIDA-CaNa_2 and BPCBG significantly increased the absorption values of U(Ⅵ)-treated testing system, and effects of CBMIDA-CaNa_2 were higher than that of BPCBG. DTPA-CaNa_3 treatment had no effects on the absorption values of U(Ⅵ)-treated testing system. Importantly, the order of competitive ability of chelating agents to mobilize the U(Ⅵ) binding to MT was consistent with the results of previous in vitro cell culture and in vivo animal decorporation experiments for U(Ⅵ). In addition, treatment with the above chelating agents had no effects on the absorption values of Zn~(2+)-treated testing system. Conclusion CBMIDA-CaNa_2 and BPCBG can chelate U(Ⅵ) with MT, and the competitive chelation ability of CBMIDA-CaNa_2 for U(Ⅵ) binding to MT is better than that of BPCBG while DTPA-CaNa_3 has no obvious effect. This competitive ELISA assay can be used as a screening method for U(Ⅵ) decorporation agents in vitro which is simple, quick and high throughput.
Objective To establish a new method for the in vitro screening of U(Ⅵ) decorporation agents by exploring the competitive combination with uranium(Ⅵ) between chelating agent and metallothionein(MT). Methods Competitive enzyme-linked immunosorbent assay(ELISA) was developed to detect the competitive ability of chelating agents to mobilize the U(Ⅵ) and Zn~(2+) binding to MT. MT antigen was coated on the surface of well of 96-well plates and then pretreated with U(Ⅵ) or Zn~(2+) and then individually treated with chelating agents of CBMIDA-CaNa_2, BPCBG and DTPA-CaNa_3 followed by reacted with MT antibody and the secondary antibody linked with horseradish peroxidase(HRP) enzyme. The absorption values were detected by spectrophotometry at 490 nm after the chromogenic reaction of OPD. Results The absorption values of this testing system increased with higher concentrations of coating MT and MT antibody, and the optimal concentration of coating MT and MT antibody were both 2 μg/mL. Similar to the effects of Zn~(2+), U(Ⅵ) treatment also reduced the absorption values of testing system, with the increase of U(Ⅵ) and Zn~(2+) concentration, and the optimal concentration of U(Ⅵ) and Zn~(2+) were both 300 μg/mL. Treatment with CBMIDA-CaNa_2 and BPCBG significantly increased the absorption values of U(Ⅵ)-treated testing system, and effects of CBMIDA-CaNa_2 were higher than that of BPCBG. DTPA-CaNa_3 treatment had no effects on the absorption values of U(Ⅵ)-treated testing system. Importantly, the order of competitive ability of chelating agents to mobilize the U(Ⅵ) binding to MT was consistent with the results of previous in vitro cell culture and in vivo animal decorporation experiments for U(Ⅵ). In addition, treatment with the above chelating agents had no effects on the absorption values of Zn~(2+)-treated testing system. Conclusion CBMIDA-CaNa_2 and BPCBG can chelate U(Ⅵ) with MT, and the competitive chelation ability of CBMIDA-CaNa_2 for U(Ⅵ) binding to MT is better than that of BPCBG while DTPA-CaNa_3 has no obvious effect. This competitive ELISA assay can be used as a screening method for U(Ⅵ) decorporation agents in vitro which is simple, quick and high throughput.
引文
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[12] Bao Y,Wang D,Li Z,et al.Efficacy of a novel chelator BPCBG for removing uranium and protecting against uranium-induced renal cell damage in rats and HK-2 cells[J].Toxicol Appl Pharmacol,2013,269(1):17-24.
[13] 暴一众.邻苯二酚类螯合剂对急性铀中毒大鼠的解毒促排效果及其作用机制研究[D].上海:复旦大学放射医学研究所,2014.
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[2] 朱寿彭,李章.铀及铀系主要核素的放射毒理学//放射毒理学[M].2版,北京:原子能出版社,1992:222-298.
[3] Klaassen C D,Liu J,Diwan B A.Metallothionein protection of cadmium toxicity[J].Toxicol Appl Pharmacol,2009,238(3):215-220.
[4] Sabolic I,Breljak D,Skarica M,et al.Role of metallothionein in cadmium traffic and toxicity in kidneys and other mammalian organs[J].Biometals,2010,23(5):897-926.
[5] Blindauer C A,Leszczyszyn O I.Metallothioneins:unparalleled diversity in structures and functions for metal ion homeostasis and more[J].Nat Prod Rep,2010,27(5):720-741.
[6] Hao Y,Ren J,Liu C,et al.Zinc protects human kidney cells from depleted uranium-induced apoptosis[J].Basic Clin Pharmacol Toxicol,2014,114(3):271-280.
[7] Hao Y,Ren J,Liu J,et al.The protective role of zinc against acute toxicity of depleted uranium in rats[J].Basic Clin Pharmacol Toxicol,2012,111(6):402-410.
[8] Tessier C,Suhard D,Rebiere F,et al.Uranium microdistribution in renal cortex of rats after chronic exposure:a study by secondary ion mass spectrometry microscopy[J].Microsc Microanal,2012,18(1):123-133.
[9] 李慧祺,梁艺怀,雷立健,等.镉、锌与金属硫蛋白抗体对金属硫蛋白的竞争结合[J].环境与职业医学,2009(2):118-120.
[10] Michon J,Frelon S,Garnier C,et al.Determinations of uranium(VI) binding properties with some metalloproteins (transferrin,albumin,metallothionein and ferritin) by fluorescence quenching[J].J Fluoresc,2010,20(2):581-590.
[11] Bao Y,Wang D,Hu Y,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 physics,2013,105(1):31-38.
[12] Bao Y,Wang D,Li Z,et al.Efficacy of a novel chelator BPCBG for removing uranium and protecting against uranium-induced renal cell damage in rats and HK-2 cells[J].Toxicol Appl Pharmacol,2013,269(1):17-24.
[13] 暴一众.邻苯二酚类螯合剂对急性铀中毒大鼠的解毒促排效果及其作用机制研究[D].上海:复旦大学放射医学研究所,2014.
[14] Bergeron R J,Wiegand J,Singh S.Desferrithiocin analogue uranium decorporation agents[J].Int J Radiat Biol,2009,85(4):348-361.