Name: screening for endocrine activity in water using commercially-available in vitro transactivation bioassays
Uploaded: 2016-12-04T13:00:00
Description: Watch this Scientific Journal Video about Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays at JoVE.com

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 throug...

<ol>
	<li>Dix, D. J., Houck, K. A., Martin, M. T., Richard, M. A., Setzer, R. W., Kavlock, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ToxCast+program+for+prioritizing+toxicity+testing+of+environmental+chemicals.">The ToxCast program for prioritizing toxicity testing of environmental chemicals.</a> Toxicol. Sci. 95 (1), 5-12 (2007).</li><li>Reif, D. M., 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=Endocrine+profiling+and+prioritization+of+environmental+chemicals+using+ToxCast+data.">Endocrine profiling and prioritization of environmental chemicals using ToxCast data.</a> Environ. Health Perspect. 118 (12), 1714-1720 (2010).</li><li>Maruya, K. 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. L., 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=Comparison+of+five+in+vitro+bioassays+to+measure+estrogenic+activity+in+environmental+waters.">Comparison of five in vitro bioassays to measure estrogenic activity in environmental waters.</a> Environ. Sci. Technol. 44 (10), 3853-3860 (2010).</li><li>Jarosova, B., 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=Europe-wide+survey+of+estrogenicity+in+wastewater+treatment+plant+effluents:+the+need+for+effect-based+monitoring.">Europe-wide survey of estrogenicity in wastewater treatment plant effluents: the need for effect-based monitoring.</a> Environ. Sci. Pollut. Res. 21 (18), 10970-10982 (2014).</li><li>Sonneveld, E., 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=Comparison+of+in+vitro+and+in+vivo+screening+models+for+androgenic+and+estrogenic+activities.">Comparison of in vitro and in vivo screening models for androgenic and estrogenic activities.</a> Toxicol. Sci. 89 (1), 173-187 (2006).</li><li>Piersma, A. H., 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=Evaluation+of+an+alternative+in+vitro+test+battery+for+detecting+reproductive+toxicants.">Evaluation of an alternative in vitro test battery for detecting reproductive toxicants.</a> Reprod. Toxicol. 38, 53-64 (2013).</li><li>Escher, B. I., 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=Benchmarking+organic+micropollutants+in+wastewater,+recycled+water+and+drinking+water+with+in+vitro+bioassays.">Benchmarking organic micropollutants in wastewater, recycled water and drinking water with in vitro bioassays.</a> Environ. Sci. Technol. 48 (3), 1940-1956 (2014).</li><li>U.S. Environmental Protection Agency (USEPA) Endocrine Disruptor Screening Program. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Prioritization of the endocrine disruptor screening program universe of chemicals for an estrogen receptor adverse outcome pathway using computational toxicology tools. , (2012).</li><li>Leusch, F. D. L., 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=Assessment+of+wastewater+and+recycled+water+quality:+a+comparison+of+lines+of+evidence+from+in+vitro,+in+vivo+and+chemical+analyses.">Assessment of wastewater and recycled water quality: a comparison of lines of evidence from in vitro, in vivo and chemical analyses.</a> Water Res. 50, 420-431 (2014).</li><li>Jia, A., Wu, S., Daniels, K. D., Snyder, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balancing+the+budget:+accounting+for+glucocorticoid+bioactivity+and+fate+during+water+treatment.">Balancing the budget: accounting for glucocorticoid bioactivity and fate during water treatment.</a> Environ. Sci. Technol. 50 (6), 2870-2880 (2016).</li><li>Huang, R., 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=Chemical+genomics+profiling+of+environmental+chemical+modulation+of+human+nuclear+receptors.">Chemical genomics profiling of environmental chemical modulation of human nuclear receptors.</a> Environ. Health Perspect. 119 (8), 1142-1148 (2011).</li><li>Mehinto, A. 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=Interlaboratory+comparison+of+in+vitro+bioassays+for+screening+of+endocrine+active+chemicals+in+recycled+water.">Interlaboratory comparison of in vitro bioassays for screening of endocrine active chemicals in recycled water.</a> Water Res. 83, 303-309 (2015).</li><li>Ternes, T. A., Joss, A., Siegrist, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scrutinizing+pharmaceuticals+and+personal+care+products+in+wastewater+treatment.">Scrutinizing pharmaceuticals and personal care products in wastewater treatment.</a> Environ. Sci. Technol. 38 (20), 392A-399A (2004).</li><li>Tang, J. Y. M., 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=Toxicity+characterization+of+urban+stormwater+with+bioanalytical+tools.">Toxicity characterization of urban stormwater with bioanalytical tools.</a> Water Res. 47, 5594-5606 (2013).</li><li>Scott, P. D., 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=An+assessment+of+endocrine+activity+in+Australian+rivers+using+chemical+and+in+vitro+analyses.">An assessment of endocrine activity in Australian rivers using chemical and in vitro analyses.</a> Environ. Sci. Pollut. Res. 21 (22), 12951-12967 (2014).</li><li>Vidal-Dorsch, D. E., Bay, S. M., Maruya, K., Snyder, S. A., Trenholm, R. A., Vanderford, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contaminants+of+emerging+concern+in+municipal+wastewater+effluents+and+marine+receiving+water.">Contaminants of emerging concern in municipal wastewater effluents and marine receiving water.</a> Environ. Toxicol. Chem. 31 (12), 2674-2682 (2012).</li><li>WateReuse Research Foundation (WRRF). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Direct potable reuse: a path forward. , (2011).</li><li>Leusch, F. D. L., 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=Assessment+of+the+application+of+bioanalytical+tools+as+surrogate+measure+of+chemical+contaminants+in+recycled+water.">Assessment of the application of bioanalytical tools as surrogate measure of chemical contaminants in recycled water.</a> Water Res. 49, 300-315 (2014).</li><li>Schriks, M., 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=Occurrence+of+glucocorticoid+activity+in+various+surface+waters+in+the+Netherlands.">Occurrence of glucocorticoid activity in various surface waters in the Netherlands.</a> Chemosphere. 93 (2), 450-454 (2013).</li><li>Suzuki, G., Sato, K., Isobe, T., Takigami, H., Brouwer, A., Nakayama, K. <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+glucocorticoid+receptor+agonist+in+effluents+from+sewage+treatment+plants+in+Japan.">Detection of glucocorticoid receptor agonist in effluents from sewage treatment plants in Japan.</a> Sci. Tot. Environ. 527-528, 328-334 (2015).</li><li>Purdom, C. E., Hardiman, P. A., Byea, V. V. J., Enoa, N. C., Tyler, C. R., Sumpter, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogenic+effects+of+effluents+from+sewage+treatment+works.">Estrogenic effects of effluents from sewage treatment works.</a> Chemistry and Ecology. 8 (4), 275-285 (1994).</li><li>Kidd, K. 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=Collapse+of+a+fish+population+after+exposure+to+a+synthetic+estrogen.">Collapse of a fish population after exposure to a synthetic estrogen.</a> Proc. Natl. Acad. Sci. 104 (21), 8897-8901 (2007).</li><li>Kojima, H., Katsura, E., Takeuchi, S., Niiyama, K., Kobayashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+of+estrogen+and+androgen+receptor+activities+in+200+pesticides+by+in+vitro+reporter+gene+assays+using+Chinese+hamster+ovary+cells.">Screening of estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells.</a> Environ. Health Perspect. 112 (5), 524-531 (2004).</li><li>Kugathas, S., Sumpter, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+glucocorticoids+in+the+environment:+First+results+on+their+potential+impacts+on+fish.">Synthetic glucocorticoids in the environment: First results on their potential impacts on fish.</a> Environ. Sci. Technol. 45, 2377-2383 (2011).</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=Development+of+a+panel+of+high-throughput+reporter-gene+assays+to+detect+genotoxicity+and+oxidative+stress.">Development of a panel of high-throughput reporter-gene assays to detect genotoxicity and oxidative stress.</a> Mutat. Res. Genet. Toxicol. Environ. Mutagen. 760, 23-32 (2014).</li><li>Cwiertny, D. M., Snyder, S. A., Schlenk, D., Kolodziej, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+designer+drugs:+when+transformation+may+not+eliminate+risk.">Environmental designer drugs: when transformation may not eliminate risk.</a> Environ. Sci. Technol. 48, 11737-11745 (2014).</li></ol>

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 lo...

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 border="0" cellpadding="0" cellspacing="0" width="826">
	<tbody>
		<tr height="42">
			<td height="42" style="height:43px;width:275px;">GeneBLAzer ER alpha DA assay kit</td>
			<td style="width:123px;">ThermoFisher</td>
			<td style="width:115px;">K1393</td>
			<td style="width:313px;">Kit includes ER division arrested (DA) cells and LiveBLAzer FRET loading kit.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:275px;">GeneBLAzer GR DA assay kit</td>
			<td style="width:123px;">ThermoFisher</td>
			<td style="width:115px;">K1391</td>
			<td style="width:313px;">Kit includes GR division arrested (DA) cells and LiveBLAzer FRET loading kit.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">PrestoBlue cell viability reagent&#160;</td>
			<td style="width:123px;">ThermoFisher</td>
			<td style="width:115px;">A-13261</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Trypan blue, 0.4% in PBS</td>
			<td style="width:123px;">Sigma-Aldrich&#160;</td>
			<td style="width:115px;">T8154</td>
			<td>Also available at ThermoFisher</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Corning 96 well black wall, clear-bottom plate</td>
			<td style="width:123px;">Corning</td>
			<td style="width:115px;">3603</td>
			<td style="width:313px;">Individually wrapped, sterile with lid</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Whatman glass fiber filters, GF/A, 1.6 &#181;M</td>
			<td style="width:123px;">Sigma-Aldrich&#160;</td>
			<td style="width:115px;">WHA1820025</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Microplate aluminum sealing film</td>
			<td style="width:123px;">E&#38;K Scientific</td>
			<td style="width:115px;">T592100</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Oasis HLB 6 cc cartridge, 200 mg sorbent</td>
			<td style="width:123px;">Waters</td>
			<td style="width:115px;">WAT106202</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">17&#946; Estradiol</td>
			<td style="width:123px;">Sigma-Aldrich&#160;</td>
			<td style="width:115px;">E2758</td>
			<td style="width:313px;">CAS #50-28-2</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Ascorbic acid</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:115px;">A61-100</td>
			<td style="width:313px;">Also available at Sigma-Aldrich</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Dexamethasone&#160;</td>
			<td style="width:123px;">Sigma-Aldrich&#160;</td>
			<td style="width:115px;">D4902</td>
			<td style="width:313px;">CAS #50-02-2</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:123px;">Sigma-Aldrich&#160;</td>
			<td style="width:115px;">D8418</td>
			<td>Molecular grade</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Solvents (acetone, hexane, methanol)</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:115px;"></td>
			<td style="width:313px;">HPLC grade</td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:275px;">Sodium azide</td>
			<td style="width:123px;">Sigma-Aldrich&#160;</td>
			<td style="width:115px;">S2002</td>
			<td style="width:313px;">Chemical is highly toxic and must be handled with caution. Use protective clothing and weigh under a fumehood. Also available at EMD Millipore.</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:275px;">Automated cell counter or hemocytometer</td>
			<td style="width:123px;">Various*</td>
			<td style="width:115px;"></td>
			<td style="width:313px;">Suppliers include Bio-Rad, Fisher Scientific, Sigma-Aldrich and ThermoFisher.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Class II biological safety cabinet</td>
			<td style="width:123px;">Various*</td>
			<td style="width:115px;"></td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">CO2 incubator</td>
			<td style="width:123px;">Various*</td>
			<td style="width:115px;"></td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:275px;">Cryogenic freezer&#160;</td>
			<td style="width:123px;">Various*</td>
			<td style="width:115px;"></td>
			<td style="width:313px;">Liquid nitrogen storage dewar is recommended.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:275px;">Fluorescence microplate reader</td>
			<td style="width:123px;">Various*</td>
			<td style="width:115px;"></td>
			<td style="width:313px;">&#160;The reader must have bottom read capabilities.</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">* No recommended source, the choice of this equipment depends on budget, frequency of use, and lab space.</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 po...

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 ...

The overall goal of this standardized procedure is to screen water samples for endocrine active chemicals, using commercially-available, receptor-based cell assays. This method can help answer key questions in the water quality monitoring field, such as, are endocrine active chemicals actually present in the sample? The main advantage of this technique is that it can analyze for a group of endocrine active compounds much faster than the current chemical methods.This technique can help diagnose water quality issues that arise from the presence of endocrine disrupting chemicals. This method provides insight into contaminants in water, but it can also be applied to other environmental matrices, such as sediment and tissues. Generally, individuals new to this technique may struggle, because it requires high precision when using pipetting techniques.To begin this procedure, prepare the water sample in stock solutions of assay-specific medium and reference chemical, as detailed in the text protocol. Store the assay-specific medium at four degrees Celsius for long-term storage. Next, add 15 microliters of the appropriate reference chemical stock to 285 microliters of assay medium in a sterile tube.Mix the sample by pipetting up and down. Then, add 200 microliters of 5%DMSO in assay medium to eight additional tubes. Transfer a 100-microliter aliquot from tube one to tube two, to begin a three-fold dilution series.Mix the diluted sample in tube two thoroughly with a pipette. Then, transfer a 100-microliter aliquot from tube two to tube three. Continue this process for each tube.Then, prepare a solvent control sample by mixing 10 microliters of DMSO in 190 microliters of assay medium. Next, collect and label four new tubes. Add 95 microliters of assay medium to the first tube.Then, add five microliters of water extract and mix thoroughly. Add 50 microliters of 5%DMSO in assay medium to the other three tubes. Then, perform a two-fold dilution by transferring 50 microliters of solution from one tube to the next.Begin by taking a vial of ER or GR division arrested cells out of the cryogenic freezer. Thaw the cells quickly by placing the vial in a 37-degree-Celsius water bath, with gentle agitation for two minutes. Then, decontaminate the vial with 70%Ethanol.Place it in a class two biological safety cabinet. Open the vial using aseptic techniques and transfer the cells into 10 milliliters of assay medium. Centrifuge at 200 times g for five minutes.When centrifugation is complete, use a sterile glass pipette to aspirate the supernatant. Then, re-suspend the cell pellet in six millileters of assay medium. Mix five microliters of cell suspension with five microliters of vital stain solution.Then, add an aliquot into a counting chamber. Count the number of live cells using a light microscope or automated cell counter. Then, estimate the density of live cells in the cell suspension.Dilute if necessary, to obtain a final density of 550, 000 live cells per milliliter of assay medium. Begin by creating a plate layout that includes a nine point assay specific calibration curve, QA/QC samples, and multiple dilutions for each water extract. Add 90 microliters of assay medium to the replicate cell free control wells.Pour the cell suspension into a sterile pipetting reservoir. Then, using a multi-channel pipette, add 90 microliters of cell suspension to the other wells. Add 10 microliters of the diluted samples to the appropriate wells.Cover the plate with a lid. Then, place the plate in a 5%C02 incubator at 37 degrees Celsius for 16 hours. Once incubation is complete, allow the plate to equilibrate to room temperature.Then next steps should be conducted in the absence of direct light. Prepare a six x loading solution according to the manufacturer's instructions. Then, add 20 microliters of the loading solution to each well.Add 10 microliters of cell viability reagent to each well, to evaluate the cytotoxicity of the diluted water extract. Seal the plate with aluminum adhesive film. Then, incubate the plate in the dark at room temperature for two hours.Once incubation is complete, add the plate to the microplate reader with bottom reading capabilities. Measure the fluorescence and analyze the data as detailed in the text protocol. In this study, four 24-hour composite samples of treated municipal waste water effluent, six grab samples of surface water from freshwater systems in Southern California, and a field blank consisting of ultra pure water were analyzed.ER activity was detected in seven of the 10 representative water samples, while GR activity was detected in four. The QA/QC performance guidelines for the estrogen receptor and glucocorticoid receptor assays can be seen here. The study results for cytotoxicity and sample precision fell well within acceptance criteria, validating the water sample measurements.The highest bioassay equivalent concentrations were found in the secondary effluents, while the GR activity of the tertiary effluent was found to be below the bio assay limit of detection. Likewise, most surface water samples exhibited no GR activity above the limit of detection and had much lower levels of ER activity than the secondary effluents. The majority of waste water treatment plant effluent samples showed a dose response above the limit of detection for both ER and GR bioassays.The representative results for the mean blue/green fluorescence ratio of these samples, plotted against the relative enrichment factor, can be seen here. This technique can be done in 24 hours, if performed properly. While attempting it, it's important to follow all the QA/QC guidelines.This technique paved the way for managers to explore alternative approaches for monitoring endocrine disrupting chemicals in water. Don't forget that working with sodium azide can be hazardous. Precautions should be taken, such as working in a properly functioning fume hood.

Research

JoVE Journal

Environment

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