Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

摘要

摘要

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been hindered partly due to a lack of standardized methods and availability of robust, user-friendly technology. In this study, commercially available, division-arrested cell lines were employed to quantitatively screen for endocrine activity of chemicals present in water samples of interest to environmental quality professionals. A single, standardized protocol that included comprehensive quality assurance/quality control (QA/QC) checks was developed for Estrogen and Glucocorticoid Receptor activity (ER and GR, respectively) using a cell-based Fluorescence Resonance Energy Transfer (FRET) assay. Samples of treated municipal wastewater effluent and surface water from freshwater systems in California (USA), were extracted using solid phase extraction and analyzed for endocrine activity using the standardized protocol. Background and dose-response for endpoint-specific reference chemicals met QA/QC guidelines deemed necessary for reliable measurement. The bioassay screening response for surface water samples was largely not detectable. In contrast, effluent samples from secondary treatment plants had the highest measurable activity, with estimated bioassay equivalent concentrations (BEQs) up to 392 ng dexamethasone/L for GR and 17 ng 17β-estradiol/L for ER. The bioassay response for a tertiary effluent sample was lower than that measured for secondary effluents, indicating a lower residual of endocrine active chemicals after advanced treatment. This protocol showed that in vitro transactivation bioassays that utilize commercially available, division-arrested cell "kits", can be adapted to screen for endocrine activity in water.

引言

Current water quality monitoring is predicated on the ability to accurately and precisely measure the occurrence of chemical contaminants as a proxy for exposure to wildlife and humans. However, this chemical-by-chemical monitoring and assessment paradigm cannot keep pace with the ever-changing chemical universe that we face. As we learn more about the fate and effects of synthetic and natural chemicals, we continue to search for measurement tools that address expected biological impacts, and that at the same time are immune to changes in chemical production, usage and environmental input. Such tools are especially relevant for understanding whether unknown or new chemicals, and transformation products, deserve our attention. Moreover, complex mixtures of chemicals present in water are poorly addressed by individual chemical monitoring. Thus, we face the challenge of modernizing the existing monitoring toolbox to better address these issues in surface waters that receive discharge of treated wastewater effluent and urban/stormwater runoff.

In recent years, bioanalytical techniques have shown promise as screening tools for water quality assessment. In particular, in'vitro bioassays that respond to chemicals acting via known, specific modes of action^1,2 are of great interest to the environmental monitoring community³. Numerous investigations have employed in vitro bioassays to quantify the endocrine activity of drinking, surface and wastewaters^{4 -6}. Moreover, a number of bioassays target molecular initiating events (e.g., receptor activation) which can potentially be linked to deleterious effects via adverse outcome pathway analyses^7,8.

The evolution of bioscreening for water quality assessment has been relatively rapid, with hundreds of different in vitro bioassay endpoints having been evaluated for their utility^9,10. Currently, only a handful of bioassays have been shown to achieve good measurement precision (within laboratories) while demonstrating the ability to differentiate among water qualities^5,6. For treated wastewater effluent in particular, the occurrence of estrogens and glucocorticoid steroids has been successfully accounted for using in vitro transactivation assays^11,12. However, most studies to date have employed bioassays whose cell lines are proprietary (and thus not widely available), require continuous care and manipulation, or both. As a result, the ability to standardize protocols, perform inter-laboratory calibration exercises, and ultimately to transfer this screening technology to the water resources community remains hindered.

At least one supplier of in vitro bioassays vetted through the U.S. ToxCast program is commercially available¹³ in easy to use "freeze and thaw" formats. These division-arrested cell "kits" have been shown to be robust in measuring the activity of chemicals extracted from water representing different levels of treatment¹⁴. Although vendor protocols are available to screen the bioactivity of individual chemicals or mixtures, some of them require modification before they can be applied to water samples. Treated wastewater effluent¹⁵, stormwater runoff¹⁶, receiving waters^17,18 and more recently recycled water^19,20 are prime examples of aqueous media that are of interest to the water quality community.

This study presents a single, standardized protocol to measure the endocrine activity in water samples using commercially available, division-arrested in vitro transactivation bioassays. We demonstrated robustness of the protocol through a comprehensive assessment of background, dose responsivity and repeatability of response for two endpoints of particular interest Estrogen and Glucocorticoid Receptor transactivation (ER and GR, respectively). The protocol was applied to screen samples of treated wastewater effluent and surface water from freshwater systems in California.

方案

1. Collect and Process Water Sample (Modified from Escher et al.⁹)

1. Fill a clean 1 L amber glass bottle containing 1 g sodium azide and 50 mg ascorbic acid to the top with water sample of interest. Store sample at 4 °C and process within 72 hr.
   NOTE: Sodium azide is highly toxic and must be handled with caution. Use protective gear (eye/face, gloves, clothing) and weigh in a properly functioning fume-hood. Do not use a metal spatula for weighing.
2. Pass the sample through a 1.6 µM glass fiber filter and then through a preconditioned Solid Phase Extraction (SPE) cartridge at a flow rate of 5-10 ml/min. Adjust the pressure of the vacuum pump to control the flow rate.
3. Vacuum dry the cartridge for 15 min.
4. Elute the cartridge with 10 ml of methanol, followed by 10 mL of acetone:hexane (1:1, v/v).
5. Concentrate eluate to ~1 ml under a gentle stream of high purity nitrogen. Solvent exchange by adding 500 µl of dimethyl sulfoxide (DMSO) and evaporating the extract down to 500 µl.
6. Transfer the extract to an amber glass autosampler vial. Store at -20 °C.

2. Prepare Dilutions of the Assay Specific Reference Chemical and Water Extract

1. Prepare 9 dilutions for the calibration curve.
   1. Make a stock solution of the assay specific reference chemical in 100% DMSO. The final concentration must be 2 µM 17β-estradiol for the ER assay, and 100 µM dexamethasone for the GR assay. Store the stock solutions at -20 °C.
   2. Add 15 µl of the appropriate reference chemical stock to 285 µl of assay medium in a sterile tube (tube #1). Mix the sample by pipetting up and down.
   3. Add 200 µl of a solution of 5% DMSO in assay medium in 8 additional tubes (tubes #2-9).
   4. Transfer a 100 µl aliquot from tube #1 to tube #2 through tube #9 to perform a 3-fold dilution series. Each diluted sample must be mixed thoroughly with a pipette before taking an aliquot and adding it to the next tube.
2. Prepare a solvent control sample by mixing 10 µl of DMSO in 190 µl of assay medium.
3. Prepare four dilutions for each water extract.
   1. In the first tube, add 5 µL of water extract in 95 µL of assay medium and mix thoroughly. Add 50 µl of 5% DMSO in assay medium in three other tubes.
   2. Perform a 2-fold dilution by transferring 50 µl of solution from one tube to the next.

3. Prepare the Cell Suspension to Conduct the FRET Bioassay

1. Prepare the assay specific medium according to the manufacturer's instructions. Assay medium must be stored at 4 °C and warmed to 37 °C in a water-bath before use.
2. Take a vial of ER or GR division-arrested cells out of the cryogenic freezer and thaw the cells quickly by placing the vial in a 37 °C water-bath for 2 min with gentle agitation. Decontaminate the vial with 70% ethanol and place it in a Class II biological safety cabinet.
3. Open the vial using aseptic techniques and transfer the cells into 10 ml of assay medium.
4. Centrifuge at 200 x g for 5 min.
5. Aspirate the supernatant using a sterile glass pipet and resuspend the cell pellet in 6 ml of assay medium.
6. Mix 5 µl of cell suspension with 5 µl of a vital stain solution and add an aliquot into a counting chamber.
7. Count the number of live cells using a light microscope or automated cell counter and estimate the density of live cells in the cell suspension. Dilute if necessary to obtain a final density of 550,000 live cells per ml of assay medium.

4. Plate Cells in a 96-well Black Wall Clear-bottom Plate and Add Diluted Water Extract

1. Create a plate layout that includes a 9-point assay specific calibration curve, QA/QC samples and multiple dilutions for each water extract. An example of a 96-well plate layout is shown in Figure 1.
2. Add 90 µl of assay medium to the replicate cell-free control wells.
3. Pour the cell suspension in a sterile pipetting reservoir and add 90 µl of cell suspension to the other wells using a multichannel pipette.
4. Add 10 µl of the diluted samples prepared during step 2 to the appropriate wells. The final concentration of DMSO per well must be 0.5% maximum.
5. Cover the plate with a lid and place it in a 5% CO₂ incubator at 37 °C for 16 hr.

Figure 1: Example of a 96-well Plate Layout. The multi-well plate is designed to include an assay specific calibration curve, 3 types of QA/QC controls (media only, cells in clean medium and cells in DMSO spiked medium) and 4 dilutions per water extract. Each control and dilution of water extract is analyzed in triplicate wells. Please click here to view a larger version of this figure.

5. Prepare the Loading Solution and Add to Each Well

1. After incubation, allow the plate to equilibrate to RT. For this FRET bioassay, steps 5.2 through 5.6 should be conducted in the absence of direct light.
2. Prepare a 6x loading solution according to the manufacturer's instructions.
3. Add 20 µl of the 6x loading solution to each well.
4. Add 10 µl of cell viability reagent to each well to evaluate the cytotoxicity of the diluted water extract.
5. Seal the plate with aluminum adhesive film.
6. Incubate the plate in the dark at RT for 2 hr.

6. Measure Cytotoxicity and Endocrine Activity Response

1. Set up the microplate reader with bottom-reading capabilities following the manufacturer's instructions.
2. For the FRET transactivation bioassay, measure fluorescence in the blue (409/460 nm, excitation (Ex)/emission (Em) wavelength) and green (409 Ex/530 Em nm) channels.
3. For the cytotoxicity assay, measure fluorescence at 560 Ex/590 Em nm.

7. Assess QA/QC Checks to Determine the Quality of the Data

1. Compare the average raw fluorescence of the cell-free (media only) and cells-only (cells in clean assay medium) controls. The media-only background fluorescence should be at least 25% lower than the response of the cells-only control.
2. Process the raw FRET data. For both 'blue' and 'green' datasets, subtract the average fluorescence of the cell-free control wells from all cell containing well. Calculate the blue/green ratio for each experimental well.
3. Compare blue/green ratio of cells-only and cells with DMSO controls. Fluorescence values of these controls should be within 15% Relative Standard Deviation (RSD).
4. Plot the assay specific calibration curve as blue/green ratio against the sample concentration expressed as a log molecular mass (log M). Calculate the slope, R² and log EC₅₀ (50% Effect Concentration). Calibration parameters should be within the expected range listed in Table 1.
5. Calculate the RSD for all triplicate wells. The variability among replicates should be less than 20%.
6. For the cytotoxicity data (560 Ex/590 Em nm), subtract the cell-free control average (i.e. media background) from all cell-containing wells.
7. Calculate the average background-subtracted fluorescence for each sample. Plot the resulting fluorescence data as a percent of the DMSO control. Sample dilutions should not exhibit more than 20% cell mortality compared to the cells-only and DMSO controls.

8. Data Analyses

1. Calculate the assay Limit of Detection (LOD) as the minimum calibration response plus two standard deviations of the mean of that response.
2. For samples showing a dose response, derive the EC₅₀ of the water extract using the slope of a linear regression of the calibration curve between 10 and 50% effect concentrations of the reference chemical.
3. Calculate the Bioanalytical Equivalent Concentration (BEQ) using the formula: BEQ = EC₅₀ of reference chemical/EC₅₀ of water sample.

结果

In the present study, 4x 24 hr composite samples of treated municipal wastewater effluent, 6 grab samples of surface water from freshwater systems in southern California and a field blank consisting of ultrapure water were selected to illustrate this protocol. 3 of the 4 effluent samples were from conventional activated sludge wastewater treatment plants ("secondary effluent"), and the fourth one from an advanced wastewater treatment plant with sand/carbon filtration added post biological...

讨论

The well documented potency of environmental estrogens, such as 17β-estradiol (E2), warrants screening for these chemicals at ng/L concentrations^23,24. In this study, the ER response for wastewater effluents (BEQ range: 2.3 to 17 ng E2/L) was somewhat higher than reported for secondary effluent from Australian WWTPs²⁰, whereas the BEQs for surface water (<0.5 to 4 ng E2/L) were within the range reported for surface and stormwater elsewhere (<1 to 11 ng E2/L)¹⁶. Despite the lo...

材料

本文使用的材料清单

姓名	公司	目录编号	评论
GeneBLAzer ER α DA 检测试剂盒	ThermoFisher	K1393	试剂盒包括 ER 分裂阻滞 （DA） 细胞和 LiveBLAzer FRET 上样试剂盒。
GeneBLAzer GR DA 检测试剂盒	ThermoFisher	K1391	试剂盒包括 GR 分裂阻滞 （DA） 细胞和 LiveBLAzer FRET 上样试剂盒。
PrestoBlue 细胞活力检测试剂	ThermoFisher	A-13261
台盼蓝，0.4% PBS	Sigma-Aldrich 溶液	T8154	也可在赛默飞世尔
康宁 96 孔黑壁、透明底板	康宁	3603	独立包装，带盖无菌
Whatman 玻璃纤维过滤器，GF/A，1.6 µM	Sigma-Aldrich
微孔板铝密封膜	E&K Scientific	T592100
Oasis HLB 6 cc 滤芯，200 毫克吸附剂	Waters	WAT106202
17β雌二醇	Sigma-Aldrich	E2758	CAS #50-28-2
抗坏血酸	Fisher Scientific	A61-100	也可在 Sigma-Aldrich
地塞米松获得	Sigma-Aldrich	D4902	CAS #50-02-2
二甲基亚砜 （DMSO）	Sigma-Aldrich	D8418	分子级
溶剂（丙酮、己烷、甲醇）	Fisher Scientific		HPLC 级
叠氮化钠	Sigma-Aldrich	S2002	化学品具有剧毒，必须谨慎处理。使用防护服并在通风橱下称重。也可在 EMD Millipore 购买。
自动细胞计数仪或血细胞计数器	各种*		供应商包括 Bio-Rad、Fisher Scientific、Sigma-Aldrich 和 ThermoFisher。
II 类生物安全柜	各种*
CO2 培养箱	各种*
低温冷冻机	各种*		液氮储存杜瓦瓶。
荧光酶标仪	各种*		Reader 必须具有 bottom 读取功能。
* 无推荐来源，该设备的选择取决于预算、使用频率和实验室空间。