Application of a nonradioactive assay for high throughput screening for inhibition of thyroid hormone uptake via the transmembrane transporter MCT8

Accepted Manuscript Application of a nonradioactive assay for high throughput screening for inhibition of thyroid hormone uptake via the transmembrane transporter MCT8

Hongyan Dong, Michael G. Wade PII: DOI: Reference:

S0887-2333(17)30014-0 doi: 10.1016/j.tiv.2017.01.014 TIV 3917

To appear in:

Toxicology in Vitro

Received date: Revised date: Accepted date:

17 October 2016 16 January 2017 19 January 2017

Please cite this article as: Hongyan Dong, Michael G. Wade , Application of a nonradioactive assay for high throughput screening for inhibition of thyroid hormone uptake via the transmembrane transporter MCT8. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Tiv(2017), doi: 10.1016/j.tiv.2017.01.014

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Application of a nonradioactive assay for high throughput screening for Inhibition of Thyroid Hormone uptake via the Transmembrane Transporter MCT8 Hongyan Dong & Michael G. Wade*

*

CR

Corresponding author, reprint requests should be addressed to.

IP

T

Environmental Health Science and Research Bureau, Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, ON, Canada, K1A 0K9

US

e-mail, [email protected] Telephone, (613) 957-7376

AC

CE

PT

ED

M

AN

Fax, (613) 941-8530

1

ACCEPTED MANUSCRIPT Abstract: Thyroid hormones (THs) play important roles in almost all physiological processes. High-throughput screening (HTS) assays are needed to screen the vast numbers of chemicals for their potential to disrupt TH signaling. The current work has confirmed the ability of a rapid

T

assay to identify substances inhibiting TH uptake through monocarboxylate transporter (MCT) 8.

IP

Perturbation of MCT8 function results in significant developmental impairments, suggesting

CR

substances inhibiting MCT8 may be important developmental toxicants. We examined the accuracy and consistency of a recently described method to identify TH inhibitors via MCT8,

US

using MDCK cells overexpressing human MCT8 gene. We confirmed the method detected T3

AN

uptake in a concentration/time-dependent manner, and this effect was blocked by substances previous reported to block TH uptake via MCT8. Assay performance was assessed extensively

M

and the system was found to have high signal dynamic range and Z' factor. The assay was also

ED

validated with a diverse set of training chemicals. This assay was then used to screen chemicals suspected to disrupt TH signalling. Other than bisphenol A (BPA), all substances tested were

PT

negative. Our results suggest that this assay could be part of a battery of screening assays to

AC

CE

predict the potential thyroid disrupting activity of chemicals.

2

ACCEPTED MANUSCRIPT Introduction: Thyroid hormones (THs) play critical roles in a diverse array of physiological processes such as metabolism, cardiac function, energy regulation and tissue development (Zoeller et al. 2007). Especially, causal relationship between TH deficiency and

T

developmental impairment is clearly defined. Disruption or even subtle impairment of TH

IP

action during critical periods of development can cause lasting effects on intelligence and

CR

behavior (Berbel et al. 2009; Li et al. 2010).

The regulation of TH production and action is complex and can be adversely

US

influenced by broad spectrum of environmental chemicals (Zoeller et al. 2007). The

AN

synthesis of TH in the thyroid gland involves the uptake of iodide into thyroid follicular cells via the sodium iodide symporter (NIS), and the incorporation of iodide into

M

thyroglobulin through the action of thyroid peroxidase (TPO). TH uptake into target cells

ED

is controlled by membrane bound transporters, such as monocarboxylate transporter (MCT) 8, MCT 10 and organic anion transport protein (OATP) 1c1. TH level in tissue is

PT

regulated by deiodinases (Dios). Dio1 (mainly in liver) and Dio2 (most TH target tissues)

CE

converts T4 (inactive prohormone) to T3 (active form). Dio3 (mainly in brain and fetal tissues) and Dio1 convert T3 or T4 into inactive T2 and reverse T3, respectively (Bianco

AC

and Kim 2006). Within the cell, TH regulates gene expression by binding to and activating TH receptors. Environmental chemicals can affect TH action by targeting any one of these processes. Significant efforts have been made to identify chemicals that disrupt NIS (Waltz et al. 2010), TPO (Paul et al. 2014; Paul Friedman et al. 2016), Dios (Renko et al. 2015) and TH receptors (Freitas et al. 2011; Sun et al. 2012). However, fewer attempts have been made to identify the chemicals affecting TH uptake into target cells via transmembrane transporters. 3

ACCEPTED MANUSCRIPT Among all the TH transmembrane transporters, MCT8 is the best understood and appears to play the most critical role in mediating T3 uptake that influences neural development in humans (Friesema et al. 2006; Friesema et al. 2004; Friesema et al. 2003). Like other members of this transporter family, MCT8 has a very narrow substrate

T

spectrum, with very high affinity and specificity for TH only (Friesema et al. 2003). The

IP

gene for MCT8 (SLC16a2) is located on the human x chromosome (Friesema et al. 2004)

CR

and is expressed in a broad spectrum of cell types including many thyroid hormoneresponsive tissues and cells. In addition, it is suspected that MCT8 plays an important role

US

in mediating the secretion of T3 and T4 from the cells of the thyroid epithelium where

AN

these are produced in response to TSH stimulation (Trajkovic-Arsic et al. 2010). Mutant forms of this gene are associated with a congenital condition characterized by abnormally

M

high serum T3 and TSH levels but generally, normal T4 levels. Such individuals also

ED

exhibit a severe psychomotor deficiency and muscle lassitude (Friesema et al. 2004; Dumitrescu et al. 2004). Several MCT8 mutant forms have been identified and the

PT

severity of the phenotype of patients carrying the mutant form is inversely proportional to

CE

the T3 transport capability of the enzyme as demonstrated in cells transfected with the various MCT8 mutant forms (Kinne et al. 2010; Kinne et al. 2009). These observations

AC

suggest that interference with MCT8 action, e.g. inhibition by chemicals, during critical periods of early brain development may lead to TH insufficiency and persistent intellectual or psychomotor deficits. By investigating the uptake of radiolabelled T3 into cells in culture, a variety of pharmaceuticals have been observed to potently inhibit T3 or T4 uptake into cells (Topliss et al. 1989; Movius et al. 1989; Ianculescu et al. 2010). Early studies did not determine the

4

ACCEPTED MANUSCRIPT identity of the T3 transporter molecule on which these substances acted. More recent studies have used cell lines expressing MCT8 as the only transporter molecule capable of transporting T3 and have identified bromsulphthalein (BSP), tyrosine kinase inhibitors (TKIs, such as sunitinib, imatinib, dasatinib and bosutinib), desipramine (DMI), genistein

T

and silymarin (Roth et al. 2010; Johannes et al. 2016; Braun et al. 2012; Braun and

IP

Schweizer 2015) as specific inhibitors of T3 transport into cells. As such, rapid assays to

CR

screen chemicals for their ability to impair T3 uptake via MCT8 inhibition may be valuable to identify substances with properties that could impair TH action. The Thyroid

US

Scoping Group of the Organization of Economic Cooperation and Development

AN

(OECD) has identified MCT8, among other mechanisms that can mediate TH action, as priority molecular target for which rapid screening assays should be developed as tools to

M

assess potential hazards of commercial substances with the aim of preventing widespread

ED

exposure to endocrine disrupting substances (OECD 2014). The current study examines the accuracy and consistency of a recently described

PT

method to test inhibition of T3 uptake via MCT8 (Jayarama-Naidu et al. 2015; Renko et

CE

al. 2012). This method relies upon a non-radioactive method of quantifying T3 uptake by measuring the rate of production of a chromogenic redox reaction of iodine with cerium

AC

and arsenate (the Sandell-Kolthoff reaction; SK). Using this detection method, we characterized the response of this assay to many substances reported to inhibit T3 uptake as well as a series of untested chemicals.

Materials and Methods:

5

ACCEPTED MANUSCRIPT Chemicals: Chemicals used for assessment, validation and application of high throughput screening (HTS) assay detecting the inhibitors of T3 uptake were of analytical grade. BSP was purchased from Alfa Aesar (Haverhill, MA, USA), 3,5Diiodothyropropionic acid (DITPA) from Santa Cruz Biotechnology, Inc. (Dallas, Texas,

T

USA), sunitinib, imatinib, dastinib and bosutinib from LC Laboratories (Woburn, MA,

IP

USA). All other chemicals were purchased from Sigma-Aldrich Canada Co. (Oakville,

CR

ON, Canada).

Cell cultures: MDCK (Kinne et al. 2009) cells stably transfected with human MCT8-

US

expression vector (MDCK-MCT8) or control vector pcDNA (MDCK-pcDNA) were kindly

AN

provided by Dr. Ulrich Schweizer, University of Bonn, Germany. Cells were cultured in 10 cm culture dishes with DMEM-F12 (1:1) medium supplemented with 10% FBS, 50 U/ml penicillin

M

and 50 µg/ml Streptomycin at 37° C and 5% CO2 incubator until 80% confluence. All

ED

experiments were conducted with cells within 6 passages of each other. T3 uptake, iodine release and SK reaction: MDCK-MCT8 or MDCK-pcDNA cells

PT

were seeded into a 96- well plate at 4 X 104/100 µl medium/well and incubated for 24 hrs as

CE

described in (Jayarama-Naidu et al. 2015) . All procedure with 96-well plate throughout the whole study was performed with Eppendorf multichannel pipet or Repeater Stream. Cells

AC

were then rinsed with warm PBS (37° C) and different concentrations of T3 were added in uptake buffer (HEPES pH 7.7 10 mM, NaCl 125 mM, CaCl2 1.3 mM, MgCl2 1.2 mM, KCl 5mM and D-glucose 5.6 mM; 100 µl/well) for 10 min to examine concentration-response relationship; or exposed to 3.3 µM T3 in uptake buffer for different time points to examine time course of T3 uptake.

6

ACCEPTED MANUSCRIPT After exposure, uptake buffer was rapidly removed and cells were washed sequentially twice with ice cold PBS containing 0.1% BSA then twice with ice cold reverse osmosis deionized water. Ammonium persulfate (APS 0.68M, 50 µl) was added to each well and the plate was sealed with Microseal “B” Adhesive Seals (Bio-Rad laboratories Ltd. Mississauga,

T

ON, Canada) and incubated for 1 hr at 90° C. The plate was then cooled to room temperature

IP

and 10 µl from each well was transferred to a new plate that contained 40 µl reverse osmosis

CR

deionized water per well. Acidic ammonium cerium (IV) sulfate solution (50 µl, 25 mM (NH4)4Ce(SO4)4, 0.5 M H2SO4) and sodium arsenite solution (50 µl, 25 mM NaAsO2, 0.5 M

US

H2SO4, 0.2 M NaCl) were added to each well and swirled to mix. Absorption at 415 nm was

AN

recorded immediately every 1 min for 30 min (Microplate Reader and SoftMax Pro Data Acquisition and Analysis Software, Molecular Devices, Sunnyvale, CA). Relative iodine

M

content was calculated as the difference in absorption values recorded at 1 min and 21 min

ED

(OD/20 min). We have found a strong linear relationship between T3 concentrations and the OD/20 min (r2=0.99) across the range of T3 levels that occurred in cell lysates from routine

PT

assay conditions (data not shown). Background signal (sourcing from other transporter, TH

CE

binding to cell or plastic surface) is negligible as shown in figure 1A. Uptake of T3 was analysed using a Hill model (SigmaPlot 12.5; Systat Software Inc., San Jose, CA, USA) from

AC

which the EC50 was calculated. Known MCT8 inhibitor BSP was used to examine the capacity of the system to identify MCT8 inhibitors. Cells were incubated with various concentrations of BSP along with T3 (3.3 µM) and T3 uptake was determined as described above. Resulting uptake data were analysed by Hill Model, as above, and concentration at half maximal inhibition (IC50) was estimated. T3 uptake assay and BSP inhibition assay were repeated at least 5

7

ACCEPTED MANUSCRIPT times. Similar results were obtained and the representative data was shown in the result section. HTS assay performance: Assay consistency for T3 uptake and inhibition by BSP was evaluated using a previously described method (Iversen et al. 2004). Uptake data was

T

generated for three plates per day on two separate days. Three plates had different layouts

IP

with 3 different concentrations which produced minimal, mid and maximal signals,

CR

respectively (Supplemental Fig.1). For T3 uptake, T3 concentrations are 0, 2 and 10 µM

US

for minimal, mid and maximal signal, respectively. For BSP inhibition of T3 (3.3 µM) uptake, BSP concentrations are 1000, 500 and 0 µM for minimal, mid and maximal signal,

AN

respectively. Signal window (SW, indicating signal dynamic range), Z’ factor (indicating signal separation, Z’= [(average of maximal signals - 3SD of maximal signals/√𝑛 ) -

M

(average of minimal signals - 3SD of minimal signals/√𝑛)] / (average of maximal signals -

ED

average of minimal signals)), variation within day and between days, as well as other

PT

parameters were calculated as suggested in the publication of Iversen et al (Iversen et al. 2004).

CE

HTS assay performance for T3 uptake was repeated twice (two sets of three plates /

AC

day for two days); while for BSP inhibition was repeated three times (three sets of three plates / day for two days). Similar results were obtained and the representative data was shown in the result section. Validation of HTS with a training set of chemicals: A training set of chemicals, which include 10 positive (Table 1) and 7 negative chemicals (Table 2), were selected based on literature. All chemicals were dissolved with 10% DMSO to make the stock solutions at 10 mM. Eight concentrations (10, 5, 2.5, 1.25, 0.625, 0.3125, 0.1536 and

8

ACCEPTED MANUSCRIPT 0.0782 mM) of each chemical were prepared using 2 times serial dilution with 10% DMSO. Cells were incubated overnight in 96-well plate (40000 cell/well) and media replaced with uptake buffer containing 3.3 µM T3 and vehicle or test chemicals at appropriate test concentrations for 10 min. Final concentration of tested chemicals: 1000,

T

500, 250, 125, 62.5, 31.25, 15.63 and 7.82 µM in 1% DMSO in a total incubation volume

IP

of 100 µl. Each 96-well plate could test 3 chemicals. Triplicate for each concentration

CR

(Supplementary Fig. 2). Both positive (BSP) and negative (DMSO vehicle) controls, were included in each plate. After 10 min incubation, cells were washed and T3 uptake was

US

assessed using SK reaction as described above. Inhibition potency of each chemical was

AN

represented as the percent of vehicle control activity (100%). Positive chemicals were repeated three times, while negative chemicals were repeated twice. Similar results were

M

obtained and the representative one was shown in the result section. Validated system was

ED

named MCT8HTS.

Application of MCT8HTS to screen chemicals: Ten chemicals suspected to be TH

PT

disruptors were selected based on literature (Table 4) and administered as described for

CE

the training set of chemicals. Their effects on MCT8 activity were determined using MCT8HTS. Each chemical was repeated three times and similar results were obtained.

AC

The representative one was shown in the result section. Cytotoxicity assay: CellTiter-Glo kit (Promega Corporation, Madison, WI) was used to detect cytotoxicity as described by manufacturer. Cells (40000 /well) were seeded in an opaque 96-well plate and cultured overnight, washed with warm PBS. Ten microliter of chemicals of serial concentrations in premade compound plate were added into 90 µl uptake buffer (as described above) and incubated for 10 min at 37° C, followed by

9

ACCEPTED MANUSCRIPT washing twice with cold PBS containing 0.1% BSA and twice by cold deionized water. Each concentration of each chemical was added into triplicate wells. Luminescence was produced by adding 100 µl Celltiter-Glo reagent and same volume of deionized water into the wells, and measured after reaction of 10 min at room temperature using a Vetaris

T

luminometer (Promega). Cell viability was represented as the percent of vehicle control

IP

(100%). Each chemical was repeated twice and similar results were obtained. The

US

CR

representative one was shown in the result section.

Results:

AN

Concentration-response and time course studies of T3 uptake into MDCK_MCT8 cells detected by SK reaction. We confirmed that the MDCK_MCT8 cells expressed the

M

human MCT8 at a very high level, while the expression of the human MCT8 in

ED

MDCK_pcDNA cells is negligible (Supplemental Fig. 3). Differential uptake of T3 was evaluated by incubating MDCK_MCT8 and MDCK_pcDNA cells with different

PT

concentration of T3 (0, 0.04, 0.12, 0.36, 1.1, 3.3, 5 and 10 µM) for 10 min. T3 uptake was

CE

concentration-dependent in MDCK_MCT8 cells (Fig.1A) with an EC50 of 1.1 µM. In contrast, uptake of T3 by MDCK_pcDNA cells was trivial; as at the highest T3

AC

concentration tested (10 µM) MCDK_pcDNA uptake represented ~1% of the uptake observed for MCDK_MCT8 cells (Fig.1A). Comparison of T3 uptake over time was examined by incubating both cell types with 3.3 µM T3 (about 80% of the response plateau) with cells sampled at various time points (up to 30 min). T3 uptake increased over time up to 30 min in MDCK_MCT8 cells, while T3 uptake in MDCK_pcDNA cells was barely noticeable at any time point (Fig. 1B). To examining the specificity of the system to

10

ACCEPTED MANUSCRIPT MCT8, we exposed the cells to DITPA, a TH analog whose entry into cells is independent of MCT8 action (Ferrara et al. 2015; Kersseboom et al. 2015). As shown in Fig. 1A, no DITPA uptake was detected up to 10 µM in either MDCK_MCT8 or MDCK_pcDNA cells. These results indicated the principle of SK reaction could be used to detect T3

T

uptake via MCT8 in this system, and importantly, the system is specific for detecting T3

IP

uptake via MCT8.

CR

Inhibition of T3 uptake by BSP. BSP is a known inhibitor for MCT8-mediated T3

US

uptake (Friesema et al. 2003). We used BSP as a positive control to determine if the system could be used to identify the MCT8 inhibitors. Our result showed that BSP

AN

inhibited T3 (3.3 µM) uptake concentration-dependently with IC50 of 250 µM (Fig. 2A). No significant cell toxicity was observed up to 1000 µM (Fig. 2B) incubated for 10 min.

M

HTS assay performance. To determine if the assay can be reliably used in a 96-well

ED

format, assay performance, reproducibility and signal uniformity were examined with a setup of 3 plates (different layout for 3 concentrations of T3 or BSP which produced min,

PT

mid and max signals) per day over two days. Signal distribution for T3 uptake was shown

CE

in Fig. 3A. Data from all plates were also used to determine assay replicability between plates and days. As shown in supplemental Table 1, drift (signal trend from left-to-right

AC

and from top-to bottom) and edge (signal difference between outer wells and center wells) effects are less than 20%, Max signal CV and mid signal CV less than 20%, min signal SD less than min signal, SW greater than 2 and Z’ factor greater than 0.4; within-day shifts and between day shifts less than 2 fold. The value of signal mean, SD, CV, drift and edge effect from all plates can be found in Supplemental Table 1. All these parameters met the criteria proposed by Dr. Iversen (Iversen et al. 2004).

11

ACCEPTED MANUSCRIPT In addition, the performance of the assay to detect MCT8 inhibition was assessed in a similar fashion using concentrations of BSP with a single concentration of T3 (3.3 µM) roughly equivalent to 80% of the response plateau. Signal distribution for BSP inhibition of T3 uptake is shown in Fig. 3B. The values of all parameters can be found in

T

Supplemental Table 2. All parameters of the system met the criteria for a HTS assay as

IP

shown in Table 3.

CR

Validatation of the HTS with known T3 uptake inhibitors and known non inhibitors of T3 uptake. Ten known T3 uptake inhibitors were chosen based on

US

indications from the literature of activity in blocking transport of T3 into any of various cell

AN

types in vitro (Table 1). All data were plotted as the percent of vehicle control (DMSO: 100% T3 uptake). A Hill model was used to fit the concentration-response data for all

M

chemicals which are shown in Fig. 4. In order to estimate the confound factor of the

ED

chemical toxicity, the results from parallel cytotoxicity assays are also depicted in a nested panel for chemicals inhibiting T3 uptake with concentration-response relationship. All

PT

substances previously reported to inhibit MCT8-mediated T3 uptake were active in our

CE

assay (Fig. 4). Sunitinib produced inhibition curve for T3 uptake, while it also induced more than 20 % cell death at concentrations greater than 32 µM (Fig. 4C). DMI produced toxicity

AC

at the two highest concentrations (500 and 1000 µM), while reducing T3 uptake at the concentrations lower than 500 µM (Fig. 4B). Despite previous reports that DPH caused a concentration dependent inhibition of T3 uptake, DPH failed to block MCT8-mediated T3 uptake at any concentration tested in current study (Fig. 4J) suggesting that this substance may block other TH transporters than MCT8. A Hill model was also used to generate Hill fit potency data: the predicted (based on Hill model calculation) and relative (based on

12

ACCEPTED MANUSCRIPT concentration-response curve) IC50 values, lowest effect concentration (LEC, represents the lowest measured concentration that yielded a significant response (greater or equal to a 20% decrease in T3 uptake)), Maximum effect (Emax, in percent activity), Hill slope and r2 value, all of which are reflected in Table 1. Based on the value of Relative Potency to BSP

T

(relative IC50 of BSP divided by relative IC50 of chemicals and multiplied by 100), the

IP

assay results demonstrated the following rank-ordered relative potency for the known T3

CR

uptake inhibitors: ICG>Dasa>Ima>Gen>Busa>BSP>Phl>DMI>Meclo. Sunitinib was not included because the relative IC50 could not be calculated with the confounder of the

US

toxicity.

AN

To further demonstrate the specificity of our model to MCT8-mediated T3 uptake, we

M

tested 7 chemicals that do not disrupt MCT8 functions based on literature (Table 2). Two amino acids, Tyr and Trp, can inhibit TH transport via competitive inhibition of amino acid transporters

ED

(LAT1 and LAT2, (Friesema et al. 2001)) but have no effect on MCT8 (Kinne et al. 2010).

PT

Neither Tyr nor Trp inhibited T3 uptake in our test system (data not shown). The other known non-MCT8 inhibitors, including 4-MBC, BP3, NIT, DAIZ and 4NP (Johannes et al. 2016) did

CE

not inhibit T3 uptake in this test either (data not shown). These results confirm the specificity of

AC

the test system for MCT8 activity. Effects of various thyroid-active substances on MCT8 activity. We examined the MCT8 inhibiting activity of several common environmental contaminants that are suspected to disrupt TH signalling (Table 4). These substances, chosen on the basis of structural similarities with T3, have been clearly demonstrated to influence thyroid physiology, but by mechanism(s) independent of MCT8. The list of substances tested includes flame retardants, plasticizer and other chemical classes. Only BPA was observed to 13

ACCEPTED MANUSCRIPT inhibit T3 uptake. Co-treatment with BPA reduced T3 uptakes to around 60% and 40% of the control at concentration of 125 µM and 250 µM, respectively (Fig. 5), although cytotoxicity was evident at the higher concentration with 79.4% viability.

T

Discussion:

IP

Currently there is little empirical evidence that chemical inhibition of MCT8 may

CR

lead to adverse health outcomes. However, given the unique sensitivity of the developing human brain to hypothyroidism (Rovet 2014; Julvez et al. 2013) and the devastating

US

neuromotor and cognitive deficits that occur in children who are unable to express a

AN

functional form of this transporter (Friesema et al. 2004; Dumitrescu et al. 2004), it is conceivable that exposure to an effective MCT8 inhibitor during critically sensitive periods

M

of brain development may result in long term reductions in mental acuity, or other

ED

neurological traits. Such effects may not result in clinical disease but, as with other causes of reduced TH signalling during brain development, may cause altered brain structure and

PT

reduce cognitive performance (Korevaar et al. 2016), and cause hyperactivity (Modesto et

CE

al. 2015). Notably, rodent models will likely be far less sensitive to the adverse effects of MCT8 inhibition than humans as mice engineered to lack a functional MCT8 do not express

AC

any evidence of altered neurological or motor function or altered brain anatomy indicative of hypothyroidism (Trajkovic et al. 2007), suggesting that rodents have multiple transporter molecules that mediate T3 transport into developing brain cells, a role that is served mainly by MCT8 in humans. As a consequence, rodent neurodevelopmental toxicity assays are unlikely to provide confirmatory neurodevelopmental data for this mechanism of action. Further work will be required to determine if human populations are exposed to substances

14

ACCEPTED MANUSCRIPT with this mode of action, beyond the pharmaceuticals used as model compounds in this study, and if such exposures have adverse effects on brain development in children. The logical first step for such further work would involve screening of a broad array of common chemicals through assay such as the MCT8HTS. The highest priority for testing would be

T

substances whose exposure results in altered circulating TH levels and/or TSH levels but

IP

whose mechanism(s) of action are poorly understood.

CR

The current study has confirmed and characterized the reliability and sensitivity of the rapid MCT8 inhibition assay first described by Jayarama-Naidu and colleagues

US

(Jayarama-Naidu et al. 2015). We have demonstrated that the assay can, with high

AN

sensitivity and specificity, identify diverse substances known to be inhibitor of the MCT8 transporter and correctly differentiate these from substances previously described to inhibit

M

cellular T3 uptake but to act via transporters other than MCT8. Our results confirmed that

ED

the MCT8HTS assay can be used in a high throughput 96 - well format to rapidly screen

disrupting substances.

PT

substances for inhibition of a potentially important yet poorly studied target of thyroid

CE

This method uses the non-radioactive SK reaction to detect cellular T3 uptake. It measures colorimetrically the chemical reaction between quadrivalent cerium and trivalent

AC

arsenic catalyzed by free iodide ions – released from T3 by reacting with ammonium persulfate – in sulfuric acid solution. This method provides advantages over the use of radio-iodinated T3 in that the timing of test run is not limited by the iodination date of the radioiodination of the T3 tracer, the rate of radioiodine decay or the health and safety considerations inherent in work with radioactive isotopes. By using SK to detect T3 associated with the test cells, the entire assay – from incubation to data collection – can be

15

ACCEPTED MANUSCRIPT completed within a few hours. However, sodium arsenite used in SK reaction could induce cancers in multiple organs and is classified as carcinogenic by International Agency for Research on Cancer of (IARC, http://monographs.iarc.fr/ENG/Monographs/vol100C/mono100C-6.pdf). Therefore, caution

T

should be taken and relevant regulations should be abided during the assay process.

IP

While the assay is very rapid, there are several considerations that may limit the

CR

throughput without very careful coordination. The short time of incubation (10 min) followed by several rapid washing steps renders the assay difficult to run in more than 1-2

US

96 well plates at a time. In addition, the rapidity of de-coloring reaction when the cerium

AN

and arsenate are added to each well and the requirement for the monitoring of the reaction kinetics rather than simply recording a final absorbance limits the number of plates that can

M

be evaluated. These limitations can be overcome through automation. A further limitation is

ED

that test substances that contain iodine – including many substances known to impair thyroid hormone physiology through other mechanisms (eg. amiodarone, iopanoic acid, and

PT

erythrosine) – will confound the assay as it is not possible to differentiate between iodine

CE

from T3 and iodine from the test agent in the SK reaction. Also, the use of SK to monitor T3 uptake results in a different concentration dependency of T3 uptake compared with that

AC

observed with 125I-labelled T3. Comparing with radio-labelled T3 – for which half maximal uptake was observed at T3 concentrations in the mid to high nM range (data not shown) – measurement of T3 uptake with SK revealed half maximal uptake at a T3 concentration in the low µM. Although this means that the assay must use a concentration of T3 (3.33 µM) that is more than an order of magnitude above T3 concentrations required for receptor activation in vitro (Freitas et al. 2011), the fact that the assay is capable of correctly

16

ACCEPTED MANUSCRIPT identifying true positive and negative substances indicates that this does not compromise the assay. However, this may indicate that the observed potency of each of the inhibitors may not reflect their true potency in vivo. The performance of MCT8HTS in our study is demonstrated to be as reliable as in

T

the recent report (Jayarama-Naidu et al. 2015). Both studies achieved relatively high Z’

IP

factor. Z’ factor is used to characterize assay reliability and indicates the ratio of assay

CR

dynamic range to variability. Higher Z’ factor means lower standard deviations and/or higher difference of signal averages produced by substance tested and control (Iversen et al.

US

2004). Furthermore, we compared the variances among observations across and between

AN

plates and over multiple days to robustly assess the inherent variability of the platform. Our work provided further evidence that this system could be used to screen chemicals that

M

affect TH signalling via MCT8. However, two studies achieved different IC50 of BSP using

ED

similar system. Jayarama-Naidu et al’s group derived similar IC50 values using both SK and isotope methods (75 μM and 84 μM, respectively), where 10 μM of T3 for 15 min

PT

incubation or 10 nM radiolabelled T3 for 5 min incubation were used, respectively. In our

CE

study, the IC50 of BSP (250 μM for 3.3 μM T3 for 10 min incubation) was about 3 times higher than their values. The difference in apparent potency of BSP may relate to the

AC

concentration of T3 used or incubation time among other possible factors. The high degree of agreement between the MCT8 inhibiting activities of diverse chemicals, reported elsewhere, and the results of our assay provide strong confirmation of the reliability of the assay platform. All TKIs tested here were demonstrated to inhibit T3 uptake via MCT8, consistent with the previous studies using the same cells (Braun et al. 2012). The concentrations of all TKI found to be active at inhibiting MCT8 in our study

17

ACCEPTED MANUSCRIPT were below levels overtly affecting cell viability, except for sunitinib. We found that concentrations of sunitinib that significantly reduced T3 uptake also significantly reduced cell viability. For example, at 64 µM, sunitinib reduced T3 uptake to 38% of control, while reducing cell viability to 60% of control. Consequently, the T3 uptake data are confounded

T

by the acute toxicity of this material. Roth et al (Roth et al. 2010) reported that DMI, a

IP

tricyclic antidepressant, inhibits T3 import into primary neurons which express MCT8 to a

CR

high degree. Our results further confirmed that DMI inhibited T3 uptake into the cells overexpressing MCT8 at non-cell-toxic concentrations. We have confirmed that Genistein,

US

an isoflavone found in soy and soy derived nutrition supplements, block T3 uptake via

AN

MCT8 (Braun and Schweizer 2015) with our results indicating an IC50 of 19.1 µM. Genistein affects TH pathway not only by inhibiting TH uptake via MCT8, but also as a

M

competitive inhibitor of other proteins that influence TH physiology. Genistein inhibits TH

ED

binding with serum transport binding proteins transthyretin (TTR) or thyroxine-binding globulin (TBG) with IC50 values of 7.9, or greater than 20 µM, respectively (Kinouchi et al.

PT

2016). Genistein also affects the activity of Dio1with IC50 of 3 µM (Renko et al. 2015). In

CE

addition, genistein is well characterized as an inhibitor of thyroid peroxidase (Divi et al. 1997; Paul et al. 2014) with an IC50 of ~1 to 4.5 M. With multiple potential antithyroid

AC

mechanisms of action, it is not surprising that consumption of genistein-containing foods has been associated with hypothyroidism (reviewed in (de Souza Dos Santos et al. 2011)). A previous study reported that phloretin, the aglycon of the plant pigment phlorizin, inhibits cellular uptake of T3 into Hep G2 cells but did not investigate the identity of the transporter involved in this cell type (Movius et al. 1989). Our results provide evidence that phloritin can block T3 uptake through inhibition of MCT8. Meclofenamic acid, Cardiogreen

18

ACCEPTED MANUSCRIPT and DPH were also demonstrated to be inhibitors of T3 uptake via unknown mechanism (Topliss et al. 1989; Movius et al. 1989; Braun et al. 2012). We found meclofenamic acid and cardiogreen inhibited T3 uptake via MCT8, while DPH did not inhibit T3 uptake in MDCK_MCT8 cells; suggesting that DPH inhibition of T3 uptake may be mediated through

T

its effect on some other transporter. We also confirmed that two amino acids (Tyr and Trp)

IP

– demonstrated previously to inhibit T3 uptake via amino acid transporters LAT1 and 2 but

CR

with no effect of T3 uptake by MCT8 – were negative in the MCT8HTS (Kinne et al. 2010). In addition, five other chemicals previously shown to have no effect on MCT8 function

US

(Johannes et al. 2016) were negative in MCT8HTS. This training set of positive and

AN

negative chemicals reveals a very high degree of concordance with effects observed elsewhere and supports the reliability of MCT8HTS.

M

We tested a variety of environmental chemicals classified as flame retardant,

ED

pesticide, plasticizer and others using the MCT8HTS. These chemicals were chosen because accumulated evidence indicates they affect TH signaling in vivo or in vitro. Although

PT

available evidence show that these substances influence TH signalling via mechanism(s)

CE

other than MCT8, none have been previously examined for their effects on the MCT8 transporter. Of all substances tested only BPA induced a reduction in T3 uptake at

AC

concentrations (125 µM) below those that reduced cell viability <80%. BPA is reported to influence TH signalling via multiple mechanism, including inhibition of NIS, reducing expression of genes involved in TH synthesis (Wu et al. 2016) and inhibiting TH binding with TH receptors (Yang and Chan 2015), but in vivo treatment with BPA led to an increase in circulating T4 (Zoeller et al. 2005). In recently published research, Johannes et al found 10 µM BPA could not inhibit T3 (10 µM) uptake with similar detection method (Johannes

19

ACCEPTED MANUSCRIPT et al. 2016). Our results are consistent with this finding and extend the conclusion that BPA is a weak MCT8 inhibitor in vitro, but the active concentration is much higher than concentrations likely to occur in vivo. In summary, we have characterized and confirmed the reliability of a rapid assay for

T

screening chemicals for their ability to block T3 uptake via the human MCT8 (MCT8HTS).

IP

In particular, we have investigated the specificity of the assay using a diverse set of known

CR

positive and negative substances, and characterized the performance of the system. Our results suggest that MCT8HTS could be part of the battery of screening assays to predict the

US

potential thyroid disrupting activity of commercial and environmental chemicals.

AN

Acknowledgements

M

We thank Dr. Ulrich Schweizer for kindly providing MDCK cells overexpressing MCT8 or

ED

control plasmids. Dr. Ella Atlas and Dr. Guillaume Pelletier provided helpful comments on the

AC

CE

grant number 810509.

PT

manuscript. This research was funded through Health Canada’s Chemicals Management Plan,

20

ACCEPTED MANUSCRIPT Figure legends: Figure 1. A. concentration-response relationship of T3 or DITPA uptake into MDCK cells. Different concentrations of T3 or DITPA were incubated with cells for 10 min. B. Time course study of T3 uptake into cells. T3 of 3.3 µM was incubated with cells up to 30 min. T3 uptake

IP

T

was detected as described in Materials and Methods. Each concentration or time point had

CR

triplicate wells in a 96-well plate. Experiment was repeated 5 times, except DITPA exposure which was repeated twice. A representative curve was shown for each experiment.

US

Figure 2. BSP inhibition of T3 (3.3 µM) uptake into MDCK_MCT8 cells. A. Different

AN

concentrations of BSP were incubated with MDCK_MCT8 cells in the presence of 3.3 µM T3 for 10 min. Experiment was repeated more than 5 times. A representative one was shown. B.

M

Viability of cells treated with different concentrations of BSP. Viability was reflected with ATP

ED

amount in the cells and detected with CellTiter-Glo 2.0. Each concentration had triplicate wells

PT

in a 96-well plate. Experiment was repeated twice and a representative curve was shown. Figure 3. A. The distribution of signals reflecting T3 uptake into the cells. T3 concentrations

CE

produced maximum, mid and minimal signals are 10, 2 and 0 µM. Experiment was repeated twice and a representative one was shown. B. The distribution of signals reflecting BSP

AC

inhibition of T3 (3.3 µM) uptake into the cells. BSP concentrations produced maximum, mid and minimal signals are 0, 250 and 1000 µM. Experiment was repeated 3 times and a representative one was shown. Figure 4. Results from a 10-positive-chemical training set. Known T3 uptake inhibitors are shown in each panel with a corresponding concentration-response curve (A – Phl; B- DMI; CSuni; D – Ima; E-Dasa; F-Busa; G-ICG; H-Gen; I-Meclo; J- DPH). Cell viabilities were shown 21

ACCEPTED MANUSCRIPT in the corresponding insert figures for the chemicals demonstrated to inhibited T3 uptake more than 80% at least at one concentration. The positive control was BSP. All chemicals were examined at the same 8 concentrations ranging from 7.8 µM to 1000 µM, except ICG from 0.5 µM to 32 µM. Red circle indicates uptake inhibition by the concentration inducing about 20%

T

cell death. Each concentration had triplicate wells in a 96 - well plate. Experiment of T3 uptake

IP

for each chemical was repeated 3 times and got similar curves. Cell viability assay for each

CR

chemical was repeated twice except DPH on which cell viability did not conducted because DPH

US

did not inhibit T3 uptake. A representative curve was shown for each chemical. Figure 5. The effect of BPA. A. Inhibition of T3 uptake by BPA at concentrations up to 250

AN

µM. Each concentration had triplicate wells in a 96 - well plate. The experiment was repeated 3 times, and a representative example was shown. Red circle indicates uptake inhibition by the

M

concentration inducing about 20% cell death. B. Cell viability affected by BPA at concentrations

ED

up to 250 µM. No cell toxicity was observed up to 125 µM. Twenty percent of cell death was

PT

observed at the 250 µM. Each concentration had triplicate wells in a 96 - well plate. Experiment

AC

CE

was repeated twice. A representative curve was shown.

22

ACCEPTED MANUSCRIPT References: Berbel P, Mestre JL, Santamaria A, Palazon I, Franco A, Graells M, Gonzalez-Torga A and de Escobar GM. Delayed neurobehavioral development in children born to pregnant women with mild hypothyroxinemia during the first month of gestation: the importance of early

IP

T

iodine supplementation. Thyroid 19: 5: 511-519, 2009.

CR

Bianco AC and Kim BW. Deiodinases: implications of the local control of thyroid hormone

US

action. J Clin Invest 116: 10: 2571-2579, 2006.

Braun D, Kim TD, le Coutre P, Kohrle J, Hershman JM and Schweizer U. Tyrosine kinase

AN

inhibitors noncompetitively inhibit MCT8-mediated iodothyronine transport. J Clin Endocrinol

M

Metab 97: 1: E100-5, 2012.

ED

Braun D and Schweizer U. Efficient Activation of Pathogenic DeltaPhe501 Mutation in Monocarboxylate Transporter 8 by Chemical and Pharmacological Chaperones. Endocrinology

PT

156: 12: 4720-4730, 2015.

CE

de Souza Dos Santos MC, Goncalves CF, Vaisman M, Ferreira AC and de Carvalho DP.

AC

Impact of flavonoids on thyroid function. Food Chem Toxicol 49: 10: 2495-2502, 2011.

Divi RL, Chang HC and Doerge DR. Anti-thyroid isoflavones from soybean: isolation, characterization, and mechanisms of action. Biochem Pharmacol 54: 10: 1087-1096, 1997.

Dumitrescu AM, Liao XH, Best TB, Brockmann K and Refetoff S. A novel syndrome combining thyroid and neurological abnormalities is associated with mutations in a monocarboxylate transporter gene. Am J Hum Genet 74: 1: 168-175, 2004.

23

ACCEPTED MANUSCRIPT Ferrara AM, Liao XH, Ye H, Weiss RE, Dumitrescu AM and Refetoff S. The Thyroid Hormone Analog DITPA Ameliorates Metabolic Parameters of Male Mice With Mct8 Deficiency. Endocrinology 156: 11: 3889-3894, 2015.

Freitas J, Cano P, Craig-Veit C, Goodson ML, Furlow JD and Murk AJ. Detection of

IP

T

thyroid hormone receptor disruptors by a novel stable in vitro reporter gene assay. Toxicol In

CR

Vitro 25: 1: 257-266, 2011.

US

Friesema EC, Docter R, Moerings EP, Verrey F, Krenning EP, Hennemann G and Visser TJ. Thyroid hormone transport by the heterodimeric human system L amino acid transporter.

AN

Endocrinology 142: 10: 4339-4348, 2001.

M

Friesema EC, Ganguly S, Abdalla A, Manning Fox JE, Halestrap AP and Visser TJ.

ED

Identification of monocarboxylate transporter 8 as a specific thyroid hormone transporter. J Biol Chem 278: 41: 40128-40135, 2003.

PT

Friesema EC, Grueters A, Biebermann H, Krude H, von Moers A, Reeser M, Barrett TG,

CE

Mancilla EE, Svensson J, Kester MH, Kuiper GG, Balkassmi S, Uitterlinden AG, Koehrle J, Rodien P, Halestrap AP and Visser TJ. Association between mutations in a thyroid

AC

hormone transporter and severe X-linked psychomotor retardation. Lancet 364: 9443: 14351437, 2004.

Friesema EC, Kuiper GG, Jansen J, Visser TJ and Kester MH. Thyroid hormone transport by the human monocarboxylate transporter 8 and its rate-limiting role in intracellular metabolism. Mol Endocrinol 20: 11: 2761-2772, 2006.

24

ACCEPTED MANUSCRIPT Ianculescu AG, Friesema EC, Visser TJ, Giacomini KM and Scanlan TS. Transport of thyroid hormones is selectively inhibited by 3-iodothyronamine. Mol Biosyst 6: 8: 1403-1410, 2010.

Iversen PW, Beck B, Chen YF, Dere W, Devanarayan V, Eastwood BJ, Farmen MW,

IP

T

Iturria SJ, Montrose C, Moore RA, Weidner JR and Sittampalam GS. HTS Assay

CR

Validation. In: Assay Guidance Manual, edited by Sittampalam GS, Coussens NP, Nelson H, Arkin M, Auld D, Austin C, Bejcek B, Glicksman M, Inglese J, Iversen PW, Li Z, McGee J,

US

McManus O, Minor L, Napper A, Peltier JM, Riss T, Trask OJ J and Weidner J. Bethesda (MD):

AN

2004.

Jayarama-Naidu R, Johannes J, Meyer F, Wirth EK, Schomburg L, Kohrle J and Renko

M

K. A Nonradioactive Uptake Assay for Rapid Analysis of Thyroid Hormone Transporter

ED

Function. Endocrinology 156: 7: 2739-2745, 2015.

PT

Johannes J, Jayarama-Naidu R, Meyer F, Katrin Wirth E, Schweizer U, Schomburg L, Kohrle J and Renko K. Silychristin, a flavonolignan derived from the milk thistle is a potent

CE

inhibitor of the thyroid hormone transporter MCT8. Endocrinology en20151933, 2016.

AC

Julvez J, Alvarez-Pedrerol M, Rebagliato M, Murcia M, Forns J, Garcia-Esteban R, Lertxundi N, Espada M, Tardon A, Riano Galan I and Sunyer J. Thyroxine levels during pregnancy in healthy women and early child neurodevelopment. Epidemiology 24: 1: 150-157, 2013.

25

ACCEPTED MANUSCRIPT Kersseboom S, Horn S, Visser WE, Chen J, Friesema EC, Vaurs-Barriere C, Peeters RP, Heuer H and Visser TJ. In vitro and mouse studies support therapeutic utility of triiodothyroacetic acid in MCT8 deficiency. Mol Endocrinol me00009999, 2015.

Kinne A, Kleinau G, Hoefig CS, Gruters A, Kohrle J, Krause G and Schweizer U. Essential

IP

T

molecular determinants for thyroid hormone transport and first structural implications for

CR

monocarboxylate transporter 8. J Biol Chem 285: 36: 28054-28063, 2010.

US

Kinne A, Roth S, Biebermann H, Kohrle J, Gruters A and Schweizer U. Surface translocation and tri-iodothyronine uptake of mutant MCT8 proteins are cell type-dependent. J

AN

Mol Endocrinol 43: 6: 263-271, 2009.

M

Kinouchi H, Matsuyama K, Kitagawa H and Kamimori H. Surface plasmon resonance assay

Biochem 492: 43-48, 2016.

ED

of inhibition by pharmaceuticals for thyroxine hormone binging to transport proteins. Anal

PT

Korevaar TI, Muetzel R, Tiemeier H and Peeters RP. Maternal thyroid function and child IQ

CE

- Authors' reply. Lancet Diabetes Endocrinol 4: 1: 18-8587(15)00471-4, 2016.

AC

Li Y, Shan Z, Teng W, Yu X, Li Y, Fan C, Teng X, Guo R, Wang H, Li J, Chen Y, Wang W, Chawinga M, Zhang L, Yang L, Zhao Y and Hua T. Abnormalities of maternal thyroid function during pregnancy affect neuropsychological development of their children at 25-30 months. Clin Endocrinol (Oxf) 72: 6: 825-829, 2010.

26

ACCEPTED MANUSCRIPT Modesto T, Tiemeier H, Peeters RP, Jaddoe VW, Hofman A, Verhulst FC and Ghassabian A. Maternal Mild Thyroid Hormone Insufficiency in Early Pregnancy and AttentionDeficit/Hyperactivity Disorder Symptoms in Children. JAMA Pediatr 169: 9: 838-845, 2015.

Movius EG, Phyillaier MM and Robbins J. Phloretin inhibits cellular uptake and nuclear

IP

T

receptor binding of triiodothyronine in human hep G2 hepatocarcinoma cells. Endocrinology

CR

124: 4: 1988-1997, 1989.

US

OECD. New scoping socument on in vitro and ex vivo assays for the identification of modulators of thyroid hormone signalling OECD: OECD, 2014.

AN

Paul Friedman K, Watt ED, Hornung MW, Hedge JM, Judson RS, Crofton KM, Houck

M

KA and Simmons SO. Tiered High-Throughput Screening Approach to Identify

ED

Thyroperoxidase Inhibitors Within the ToxCast Phase I and II Chemical Libraries. Toxicol Sci 151: 1: 160-180, 2016.

PT

Paul KB, Hedge JM, Rotroff DM, Hornung MW, Crofton KM and Simmons SO.

CE

Development of a thyroperoxidase inhibition assay for high-throughput screening. Chem Res

AC

Toxicol 27: 3: 387-399, 2014.

Renko K, Hoefig CS, Hiller F, Schomburg L and Kohrle J. Identification of iopanoic acid as substrate of type 1 deiodinase by a novel nonradioactive iodide-release assay. Endocrinology 153: 5: 2506-2513, 2012.

27

ACCEPTED MANUSCRIPT Renko K, Schache S, Hoefig CS, Welsink T, Schwiebert C, Braun D, Becker NP, Kohrle J and Schomburg L. An Improved Nonradioactive Screening Method Identifies Genistein and Xanthohumol as Potent Inhibitors of Iodothyronine Deiodinases. Thyroid 25: 8: 962-968, 2015.

Roth S, Kinne A and Schweizer U. The tricyclic antidepressant desipramine inhibits T3 import

IP

T

into primary neurons. Neurosci Lett 478: 1: 5-8, 2010.

CR

Rovet JF. The role of thyroid hormones for brain development and cognitive function. Endocr

US

Dev 26: 26-43, 2014.

Sun H, Si C, Bian Q, Chen X, Chen L and Wang X. Developing in vitro reporter gene assays

AN

to assess the hormone receptor activities of chemicals frequently detected in drinking water. J

M

Appl Toxicol 32: 8: 635-641, 2012.

ED

Topliss DJ, Kolliniatis E, Barlow JW, Lim CF and Stockigt JR. Uptake of 3,5,3'triiodothyronine by cultured rat hepatoma cells is inhibitable by nonbile acid cholephils,

PT

diphenylhydantoin, and nonsteroidal antiinflammatory drugs. Endocrinology 124: 2: 980-986,

CE

1989.

AC

Trajkovic M, Visser TJ, Mittag J, Horn S, Lukas J, Darras VM, Raivich G, Bauer K and Heuer H. Abnormal thyroid hormone metabolism in mice lacking the monocarboxylate transporter 8. J Clin Invest 117: 3: 627-635, 2007.

Trajkovic-Arsic M, Muller J, Darras VM, Groba C, Lee S, Weih D, Bauer K, Visser TJ and Heuer H. Impact of monocarboxylate transporter-8 deficiency on the hypothalamuspituitary-thyroid axis in mice. Endocrinology 151: 10: 5053-5062, 2010.

28

ACCEPTED MANUSCRIPT Waltz F, Pillette L and Ambroise Y. A nonradioactive iodide uptake assay for sodium iodide symporter function. Anal Biochem 396: 1: 91-95, 2010.

Wu Y, Beland FA and Fang JL. Effect of triclosan, triclocarban, 2,2',4,4'-tetrabromodiphenyl ether, and bisphenol A on the iodide uptake, thyroid peroxidase activity, and expression of genes

IP

T

involved in thyroid hormone synthesis. Toxicol In Vitro 32: 310-319, 2016.

CR

Yang J and Chan KM. Evaluation of the toxic effects of brominated compounds (BDE-47, 99,

US

209, TBBPA) and bisphenol A (BPA) using a zebrafish liver cell line, ZFL. Aquat Toxicol 159: 138-147, 2015.

AN

Zoeller RT, Bansal R and Parris C. Bisphenol-A, an environmental contaminant that acts as a

M

thyroid hormone receptor antagonist in vitro, increases serum thyroxine, and alters

ED

RC3/neurogranin expression in the developing rat brain. Endocrinology 146: 2: 607-612, 2005.

Zoeller RT, Tan SW and Tyl RW. General background on the hypothalamic-pituitary-thyroid

AC

CE

PT

(HPT) axis. Crit Rev Toxicol 37: 1-2: 11-53, 2007.

29

ACCEPTED MANUSCRIPT Fig. 1 A.

1.8

1.6

1.6

1.4

1.2

T3 in MDCK_MCT8 T3 in MDCK-pcDNA DITPA in MDCK_MCT8 DITPA in MDCK_pcDNA

0.8 0.6

0.8

A

0.4

0.2

0.2

D E

0.0

1

10

M 0.0

-0.2 0

T P

Concentration ( M)

U N

0.6 0.4

0.1

I R

C S

1.0

1.0

0.01

T P

MDCK_MCT8 MDCK_pcDNA

1.2

OD/20min

1.4

OD/20min

B.

10

20

Time (min)

E C

C A

30

30

ACCEPTED MANUSCRIPT Fig. 2.

A.

B.

T P

I R

120 100

100

C S

80

% of DMSO

% of DMSO

80

60

U N

60

A

40

40

20

D E

0 IC50

100

T P

BSP ( M)

M

20

0 10

1000

100

BSP

E C

C A

31

1000

M)

ACCEPTED MANUSCRIPT Fig. 3.

A.

B.

T P

I R

C S

A

U N

D E

M

T P

E C

C A

32

ACCEPTED MANUSCRIPT Fig. 4.

T P

I R

C S

A

U N

D E

M

T P

E C

C A

33

ACCEPTED MANUSCRIPT Fig. 5. A.

B.

T P

I R

140

100

C S

120

80

U N

% of DMSO

% of DMSO

100

60

40

D E

20

T P

0 2

4

8

16

BPA

32

62

E C

M)

125

80

M

A 60 40 20 0

16

250

32

62

BPA

C A

34

M)

125

250

ACCEPTED MANUSCRIPT

Source

Drug Drug Drug Drug Natural product Drug Drug Natural product Drug Drug

Table 1. Hill Model Fit and Potency Data for 10 Positive T3 uptake Inhibitors in the Training Set Relative Ab Predicted IC Relative Emax (% LEC potency to Chemicals br. Mechanism [Ref] 50 (µM) IC50 (µM) control) (µM) BSP Unknown (Topliss et Cardiogreen ICG al. 1989) 2.4 4 32 2 6250 Das MCT8 (Braun et al. Dasatinib a 2012) 13.7 19 22 7.8 1316 Su MCT8 (Braun et al. Sunitinib ni 2012) 16.4 27 (?) 20 7.8 926 Im MCT8 (Braun et al. Imatinib a 2012) 7.1 31 28 7.8 806 Ge MCT8 (Braun and Genistein n Schweizer 2015) 19.1 31 37.6 16 806 Bus MCT8 (Braun et al. Bosutinib a 2012) 88 170 39 16 147 Bromosulfale BS MCT8 (Jayaramain P Naidu et al. 2015) 225 250 28.8 63 100 Unknown (Movius Phloretin Phl et al. 1989) 298.48 500 37 63 50 DM MCT8 (Roth et al. Desipramine I 2010) 304 505 40 125 50 Meclofenami Me Unknown (Topliss et c acid clo al. 1989) 174.67 >1000 76 500 <25

T P

I R

C S

U N

A

D E

M

T P

5,5Diphenylhyd antoin

E C

C A

DP Unknown (Topliss et Drug H al. 1989) N/A N/A 93 Predicted IC50, derived from Sigmaplot, Standard Curves, Hill model. Relative IC50, corresponding to the 50% response level on the y axis Emax (% Control), maximum inhibition response LEC, Lowest effect concentration, the lowest concentration demonstrating a >=20% inhibition.

35

N/A

N/A

Hill slope

-2.6

R2 0.9 96 0.9 90 0.9 90 0.9 45 0.9 16 0.9 70 0.9 88 0.9 97 0.9 97 0.9 66

-0.1

0.8 98

-2.3 -1.5 -1.2 -10 -4.4 -1.5 -0.8 -0.3 -0.7

Validati on MCT8 Validat ed ? Validat ed Validat ed Validat ed Validat ed MCT8 Validat ed MCT8 not via MCT8

ACCEPTED MANUSCRIPT

Table 2. Known Negative Substances Tested in MCT8HTS

Amino acid

Tyrosine

Tyr

Amino acid

Tryptophan 4methylbenzyliden campher

Trp

Refs (Kinne et al. 2010) (Kinne et al. 2010) (Johannes et al. 2016)

4-MBC BP3

Herbicide

Nitrofen

NIT

Isoflavone Detergent additives

Daidzein

DAIZ

AN

US

Benzophenon 3

(Johannes et al. 2016) (Johannes et al. 2016) (Johannes et al. 2016) (Johannes et al. 2016)

CR

Ultraviolet (UV) filters Ultraviolet (UV) filters

Abbr.

4NP

AC

CE

PT

ED

M

4-nonylphenol

36

Observed Effect in MCT8HTS

T

Name

IP

Chemical Class

negative negative

negative negative negative negative negative

ACCEPTED MANUSCRIPT Table 3. Acceptance Criteria and Check List Intra-Plate Tests

Meets Criterion? T3 uptake

Yes Yes

Yes

Yes

T

Yes Yes

Yes

Yes Yes

Yes Yes

Yes

Yes

Yes

Yes

Yes

Yes

CR

IP

Yes

US

1 Check for drift / edge effects in all plates<20% 2 All max (HI) signal CV's < 20% All mid signal (unnormalized) CV's < 3 20% All normalized mid signal (mid %) SD's < 4 20 All min (LO) SD's < Min(max (HI) SD, mid 5 SD)* 6 All SW's >2 All Z' Factors > 0.4 (and < 1 ; must pass 6 7 or 7)

BSP inhibition of T3 uptake

AN

Inter-Plate Tests

M

1 All within-day fold shifts < 2 All Average (between-)Day fold shifts < 2 2

AC

CE

PT

ED

*Indicates SD for all minimal signals should less than the smallest SD among all SDs for maximal signals and mid signals.

37

ACCEPTED MANUSCRIPT Table 4. Chemicals screened with MCT8HTS Abbr.

Triclosan

TCS ETU

Ethylenethiourea

Flame retardant Flame retardant metabolite

Flame retardant

Brominated diphenyl ether 209 Diphenyl phosphate bistribromophenox yethane

IP

T

(Ibhazehiebo et al. 2011a; Ibhazehiebo et al. 2011b) (Szabo et al. 2009)

DE-71

negative negative

(Yang and Chan 2015) BDE 209 DpHP

(Behl et al. 2015; Kojima et al. 2013)

BPA

Warfarin

PT CE AC

38

negative negative

(Johnson et al. 2013)

BTBPE

ED

Plasticizer Bisphenol A Pesticide/anticoag ulant drug Warfarin

HBCD

negative

AN

Flame retardant

Commercial penta mixture

negative

(Freitas et al. 2011) TBBPA

M

Flame retardant

Tetrabromobisp henol A Hexabromocycl ododecane

Flame retardant

Result negative

(Maranghi et al. 2013)

CR

Fungicide

Name

US

Chemical Class Antimicrobial agent

Refs (Effect on TH signalling) (Wu et al. 2016)

negative

(Gentilcore et al. 2013) (Khan et al. 2014; Macchia 2000)

Positive negative

ACCEPTED MANUSCRIPT Highlights

T IP CR US AN M ED PT

 

CE



Confirmed that the nonradioactive SK reaction can detect thyroid hormone (TH) uptake via MCT8. Confirmed that the nonradioactive SK reaction can identify the TDCs inhibiting TH uptake via MCT8. Several potential TDS were tested using this model. Bisphenol A inhibits TH uptake via MCT8 albeit only at high concentrations.

AC



39