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.

1. Collect and Process Water Sample (Modified from Escher et al.9)
<ol>
	<li>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 &#176;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.</li>
	<li>Pass the sample through a 1.6 &#181;M 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.</li>
	<li>Vacuum dry the cartridge for 15 min.</li>
	<li>Elute the cartridge with 10 ml of methanol, followed by 10 mL&#160;of acetone:hexane (1:1, v/v).</li>
	<li>Concentrate eluate to ~1 ml under a gentle stream of high purity nitrogen. Solvent exchange by adding 500 &#181;l of dimethyl sulfoxide (DMSO) and evaporating the extract down to 500 &#181;l.</li>
	<li>Transfer the extract to an amber glass autosampler vial. Store at -20 &#176;C.</li>
</ol>
2. Prepare Dilutions of the Assay Specific Reference Chemical and Water Extract
<ol>
	<li>Prepare 9 dilutions for the calibration curve.
	<ol>
		<li>Make a stock solution of the assay specific reference chemical in 100% DMSO. The final concentration must be 2 &#181;M 17&#946;-estradiol for the ER assay, and 100 &#181;M dexamethasone for the GR assay. Store the stock solutions at -20 &#176;C.</li>
		<li>Add 15 &#181;l of the appropriate reference chemical stock to 285 &#181;l of assay medium in a sterile tube (tube #1). Mix the sample by pipetting up and down.</li>
		<li>Add 200 &#181;l of a solution of 5% DMSO in assay medium in 8 additional tubes (tubes #2-9).</li>
		<li>Transfer a 100 &#181;l 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.</li>
	</ol>
	</li>
	<li>Prepare a solvent control sample by mixing 10 &#181;l of DMSO in 190 &#181;l of assay medium.</li>
	<li>Prepare four dilutions for each water extract.
	<ol>
		<li>In the first tube, add 5 &#181;L&#160;of water extract in 95 &#181;L of assay medium and mix thoroughly. Add 50 &#181;l of 5% DMSO in assay medium in three other tubes.</li>
		<li>Perform a 2-fold dilution by transferring 50 &#181;l of solution from one tube to the next.</li>
	</ol>
	</li>
</ol>
3. Prepare the Cell Suspension to Conduct the FRET Bioassay
<ol>
	<li>Prepare the assay specific medium according to the manufacturer&#39;s instructions. Assay medium must be stored at 4 &#176;C and warmed to 37 &#176;C in a water-bath before use.</li>
	<li>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 &#176;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.</li>
	<li>Open the vial using aseptic techniques and transfer the cells into 10 ml of assay medium.</li>
	<li>Centrifuge at 200 x g for 5 min.</li>
	<li>Aspirate the supernatant using a sterile glass pipet and resuspend the cell pellet in 6 ml of assay medium.</li>
	<li>Mix 5 &#181;l of cell suspension with 5 &#181;l of a vital stain solution and add an aliquot into a counting chamber.</li>
	<li>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.</li>
</ol>
4. Plate Cells in a 96-well Black Wall Clear-bottom Plate and Add Diluted Water Extract
<ol>
	<li>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.</li>
	<li>Add 90 &#181;l of assay medium to the replicate cell-free control wells.</li>
	<li>Pour the cell suspension in a sterile pipetting reservoir and add 90 &#181;l of cell suspension to the other wells using a multichannel pipette.</li>
	<li>Add 10 &#181;l of the diluted samples prepared during step 2 to the appropriate wells. The final concentration of DMSO per well must be 0.5% maximum.</li>
	<li>Cover the plate with a lid and place it in a 5% CO2 incubator at 37 &#176;C for 16 hr.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/54725/54725fig1.jpg" /> 
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. <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54725/54725fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Prepare the Loading Solution and Add to Each Well
<ol>
	<li>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.</li>
	<li>Prepare a 6x loading solution according to the manufacturer&#39;s instructions.</li>
	<li>Add 20 &#181;l of the 6x loading solution to each well.</li>
	<li>Add 10 &#181;l of cell viability reagent to each well to evaluate the cytotoxicity of the diluted water extract.</li>
	<li>Seal the plate with aluminum adhesive film.</li>
	<li>Incubate the plate in the dark at RT for 2 hr.</li>
</ol>
6. Measure Cytotoxicity and Endocrine Activity Response
<ol>
	<li>Set up the microplate reader with bottom-reading capabilities following the manufacturer&#39;s instructions.</li>
	<li>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.</li>
	<li>For the cytotoxicity assay, measure fluorescence at 560 Ex/590 Em nm.</li>
</ol>
7. Assess QA/QC Checks to Determine the Quality of the Data
<ol>
	<li>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.</li>
	<li>Process the raw FRET data. For both &#39;blue&#39; and &#39;green&#39; 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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Calculate the RSD&#160;for all triplicate wells. The variability among replicates should be less than 20%.</li>
	<li>For the cytotoxicity data (560 Ex/590 Em nm), subtract the cell-free control average (i.e. media background) from all cell-containing wells.</li>
	<li>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.</li>
</ol>
8. Data Analyses
<ol>
	<li>Calculate the assay Limit of Detection (LOD) as the minimum calibration response plus two standard deviations of the mean of that response.</li>
	<li>For samples showing a dose response, derive the EC50&#160;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.</li>
	<li>Calculate the Bioanalytical Equivalent Concentration (BEQ) using the formula: BEQ = EC50 of reference chemical/EC50 of water sample.</li>
</ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tiered,+integrated+biological+and+chemical+monitoring+framework+for+contaminants+of+emerging+concern+(CECs)+in+aquatic+ecosystems.">A tiered, integrated biological and chemical monitoring framework for contaminants of emerging concern (CECs) in aquatic ecosystems.</a> Integr. Environ. Assess. Manag. , (2015).</li><li>Van der Linden, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+multiple+hormonal+activities+in+wastewater+effluents,+surface+water,+using+a+panel+of+steroid+receptor+CALUX+bioassays.">Detection of multiple hormonal activities in wastewater effluents, surface water, using a panel of steroid receptor CALUX bioassays.</a> Environ. Sci. Technol. 42 (15), 5814-5820 (2008).</li><li>Leusch, F. D. 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The authors have nothing to disclose.

The well documented potency of environmental estrogens, such as 17&#946;-estradiol (E2), warrants screening for these chemicals at ng/L concentrations23,24. 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 (&#60;0.5 to 4 ng E2/L) were within the range reported for surface and stormwater elsewhere (&#60;1 to 11 ng E2/L)16. Despite the low levels of ER activity measured in the surface water samples, this assay represents a relevant screening endpoint for water quality assessment. Estrogens such as bisphenol A and alkylphenol surfactants are often detected in the aquatic environment, but they can be difficult to quantify using conventional analytical chemistry. ER-cell assays may also prove useful for screening of currently and newly registered pesticides, as shown by Kojima et al25.
The GR activity exceeded the ER activity by an order of magnitude in all 4 effluent samples analyzed in this study (Table 2). This trend is consistent with previous studies on wastewater effluent14, and the range for GR-BEQs reported here is slightly higher but comparable to that reported for other secondary effluents using the same in vitro bioassay12. Using a different cell bioassay, the range for GR-BEQs for effluent from wastewater treatment plants in Japan was lower (&#60;3 to 78 ng Dex/L) than those reported for secondary effluent in the present study22. With the exception of the single surface water sample that registered a maximum GR response of 30 ng Dex/L (Table 2, sample E), the GR activity in the remaining surface water samples was consistent with the low activity reported for Dutch surface waters21. However, the environmental impact for GR active chemicals is less well characterized than for ER active chemicals26, making assessment of the potential for higher order effects a challenge.
As with conventional chemical analysis, adherence to written protocols and validation of measurements using a performance-based QA/QC approach maximizes data quality and robustness. Although validation of cell bioassays adapted for water quality assessment will continue to evolve and improve, measurement of the QA/QC parameters first outlined in Mehinto et al.14 and applied to this study showed that our results were well within prespecified guideline values (Table 1). The calibration curve parameters for these bioassays mirror those used for calibrating other analytical protocols (e.g., GC-MS), with the addition of criteria that assess the viability of the cells (e.g., cytotoxicity) representing the main difference. Another operational difference made possible using cells in a high throughput format is the inclusion of multiple sample dilutions that illustrate the concentration dependent response expected when bioactivity is measured (Figure. 2). In reviewing the sample measurements, the lower ER and GR responses for the tertiary effluent (sample D) compared to the three secondary effluent samples (A-C) provides additional evidence of the relative accuracy of our bioassay screening results in Table 2. Incorporation of control or reference samples that represent the matrix of interest, in this case water, would further enhance the ability to compare bioassay results within and especially across measuring entities.
The responses of the ER and GR transactivation cell lines utilized in this study were expressed relative to the activity of the strong agonists E2 and dexamethasone, respectively. This allows for a quantitative estimate of bioactivity expressed as a Bioassay Equivalent Concentration (BEQ). This further allows for investigation of chemicals that contribute to the bioassay response, i.e. a direct comparison of BEQs with concentrations of individual agonists determined via conventional analytical chemistry. This concept underlines the added utility of bioscreening in directing diagnostic investigation of causative agents, also known as effects-directed analysis.
Since SPE is routinely used to isolate a wide range of pharmaceuticals and other &#34;emerging contaminants&#34; from water, we employed a previously published protocol9, 20 that featured a sorbent designed to capture both hydrophilic and hydrophobic chemicals in water. Although we assume quantitative capture of all bioactive organic compounds using this technique, additional work is needed to fully standardize and validate the performance of such extraction methods. The type of cell assay employed in this study may present another limitation. These assays measure the interactions between contaminants and nuclear receptors and do not take into account other possible interactions, such as chemical-membrane receptor. Thus, it is possible that the screening responses measured here were underestimated. Like sample extraction, there are no standardized methods to analyze cell assay data and different approaches may produce different results. The linear regression used in this study to model the response for the lower half of the calibration curve may not be appropriate for all cell assays, particularly if modeled response is extended beyond the relatively narrow range specified in the present study. Additional investigation into the above issues is needed to understand the extent to which these tools can be applied as a robust monitoring tool.
Whereas much of the groundwork is being laid for ER and GR as screens for endocrine active chemicals, researchers are broadening the scope of bioanalytical tools to include other relevant mode of actions, e.g., androgenicity, genotoxicity, neurotoxicity and immunotoxicity9,27. A more complete toolbox will enhance the ability to screen for bioactive chemicals, including transformation products of known and/or unknown chemicals that can occur28, now and into the future, in various aquatic environments19.

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&#39;vitro bioassays that respond to chemicals acting via known, specific modes of action1,2 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 wastewaters4 -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 analyses7,8.
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 utility9,10. Currently, only a handful of bioassays have been shown to achieve good measurement precision (within laboratories) while demonstrating the ability to differentiate among water qualities5,6. For treated wastewater effluent in particular, the occurrence of estrogens and glucocorticoid steroids has been successfully accounted for using in vitro transactivation assays11,12. 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 &#34;freeze and thaw&#34; formats. These division-arrested cell &#34;kits&#34; 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 waters17,18 and more recently recycled water19,20 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.

<table><tbody><tr><td>GeneBLAzer ER alpha DA assay kit</td><td>ThermoFisher</td><td>K1393</td><td>Kit includes ER division arrested (DA) cells and LiveBLAzer FRET loading kit.</td></tr><tr><td>GeneBLAzer GR DA assay kit</td><td>ThermoFisher</td><td>K1391</td><td>Kit includes GR division arrested (DA) cells and LiveBLAzer FRET loading kit.</td></tr><tr><td>PrestoBlue cell viability reagent&amp;nbsp;</td><td>ThermoFisher</td><td>A-13261</td><td></td></tr><tr><td>Trypan blue, 0.4% in PBS</td><td>Sigma-Aldrich&amp;nbsp;</td><td>T8154</td><td>Also available at ThermoFisher</td></tr><tr><td>Corning 96-well black wall, clear-bottom plate</td><td>Corning</td><td>3603</td><td>Individually wrapped, sterile with lid</td></tr><tr><td>Whatman glass fiber filters, GF/A, 1.6 &amp;micro;M</td><td>Sigma-Aldrich&amp;nbsp;</td><td>WHA1820025</td><td></td></tr><tr><td>Microplate aluminum sealing film</td><td>E&amp;amp;K Scientific</td><td>T592100</td><td></td></tr><tr><td>Oasis HLB 6 cc cartridge, 200 mg sorbent</td><td>Waters</td><td>WAT106202</td><td></td></tr><tr><td>17&amp;beta; Estradiol</td><td>Sigma-Aldrich&amp;nbsp;</td><td>E2758</td><td>CAS #50-28-2</td></tr><tr><td>Ascorbic acid</td><td>Fisher Scientific</td><td>A61-100</td><td>Also available at Sigma-Aldrich</td></tr><tr><td>Dexamethasone&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>D4902</td><td>CAS #50-02-2</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>D8418</td><td>Molecular grade</td></tr><tr><td>Solvents (acetone, hexane, methanol)</td><td>Fisher Scientific</td><td></td><td>HPLC grade</td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich&amp;nbsp;</td><td>S2002</td><td>Chemical is highly toxic and must be handled with caution. Use protective clothing and weigh under a fume-hood. Also available at EMD Millipore.</td></tr><tr><td>Automated cell counter or hemocytometer</td><td>Various*</td><td></td><td>Suppliers include Bio-Rad, Fisher Scientific, Sigma-Aldrich and ThermoFisher.</td></tr><tr><td>Class II biological safety cabinet</td><td>Various*</td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Various*</td><td></td><td></td></tr><tr><td>Cryogenic freezer&amp;nbsp;</td><td>Various*</td><td></td><td>Liquid nitrogen storage dewar is recommended.&amp;nbsp;</td></tr><tr><td>Fluorescence microplate-reader</td><td>Various*</td><td></td><td>&amp;nbsp;The reader must have bottom read capabilities.</td></tr><tr><td>* No recommended source, the choice of this equipment depends on budget, frequency of use, and lab space.</td><td></td><td></td><td></td></tr></tbody></table>

screening for endocrine activity in water using commercially-available in vitro transactivation bioassays

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 17&#946;-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 &#34;kits&#34;, can be adapted to screen for endocrine activity in water.

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&#160;effluent samples were from conventional activated sludge wastewater treatment plants (&#34;secondary effluent&#34;), and the fourth one from an advanced wastewater treatment plant with sand/carbon filtration added post biological treatment (&#34;tertiary effluent&#34;). Surface water samples were collected from watersheds representing different land use (open, agricultural and urban).
Dilutions of the water extracts showed no apparent cytotoxicity with less than 20% cell mortality observed compared to the DMSO controls (Table 1). Cell-free (media only) background was low, and the comparison between cells-only and cells with DMSO responses indicated that the solvent had no measurable impact on the fluorescence measurements. Parameters of the assay specific calibration curves, including slope and log EC50, were within the range of acceptable historical values (Table 1) demonstrating a good reproducibility of the calibration curves over time. An example of acceptable calibration curve for the ER assay is shown in Figure 2. The selection of 17&#946;-estradiol and dexamethasone as reference chemicals was based on the strength of assay response, likelihood of occurrence in treated wastewater and historical use by other investigators20-22. The variability in the response among triplicate samples was less than 20% RSD. Thus, all data were deemed to be of acceptable quality, and were subsequently used to estimate the ER and GR bioactivity (expressed as BEQ in ng/L) in wastewater and surface water samples.
A Relative Enrichment Factor (REF) of 5 to 10 and 4&#160;dilutions per sample appeared suitable for screening of the water samples analyzed in the present study. The majority of WWTP effluent samples showed a dose response above LOD for both ER and GR bioassays; the lowest dilution had a response within the calibration curve and the highest dilution was typically below LOD (Figure 3). However, surface water extracts did not show strong ER or GR activity. Despite an REF of 10, the fluorescent responses were often close to or below the LOD. The absence of bioassay responses for the field blank confirmed that the detections noted above LOD were not the result of sampling or procedural lab artifacts.
ER and GR activities were detected in 7 and 4 of the 10 representative water samples, respectively (Table 2), with the highest of BEQs of 17 ng E2/L and 392 ng Dex/L found in the secondary effluents. The GR activity in the sample of tertiary effluent was below the bioassay LOD. Likewise, most surface water samples exhibited no GR activity above LOD and much lower levels of ER activity than the secondary effluents.
<img alt="Figure 2" src="/files/ftp_upload/54725/54725fig2.jpg" /> 
Figure 2: Example of Calibration Curve for the ER Assay. The dose-response curve, plotted as the mean fluorescence ratio (&#177; SEM), is sigmoidal and used to determine the EC10 and EC50 values. This portion of the curve is fitted using linear regression and used to derive a bioassay equivalent concentration. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54725/54725fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54725/54725fig3.jpg" /> 
Figure 3: Mean Blue/green Fluorescence Ratio (&#177; SEM) of Water Samples Plotted Against theRrelative Enrichment Factor. Bioassay Equivalent Concentrations (BEQ) are calculated for samples whose dilution series show a concentration dependent-response with a minimum of two points above the limit of detection (LOD). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54725/54725fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="2">Estrogen Receptor (ER) Assay</td>
			<td colspan="2">Glucocorticoid Receptor (GR) Assay</td>
		</tr>
		<tr>
			<td></td>
			<td>Study results&#160;</td>
			<td>Acceptance criteria&#160;</td>
			<td>Study results&#160;</td>
			<td>Acceptance criteria&#160;</td>
		</tr>
		<tr>
			<td>Calibration parameters</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Mean log EC50 (M)</td>
			<td>-9.6</td>
			<td>-10.1 to -9.1</td>
			<td>-8.5</td>
			<td>-9.0 to -8.0</td>
		</tr>
		<tr>
			<td>Mean slope</td>
			<td>1.1</td>
			<td>0.9 to 1.5</td>
			<td>2.0</td>
			<td>1.8 to 2.4</td>
		</tr>
		<tr>
			<td>R2</td>
			<td>0.99</td>
			<td>&#62;0.95</td>
			<td>0.99</td>
			<td>&#62;0.95</td>
		</tr>
		<tr>
			<td>QA/QC controls</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Media background</td>
			<td>yes</td>
			<td>media only &#60;cell+media</td>
			<td>yes</td>
			<td>media only &#60;cell+media</td>
		</tr>
		<tr>
			<td>Cytotoxicity (% mortality)</td>
			<td>0%</td>
			<td>&#60;20% mortality</td>
			<td>0 to 5%</td>
			<td>&#60;20% mortality</td>
		</tr>
		<tr>
			<td>Sample precision (%RSD)</td>
			<td>2 to 18%</td>
			<td>&#60;20%</td>
			<td>4 to 13%</td>
			<td>&#60;20%</td>
		</tr>
	</tbody>
</table>
Table 1: Summary of QA/QC Results From Bioanalytical Screening of Treated Wastewater Effluent and Surface Water Samples.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample ID and Description</td>
			<td>ER-BEQ (ng E2/L)</td>
			<td>GR-BEQ (ng Dex/L)</td>
		</tr>
		<tr>
			<td>A &#8211; final effluent from full secondary treatment plant</td>
			<td>6.4</td>
			<td>248</td>
		</tr>
		<tr>
			<td>B &#8211; final effluent from full secondary treatment plant</td>
			<td>13</td>
			<td>392</td>
		</tr>
		<tr>
			<td>C &#8211; final effluent from full secondary treatment plant</td>
			<td>17</td>
			<td>236</td>
		</tr>
		<tr>
			<td>D &#8211; final effluent from tertiary treatment plant</td>
			<td>2.3</td>
			<td>&#60;22</td>
		</tr>
		<tr>
			<td>E &#8211; surface water from agricultural area</td>
			<td>&#60;0.5</td>
			<td>30</td>
		</tr>
		<tr>
			<td>F &#8211; surface water from agricultural area</td>
			<td>&#60;0.5</td>
			<td>&#60;22</td>
		</tr>
		<tr>
			<td>G &#8211; surface water from urban area</td>
			<td>4</td>
			<td>&#60;22</td>
		</tr>
		<tr>
			<td>H &#8211; surface water from urban area</td>
			<td>0.9</td>
			<td>&#60;22</td>
		</tr>
		<tr>
			<td>I &#8211; surface water from open field</td>
			<td>0.8</td>
			<td>&#60;22</td>
		</tr>
		<tr>
			<td>J &#8211; surface water from open field</td>
			<td>&#60;0.5</td>
			<td>&#60;22</td>
		</tr>
		<tr>
			<td>K &#8211; field blank (ultrapure water)</td>
			<td>&#60;0.5</td>
			<td>&#60;22</td>
		</tr>
	</tbody>
</table>
Table 2: Bioassay Equivalent Concentrations for Estrogen Receptor (ER-BEQ) and Glucocorticoid Receptor (GR-BEQ) activity. Three types of samples collected in California (USA) are analyzed: municipal wastewater effluent (A-D), surface water (E-J), and field blank (K).

Watch this Scientific Journal Video about Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays at JoVE.com

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

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.

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

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been ...

screening-for-endocrine-activity-water-using-commercially-available

Nanyang Technological University

Research

JoVE Journal

Environment

7.5K Views.  Southern California Coastal Water Research Project Authority. 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.

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

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.

Endocrine Disrupting Compounds (EDC) pose a substantial risk to the aquatic environment. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. The methodology provided in this paper allows for an assessment of the efficacy and suitability of wastewater treatment processes with respect to EDC removal.

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in ...

Using Tg(Vtg1:mcherry) Zebrafish Embryos to Test the Estrogenic Effects of Endocrine Disrupting Compounds

There are many endocrine disrupting compounds (EDC) in the environment, especially estrogenic substances. The detection of these substances is difficult due to their chemical diversity; therefore, increasingly more effect-detecting methods are used, such as estrogenic effect-sensitive biomonitor/bioindicator organisms. These biomonitoring organisms include several fish models. This protocol covers the use of zebrafish Tg(vtg1: mCherry) transgenic line as a biomonitoring organism, including the propagation of fish and the treatment of embryos, with an emphasis on the detection, documentation, and evaluation of fluorescent signals induced by EDC. The goal of the work is the demonstration of the use of the Tg(vtg1: mCherry) transgenic line embryos to detect estrogenic effects. This work documents the use of transgenic zebrafish embryos Tg(vtg1: mCherry) for the detection of estrogenic effects by testing two estrogenic substances, &#945;- and &#946;-zearalenol. The described protocol is only a basis for designing assays; the test method can be varied according to the test endpoints and the samples. Moreover, it can be combined with other assay methods, thereby facilitating the future use of the transgenic line.

Using TgVtg1:mcherry Zebrafish Embryos to Test the Estrogenic Effects of Endocrine Disrupting Compounds

Present here is a detailed protocol for the use of zebrafish embryos Tg(vtg1: mCherry) for the detection of estrogenic effects. The protocol covers the propagation of the fish and treatment of embryos, and emphasizes the detection, documentation, and the evaluation of fluorescent signals induced by endocrine disrupting compounds (EDC).

There are many endocrine disrupting compounds (EDC) in the environment, especially estrogenic substances. The detection of these substances is difficult ...

Screening for Phytoestrogens using a Cell-based Estrogen Receptor  &#946; Reporter Assay

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in the vertebrates that consume them, such as endocrine function. Phytoestrogens, the most well studied endocrine-active phytochemicals, directly interact with the hypothalamo-pituitary gonadal axis of the vertebrate endocrine system. Here we present the novel use of a cell-based assay to screen plant extracts for the presence of compounds that have estrogenic biological activity. This assay uses mammalian cells engineered to highly express estrogen receptor beta (ER&#946;) and that have been transfected with a luciferase gene. Exposure to compounds with estrogenic activity results in the cells producing light. This assay is a reliable and simple way to test for biological estrogenic activity. It has several improvements over transient transfection assays, most notably, ease of use, the stability of the cells, and the sensitivity of the assay.

We have optimized a commercially available estrogen receptor &#946; reporter assay for screening human and nonhuman primate foods for estrogenic activity. We validated this assay by showing that the known estrogenic human food soy registers high, while other foods show no activity.

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in ...

Biochemistry

In Silico Modeling Method for Computational Aquatic Toxicology of Endocrine Disruptors: A Software-Based Approach Using QSAR Toolbox

Computational analyses of toxicological processes enables high-throughput screening of chemical substances and prediction of their endpoints in biological systems. In particular, quantitative structure-activity relationship (QSAR) models have been increasingly applied to assess the environmental effects of a plethora of toxic materials. In recent years, some more highlighted types of toxicants are endocrine disruptors (EDs, which are chemicals that can interfere with any hormone-related metabolism). Because EDs may significantly affect animal development and reproduction, rapidly predicting the adverse effects of EDs using in silico techniques is required. This study presents an in silico method to generate prediction data on the effects of representative EDs in aquatic vertebrates, particularly fish species. The protocol describes an example utilizing the automated workflow of the QSAR Toolbox software developed by the Organization for Economic Co-operation and Development (OECD) to enable acute ecotoxicity predictions of EDs. As a result, the following are determined: (1) calculation of the numerical correlations between the concentration for 50% of lethality (LC50) and octanol-water partition coefficient (Kow), (2) output performances in which the LC50 values determined in experiments are compared to those generated by computations, and (3) the dependence of estrogen receptor binding affinity on the relationship between Kow and LC50.

Quantitative structure-activity relationship (QSAR) modeling is a representative bioinformatics-assisted method in toxicological screening. This protocol demonstrates how to computationally assess the risks of endocrine disruptors (EDs) in aquatic environments. Utilizing the OECD QSAR Toolbox, the protocol implements an in silico assay for analyzing toxicity of EDs in fish.

Computational analyses of toxicological processes enables high-throughput screening of chemical substances and prediction of their endpoints in biological ...

Bioengineering

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

Biology

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

A Rapid In Vivo Bioassay for Developmentally Active Enhancers

A potential regulatory sequence (PRS) can be identified by several approaches including conservation analysis and transcription factor-specific chromatin immunoprecipitation followed by next generation sequencing (TF ChIP-seq). TF ChIP-seq generates a large data set (TF-regulatome) that includes PRSs and background interactions. These approaches require secondary low throughput validation in model systems that replicate the temporal and spatial specificity of the PRS. 
In this report, a protocol is described to rapidly validate activity of PRSs in their developmental context. Following selection of candidate PRSs, the associated sequences are isolated, cloned into a GFP reporter construct, and then transfected into chick embryos. For PRSs that are anticipated to be functional during early development, from gastrulation through early organogenesis, whole embryo electroporation (EP) is recommended. With this technique transfection, incubation and monitoring occurs ex ovo during early gastrulation. The entire embryo is transfected and as structures form, they can be monitored live for PRS activity. This ex ovo approach supports development up to early limb bud outgrowth. 
If the functional activity of a PRS is expected to be later in development or if the investigation is to ascertain its activity during the development of a specific organ, targeted regional electroporation (TREP) is used. This approach requires injection of construct DNA directly into the developing organ, precursor or adjacent cavity prior to electroporation. An approach to transfect the presumptive limb bud using TREP is also outlined and illustrated. TREP supports a rapid organ-specific validation of a PRS' activity. Both techniques can also be used to investigate regulatory domains by site-directed mutagenesis of the PRS-reporter construct. 
The chick as a bioassay offers a rapid effective tool for PRS validation, functional domain determination, and further characterization of the regulatory events responsible for development.

With the increased use of transcription factor-specific ChIP-seq technology, techniques to validate potential regulatory regions are critical. The authors describe a rapid chick bioassay to validate the activity of regulatory sequences functioning during development.

A potential regulatory sequence (PRS) can be identified by several approaches including conservation analysis and transcription factor-specific chromatin ...

Imaging of Thyroid Hormone Action Indicator Mice to Assess Endocrine Disruption

In vivo Characterization of Endocrine Disrupting Chemical Effects via Thyroid Hormone Action Indicator Mouse

Thyroid hormones (TH) play a critical role in cell metabolism and tissue function. TH economy is susceptible to endocrine disrupting chemicals (EDCs) that can disturb hormone production or action. Many environmental pollutants are EDCs, representing an emerging threat to both human health and agricultural production. This has led to an increased demand for proper test systems to examine the effects of potential EDCs. However, current methodologies face challenges. Most test systems use endogenous markers regulated by multiple, often complex regulatory processes, making it difficult to distinguish direct and indirect effects. Moreover, in vitro test systems lack the physiological complexity of EDC metabolism and pharmacokinetics in mammals. Additionally, exposure to environmental EDCs usually involves a mixture of multiple compounds, including in vivo generated metabolites, so the possibility of interactions cannot be ignored. This complexity makes EDC characterization difficult. The Thyroid Hormone Action Indicator (THAI) mouse is a transgenic model that carries a TH-responsive luciferase reporter system, enabling the assessment of tissue-specific TH action. One can evaluate the tissue-specific effects of chemicals on local TH action by quantifying luciferase reporter expression in tissue samples. Furthermore, with in vivo imaging, the THAI mouse model allows for longitudinal studies on the effects of potential EDCs in live animals. This approach provides a powerful tool for testing long-term exposure, complex treatment structures, or withdrawal, as it enables the assessment of changes in local TH action over time in the same animal. This report describes the process of in vivo imaging measurements on THAI mice. The protocol discussed here focuses on developing and imaging hyper- and hypothyroid mice, which can serve as controls. Researchers can adapt or expand the treatments presented to meet their specific needs, offering a foundational approach for further investigation.

Author Spotlight: In Vivo Assessment of Thyroid Hormone Disruption Using the THAI Mouse Model

The Thyroid Hormone Action Indicator mouse model was developed to enable tissue-specific quantification of local thyroid hormone action using its endogenous regulatory machinery. Recently, it has been shown that the model is suitable for characterizing endocrine-disrupting chemicals interacting with thyroid hormone economy, both by ex vivo and in vivo methodologies.

Thyroid hormones (TH) play a critical role in cell metabolism and tissue function. TH economy is susceptible to endocrine disrupting chemicals (EDCs) that ...

Neuroscience

Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory

The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine disruption. Estrogenic ligands include both natural and manmade &#34;Environmental Estrogens&#34; (EEs) found in many consumer goods including Personal Care Products (PCPs), plastics, pesticides, and foods. EEs disrupt hormone signaling in humans and other animals, potentially reducing fertility and increasing disease risk. Despite the importance of EEs and other Endocrine Disrupting Chemicals (EDCs) to public health, endocrine disruption is not typically included in undergraduate curricula. This shortcoming is partly due to a lack of relevant laboratory activities that illustrate the principles involved while also being accessible to undergraduate students. This article presents an optimized YES for quantifying ligands in personal care products that bind estrogen receptors alpha (ER&#945;) and/or beta (ER&#946;). The method incorporates one of the two colorimetric substrates (ortho-nitrophenyl-&#946;-D-galactopyranoside (ONPG) or chlorophenol red-&#946;-D-galactopyranoside (CPRG)) that are cleaved by &#946;-galactosidase, a 6-day refrigerated incubation step to facilitate use in undergraduate laboratory courses, an automated application for LacZ calculations, and R code for the associated 4-parameter logistic regression analysis. The protocol has been designed to allow undergraduate students to develop and conduct experiments in which they screen products of their choosing for estrogen mimics. In the process, they learn about endocrine disruption, cell culture, receptor binding, enzyme activity, genetic engineering, statistics, and experimental design. Simultaneously, they also practice fundamental and broadly applicable laboratory skills, such as: calculating concentrations; making solutions; demonstrating sterile technique; serially diluting standards; constructing and interpolating standard curves; identifying variables and controls; collecting, organizing, and analyzing data; constructing and interpreting graphs; and using common laboratory equipment such as micropipettors and spectrophotometers. Thus, implementing this assay encourages students to engage in inquiry-based learning while exploring emerging issues in environmental science and health.

This article presents an optimized yeast estrogen screen for quantifying ligands in Personal Care Products (PCPs) that bind estrogen receptors alpha (ER&#945;) and/or beta (ER&#946;). The method incorporates two colorimetric substrate options, a six-day refrigerated incubation for use in undergraduate courses, and statistical tools for data analysis.

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

Summary

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In vivo Characterization of Endocrine Disrupting Chemical Effects via Thyroid Hormone Action Indicator Mouse

Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory