(NIS) Inhibitors in ToxCast Phase I Chem - ACS Publications

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Ecotoxicology and Human Environmental Health

High-Throughput Screening and Quantitative Chemical Ranking for Sodium Iodide Symporter (NIS) Inhibitors in ToxCast Phase I Chemical Library Jun Wang, Daniel Hallinger, Ashley Murr, Angela Buckalew, Steven O'Neal Simmons, Susan C. Laws, and Tammy Stoker Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b06145 • Publication Date (Web): 03 Apr 2018 Downloaded from http://pubs.acs.org on April 3, 2018

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Environmental Science & Technology

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High-Throughput Screening and Quantitative Chemical Ranking for

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Sodium Iodide Symporter (NIS) Inhibitors in ToxCast Phase I Chemical

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Library

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Jun Wang1,2, Daniel R. Hallinger2, Ashley S. Murr2, Angela R. Buckalew2, Steven O. Simmons3,

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Susan C. Laws2,*, Tammy E. Stoker2,*

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1

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37831, USA; 2Endocrine Toxicology Branch, Toxicity Assessment Division, National Health

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and Environmental Effects Research Laboratory, Office of Research and Development, U.S.

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Environmental Protection Agency, Research Triangle Park, NC, 27711, USA; 3National Center

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for Computational Toxicology, Office of Research and Development, U.S. Environmental

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Protection Agency, Research Triangle Park, NC, 27711, USA

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*Corresponding authors: [email protected] (Phone: 919-541-0173 Fax: 919-541-5138) and

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[email protected] (Phone: 919-541-2783 Fax: 919-541-5138)

Oak Ridge Institute for Science and Education, U.S. Department of Energy, Oak Ridge, TN

1 ACS Paragon Plus Environment


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Abstract

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Thyroid uptake of iodide via the sodium-iodide symporter (NIS) is the first step in the

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biosynthesis of thyroid hormones that are critical for health and development in humans and

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wildlife. Despite having long been a known target of endocrine disrupting chemicals such as

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perchlorate, information regarding NIS inhibition activity is still unavailable for the vast majority

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of environmental chemicals. This study applied a previously validated high-throughput approach

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to screen for NIS inhibitors in the ToxCast phase I library, representing 293 important

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environmental chemicals. Here 310 blinded samples were screened in a tiered-approach by an

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initial single-concentration (100µM) radioactive-iodide uptake (RAIU) assay, followed with 169

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samples further evaluated in multi-concentration (0.001µM-100µM) testing in parallel RAIU and

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cell viability assays. A novel chemical ranking system that incorporates multi-concentration

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RAIU and cytotoxicity responses was also developed as a standardized method for chemical

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prioritization in current and future screenings. Representative chemical responses and thyroid

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effects of high-ranking chemicals are further discussed. This study significantly expands current

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knowledge of NIS inhibition potentials in environmental chemicals, and provides critical support

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to U.S.EPA’s Endocrine Disruptor Screening Program (EDSP) initiative to expand coverage of

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thyroid molecular targets as well as the development of thyroid adverse outcome pathways

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(AOPs).


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Introduction

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The presence of endocrine disrupting chemicals (EDCs) in the environment continues to be a top

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public health concern not only for effects on the regulation of androgen and estrogen pathways,

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but also thyroid hormone homeostasis. Thyroid hormones [TH; i.e., thyroxine (T4) and

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triiodothyronine (T3)] regulate an array of physiological processes that are essential for

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metabolism, cardiovascular function, bone maintenance, as well as fetal and post-natal

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neurodevelopment.1-3 Over the past two decades, a number of structurally diverse xenobiotics

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have been shown to interfere with TH homeostasis and result in physiological and morphological

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perturbations.4-7 Further work in this area has identified multiple molecular targets of chemical-

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mediated thyroid disruption including the regulation of circulating TH through feedback

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mechanisms within the hypothalamic-pituitary-thyroid (HPT) axis, TH synthesis and secretion,

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TH distribution and transport, TH metabolism, and TH receptor binding and action.8-13 These

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studies demonstrate a need for a better understanding of the structural characteristics of

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chemicals with thyroid disrupting activity, the potential for environmental exposures, and the

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possible health risks to humans and wildlife. To address these concerns, the U.S. EPA’s

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Endocrine Disruptor Screening Program (EDSP21, https://www.epa.gov/endocrine-disruption) in

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concert with the U.S. EPA’s Office of Research and Development (ORD) recently expanded the

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coverage of molecular targets for thyroid disruption by developing and implementing the use of

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high-throughput screening (HTS) assays to identify inhibitors of TH synthesis (e.g.,

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sodium/iodide symporter (NIS) and thyroid peroxidase) and T4 metabolism (e.g., deiodinases).

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Methods for a thyroid peroxidase assay were previously described and used to screen chemicals

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in ToxCast chemical libraries.14, 15 In addition, our laboratory recently demonstrated the utility



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of another HTS assay to detect chemicals that disrupt NIS-mediated transport of extracellular

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iodide across the cellular membrane.16

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NIS is a glycoprotein with 13 transmembrane helices that actively transports iodide (I-) into

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thyroid follicular cells.17-19 This activity relies on the Na+ electrochemical gradient maintained by

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Na+/K+ ATPases with an electrogenic stoichiometry of 2 Na+ per I-.20,

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physiological conditions, NIS can concentrate iodide into the thyroid gland 20- to 40-fold greater

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than serum levels.22 Malfunction of the NIS protein is known to disrupt TH homeostasis; patients

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with various gene mutations leading to aberrant NIS protein have been diagnosed with

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hypothyroidism.23 NIS activity can also be disrupted by xenobiotic chemicals. A well-known

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example is perchlorate (ClO4−), which is a widespread environmental contaminant that has been

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detected in soil, ground/surface water, food, and consumer products across the world24-28.

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Perchlorate, along with several other environmental anions including thiocyanate (SCN−) and

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nitrate (NO3−), have been demonstrated to competitively inhibit I- uptake by NIS and disrupt TH

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synthesis in humans

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known target of endocrine disruption, studies on NIS inhibition activity have been largely

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restricted to only a few environmental chemicals. However, two studies have recently identified

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triclosan, triclocarban, BDE-47, bisphenol A34, and several small drug-like organic molecules as

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NIS-mediated iodide transport inhibitors35, suggesting that NIS inhibition activity extends

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beyond traditionally known anions to the more complex organic compounds. Considering the

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pivotal role of NIS in the thyroid hormone system, there is an urgent need to expand the

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knowledge of NIS inhibition potentials to a broader range of environmental chemicals.

29-31

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Under normal

and multiple species of vertebrates32, 33. Despite having long been a


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In this study, we applied a previously validated HTS approach to screen the 293 ToxCast

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(https://www.epa.gov/chemical-research/toxicity-forecasting) phase I chemicals and developed a

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specific chemical ranking approach to assist with chemical prioritization in current and future

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screenings. ToxCast phase I library contains a diverse collection of environmental chemicals

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(mostly pesticides and antimicrobials) that are currently under the U.S. EPA’s regulatory

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purview36, as well as a subset of chemicals that have been extensively tested in the Agency’s

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EDSP Tier I Screening Battery.37 This study significantly expands our current knowledge of

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environmentally-relevant chemicals with NIS inhibition potential and contributes to a broader

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understanding of thyroid disruptive mechanisms. In addition, the results support the development

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of adverse outcome pathways (https://aopwiki.org/) related to thyroid hormone disruption and

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Organization for Economic Co-operation and Development (OECD) test guidelines38 to assess

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thyroid disruptive chemicals. With an accompanied R package made publicly available, the

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newly developed chemical ranking approach could also be extended as a standardized method to

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prioritize chemicals for other HTS studies.



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Materials and Methods

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Chemicals

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Chemical names, CAS numbers, and maximum concentrations tested are shown in Table S1 (test

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chemicals) and Table S2 (assay controls). All control chemicals were initially solubilized in

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DMSO (EMD Millipore Corp., Darmstadt, Germany) at 20mM and included sodium perchlorate

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(NaClO4; RAIU assay positive control), sodium nitrate (NaNO3; RAIU assay EC80 control),

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sodium thiocyanate (NaSCN; RAIU assay EC20 control), 2,4-dichlorophenoxyacetic acid (2,4-D;

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RAIU assay negative control), and 2,3-dichloro-1,4-napthoquinone (DCNQ; cell viability assay

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positive control) (Sigma Aldrich, St. Louis, MO).

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Chemicals of the ToxCast phase I_v2 library were obtained from the National Center of

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Computational Toxicology (NCCT), US EPA, Research Triangle Park, NC, USA.36

Each

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chemical was solubilized in DMSO (≤ 20mM, Table S1) and provided as 310 blinded samples in

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five 96-well plates (62 samples per plate) (Evotec Inc., South San Francisco, CA). Stock

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chemical plates were visually examined under microscope to check for solubility/precipitate in

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each well (Table S1). Of the 310 samples, there were 293 unique chemicals; the remaining 17

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served as internal quality control replicates of 12 chemicals randomly distributed among the five

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plates. All chemicals were transferred to bioassay plates using a BioMek FX Automated

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Laboratory Workstation (Beckman Coulter, Indianapolis, IN) equipped with a stainless steel high

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density replicating (HDR) tool (96-pin) to transport 0.35µL per well for the RAIU and cell

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viability assays.


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Radioactive Iodide Uptake (RAIU) and Cell Viability Assays

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RAIU and cell viability assays were conducted with low passage hNIS-HEK293T-EPA cells (
50%, absolute EC50

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(absEC50) were also reported. absEC50 was determined as the log concentration where the

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modeled activity equals 50% of control activity. absEC80 was determined as the log

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concentration where the modeled activity equals 80% of control activity.

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For chemicals that demonstrated significant cytotoxicity (exceeding 3bMAD of cell viability

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assay, 17.7%), the concentration where a significant reduction in cell viability for each chemical

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was determined and referred to as the cytotox-point. The cytotox-point is the equivalent of

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absEC82.3, the log concentration where the modeled activity equals the cutoff value for

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significant cytotoxicity (82.3% of control viability).

( ) |

, where σpos and µpos are the standard deviation and



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Chemical ranking score

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To prioritize the chemicals for potential NIS inhibition activity and further evaluation, a new

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scoring system was developed based on two metrics that take into account the confounding

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impact of cytotoxicity on identifying RAIU inhibition activity: 1) toxicity-adjusted area (TAA)

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and 2) the difference of median responses of RAIU and cell viability at maximum tested

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concentration (Median-Difference) (Figure1). Ranking analysis was only performed if a

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chemical produced significant RAIU inhibition in multi-concentration screening. To obtain TAA

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(gray stripe area illustrated in Figure 1), RAIU inhibition area and cytotoxicity area were first

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calculated. RAIU inhibition area was defined by the RAIU 3bMAD (23.8%) significant

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threshold horizontal line (top border), maximum concentration vertical line (right border), and

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the RAIU dose-response curve. Cytotoxicity area was defined with the same top and right

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borders and the cell viability dose-response curve. TAA was then obtained by subtracting the

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cytotoxicity area from the RAIU inhibition area. Therefore, the numeric value of TAA is

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penalized when a chemical demonstrates strong cytotoxicity. Median-Difference was calculated

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using the median of cell viability responses minus the median of RAIU responses at the

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maximum tested concentration (usually 100 µM). Larger Median-Difference values represent

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larger separations between RAIU and cell viability.

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To rank test chemicals, the well-documented NIS inhibitor, NaClO4, was chosen as the reference

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chemical to normalize the TAA and Median-Difference of each test chemical. Specifically, the

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TAA and Median-Difference values of the NaClO4 positive control included on each of the 54

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multi-concentration testing plates were first calculated to obtain the median of NaClO4 TAA and

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Median-Difference (150.03 and 95.67 respectively). The TAA and Median-Difference of test


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chemicals were normalized as the percentage of the median NaClO4 TAA and Median-

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Difference separately and then summed to obtain a chemical ranking score. The ranking score of

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200 represents the potency of the reference NaClO4 (Figure 1A). Figure 1B and 1C show the

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dose-response of two test chemicals with lower level ranking scores due to less RAIU inhibition

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and increased cytotoxicity level.

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Functions for the calculation of the ranking score, TAA, and Median-Difference, along with

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dose-response modeling and visualization are made available in the R package ‘toxplot’

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(https://cran.r-project.org/package=toxplot).



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Results and Discussion

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Assay performance and quality control

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Performance of the RAIU and cell viability assays was monitored for each assay plate through

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CV of DMSO vehicle control, Z’ and AC50 for the positive control chemicals NaClO4/DCNQ

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(Table S3). Fifteen plates in single-concentration RAIU assay and 54 plates were used in multi-

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concentration RAIU and cell viability assays, respectively. Both RAIU and cell viability assays

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performed well with excellent dynamic range, reproducibility, and reliability during both single

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and multi-concentration screening. CV of DMSO were ≤ 11.5% and the standard deviation of

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control AC50 were ≤0.13 (logM). The Z’ scores were also consistently above 0.64 in RAIU

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screening. Additional controls including NaNO3, NaSCN, and 2,4-D (RAIU assay EC80, EC20

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and negative controls) also showed great consistency with expected responses in the RAIU and

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cell viability assays with small variances across the entire screening (Table S4).

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The phase I_v2 chemical library consisted of 310 blinded samples that included 293 unique

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chemicals. Twelve of these chemicals were internally replicated (7 chemicals replicated twice

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and 5 chemicals replicated three times) to assess assay reproducibility. The robustness of the

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RAIU assay is shown in Figure S2a, where all 12 replicated chemicals, excluding bisphenol A,

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produced highly reproducible results. Of the 12 replicated chemicals, 10 exceeded the 20%

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inhibition threshold and were subsequently tested in multi-concentration screening. The AC50 for

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each replicate of the 10 chemicals was calculated to assess the reproducibility of the RAIU assay

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in the multi-concentration setting (Figure S2b, Table S5). These 10 internal replicate samples


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had highly reproducible AC50 (logM) values with the maximum variation range less than 0.25

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(logM).

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Single-concentration screening

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To facilitate the screening process, the 310 blinded samples were first tested in the RAIU assay

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at their maximum permissible concentration (typically 100µM, Table S1) to select potentially

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active compounds for multi-concentration evaluation. Single-concentration RAIU screening

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results for all samples were ordered by increasing inhibitory median responses and plotted as

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median and maximum/minimum responses for each sample (Figure S3). These samples

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demonstrated a wide range of iodide uptake inhibition activity in the assay with median

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responses ranging from 2.8% to 116.6% of maximum iodide uptake relative to the DMSO

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control. Of the 310 samples, 169 samples (54.5%) produced over the 20% inhibition threshold

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and were subjected to multi-concentration testing (Table S1).

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All chemical samples were also tested with Sandell-Kolthoff reactions41 to determine the

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presence of any contaminant iodide that may lead to potential false positive RAIU inhibition (see

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SI Part I). Only two chemicals (iodosulfuron-methyl-sodium and 3-iodo-2-propynyl-N-

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butylcarbamate) produced positive SK reactions, suggesting that overall iodide contamination

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was not introducing false positive results in the RAIU assay.

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Multi-concentration testing

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The 169 blinded chemical samples with ≥20% inhibition in single-concentration RAIU screening

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were further tested at 6 concentrations (0.001µM – 100µM) in parallel RAIU and cell viability



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assays. Dose-response curves for test chemicals and positive controls (NaClO4 and DCNQ) are

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available in SI. The significant activity threshold (3bMAD, calculated across all 54 assay plates

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in multi-concentration screening) for the cell viability and RAIU assays were 17.7% and 23.8%,

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respectively. Among the 169 tested samples, 137 showed significant RAIU inhibition activity

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(>23.8% inhibition). Within the 137 samples with significant RAIU inhibition activity, 26

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samples exhibited no change in cell viability, while 111 displayed a cytotoxic response at one or

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more concentrations tested.

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In reporting the RAIU and cell viability responses of each test chemical, several activity metrics,

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including AC50, absEC50, and cytotox-point (Table 1 and Table S1), were used together to aid in

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the interpretation of the assay results. AC50 and absEC50 indicate the chemical potency from two

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different perspectives, as they differ in the inhibition activity level each represents. absEC50 is

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the concentration that causes 50% inhibition, while AC50 is the concentration that triggers half-

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maximal inhibition, which is 100, 17 had ranking

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scores between 50 and 100, and 116 had ranking scores KClO4>NaClO4>NaBF4>NaSCN) is also in full

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agreement with previously reported potencies.16

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Cytotoxicity information is critical for interpreting the observed inhibition in the RAIU assay. A

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traditional toxicological approach would eliminate testing results for all concentrations with

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significant cytotoxicity. If using this approach, 46 chemicals in this study produced significant

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RAIU inhibition without significant cytotoxicity at one or two concentrations (Table 1, S1 and

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Figure 2). Among the 46 chemicals, 5 of them produced >50% RAIU inhibition at concentrations

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where no cytotoxicity was observed. However, simply excluding RAIU data points where

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significant cytotoxicity is observed may also cause high false negative rate that is undesirable in

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HTS, especially for chemicals that only produced borderline cytotoxicity. Although it is

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impossible to completely dissociate RAIU and cytotoxic activities, a chemical with stronger

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cytotoxicity at lower concentrations is less likely to be an RAIU inhibitor than one with less or

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no cytotoxicity, given the same level of observed RAIU inhibition. The new ranking system was

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designed with this rationale to provide a continuous metric that adjusts cytotoxic chemicals

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without completely excluding them, thereby reducing the chance of false negatives. For example,

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a chemical such as triphenyltin hydroxide (Figure 3a) that had significant and strong RAIU

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inhibition but with low cytotoxicity (at 1E-5M and 1E-4M) will not only be included, but also

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receive a high ranking score.

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As described previously, the ranking score incorporates cytotoxicity responses when calculating

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the two underlying metrics, TAA and Median-Difference. Unlike other single-point metrics such

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as AC50, the TAA incorporates levels of RAIU inhibition and cytotoxicity at all tested

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concentrations and the shape of the dose-response curves. The addition of Median-Difference as 16 ACS Paragon Plus Environment

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a component in the ranking score further promotes chemicals with high RAIU inhibition and low

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cytotoxicity at the highest concentration so that chemicals like PFOS, oxyfluorfen, and

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cypronidil (Figure 3a) were ranked higher than using TAA alone. These features of the ranking

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system enhance the quality of chemical prioritization and it produced a vastly different ranking

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compared to using the AC50 or absEC50 of RAIU response, as ranking scores had only moderate

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correlation with AC50 or absEC50 for the 137 chemicals (Spearman’s rank correlation, r = 0.46

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and 0.41, p 100, 17 having ranking score between 50 and 100, and 116 having ranking score

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(NIS) Inhibitors in ToxCast Phase I Chem - ACS Publications

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