Impacts of Unregulated Novel Brominated Flame Retardants on

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Impacts of Unregulated Novel Brominated Flame Retardants on Human Liver Thyroid Deiodination and Sulfotransferation Tristan Alexander Smythe, Craig M Butt, Heather M Stapleton, Kerri Pleskach, Geemitha Ratnayake, Chae Yoon Song, Nicole Riddell, Alex Konstantinov, and Gregg Thomas Tomy Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 25 May 2017 Downloaded from http://pubs.acs.org on May 29, 2017

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Impacts of Unregulated Novel Brominated Flame Retardants on Human Liver Thyroid Deiodination and Sulfotransferation Tristan A. Smythe1, Craig M. Butt2, Heather M. Stapleton2, Kerri Pleskach3, Geemitha Ratnayake4, Chae Yoon Song5, Nicole Riddell6, Alex Konstantinov6 and Gregg T. Tomy1*

1

Department of Chemistry, University of Manitoba, Winnipeg, MB, R3T 2N2 Canada

2

Nicholas School of the Environment, Duke University, Durham, NC, 27708 USA

3

Department of Fisheries and Oceans Canada, Freshwater Institute, Winnipeg, MB, R3T 2N6 Canada

4

Fort Richmond Collegiate, Winnipeg, MB, R3T 3B3

5

Department of Chemistry, McGill University, Montreal, QC, H3A 0G4 Canada

6

Wellington Laboratories Inc., 345 Southgate Drive, Guelph, ON, N1G 3M5 Canada

*To whom correspondence should be addressed: Gregg Tomy; email: [email protected]; ph: 204-474-8127; fax: 204-474-7608 594 Parker Bldg., University of Manitoba, R3T 2N2 Word count: Number of words = 4786 Number of Figures = 4

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ABSTRACT 1

The inhibitory effects of five novel brominated flame retardants:

1,2-bis(2,4,5-

2

tribromophenoxy)ethane (BTBPE), decabromodiphenylethane (DBDPE), 2-ethylhexyl-

3

2,3,4,5-tetrabromobenzoate (EH-TBB), bis(2-ethylhexyl)tetrabromophthalate (BEH-

4

TEBP), and β-tetrabromoethylcyclohexane (β-TBECH) on thyroid hormone deiodinase

5

(DIO) and sulfotransferase (SULT) activity were investigated using human in vitro liver

6

microsomal and cytosolic bioassays. Enzymatic activity was measured by incubating

7

active human liver sub-cellular fractions with thyroid hormones (T4 and rT3 separately)

8

and measuring changes in thyroid hormone (T4, T3, rT3, and 3,3’-T2) concentrations.

9

Only DBDPE showed inhibition of both outer and inner ring deiodination (O and IRD) of

10

T3 and 3,3′-T2 formation from T4 respectively, with an estimated IC50 of 160 nM; no

11

statistically significant inhibition of SULT activity was observed. ORD inhibition of 3,3′-

12

T2 formation from rT3 was also observed (IC50 ~100 nM). The kinetics of T4 O and IRD

13

were also investigated, although a definitive mechanism could not be identified as the

14

Michaelis-Menten parameters and maximal rate constants were not significantly

15

different. Concentrations tested were intentionally above expected environmental levels,

16

and this study suggests that these NBFRs are not potent human liver DIO and SULT

17

inhibitors. To our knowledge, DBDPE is the first example of a non-hydroxylated

18

contaminant inhibiting DIO activity, and further study of the mechanism of action is

19

warranted.

20 21

1. Introduction

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Brominated flame retardants (BFRs) are a class of compounds used extensively in the

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plastics and textile industries. Many BFRs are classified as high production volume

24

chemicals by the Organization for Economic Co-operation and Development (OECD),

25

produced in quantities greater than 1000 tonnes per year in at least one member country.1

26

They are used in commercial products to slow and mitigate the spread of fire, and to meet

27

flammability standards;2,3 polybrominated diphenyl ethers (PBDEs) are a specific

28

subclass of BFRs. In the past 15 years, increasing evidence has emerged demonstrating

29

the persistence (P) and bioaccumulation (B) of PBDEs in both aquatic and terrestrial

30

environments,4–7 as well as their inherent toxicities (iT) to marine8,9 and land10,11 animals.

31

In 2014, at the 9th annual meeting of the United Nations Stockholm Convention (UN-SC),

32

UN members agreed that decabromodiphenyl ether (BDE-209) warranted global

33

regulatory action due to its P, B & iT characteristics, and it is currently scheduled to join

34

both the penta- and octaBDE formulations on the list of regulated persistent organic

35

pollutants (POPs) under the UN-SC.12

36 37

These regulations have led to an increase in the demand for alternative, or novel BFRs

38

(NBFRs). Firemaster® 550 (FM 550) is a widely used example, intended to replace

39

pentaBDE, containing triphenyl phosphate (TPHP), isopropylated triphenyl phosphate

40

(TIPPP),

41

ethylhexyl)tetrabromophthalate

42

tribromophenoxy)ethane (BTBPE) is being used as a replacement for the octaBDE

43

mixture,14 and it is believed that decabromodiphenyl ethane (DBDPE) will replace

44

decaBDE after its imminent phase out.15

2-ethylhexyl-2,3,4,5-tetrabromobenzoate (BEH-TEBP).13

(EH-TBB) Similarly,

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and

bis(2-

1,2-bis(2,4,6-

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Previous in vitro studies by Butt et al. showed that several halogenated organic

47

contaminants (HOCs), including numerous hydroxylated PBDEs, negatively impacted

48

hepatic thyroid-regulating deiodinases16 (DIO) and sulfotransferases17 (SULT),

49

suggesting the potential for the disruption of thyroid homeostasis. The working

50

hypothesis for these studies has been based on structural mimicry between HOCs/PBDEs

51

and endogenous thyroid hormones (THs). Figure 1 illustrates the process of TH

52

deiodination.

53 54

While chemical mimicry is a contributing factor for endocrine disruption, earlier studies

55

on structurally dissimilar contaminants have demonstrated that this is not the only factor

56

that can affect thyroid metabolism.18 Palace et al.19 showed that several HBCDs altered

57

glucuronidation and deiodination pathways in juvenile rainbow trout (Oncorhynchus

58

mykiss), although to our knowledge no further and more specific studies have been done

59

on the activity or mechanisms therein. Therefore, it is still of interest to study chemically

60

dissimilar NBFRs for their activities within these systems.

61 62

Recent studies have shown that many of these NBFRs are commonly detected in indoor

63

dust, and inadvertent dust ingestion is considered the primary route of exposure to

64

humans in Canada and the United States.20–22 In conjunction with the aforementioned

65

studies on HOC effects on DIO and SULT, there is a strong interest in studying NBFRs

66

and their impact on endocrine endpoints to further investigate their potential for toxicity

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and effects on human health. In this study, both DBDPE and BTBPE were examined due

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β-

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to their increasing use and similarity in structure to PBDEs. The activity of

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tetrabromoethylcyclohexane ( β -TBECH) was also investigated because it has been

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shown to disrupt the thyroid axis in juvenile brown trout (Salmo trutta).23 Previous work

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by Patisaul et al. demonstrated that FM 550 may act as an endocrine disruptor in rats

72

under high exposures.24 Both EH-TBB and BEH-TEBP are included in the FM 550

73

commercial mixture, and were included in this study. Figure 2 depicts the chemical

74

structures of the compounds studied.

75 76

Using pooled and commercially available human liver microsomes (HLMs) and cytosol

77

(HLC), the effect of NBFRs on both DIO and SULT activities in human liver tissues was

78

studied using an established in vitro bioassay.16,17 Concentrations greater than that

79

reported in the environment were chosen initially to determine if effects might be

80

observed at higher exposure concentrations. In addition, a novel micro-liquid

81

chromatography high-resolution mass spectrometry ( μ -LC HR-MS) method was

82

developed and used to measure DIO and SULT activities.

83 84

2. Materials and Methods

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2.1 Chemical Reagents. 3,3′,5,5′-tetraiodothyronine (T4, > 96%), 3,3′,5-triiodothyronine

86

(T3, > 97%), 3,3′,5′-triiodothyronine (rT3, > 97%), monopotassium phosphate (> 99%),

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dipotassium phosphate (> 98%), dimethylsulfoxide (DMSO, > 99.9%), toluene (>

88

99.9%), 3′-phosphoadenosine 5′-phosphosulfate lithium salt (PAPS, > 60%), β -

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nicotinamide adenine dinucleotide 2’-phosphate reduced tetrasodium salt hydrate (β-

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NADPH, > 95%), and DL-dithiothreitol (DTT, > 95%) were purchased from Sigma

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Aldrich (Oakville, ON, CA). 3,3′-diiodothyronine (3,3′-T2, > 95%) was purchased from

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Cayman Chemical (Ann Arbor, MI, USA). EH-TBB (> 98%), DBDPE (> 98%), BTBPE

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(> 98%), β-TBECH (> 98%), and BEH-TEBP (> 98%) were supplied by Wellington

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Laboratories Inc. (Guelph, ON, CA). 10 M HCl, and HPLC grade (> 99.9%) water,

95

methanol, and acetone were purchased from Fisher Scientific (Ottawa, ON, CA). Nonane

96

was purchased from Caledon Laboratory Chemicals (> 99%, Georgetown, ON, CA).

97

Additional PAPS was also purchased from Santa Cruz Biotechnology (> 60%, Dallas,

98

TX, USA). Stable carbon-13 isotope standards (13C6-T4, 13C6-T3, 13C6-rT3, and 13C6-3,3′-

99

T2 in methanol, > 95%) were purchased from Sigma-Aldrich. 0.1 M potassium phosphate

100

buffers were prepared from the mono and dipotassium phosphate salts and water at pHs

101

7.2 and 7.4 separately.

102 103

2.2 In Vitro Bioassays. Details of the in vitro bioassays can be found in Butt et al.16,17 In

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brief, for the deiodinase assays, HLMs (pooled from 50 donors of mixed gender, Fisher

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Scientific, Ottawa, ON, CA), were diluted to 1 mg protein/mL in 0.1 M potassium

106

phosphate buffer (pH 7.4), 10 mM DTT, 100 µM NADPH, with 1 µM T4 (total volume =

107

1 mL). BEH-TEBP (3.75 to 3750 nM), β-TBECH (6.00 to 24,000 nM), and EH-TBB

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(4.81 to 19,200 nM) were spiked at varying concentrations, and carried in DMSO;

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BTBPE from 3.64 to 364 nM in 9:1 nonane:toluene; DBDPE from 0.13 to 260 nM in

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toluene. A complete list of concentrations used for each NBFR can be found in SI.

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Samples were incubated with 0.5% total volume (5 µL) of carrier solvent. Assays were

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performed as described by Butt et al.16 with the following alteration: reactions were

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performed at 37°C in a shaking incubator (Innova 43, Eppendorf, Mississauga, ON, CA)

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at 115 rpm for 1 hr. Following quenching with 1 mL ice-cold methanol, 25 ng of an

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isotope dilution mixture (ISD mix, 13C6-T4,

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into the resulting solutions. Extraction from protein, and solid-phase extraction (SPE)

117

were performed identically to Butt et al.16 Each concentration level was performed in

118

triplicate. DBDPE assays were repeated using 1000, 5000, and 10,000 nM rT3 to monitor

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rT3 ORD.

13

C6-T3,

13

C6-rT3,

13

C6-3,3′-T2) was spiked

120 121

For the sulfotransferase assays, HLC was pooled from 50 donors of mixed gender

122

(Xenotech, Kansas City, KS, USA), and diluted to 0.25 mg protein/mL in 0.1 M

123

potassium phosphate buffer (pH 7.2), 50 µM PAPS, with 1 µM 3,3′-T2 (total volume =

124

200 µL). The NBFR concentrations used were identical to that of the DIO assays, except

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for DBDPE (only 0.13 to 130 nM DBPDE was used). All incubations were performed

126

with 0.5% total volume (1 µL) of carrier solvent. All samples were run in triplicate.

127

Assays were performed as described by Butt et al.,17 using a shaking incubator at 37°C

128

and 115 rpm for 30 min. Following quenching with 800 µL of ice-cold 0.1 M HCl, 6.25

129

ng of

130

described by Butt et al.17

13

C6-T2 was spiked in as an internal standard. SPE clean-up was performed as

131 132

T4 DIO kinetics were studied by measuring outer ring and inner ring deiodination (ORD

133

and IRD) of T4 at 10, 1000, 5000, and 10,000 nM, as a function of DBDPE concentration

134

(0.13, 1.3, 13, 64, 130, and 260 nM).

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2.3 Instrumental Analysis. Separation and high-resolution mass spectral analysis was

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performed using an Eksigent MicroLC 200 coupled to an AB SCIEX 5600 HR-QTOF-

138

MS system (Concord, ON, CA). Chromatography was performed using an Eksigent

139

HALO C18 column (50 x 0.5 mm, 2.7 µm particle size, 90 Å pore size). Methanol and

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water (each with 0.1% acetic acid) were selected as the mobile phases. Column

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temperature was set to 30°C, and all samples were injected at 2 µL with 25 µL/min flow

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rates. Initial conditions of 80:20 water:methanol were held for 0.5 min before increasing

143

to 100% methanol over 3.0 min. The gradient was then returned to initial conditions over

144

0.1 min and held for 0.9 min for a total run time of 4.5 min (Figure S1). TOF-MS data

145

was acquired from 100 to 1000 m/z using electrospray ionization (ESI) in the negative

146

mode, with a capillary voltage of 4500 V. Nitrogen was used as the collision, nebulizer,

147

and drying gas; gas temperature was set to 350°C with a nebulizer flow of 9 L/min. The

148

exact masses utilized are listed in Table S1. The MS system was calibrated to within 2-5

149

ppm daily on m/z 792.4598 via direct infusion using the provided calibrant delivery

150

system (CDS) in the atmospheric pressure chemical ionization (APCI) mode per

151

manufacturer instructions.

152 153

Data acquired were processed using SCIEX MultiQuant software. Analyte responses

154

were determined via isotope-dilution, and normalized with respect to internal standard

155

response factors. In the absence of

156

normalized to the signal of 13C6-3,3′-T2.

13

C6-3,3′-T2-sulfate, 3,3′-T2-sulfate (3,3′-T2S) was

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Deiodinase and sulfotransferase activities were calculated by determining the ratio of the

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mass of product formed with respect to the mass of substrate (T4, rT3, or 3,3′-T2) in each

160

sample. The percent inhibition was then calculated by normalizing this ratio to that

161

obtained in the active controls. Regression analyses were performed using the “sigmoidal

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dose-reponse” model in SigmaPlot (version 13.0, Systat Software Inc., Chicago, IL).

163

Statistical differences between dose levels were assessed using one-way ANOVA (p
98% purity), approximately 37.5

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nM of the hydroxylated isomers would have been present in the highest concentration

275

samples tested (3.75 µM). As a result, the inhibition observed at this concentration

276

(~20%) could be attributed to the presence of hydroxylated side products. However, this

277

could not be confirmed analytically.

278 279

Because T4 substrate concentrations > 10 µM could not be accurately measured due to

280

aqueous solubility limits, there was significant variability in the acquired Km parameters.

281

As a result, no statistically significant (p < 0.05) differences in the Km parameters were

282

observed. While blocking of the active site and/or structural confirmation changes of the

283

protein are possible, these results are inconclusive and do not definitively suggest either

284

competitive or noncompetitive inhibition.

285 286

3.4 Implications. Thyroid hormones (THs) are critical in the regulation of metabolism,

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and in the growth and development of humans, and wildlife. In humans, T4 is primarily

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secreted by the thyroid gland, while rT3 and 3,3′-T2 are only produced through

289

deiodination in peripheral tissues.32 T4 is an inactive pro-hormone and must first be

290

deiodinated at the 5′ position to produce physiologically active T3; this typically occurs

291

in peripheral tissues such as the liver and brain.32 Sulfotransferase induced conjugation of

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thyroid hormones (with a preference for 3,3′-T2) to the sulfate derivatives facilitates

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further deiodination for the purpose of iodine recycling, increasing water-solubility, and

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facilitating excretion of the residual THs (biliary and/or urinary).32,33 The relative

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concentrations of active to inactive TH is important for the hypothalamic-pituitary-

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thyroid axis in the regulation of TH production. Increasing or decreasing TH levels, may

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lead to physiological changes such as changes in follicular cells in the thyroid

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gland,19,23,34 and can occur via one (or more) of many processes. While inhibition of

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deiodination and/or sulfotransferation may disrupt regular TH levels, any changes in the

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regulation or activities of other TH system enzymes can have similar effects. For

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example, several BFRs and their respective metabolites have been shown to interact with

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androgen receptors and transthyretin (TTR),35 as well as T4 binding globulin (TBG).36

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TTR is responsible for transport of T4 across the blood-brain barrier, as well as transport

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from the placenta to the fetus during pregnancy.37 TBG is the primary transport protein

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for TH in the blood, with the majority of TH bound by either TBG or TTR.38 Competition

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or interference with TH binding to TTR and TBG may therefore alter the amount of free

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TH available. Due to the complexity of the TH system, a large number of targets for

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exogenous TH disrupting chemicals exist, for which many in vitro assays have been

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developed to address the individual mechanisms.37 The present study examines

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specifically the effects of selected NBFRs on deiodination and sulfotransferation in

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human hepatic O and IRD activity.

312 313

The similarity in structure of several flame retardants to the endogenous THs has

314

previously been linked to the disruption of thyroid homeostasis. It has been reported that

315

halogenated organic contaminants (HOCs) with hydroxyl groups between two halogen

316

substituents on a diphenyl, such as tetrabromobisphenol A (TBBPA) and some OH-

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BDEs, act as the most potent inhibitors of deiodinases and sulfotransferases.16,17

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However, non-analogous compounds have also been shown to negatively impact the

319

thyroid system through different mechanisms.19 HBCDs were shown to induce

320

concomitant epithelial cell hypertrophy in juvenile rainbow trout (Oncorhynchus mykiss)

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altering circulating levels of endogenous THs.19 Similar results were found for exposure

322

to β-TBECH in juvenile brown trout (Salmo trutta).23 While little to no inhibitory activity

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was observed for β-TBECH, BEH-TEBP, EH-TBB, and BTBPE at the doses tested, there

324

is still a concern based on previous studies that they may interact with other aspects of the

325

hypothalamic-pituitary-thyroid system. For example, an in vivo rat study by Patisaul et

326

al.24 showed that exposure to Firemaster 550® (containing EH-TBB, BEH-TEBP, TPHP,

327

and TIPPP), through both gestation and lactation, caused significant increases in dam

328

serum T4 levels, likely through increases in TH transport proteins TTR and/or TBG. This

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is believed to be the cause of the observed developmental abnormalities such as early

330

puberty and metabolic dysfunction. Porcine esterases have been reported to bioactivate

331

BEH-TEBP into mono-(2-ethylhexyl) tetrabromophthalate, which was shown to

332

significantly decrease serum T3 levels in rats which caused maternal hepatotoxicity.39 β-

333

TBECH exposure has been shown to disrupt the thyroid axis in juvenile brown trout

334

(Salmo trutta),23 lead to changes in the expression of thyroid and estrogen receptors in

335

chicken LMH cells,40 and to affect gene regulation and cause developmental

336

abnormalities in in vivo zebrafish (Danio rerio) studies.41

337 338

DBDPE showed significant inhibition in the production of both T3 and 3,3′-T2 in DIO

339

assays (Figure 3). Our studies suggest that DBDPE may be of similar potency as an

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inhibitor as hydroxylated PBDEs. Butt et al. reported 5′-OH-BDE-99 to have an IC50 of

341

400 nM16 in the formation of T3 from T4 ORD, while our results show unhydroxylated

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DBDPE possessing an IC50 on the order of 100-200 nM. However, considering the low

343

predicted aqueous solubility of DBDPE and previous environmental measurements (non-

344

detect, < 16 nM7), these concentrations for DBDPE are likely not environmentally

345

significant. Both previous studies on PBDEs in in vitro DIO16 and SULT17 assays

346

demonstrated that the presence of the hydroxyl group is critical for PBDE inhibition, with

347

PBDEs producing little to no inhibition. Despite the similarity in structure of BTBPE to

348

DBDPE, BTBPE did not exhibit any inhibitive properties in DIO at comparable doses.

349

BTBPE more closely resembles PBDEs, having two ether linkages instead of one. As a

350

result, it is possible that if BTBPE were DIO active, it would behave similarly to PBDEs

351

and require hydroxylation for potency. In an earlier study involving the in vivo exposure

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of BTBPE to juvenile rainbow trout (Oncorhynchus mykiss), no debrominated or

353

hydroxylated metabolites of BTBPE were detected, and the thyroid axis did not appear to

354

be significantly affected.42 This is consistent with the lack of inhibition observed here,

355

even at significantly higher doses.

356 357

Although the doses tested here are higher than the aqueous solubilities for each

358

compound, concentrations measured in serum are sometimes measured higher than the

359

aqueous solubility. Stapleton et al. previously reported up to 63.8 ng/g lipid of BDE-209

360

in serum in a North Carolina toddler cohort study,43 or approximately 0.4 nM BDE-209.

361

However, the estimated aqueous solubility is on the order of 1 x10-8 nM (calculated using

362

the EPI SuiteTM program). This discrepancy is likely due to enhanced solubility in

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serum/blood from proteins and other lipophilic compounds; thus, there is an interest in

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examining NBFRs at these higher concentrations.

365 366

HOCs containing biphenyl, diphenyl ether, or similar moieties are typically metabolized

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through a meta-para epoxide (arene oxide) intermediate.44 DBDPE is fully brominated

368

which prevents this reaction from occurring. Additionally, while Butt et al. did not

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monitor the production of hydroxylated metabolites in their in vitro DIO study, they

370

showed that although BDE-99 did not exhibit any inhibition, 5-,5′-, and 6′-OH-BDE-99

371

did, suggesting the lack of significant formation of the relevant BDE-99 metabolites

372

during the assays.16 It is assumed that this is also true in the present study. Although no

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attempt was made to examine possible debromination products, it is believed that it is

374

unlikely that DBDPE underwent both a debromination and hydroxylation reaction in

375

vitro in the duration of the experiment. To our best knowledge, this is the first report of a

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non-hydroxylated compound exhibiting inhibition of these enzymes. Due to the

377

hydrophobic nature of DBDPE, it is possible that this is simply a result of non-specific

378

binding to the deiodinase allosterically, although similar inhibition was not observed for

379

BEH-TEBP despite being similarly hydrophobic. This study did not investigate this

380

specific interaction further, and the results do not definitively suggest any specific

381

mechanism. The kinetic assays of this study were inconclusive with regards to deducing a

382

mechanism of inhibition.

383 384

3.5 Environmental Significance. The results presented here for the inhibition of human

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liver deiodinases and sulfotransferases adds to the body of literature surrounding the

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potential impacts of NFBRs in the environment. This study suggests that SULT inhibition

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by β-TBECH, BEH-TEBP, EH-TBB, BTBPE, and DBDPE are not likely to affect the

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human thyroid axis. Similarly, no significant inhibition of DIO was observed for all

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NBFRs except for DBDPE, suggesting the possibility of DBDPE interacting with human

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liver deiodinases at high concentrations. To our best knowledge, only one study has

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reported the detection of measurable quantities of DBDPE in human blood serum

392

samples, although with very low detection frequencies (5.9% across 102 serum

393

samples).45 This is consistent with other studies where measurable quantities of DBDPE

394

could not be detected in human blood serum samples.46–48 While DBDPE has been shown

395

to bioaccumulate in multiple freshwater environments in both Canada7 and China,49 and

396

has been detected in both indoor and outdoor environments globally,50 the concentrations

397

used in this study were significantly greater than what is environmentally significant.

398

Regardless, our results indicate that DBDPE can have a direct impact on the

399

hypothalamic-pituitary-thyroid axis, and it is of interest to examine the possible

400

mechanism further. Butt et al. previously reported IC50 values for inhibition of T3

401

formation in the presence of several different HOCs at or significantly greater than 1 µM

402

(e.g. TBBPA IC50 = 2.1 µM), with the lowest measured IC50 of 440 nM for 5′-OH BDE

403

99.16 To our knowledge this makes our estimated IC50 of ~160 nM the lowest value

404

observed for a BFR at this time. However, further studies are needed to examine other

405

impacts DBDPE may have on the endocrine system of wildlife species. Futhermore, no

406

detailed studies have been done on the kinetics of DIO inhibition by other BFRs to our

407

knowledge, and it is of interest to compare the observed rates of PBDE inhibition vs.

408

DBDPE, particularly due to the lack of hydroxyl functionality in DBDPE. Such

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comparison studies may help better elucidate a possible mechanism of inhibition.

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Because both DIO and SULT activity are critical to the functionality and development in

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other tissues in addition to the liver, further investigation of the effects of DBDPE in

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these tissues (e.g. brain, placenta) is warranted. There remains a significant gap for many

413

NBFRs on their mechanistic modes of action within the TH system, and additional

414

studies for other in vitro endpoints may help in the understanding of in vivo test results.

415 416

Acknowledgements

417

Dr. Vince Palace (International Institute for Sustainable Development – Experimental

418

Lakes Area) is thanked for discussion on mechanisms for thyroid hormone disruption. KP

419

is now with the Canadian Grain Commission. CMB is now with SCIEX. This work was

420

supported by a Natural Sciences and Engineering Research Council (NSERC) Discovery

421

Grant to GTT and an NSERC-Undergraduate Student Research Award to TAS.

422 423

Supplementary Information

424

The following tables and figures can be found in SI: Table S1. Negative mode time-of-

425

flight ions selected on AB SCIEX 5600 HR-QTOF-MS system. Table S2. MRM

426

transitions selected on API 2000 Triple Quadrupole system; acquired in the negative

427

mode. Figure S1. a) Representative extracted ion chromatogram (XIC) of labeled

428

internal standards in the BEH-TEBP active control (1) sample; T3 peak at 2.51 min, rT3

429

at 2.65 min. b) XICs for both native 3,3′-T2 and 3,3′-T2S of BEH-TEBP active control

430

(2). Figure S2. Inhibition of T3 and 3,3′-T2 production resulting from the incubation of

431

human liver microsomes with 1 µM T4, and NBFRs at varying concentrations according.

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T3 formation in the presence of BEH-TEBP is the only assay to present statistically

433

significant (p < 0.05) inhibition at the highest concentration; R2 = 0.9780. L3 BTBPE

434

point corresponds to n = 1, thus no statistical analysis can be done on these points. Error

435

bars represent 1 standard deviation. Figure S3. Inhibition of T3 and 3,3′-T2 production

436

resulting from the incubation of human liver microsomes with 5 and 10 µM T4, and

437

DBDPE at varying concentrations. 10 µM L6 n = 1. Error bars represent 1 standard

438

deviation. Figure S4. Inhibition of 3,3′-T2 production resulting from the incubation of

439

human liver microsomes with 5 and 10 µM rT3, and DBDPE at varying concentrations.

440

10 µM L5 n = 2. Error bars represent 1 standard deviation. Figure S5. Inhibition of 3,3′-

441

T2S production resulting from the incubation of human liver cytosol with 1 µM 3,3′-T2,

442

and NBFRs at varying concentrations. Error bars represent 1 standard deviation. Figure

443

S6. Inhibition of 3,3′-T2S production resulting from the incubation of human liver

444

cytosol with 1 µM 3,3′-T2, and DBDPE at varying concentrations. There is no statistical

445

difference between concentration levels (n = 3, ANOVA p < 0.05). Error bars represent 1

446

standard deviation.

447 448

References

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Figure Captions Figure 1. Stepwise deiodinase reactions investigated in the present study. Type I, II, and III refer to specific isoforms of DIO enzymes. Figure 2. Chemical structures of the NBFRs examined, and BDE-209 as a PBDE reference structure, are presented. Figure 3. Inhibition of T3 and 3,3′-T2 production resulting from the incubation of human liver microsomes with 1 µM T4 (and rT3), and DBDPE at varying concentrations, n = 3. Error bars represent 1 standard deviation. Figure 4. Formation rate (pmol/min/mg protein) of T3 and 3,3′-T2 from incubation of T4. Maximum reaction rate (Vmax) obtained from “one site saturation” regression model in SigmaPlot 13.0. Data points represent the mean (n = 3); error bars represent 1 standard error.

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Figure 1. Stepwise deiodinase reactions investigated in the present study. Type I, II, and III refer to specific isoforms of DIO enzymes. Carbons 1-6 and 1′-6′ represent inner and outer ring carbons respectively.

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β

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Figure 2. Chemical structures of the NBFRs examined, and BDE-209 as a PBDE reference structure, are presented.

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R2 = 0.9455

R2 = 0.7209

663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678

R2 = 0.9843

log DBDPE concentration (nM) Figure 3. Inhibition of T3 and 3,3′-T2 production resulting from the incubation of human liver microsomes with 1 µM T4 (and rT3), and DBDPE at varying concentrations, n = 3. Error bars represent 1 standard deviation. .

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Figure 4. Formation rate (pmol/min/mg protein) of T3 and 3,3′-T2 from incubation of T4. Maximum reaction rate (Vmax) obtained from “one site saturation” regression model in SigmaPlot 13.0. Data points represent the mean (n = 3); error bars represent 1 standard error.

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