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Ecotoxicology and Human Environmental Health
High-throughput Effect-Directed Analysis using downscaled in vitro reporter gene assays to identify endocrine disruptors in surface water Nick Zwart, Shan Li Nio, Corine J Houtman, Jacob De Boer, Jeroen Kool, Timo Hamers, and Marja H. Lamoree Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b06604 • Publication Date (Web): 16 Mar 2018 Downloaded from http://pubs.acs.org on March 20, 2018
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High-throughput Effect-Directed Analysis using downscaled in vitro reporter
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gene assays to identify endocrine disruptors in surface water
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Nick Zwart , Shan Li Nio , Corine J. Houtman , Jacob de Boer , Jeroen Kool , Timo Hamers , Marja H. Lamoree
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Department Environment & Health, VU University, Amsterdam, The Netherlands 2
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The Water Laboratory, Haarlem, The Netherlands
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Biomolecular Analysis group, VU University, The Netherlands
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E-mail contact:
[email protected] 11 12
Effect-Directed Analysis (EDA) is a commonly used approach for effect-based identification of endocrine
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disruptive chemicals in complex (environmental) mixtures. For routine toxicity assessment of e.g. water
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samples however, current EDA approaches are considered time consuming and laborious. We achieved
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faster EDA and identification, by downscaling of sensitive cell-based hormone reporter gene assays and
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increasing fractionation resolution to allow testing of smaller fractions with reduced complexity. The
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high-resolution EDA approach is demonstrated by analysis of four environmental passive sampler
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extracts. Downscaling of the assays to 384-well format allowed analysis of 64 fractions in triplicate (or
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192 fractions without technical replicates) without affecting sensitivity compared to the standard 96-
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well format. Through a parallel exposure method, agonistic and antagonistic androgen- and estrogen
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receptor activity could be measured in a single experiment following a single fractionation. From sixteen
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selected candidate compounds, identified through non-targeted analysis, thirteen could be confirmed
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chemically and ten were found biologically active of which the most potent non-steroidal estrogens
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were identified as oxybenzone and piperine. The increased fractionation resolution and the higher
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throughput that downscaling provides, allows for future application in routine high-resolution screening
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of large numbers of samples in order to accelerate identification of (emerging) endocrine disruptors.
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Introduction
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Endocrine disruption is an important endpoint in toxicological and environmental screening as well as
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the water quality control of the drinking water production process. Endocrine disruptive chemicals
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(EDCs) like estrogens1 and androgens2 have been detected in the aquatic environment as pollutants. A
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large portion of EDCs are emitted into the aquatic environment through urban (steroid hormones, flame
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retardants, plasticizers) or industrial wastewater,3 agricultural runoff4 or deposition after combustion
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(PAHs).5 Subsequent exposure can lead to disrupted signaling of endogenous hormones. Further liver
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metabolism can enhance receptor binding potency of (inactive) pollutants and increase the risk of
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endocrine disruption.6 While well characterized potent EDCs are actively monitored, unknown
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compounds with endocrine disruptive potency, including their metabolites, transformation products and
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degradation products, remain to be discovered. The ability to detect and identify relevant and yet
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unknown EDCs is essential to efforts aimed at reducing their presence in the aquatic environment and
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reducing human exposure.
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Cell-based reporter gene assays have been used in Effect-Directed Analysis (EDA) for the identification of
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(emerging) EDCs in environmental samples.7–9 Via EDA, compounds not analyzed by routine (chemical)
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analysis are identified based on their biological activity in reporter bioassays. Activity measured by the
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reporter gene assays in particular fractions collected during chromatographic separation can be
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correlated to mass spectrometry data. A response in one or more fractions can direct efforts to identify
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the compound responsible for the observed activity to a limited number of corresponding masses on the
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mass chromatogram.
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Reducing fraction complexity through high-resolution fractionation decreases the number of
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compounds and masses to be identified per fraction, but increases the total number of fractions.
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Luciferase reporter gene cell lines, while showing high sensitive towards their respective (ant)agonists,
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are usually performed in a 96-well plate format which limits the number of samples or fractions that can
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be analyzed simultaneously.
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This study focused on improvement of the current EDA approach by increasing throughput and
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resolution to allow for faster identification as a step forward to a more routine application of EDA in
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future (surface) water quality assessment.
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Firstly, for high-resolution EDA of endocrine disruptive chemicals, an androgen receptor (AR) (AR-
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EcoScreen),
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EcoScreen GR-KO),11 aryl hydrocarbon receptor (AhR) receptor (DR-Luc)12 and estrogen receptor (ER)
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(VM7Luc4E2, formerly known as BG1Luc4E2; further referred to as ER-Luc)13 reporter gene assay were
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downscaled from 96- to 384-well plate format. Throughput is further improved by introducing a method
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for parallel exposure of multiple endpoints with samples or fractions from a single source plate. In
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addition, a metabolic system was incorporated in the downscaled assays, to allow for formation and
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detection of active metabolites from inactive or less active pollutants.
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Secondly, downscaled assays, using the parallel exposure method, were applied in an EDA approach to
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analyze four passive sampler extracts which were separated by UPLC and collected as either 64 or 192
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fractions. The mass spectra (recorded in parallel) were analyzed at retention times that correlated with
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a recently developed AR-EcoScreen glucocorticoid receptor (GR) knockout mutant (AR-
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active fractions to select masses for identification. A qualitative non-targeted screening was performed
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and selected candidates were confirmed chemically and biologically.
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Materials and methods
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Materials
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Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) medium with glutamax, phenol-
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free DMEM/F12 medium with L-glutamine, low glucose phenol-free DMEM medium, and fetal bovine
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serum were obtained from Gibco (Eggenstein, Germany), penicillin/streptomycin, G418, hygromycin,
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zeocin, ATP, co-enzyme A, formic acid, acetonitrile (HPLC grade) and methanol (Chromasolv) from Sigma
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(Zwijndrecht, The Netherlands), luciferin from Promega (Fitchburg, WI, USA), DTT (dithiothreitol) from
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Duchefa (Haarlem, The Netherlands) and Aroclor 1254 induced rat liver S9 fraction was obtained from
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MP Biomedicals (Santa Ana, USA). Water was purified on a Milli-Q Reference A+ purification system
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(Millipore, Bedford, MA, USA). Reference compounds used for validation of the downscaled test
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methods and candidate compounds for confirmation of hits were obtained from various suppliers (see
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Supporting Information Table S1) and were dissolved in DMSO (Acros, Geel, Belgium).
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Cell culture conditions
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AR-EcoScreen (CHO-K1), exhibiting residual GR sensitivity, and AR-EcoScreen GR-KO (CHO-K1) cells, with
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exclusive AR sensitivity, were maintained as described by Satoh et al.10 ER-Luc (MCF7 human breast
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carcinoma) cells were maintained as described by Rogers and Denison.13 Briefly, cells were cultured at
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37 °C with 5% CO2 in DMEM/F12 medium (with 10% fetal bovine serum and 1% penicillin/streptomycin),
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further referred to as culture medium, and sub-cultured twice weekly. DR-Luc cells were maintained and
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exposed as described in Supporting Information, Section S1.1.
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Reporter gene assay protocols
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Reporter gene assays were performed in transparent polystyrene CellStar 96-well plates (655160)
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(Greiner Bio-One, Alphen aan den Rijn, The Netherlands) (as the standard format to compare against the
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downscaled format) or in white µclear polystyrene CellStar 384-well plates (781098) (Greiner Bio-One,
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Alphen aan den Rijn, The Netherlands) (downscaled format) using the same cell lines. To maintain the
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cell-to-surface-area ratio during seeding and compensate for smaller well size, reaction volumes used in
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the downscaled format were approximately 3-fold lower compared to the standard format (see
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Supporting Information Table S2). Potential effects of medium evaporation on the measurement were
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reduced by filling the outer ring with 100 µL (96-well plates) or two outer rings with 34 µL (384-well
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plates) sterile milliQ water which resulted in 60 or 240 wells available for measurements, respectively.
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Prior to exposure, trypsinated cells were resuspended in phenol-free DMEM/F12 L-glutamine (AR-
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EcoScreen and AR-EcoScreen GR-KO), low glucose phenol-free DMEM (ER-Luc) medium (with 5%
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charcoal stripped fetal bovine serum), further referred to as AR-EcoScreen or ER-Luc assay medium,
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respectively, and seeded at 200.000 cells/mL in 100 µL (96-well plates) or 34 µL aliquots (384-well
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plates). Plates were incubated for 24 hours at 37 °C and 5% CO2. In each experiment (n=1), seeded cells
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were exposed at t=0 in triplicate to compounds dissolved in 100 µL (96-well plates) or 34 µL assay
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medium (384-well plates), at a final DMSO concentration of 1 or 5 µL/mL for the AR-EcoScreen (and GR-
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KO) or ER-Luc cells, respectively, using a single (96-well plates) or 8-channel (384-well plates) manual
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pipette. Reference compound dilution series were prepared in DMSO and, prior to exposure, diluted to
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exposure concentrations (see Supporting Information Table S1) in the respective assay medium for each
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cell type at a final DMSO concentration of 1 or 5 µL/mL for the AR-EcoScreen (and GR-KO) or ER-Luc
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cells, respectively. In antagonism experiments, cells were additionally exposed to compounds or
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fractions in the presence of 200 pM DHT in AR-EcoScreen (and GR-KO) or 4 pM 17β-estradiol (E2) in ER-
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Luc by spiking assay medium with 5 µM DHT and 0.1 µM E2 in DMSO, respectively. Cytotoxicity was
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measured in the exposed cells by adding resazurin dissolved in PBS at t=22 h leading to a final
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concentration of 21 nM and measuring fluorescence (λex=530 nm; λem=590 nm) immediately after
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addition and at t=24 h. Conversion rate of resazurin into resorufin in cells exposed to test substances
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was compared to the conversion rate in cells exposed to vehicle (DMSO) only. At t=24 h, medium was
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aspirated and cells were lysed for 10 minutes on an orbital shaker at 700 rpm with 50 µL (96-well plates)
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or 17 µL lysis mix (25 mM TRIS (pH 7.8), 2 mM DDT, 2 mM 1,2-cyclohexylenedinitrilotetraacetic acid
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(CDTA), 10% Glycerol and 1% Triton-X100) (384-well plates). Luminescence was measured on a
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Varioskan luminometer (Thermo Fisher Scientific, Waltham, MA, USA) for one second after injection of
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100 µL or 34 µL of glow-mix (2 mM Trycin, 1.07 mM C4H2Mg5O14, 2.67 mM MgSO4, 0.1 mM EDTA, 33.3
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mM DTT, 270 mM Co-enzyme A, 470 mM Luciferin, and 530 mM ATP) followed by quenching of the
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reaction with 100 µL (96-well plates) or 34 µL 0.1 M NaOH (384-well plates).
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Biotransformation
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Prior to exposure of cells seeded on a 384-well plate in the downscaled format, compounds or fractions
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were incubated for 90 minutes at 37 °C in 50 µL DMEM phenol-free low glucose medium (with a final
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volume of 3.6% DMSO (v:v) to ensure solubility of the concentrated compounds during metabolism) in
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96-well plates, with or, as a control, without addition of 1.7 µL S9-mix (300 µL rat liver S9 fraction per
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mL, 33 mM KCl, 8mM MgCl2∙6H2O, 6.5 mM glucose-6-phosphate, 4 mM NADP, 100 mM sodium
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phosphate buffer pH 7.4), to the 50 µL reaction volume at a final concentration of 33 µL S9-mix per mL
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reaction volume (0.2 mg protein per mL reaction mixture), for generation of metabolites. Incubations
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were performed on single compounds at concentrations from 1.81 to 109 µM (BPA), 0.181 to 181 µM
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(flutamide) or 1.81 to 181 µM (tamoxifen). After incubation, 216 µL DMEM phenol-free low glucose
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medium was added to the reactions, reducing DMSO concentrations to 0.7% and S9-mix concentrations
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to 6.4 µL/mL. Cells seeded on 384-well plates were prepared for exposure by adding 24 µL of their
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respective assay medium to the aspirated cells. In each experiment (n=1) 10 µL of diluted
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biotransformation reaction mixtures were added to the cells in triplicate to reach the final volume of 34
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µL with a maximum DMSO concentration of 0.2% and an S9-mix concentration of 1.9 µL per mL
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exposure medium.
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Sample preparation
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Adsorption-based Speedisk (SD) and partitioning-based silicone rubber (SR) passive samplers were
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deployed for a period of six weeks between August and October 2014 in the river Meuse at Eijsden and
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in the effluent stream of an ultra-low loaded activated sludge municipal wastewater treatment plant
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(WWTP) with 25% industrial contribution serving a population equivalent of approximately 300.000, in
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the Netherlands. Briefly, sample preparation consisted of dichloromethane extraction of SD and hexane
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extraction of SR samplers. SD and SR extracts were solvent-exchanged from dichloromethane and
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hexane, respectively, by evaporation under a flow of nitrogen at room temperature and redissolving the
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samples in 1:2:3 (v:v:v) MeOH:ACN:H2O to 8.4 SD/mL and 60 g SR/mL, respectively.
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LC-MS analysis and fractionation
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Fractionation of SD and SR sample extracts was performed on a Kinetex C18 (100 x 2.1 mm, 1.7 µm
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particle size) column using an Agilent Infinity 1290 UPLC pump and autosampler. Extracts were injected
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(10 µL) at a flowrate of 250 µL/min in 80% mobile phase A (100% H2O + 0.1% formic acid) and 20%
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mobile phase B (100% ACN + 0.1% formic acid). The solvent gradient increased to 80% mobile phase B
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over 15 minutes and was subsequently kept as such for 13 minutes. Post-column, the flow was split in a
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9:1 ratio on a Quicksplit adjustable flow splitter (ASI, Richmond, CA, USA) with 9 parts being diverted to
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a nanofraction collector and 1 part to a microTOF II time-of-flight mass spectrometer (Bruker Daltonics,
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Billerica, MA, USA). The MS was equipped with an ESI source set to positive mode and scanned masses
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from 50 m/z to 3000 m/z at 10 Hz. Corona and capillary voltages were set to 500 and 4500 V
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respectively. Nebulizer pressure was kept at 2 bar and nitrogen drying gas flow was kept at 6 L/min.
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Exposure of fractionated extracts
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In each experiment (n=1), sample fractions were collected in either 96 (64 fractions collected at 26
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second intervals) or 384-well plates (192 fractions collected at 9 second intervals) filled with 10 µL or 4
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µL 10% DMSO in milliQ water, respectively, as keeper to increase recoveries.14 Collected fractions were
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dried in a Centrivap concentrator (Labconco Corp., Kansas City, MO, USA) for 5 hours at 25°C and
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redissolved in 50 µL or 12 µL ER-Luc assay medium supplemented with 3.6% DMSO added to improve
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solubility of compounds, in 96- or 384-well plates respectively, for 10 minutes at 700 rpm. Redissolved
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fractions were diluted with 216 µL or 50 µL ER-Luc assay medium, lowering the DMSO concentration to
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0.7% (considering potential residual DMSO used as keeper negligible), in 96- or 384-well plates
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respectively. Cells seeded on 384 well plates in the downscaled format were prepared for exposure to
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fractions by aspirating the medium and adding 24 µL AR-EcoScreen assay medium to AR-EcoScreen (and
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GR-KO) cells or 24 µL ER-Luc assay medium with 0.43% DMSO to ER-Luc cells. Cells were exposed to a
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single concentration by adding 10 µL of the redissolved fraction to prepared cells, in triplicate for
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measuring 64 fractions or in a single well for measuring 192 fractions, using a digital stepper- or manual
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8-channel pipette. This resulted in a final DMSO concentration of 0.2% (2 µL/mL) in AR-EcoScreen (and
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GR-KO) and 0.5% (5 µL/mL) in ER-Luc cells during exposure compared to 0.1% and 0.5% DMSO during
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exposure, respectively, described in in the reporter gene assay protocol. As a result from the addition of
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10 µL dissolved fraction in ER-Luc assay medium to 24 µL AR-EcoScreen assay medium, AR-EcoScreen
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(and GR-KO) cells were exposed in 29.4% ER-Luc and 70.6% AR-EcoScreen assay medium. From each
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fraction plate, multiple seeded 384-well plates were exposed to measure different endpoints in parallel.
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Reference compound dilution series prepared in DMSO were diluted in the same assay medium
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composition and at the same DMSO concentration at which cells were exposed to fractions. Cells were
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exposed, in triplicate, to reference compounds by adding 34 µL of the diluted compounds to aspirated
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cells.
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Data analysis
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Bioassay results were analyzed in Prism 5.04 (Graphpad Software Inc, San Diego, CA). For each serial
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dilution data set, D'Agostino-Pearson test was used to test data normality, and Levene’s test to test
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homogeneity of variance (significance P < 0.05). Dose response curves of reference compounds and
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candidate compounds were fitted with a four parametric logistic function [Y=A+(B-A)/(1+(x/C)^D)],
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where A and B denote minimal and maximal response respectively, C is the EC50 or IC50, D is the
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hillslope and x represents the tested concentration. Significant differences (P
