在用水筛查内分泌活动市售的<em>体外</em>式激活生物测定
Summary
A protocol to screen for endocrine activity in organic extracts of water samples, including treated wastewater effluent and surface (receiving) water, was adapted using commercially available division-arrested (“freeze and thaw”) in vitro transactivation bioassays.
Abstract
体外转录生物测定表明希望作为水质监测工具，但他们采用和广泛应用已部分阻碍由于缺乏规范的方法和强大的，用户友好的技术可用性。在这项研究中，使用可商购的，分裂停滞的细胞系，以定量筛选本感兴趣的环境质量的专业人员水样中的化学物质的内分泌活性。其中包括全面的质量保证/质量控制一个单一的，标准化的协议（QA / QC）检查是为雌激素和糖皮质激素受体的活性（ER和GR分别）使用基于细胞的荧光共振能量转移（FRET）分析开发。在加利福尼亚州（美国）处理城市废水和地表水从淡水系统的样品，采用固相萃取提取和使用标准化原为内分泌活动分析山坳。背景和特定端点引用的化学物质的剂量 – 反应满足必要的可靠的测量QA / QC准则。地表水样品的生物测定筛选反应，主要是检测不到的。与此相反，从二级处理厂流出物样品具有最高的可测量的活性，与估计的生物测定当量浓度（BEQs）到392纳克地塞米松/升的GR和17纳克17β雌二醇/升的ER。为三级污水样品的生物测定的反应比对二级污水测量低，说明经过深度处理一个较低的残余内分泌活性化学物质。这个协议表明 ，利用市售的体外转录生物测定，除法被捕细胞“试剂盒”，可适于筛选在水中内分泌活动。
Introduction
当前水质监测的前提是要准确和精确地测量化学污染物的产生，作为暴露于野生动物和人类的代理的能力。然而，这种化学物质通过化学监测和评估模式不能跟上，我们所面临的不断变化的化学世界。当我们了解的命运和合成和天然化学物质的影响，我们继续寻找测量工具，解决预期生物的影响，而在同一时间的免疫化学品生产，使用和环保投入的变化。这样的工具是特别了解相关未知或新的化学品，并转化产物是否值得我们注意。此外，存在于水中的化学物质的复杂混合物由个体化学监测不善处理。因此，我们面临的现有监控工具箱地表水THA这些问题，现代化，更好地解决的挑战收不到处理后的废水污水和城市/雨水径流排放。
近年来，生物分析技术已经表明希望作为筛选工具水质评估。如果对通过称为起作用的化学反应尤其in'vitro生物测定，动作1,2的具体模式是极大的兴趣，环境监测社区3。大量调查在体外生物测定用来量化饮用水，地表废水4-6的内分泌活动。此外，一些生物测定目标分子发起事件（ 例如，受体活化），其可以潜在地通过不利后果通路连接于有害影响分析7,8。
生物筛选水质评价的发展一直相对较快，与数百种不同的体外生物分析端点已被评估他们的实用9,10。目前，仅生物测定的少数已显示以达到良好的测量精度（实验室内）同时展现水质5,6-之间区分的能力。特别是对于处理后的废水污水，雌激素和糖皮质类固醇的发生已经成功占据了利用体外转录实验11,12。然而，大多数研究迄今为止已采用的生物测定，其细胞系是专有的（并且因此没有被广泛可得），需要连续护理和操纵，或两者兼而有之。这样一来，能够标准化的协议，进行实验室间的校准练习，并最终该筛选技术转移到水资源的社会仍然受阻。
通过美国ToxCast程序审核体外生物检测中的至少一个供应商是容易商购的13用“冻融4;格式。这些除法被捕细胞“试剂盒”已被证明是一种用于测定从水中提取的化学品代表不同程度的处理14的活性健壮。虽然商协议可用来筛选个别化学品或混合物的生物活性，其中一些需要修改，才可以被应用到的水样。处理后的废水出水15，雨水径流16，受纳水体17,18和最近的循环水19,20是感兴趣的水质社区水介质的最好的例子。
这项研究提供了一个单一的，标准化的协议使用市售测量水样中的内分泌活动，除被捕体外转录生物测定。我们通过后台，剂量响应和可重复性响应为TW的全面评估证明该协议的健壮性特别感兴趣雌激素与糖皮质激素受体转录（ER和GR，分别）邻端点。该协议在加州应用于处理后的废水污水和地表水幕样本淡水系统。
Protocol
Representative Results
Discussion
环境雌激素，如17β雌二醇（E2），这些化学品在纳克/升浓度23,24权证筛查的有案可稽效力。在这项研究中，废水排放对ER响应（BEQ范围：2.3至17毫微克的E2 / L）的比报道的从澳大利亚污水处理厂20二级出水稍高，而BEQs地表水（<0.5至4纳克E2 / L的）为报道的表面和雨水别处（<1至11毫微克的E2 / L），16的范围内。尽管地表水样本中测得ER活动水平低，此法代表了水质评价是?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Funding was provided by State Water Resources Control Board (Agreements No. 10-096-250 and 14-090-270). We thank S. Abbasi, M. Connor, S. Engelage, K. North, J. Armstrong, S. Asato, M. Dojiri, D. Schlenk, S. Snyder, S. Westerheide, B. Escher, F. Leusch, G. Pelanek, K. Bi, and J. Printen. The authors declare no conflict of interest, and reference to trade names does not imply endorsement.
Materials
| GeneBLAzer ER alpha DA assay kit | ThermoFisher | K1393 | Kit includes ER division arrested (DA) cells and LiveBLAzer FRET loading kit. |
| GeneBLAzer GR DA assay kit | ThermoFisher | K1391 | Kit includes GR division arrested (DA) cells and LiveBLAzer FRET loading kit. |
| PrestoBlue cell viability reagent | ThermoFisher | A-13261 | |
| Trypan blue, 0.4% in PBS | Sigma-Aldrich | T8154 | Also available at ThermoFisher |
| Corning 96 well black wall, clear-bottom plate | Corning | 3603 | Individually wrapped, sterile with lid |
| Whatman glass fiber filters, GF/A, 1.6 µM | Sigma-Aldrich | WHA1820025 | |
| Microplate aluminum sealing film | E&K Scientific | T592100 | |
| Oasis HLB 6 cc cartridge, 200 mg sorbent | Waters | WAT106202 | |
| 17β Estradiol | Sigma-Aldrich | E2758 | CAS #50-28-2 |
| Ascorbic acid | Fisher Scientific | A61-100 | Also available at Sigma-Aldrich |
| Dexamethasone | Sigma-Aldrich | D4902 | CAS #50-02-2 |
| Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D8418 | Molecular grade |
| Solvents (acetone, hexane, methanol) | Fisher Scientific | HPLC grade | |
| Sodium azide | Sigma-Aldrich | S2002 | Chemical is highly toxic and must be handled with caution. Use protective clothing and weigh under a fumehood. Also available at EMD Millipore. |
| Automated cell counter or hemocytometer | Various* | Suppliers include Bio-Rad, Fisher Scientific, Sigma-Aldrich and ThermoFisher. | |
| Class II biological safety cabinet | Various* | ||
| CO2 incubator | Various* | ||
| Cryogenic freezer | Various* | Liquid nitrogen storage dewar is recommended. | |
| Fluorescence microplate reader | Various* | The reader must have bottom read capabilities. | |
| * No recommended source, the choice of this equipment depends on budget, frequency of use, and lab space. | |||
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