Thyroid hormone-disrupting potentials of major benzophenones in two

Subscriber access provided by Kaohsiung Medical University

Ecotoxicology and Human Environmental Health

Thyroid hormone-disrupting potentials of major benzophenones in two cell lines (GH3 and FRTL-5) and embryo-larval zebrafish Jungeun Lee, Sujin Kim, Young Joo Park, Hyo-Bang Moon, and Kyungho Choi Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b01796 • Publication Date (Web): 11 Jul 2018 Downloaded from http://pubs.acs.org on July 12, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31

Environmental Science & Technology

1

Thyroid hormone-disrupting potentials of major benzophenones in two cell lines (GH3

2

and FRTL-5) and embryo-larval zebrafish

3 4

Jungeun Lee,† Sujin Kim,† Young Joo Park,‡ Hyo-Bang Moon,§ Kyungho Choi*,†,∥

5

†

6

University, Seoul 08826, Republic of Korea

7

‡

8

University College of Medicine, Seoul 03080, Korea

9

§

Department of Environmental Health Sciences, School of Public Health, Seoul National

Department of Internal Medicine, Seoul National University Hospital and Seoul National

Department of Marine Sciences and Convergent Technology, Hanyang University, Ansan

10

15588, Republic of Korea

11

∥

12

Korea

13

* Corresponding author

14

E-mail address: [email protected] (K. Choi).

15

(Tel) 82-2-880-2738

16

(Fax) 82-2-745-9104

Institute of Health and Environment, Seoul National University, Seoul 08826, Republic of

ACS Paragon Plus Environment


17

Page 2 of 31

Abstract

18

Benzophenones (BPs) have been widely used in personal care products (PCPs) such as UV

19

protectants. Sex endocrine-disrupting effects have been documented for some BPs, but,

20

significant knowledge gaps are present for their thyroid-disrupting effects. To investigate the

21

thyroid-disrupting potential of BPs, a rat pituitary (GH3) and thyroid follicle (FRTL-5) cell

22

line were employed on six BPs, i.e., benzophenone (BP), benzophenone-1 (BP-1),

23

benzophenone-2

24

benzophenone-8 (BP-8). Subsequently, zebrafish (Danio rerio) embryo exposure was

25

conducted for three potent BPs that were identified based on the transcriptional changes

26

observed in the cells. In GH3 cells, all BPs except BP-4 down-regulated the Tshβ, Trhr, and

27

Trβ genes. In addition, some BPs significantly up-regulated the Nis and Tg genes while

28

down-regulating the Tpo gene in FRTL-5 cells. In zebrafish embryo assay conducted for BP-

29

1, BP-3, and BP-8, significant decreases in whole-body T4 and T3 level were observed at 6

30

day post-fertilization (dpf). The up-regulation of the dio1 and ugt1ab genes in the fish

31

suggests that decreased thyroid hormones are caused by changing metabolism of the

32

hormones. Our results show that these frequently used BPs can alter thyroid hormone

33

balances by influencing the central regulation and metabolism of the hormones.

(BP-2),

benzophenone-3

(BP-3),

benzophenone-4

(BP-4),

and

34 35

Keywords: benzophenones, UV-filter, GH3 cell line, FRTL-5 cell line, zebrafish, thyroid

36

hormone, endocrine disruption


Page 3 of 31


37

1. Introduction

38

Benzophenones (BPs) have been frequently used as UV protection agents in personal care

39

products (PCPs) such as sunscreen, nail polish, lipsticks, shampoo, and hand sanitizer. As the

40

frequency of their use has increased, their detection in the environment and biota has been

41

more frequently reported. One good example is BP-3, which is one of the most heavily used

42

BP UV filters. This chemical has been widely detected in environmental media such as

43

surface water and wastewater around the world.1-3 BP-3 and its structural analogs have also

44

been reported in human specimens such as urine, serum, breast milk, adipose tissue, and

45

placental tissue.4,5

46

BPs have been shown to have sex endocrine-disrupting potentials in many experimental

47

animals. For example, BP-3 has been reported to cause estrogenic effects in Japanese medaka

48

(Oryzias latipes), rainbow trout (Oncorhynchus mykiss), and rats.6-8 Similar estrogenic effects

49

were reported for BP-1, a major metabolite of BP-3 and a UV protection agent.9-11 However,

50

for other BPs, e.g., BP-8, toxicological information related to endocrine disruption is limited.

51

Moreover, most of the endocrine-disrupting effects of BPs have been focused on sex hormone

52

disruption.

53

Thyroid hormones play crucial roles in the development, growth and energy metabolism of

54

humans and animals. Therefore, a disruption or alteration in thyroid function would cause

55

various types of adverse effects to organisms, i.e., somatic and brain growth retardation,

56

developmental defects, and structural abnormalities.12,13 The thyroid hormone system is

57

therefore tightly regulated by a negative feedback system involving the hypothalamic-

58

pituitary-thyroid (H-P-T) axis. Many environmental chemicals have been suggested to disrupt

59

the regulations of thyroid hormone homeostasis by affecting the synthesis, transportation, and

60

metabolism of thyroid hormones.14,15 BP-2 is one of the best-known thyroid disruptors in

61

both in vitro and in vivo studies.16-18 BP-2 exposure led to a decrease in serum T4 and T3



62

levels in rats.16,17 Thyroid peroxidase (TPO) is reported to be inhibited by exposure to BP-2,

63

and this exposure could lead to a decrease in thyroid hormone levels.15,17,19 However,

64

information on thyroid disruption by BPs other than BP2 is scarce, and the mechanisms of

65

thyroid disruption are generally focused on TPO inhibition.

66

Epidemiological evidence suggesting the thyroid hormone-disrupting potentials of BP-3 in

67

human populations is accumulating. Among the general USA population, urinary BP-3

68

concentration was associated with decreased total T4 in males (n=960), and with decreased

69

free and total T4 in females (n=869).20 Among pregnant women (n=106), the concentration of

70

BP-3 in urine was similarly negatively associated with serum free T3 level.21 Experimental

71

evidence of the thyroid-disrupting effects of BPs other than BP-2, however, is rare.

72

Significant knowledge gaps are present for thyroid hormone-disrupting effects and associated

73

mechanisms for structural analogues of BP-2.

74

The objective of this study is to evaluate thyroid-disrupting effects of major BPs. For this

75

purpose, six BPs frequently used in consumer products were chosen and screened for thyroid-

76

disrupting potentials employing two in vitro test models, i.e., GH3 and FRTL-5 cells. GH3 is

77

a rat pituitary carcinoma cell line that has been utilized as T-screen assay based on T3-

78

dependent cell proliferation.22,23 FRTL-5, a rat thyroid follicular cell line, has frequently been

79

utilized to evaluate the sodium/iodide symporter (NIS)-mediated iodide uptake.24-26 Recently,

80

these two cell lines have been employed to understand mechanisms of thyroid-disrupting

81

chemicals by measuring transcriptional changes in genes that are key for central regulation27-

82

29

83

results of these two in vitro assays, and they were subsequently tested for thyroid-disrupting

84

potentials and related mechanisms in the H-P-T axis using zebrafish larvae. The results of the

85

present study will help identify BPs with potential thyroid-disrupting effects, and expand the

86

understanding of the associated mechanisms.

and synthesis of thyroid hormones.28-30 Then, three potent BPs were identified based on the


Page 4 of 31

Page 5 of 31


87



88

2. Materials and Methods

89

2.1 Chemicals

90

Page 6 of 31

Six BPs, i.e., BP (benzophenone, CAS no. 119-61-9, ≥ 99% purity), BP-1 (2,4-

91

dihydroxybenzophenone,

92

tetrahydroxybenzophenone, CAS no. 131-55-5, 97% purity), BP-3 (2-hydroxy-4-

93

methoxybenzophenone,

94

methoxybenzophenone-5-sulfonic acid, CAS no. 4065-45-6, ≥ 97% purity), and BP-8 (2,2′-

95

dihydroxy-4-methoxybenzophenone, CAS no. 131-53-3, 98% purity) were purchased from

96

Sigma-Aldrich (St. Louis, MO, USA). Structural information and the molecular weight of

97

each BP are shown in Table S1. T3 (Triiodothyronine, CAS no. 6893-02-3) and TSH

98

(Thyroid-stimulating hormone, CAS no. 9002-71-5), which were used as positive controls for

99

in vitro assays, were purchased from Sigma-Aldrich. As a solvent, dimethyl sulfoxide

100

(DMSO, CAS no. 67-68-5) was used. Hybri-MaxTM grade DMSO (≥ 99.7% purity) was

101

purchased from Sigma-Aldrich, and for fish exposure, DMSO with ≥ 99% purity was

102

purchased from Junsei Chemical Co. (Tokyo, Japan)

CAS

CAS

no.

no.

131-56-6,

131-57-7,

99%

98%

purity),

purity),

BP-2

BP-4

(2,2′,4,4′-

(2-hydroxy-4-

103 104

2.2 GH3 Cell Culture and Exposure

105

The GH3 cell line was obtained from Korean Cell Line Bank (Seoul, Korea) and was

106

maintained at 37◦C with 5% CO2. The cells were grown in a Dulbecco’s modified Eagle’s

107

medium/Ham’s F-12 nutrient mixture (Sigma–Aldrich) that was supplemented with 10% fetal

108

bovine serum (FBS; Gibco®, LifeTechnologies, Carlsbad, CA, USA) following the protocol

109

used by Kim et al. (2015).28 For exposure, GH3 cells were seeded in 24-well plates at

110

2.0×105 cells/well and then incubated for 20 h. To circumvent potential confounding effects

111

from steroid hormones and growth factors that are present in the serum (FBS), the growth

112

medium was changed to serum-free medium, which contains 1% BD ITS+ premix (BD


Page 7 of 31


113

Biosciences, Franklin Lakes, NJ, USA), 4 hours before the exposure. After incubation for 4 h,

114

the cells were exposed to different dose ranges of BPs. Based on a preliminary cytotoxicity

115

assay using WST-1 cell proliferation reagent (Roche Applied Science, Mannheim, Germany),

116

the concentrations beyond which steep decline in cell proliferation was observed were

117

excluded in order to circumvent possible confounding observations due to cytotoxicity

118

(Figure S1). The test concentrations were as follows: 0, 3.2, 10, 32, and 100 µM (or 0.6-18.2

119

mg/L) for BP; 0, 1, 3.2, 10, and 32 µM (0.2-6.9 mg/L) for BP-1; 0, 0.32, 1, 3.2, and 10 µM

120

(0.1-2.5 mg/L) for BP-2; 0, 3.2, 10, 32, and 100 µM (0.7-22.8 mg/L) for BP-3; 0, 10, 32, 100,

121

and 320 µM (3.1-98.7 mg/L) for BP-4; and 0, 3.2, 10, 32, and 100 µM (0.8-24.4 mg/L) for

122

BP-8. T3 was used as a positive control at 1 nM, and was treated in each set of exposures for

123

verification. The test doses were prepared in triplicate (n=3) for each treatment (0.1% v/v

124

DMSO). The results presented are based on three independent biological replicates using

125

cells from the same origin with different passage numbers.

126 127

2.3 FRTL-5 Cell Culture and Exposure

128

FRTL-5 cells were maintained at 37°C in a 5% CO2 atmosphere. FRTL-5 cells were

129

cultured in Coon's modified Ham's F-12 medium (Sigma-Aldrich) supplemented with 10%

130

calf serum (Gibco®) and a mixture of 6 hormones (6H medium) that included insulin (1

131

µg/mL), transferrin (5 µg/mL), somatostatin (10 ng/mL), Gly-His-Lys acetate (10 ng/mL),

132

hydrocortisone (10 nM), and thyroid stimulating hormone (TSH, 1 mU/mL), following the

133

protocol used by Kim et al. (2015).28 All the hormones used in the medium were purchased

134

from Sigma–Aldrich. The 6H medium was supplemented with L-glutamine (2 mM; Gibco®)

135

and MEM non-essential amino acids (1 mM; Gibco®). FRTL-5 cells were seeded in 24-well

136

plates at 8.0 × 104 cells/well and incubated for 24 h with 6H medium. After 24 h, the medium

137

was exchanged to 5H medium (6H medium without TSH) and incubated for 24 h. The



138

exposure medium was prepared with 0.1% chemical stock in 5H medium. The cells were then

139

dosed with a series of concentrations of each chemical: 0, 10, 32, 100, and 320 µM (1.8-58.3

140

mg/L) for BP; BP-1 (2.1-68.6 mg/L); BP-2 (2.5-78.8 mg/L); BP-3 (2.3-73.0 mg/L); and BP-4

141

(3.1-98.7 mg/L); and 0, 3.2, 10, 32, and 100 µM (0.8-24.4 mg/L) for BP-8. These

142

experimental doses were determined based on preliminary range-finding tests at non-

143

cytotoxic doses based on the same method that was used for GH3 cells (Figure S2). TSH (10

144

mU/mL) was used as a positive control.28 The cells were exposed to several doses of each

145

chemical for 24 h, with three technical replicates (n=3) and three biological replicates.

146 147

2.4 Zebrafish Embryo-larval Exposure

148

Based on responses observed in GH3 and FRTL-5 cells, three potent BPs, i.e., BP-1, BP-3,

149

and BP-8, were selected for zebrafish embryo-larval exposure. Wild-type zebrafish have been

150

in-house cultured in the Environmental Toxicology Laboratory at Seoul National University

151

(Seoul, Korea). Fertilized eggs were obtained by mating sexually mature adult fish. A total of

152

250 eggs per replicate were randomly distributed into 500 mL glass beakers that contained

153

300 mL exposure media within 5 h after fertilization. Each exposure group contained four

154

replicates (n=4), and a solvent control group included eight replicates (n=8). Based on a

155

preliminary range finding test, concentrations which did not affect survival or hatchability of

156

the fish were determined and were employed to choose the highest exposure concentration

157

for each chemical, i.e., 1000 µg/L for BP-1, and 320 µg/L for BP-3 and BP-8 (Table S2). The

158

exposure concentrations for each chemical were determined at 0, 100, 320, and 1000 µg/L for

159

BP-1; 0, 32, 100, and 320 µg/L for BP-3; and 0, 32, 100, and 320 µg/L for BP-8. The

160

exposure medium was prepared with dechlorinated water with DMSO stock (0.005% v/v)

161

and was replaced daily until 6 days post-fertilization (dpf) of exposure. During the exposure,

162

water temperature of approximately 27 ± 1◦C and a photoperiod of 14 L: 10 D were


Page 8 of 31

Page 9 of 31


163

maintained. Hatchability and survival of the embryo and larval fish were recorded daily.

164

Water quality parameters, such as dissolved oxygen, temperature, pH, and conductivity, were

165

measured regularly after renewal of exposure media (for water chemistry, refer to Table S4).

166

At 6 dpf, larvae were euthanized with ice water and collected into e-tubes. Before collecting

167

larvae, the weight of empty e-tubes was measured separately. A total of 20 larvae were

168

randomly sampled for gene analysis, and another 180 larvae were employed for thyroid

169

hormone measurement. Immediately after collection, water was removed from the e-tube

170

with a pipette, and the total weight of the e-tube, including the 180 larvae, was measured to

171

determine the wet weight of zebrafish larvae. The fish samples were stored at −80◦C until

172

being used for further analysis.

173 174

2.5 Thyroid Hormone Extractions and Measurement

175

For thyroid hormone extraction, zebrafish larvae (n=180 per replicate) were homogenized

176

using a motor driven tissue grinder in 110 µL ELISA buffer (Gingko Bioscience, China). The

177

homogenates were sonicated for 10 min at 4◦C and centrifuged at 5000×g for an additional 10

178

min at 4◦C. The supernatant was collected and stored at −80◦C until analysis. T4 and T3 levels

179

were measured using enzyme-linked immunosorbent assays (ELISA) following the protocol

180

used by Yu et al. (2010) with minor modifications.31 The test kits (Cat no. CEA452Ge for T4;

181

Cat no. CEA453Ge for T3) were purchased from Cloud-Clone Corp. (Wuhan, China). The

182

detection limits for T4 and T3 were 1.42 ng/mL and 47.2 pg/mL, respectively. Measurement

183

was conducted by a plate reader (Tecan Infinite® 200, Tecan Group Ltd., Mändorf,

184

Switzerland) following the manufacturer’s instructions.

185 186 187

2.6 RNA Isolation and Quantitative Real-time Polymerase Chain Reaction (qRT-PCR) To isolate RNA for GH3 and FRTL-5 assays, the cells were washed twice with ice-cold



188

phosphate-buffered saline (PBS) and lysed with lysis buffer. For zebrafish exposure, 20

189

larvae were ground in lysis buffer with the tissue grinder. RNA was immediately isolated

190

using the RNeasy Mini Kit (Qiagen, Hilden, Germany) following each experiment. The

191

quality and concentration of RNA were measured with an Epoch Take 3 spectrophotometer

192

(Biotek, Bad Friedrichshall, Germany). The quality of RNA was checked by conducting

193

absorbance measurements (260/280 ratio > 1.8), and the possible presence of contaminants,

194

such as protein, was determined. After dilution of mRNA to make the same concentration in

195

each sample, complementary DNAs (cDNAs) were synthesized using the iScriptTM cDNA

196

synthesis kit (BioRad Hercules, CA, USA). For quantitative real-time PCR (qRT-PCR), 20

197

µL of the qRT-PCR reaction mix was combined with 10 µL of LightCycler® 480 SYBR

198

Green I Master mix (Roche Diagnostics Ltd., Lewes, UK), 1.0 µL of each PCR primer (10

199

pmol), 6 µL of purified PCR-grade water, and 2 µL of the cDNA diluted 1:4 with water. From

200

a set of candidates, Gapdh, β-actin, and 18s genes were selected as housekeeping genes in

201

GH3, FRTL-5 cell assays, and zebrafish larvae gene analysis, respectively (Tables S7 and

202

S8). The primer sequences used in this study are shown in supplementary Table S9. In GH3

203

cells, three genes involved in central regulation of the thyroid system were observed,28,29

204

which include Thyrotropin-releasing hormone receptor (Trhr), Thyroid stimulating hormone

205

beta (Tshβ), and Thyroid hormone receptor beta (Trβ). In FRTL-5 cells, four genes

206

responsible for thyroid hormone synthesis were analyzed in the cells,28-30 which include

207

Thyroid-stimulating hormone receptor (Tshr), Sodium/iodide symporter (Nis), Thyroglobulin

208

(Tg), and Thyroid peroxidase (Tpo). In zebrafish larvae, transcriptional changes of eight

209

genes which play key roles in the H-P-T axis were analyzed.28,31,32 These genes were tshβ for

210

central regulation; solute carrier family 5 member 5 (slc5a5, i.e., nis), tg, and tpo for thyroid

211

hormone synthesis; paired box protein 8 (pax8) for thyroid development; and deiodinase 1

212

(dio1), deiodinase 2 (dio2), and uridine diphosphate glucuronosyltransferase 1 family a, b


Page 10 of 31

Page 11 of 31


213

(ugt1ab) for thyroid hormone metabolism. qRT-PCR was performed using LightCycler 480

214

(Roche Applied Science, Indianapolis, IN, USA). The thermal cycle profile was as follows:

215

pre-incubation at 95◦C for 10 min, 40 cycles of amplification at 95◦C for 10 s, 85◦C for 20 s,

216

and 72◦C for 20 s. The threshold cycle (Ct) was determined for each reaction and normalized

217

to the housekeeping gene using the 2−∆∆Ct method.33

218 219

2.7 Statistical Analysis

220

The normality of data distribution and homogeneity of variances were analyzed by Shapiro–

221

Wilk’s test and Levene’s test, respectively. Depending on the distribution, one-way analysis

222

of variance (ANOVA) with Dunnett’s test or Dunnett’s T3 post hoc test was used for

223

comparison among control and treatments. Linear regression analysis was conducted for

224

trend analysis. p values less than 0.05 were considered significant. Mean values were

225

expressed with standard error of the mean (SEM) for all data. IBM SPSS 20.0 for Windows

226

(SPSS Inc., Chicago, IL, USA) was used for data analysis.

227



228

3. Results

229

3.1 Alterations of Gene Expression in GH3 Cells

230

After exposure to all tested BPs, similar transcriptional changes, but with different

231

potencies, were observed for major thyroid hormone-regulating genes in GH3 cell assays

232

(Figure 1). Significant down-regulation of the Tshβ, Trhr, and Trβ genes was observed

233

following exposure to BP-1 and BP-2, even at doses of 10 µM and below. Similarly, BP, BP-

234

3, and BP-8 significantly down-regulated all three genes at doses around 32 µM. This down-

235

regulating pattern of the Tshβ, Trhr, and Trβ genes was similar to those observed following

236

T3 exposure (Figure S3). For BP-4, slight but significant down-regulation was observed only

237

for the Trβ gene.

238 239

3.2 Alterations of Gene Expression in FRTL-5 Cells

240

In the FRTL-5 cells, exposure to most BPs up-regulated the Nis and Tg genes and down-

241

regulated the Tpo gene (Figure 1). The Nis gene was significantly up-regulated following

242

exposure to BP, BP-1, BP-3, and BP-8. Significant up-regulation of the Tg gene was observed

243

after exposure to BP and BP-3. Although statistical significance was not reached, the change

244

in Tg gene transcription was relatively high, i.e., > 3.0-fold change after exposure to BP-1

245

and BP-8. In addition, exposure to BP-1, BP-2, BP-3, and BP-8 significantly down-regulated

246

the Tpo gene. Especially, BP-3 and BP-8 significantly down-regulated the Tpo gene at doses

247

relatively lower than the other tested BPs. However, none of the tested BPs affect the

248

transcription level of the Tshr gene. In addition, BP-4 did not affect expression of any tested

249

genes after exposure. Exposure to TSH significantly up-regulated Nis by up to 9.6-fold and,

250

at the same time, significantly down-regulated the Tshr, Tg, and Tpo genes (Figure S4).

251 252

3.3 Effects on Zebrafish Embryos and Larvae


Page 12 of 31

Page 13 of 31

253


3.3.1 Thyroid Hormones in Zebrafish Larvae

254

Exposure to BP-1, BP-3, and BP-8 until 6 dpf decreased whole-body T4 and T3 levels in

255

zebrafish larvae (Figure 2). Zebrafish larvae treated with BP-3 and BP-8 showed significant

256

decrease of T3 levels, but not T4 levels, at test concentrations as low as 32 µg/L. In contrast,

257

BP-1 significantly decreased both T4 and T3 levels of the fish larvae at 320 and 1000 µg/L.

258

However, BP-1, BP-3, or BP-8 exposure until 6 dpf did not affect the embryo and larval

259

survival, hatchability, or body weight of zebrafish (Table S3).

260 261

3.3.2 Transcriptional Changes Related to the Thyroid System in Zebrafish Larvae

262

Significant up-regulation of thyroid-related genes was observed after exposure to BP-3 or

263

BP-8, but not after BP-1 exposure (Figure 2). BP-3 significantly up-regulated the tg, dio1,

264

and ugt1ab genes, and BP-8 significantly up-regulated all genes except tshβ in the 100 µg/L

265

group. Although statistical significance was not always observed, all genes analyzed in this

266

study were up-regulated by > 1.5-fold after exposure to BP-3 or BP-8. Especially for BP-3,

267

all measured genes exhibited a significant trend of up-regulation (p value < 0.05, trends are

268

not shown). Following BP-1 exposure, thyroid hormone levels were significantly changed,

269

but associated changes in thyroid hormone-related genes were not significant.



270

4. Discussion

271

The responses of a rat pituitary cell (GH3 cell) and a rat thyroid gland cell (FRTL-5 cell)

272

following exposure to six BPs (Figure 1) clearly show that the tested BPs could influence

273

these key organs that regulate thyroid hormone balance. Changes in thyroid hormone levels

274

in zebrafish larvae following exposure to three BPs, i.e., BP-1, BP-3, and BP-8 (Figure 2),

275

also support the thyroid-disrupting potentials of these chemicals. The results of the present

276

study suggest that most commonly used BPs could disrupt the thyroid system in a manner

277

similar to BP-2. In addition, the results of this study are in agreement with previous studies,

278

which reported negative associations between thyroid hormone levels and BP-3 exposure in

279

human populations.20,21

280 281

4.1 Thyroid-disrupting Potentials of BPs in GH3 and FRTL-5 Cells

282

Down-regulations of the Trhr, Tshβ, and Trβ genes in GH3 cells by BPs were comparable to

283

the responses observed following the exposure to T3, suggesting that BPs may act similar to

284

T3 on the rat pituitary gland (Figures 1 and S3). A decrease in thyrotropin-releasing hormone

285

(TRH) receptor, encoded by Trhr, might cause reduced sensing of TRH signals.34 As TRH

286

stimulates the pituitary gland to release TSH, down-regulation of the Trhr gene may partly

287

explain the thyroid-lowering effects of the BPs tested in this study. A previous study

288

employing rat models reported that decreases in thyroid hormones by PCB153 exposure were

289

also mediated by down-regulation of Trhr gene.35 Further mechanistic studies should be

290

performed to confirm the proposed mechanisms and the effects of BPs on TRH signaling.

291

Thyroid hormone, especially bioactive T3, interacts with thyroid hormone receptors (TRs) to

292

exert its effect on the target tissue or organ. Therefore, down-regulation of the Trβ gene in

293

GH3 cell following the exposure to BPs may also suggest possible T3-like activities of the

294

test BPs.


Page 14 of 31

Page 15 of 31


295

Down-regulation of the Tpo gene in FRTL-5 cells by most BPs indicates that these BPs can

296

disrupt the thyroid system in a manner similar to BP-2 (Figure 1). TPO is an enzyme

297

involved in the coupling of iodide to thyroglobulin, or iodine organification.34 BP-2 is

298

reported to decrease thyroid hormone through inhibiting TPO activities.16-18,36 Inhibition of

299

TPO activity by major BPs can be explained to certain extent by their structural

300

characteristics. Previous studies have reported that flavonoids, which contain a resorcinol

301

moiety, showed inactivation of the TPO enzyme by covalent binding to TPO.37,38 Therefore,

302

free resorcinol moieties in the structure of BP-1 and BP-2 could account for their TPO

303

inhibition activity, while for BP-3 and BP-8 the methyl group on the resorcinol hydroxyl

304

group might reduce TPO inhibition. However, inconsistent effects on TPO activities were

305

also reported among several BPs, e.g., BP-1, BP-3, and BP-8, in previous studies employing

306

Amplex UltraRed (rat thyroid microsomes and a fluorescent peroxidase substrate) and FTC-

307

238 cell.18,36,39 These inconsistent observations could be partly explained by the different

308

experimental conditions and models employed. The previous studies used either isolated

309

molecules (i.e., TPO) or a follicular thyroid carcinoma cell line of human origin to assess

310

TPO activity. It should also be noted that changes in Tpo regulation observed in the present

311

study should be interpreted with caution, as down-regulation of the Tpo gene in FRTL-5 cells

312

following exposure to TSH (Figure S4) contradicts the reports of previous studies.28,29

313

However, down-regulation of the Tpo gene by exposure to BPs observed in this study (Figure

314

1) is comparable to those of previous studies that reported inhibition of TPO activities by BP-

315

2.17,18,36,39

316

Steep up-regulation of the Nis gene by most BPs except BP-4 is not comparable to the

317

reports of previous studies, which observed no changes in iodide uptake activity in the same

318

cell line.17,18 The NIS mediates the uptake of iodide into the thyroid follicular cell, which

319

contribute to subsequent iodination of TG protein by TPO for synthesis of thyroid hormones.



320

NIS has been known to be regulated post-transcriptionally,40,41 therefore iodide uptake may

321

not be directly influenced through transcriptional changes of Nis gene.

322 323

4.2 Thyroid-disrupting Potentials of BPs in Zebrafish Larvae

324

The decreases of thyroid hormones following exposure to BP-1, BP-3, and BP-8 observed in

325

the larval fish (Figure 2) indicate that these BP analogs disrupt thyroid hormones through the

326

same manner as BP-2. Experimental evidence showing that BP-2 exposure causes a decrease

327

in thyroid hormone levels has accumulated. BP-2 lowered the levels of intra-follicular T4 in

328

zebrafish larvae (EC50=4.70 µM, 1.16 mg/L; exposed between 2 dpf and 5 dpf).42 In

329

ovariectomized adult rats, after 5 days of oral exposure to BP-2, significant decreases of

330

serum T4 and T3 levels were reported.16,17 In human populations, similar observations have

331

been reported. BP-3 measured in urine showed a negative association with free T3 levels in

332

the serum of pregnant women (n=106)21 and with T4 levels in the serum of the general USA

333

population (n=1829).20 However, since embryo-larval zebrafish depend on maternal T4 from

334

yolk and start endogenous synthesis of T4 approximately at 72 hpf, changes in thyroid

335

regulating genes measured during the early life stages should be interpreted with caution.43,44

336

Up-regulation of the slc5a5, tg, and tpo genes may be seen as compensatory effects by

337

negative feedback regulation in zebrafish larvae (Figure 2). Since all of these genes are

338

involved in thyroid hormone synthesis, decreased T4 and T3 levels could stimulate negative

339

feedback regulation to produce more thyroid hormones. It is interesting to observe up-

340

regulation of the tpo gene in the zebrafish larvae, considering that BP-2 has been widely

341

reported to inhibit TPO activity in previous studies. Our observation may be due to the

342

difference between gene transcription and protein activity: TPO inhibition could occur

343

despite up-regulation of the tpo gene, as it is generally accepted that transcriptional changes

344

are more transient and are not always directly linked to the changes in protein activity.45


Page 16 of 31

Page 17 of 31


345

Other genes involved in stimulation of the thyroid hormone synthesis (tshβ) or development

346

of the thyroid gland (pax8) showed up-regulation following exposure to BP-3 and BP-8,

347

which also indicates the compensatory efforts against decreased thyroid hormone levels.

348

Up-regulation in the dio1 and ugt1ab genes following exposure to BP-3 and BP-8 in

349

zebrafish larvae is in line with the decreases of thyroid hormone levels in the fish (Figure 2).

350

The Dio1 plays a critical role in both activation and inactivation of thyroid hormones, most

351

effectively being involved in clearance of inactive metabolite rT3.46 Previous research

352

reported that an increase in Dio1 activity could explain degradation of thyroid hormones in

353

T4-fed tilapias.47 Furthermore, up-regulation of the hepatic dio1 gene was observed in

354

hypothyroid tilapia,48 which is in line with our observation. UGT is an enzyme responsible

355

for the glucuronidation of T4, which will lead to its biliary excretion. Therefore, increased

356

ugt1ab transcription caused by exposure to BPs could lead to enhanced excretion of thyroid

357

hormones and result in decreased thyroid hormone levels in zebrafish larvae. Previous studies

358

also suggested that UGT could reduce thyroid hormone levels through T4 glucuronidation in

359

both animal and in vitro models.49-51

360

BP-1 appeared to disrupt the thyroid system in zebrafish larvae in different manner than BP-

361

3 or BP-8, since we could not observe any significant regulatory changes of the genes,

362

including the ugt1ab gene, after exposure to BP-1 (Figure 2). The underlying reason for

363

thyroid hormone decrease by BP-1 is not clear. Sex hormone-disrupting effects of BP-1 could

364

be one of possible reasons that are potentially associated with the observed thyroid hormone-

365

lowering effects. BP-1 is reported to have greater binding affinity to estrogen receptor than

366

BP-3 and BP-8.9,52,53 Although conflicting evidence has been reported regarding to the link

367

between estrogen and thyroid hormone levels, experimental observations imply a possible

368

interaction between sex and thyroid hormone axes. For instance, fish treated with estradiol

369

showed decreases in thyroid hormone levels in plasma.54-56 However, whether the effects on



370

thyroid hormones by BP-1 could be explained by potential interactions between sex and

371

thyroid hormone axes warrants further mechanistic investigation.

372

While the directions of regulatory changes in major thyroid-related genes were similar,

373

those for a couple of genes such as tshβ and tpo, were not consistent between the zebrafish

374

larvae and the rat cells (Figures 1 and 2). This discrepancy between the in vivo and the in

375

vitro models can be partly explained by the presence of feedback mechanisms to regain

376

homeostasis in the fish, which are not available in the cell lines. In addition, dissimilarities in

377

thyroid systems, such as thyroid hormone receptors, among species may be also noted as a

378

possible reason,34,57 while the thyroid system is generally considered to be evolutionarily

379

conserved across species.34

380 381

4.3 Environmental Relevance and Implications

382

Our observations of thyroid hormone-disrupting potentials of major BPs in both zebrafish

383

larvae and rat cell models show that some of these commonly used sunscreen chemicals may

384

lead to adverse health consequences in aquatic ecosystem. In the present study, a significant

385

decrease of T3 was observed after BP-3 exposure at a concentration of 32 µg/L (or 27 µg/L

386

measured concentration), which is within an order of magnitude of difference higher than the

387

maximum level reported in ambient water, i.e., 5,429 ng/L in seawater from Hong Kong.3 In

388

wastewater influent, BP-3 has been detected at up to 10,400 ng/L in San Diego County in the

389

USA.58 BP-3 has been more widely detected in water environments compared to other BPs.59

390

Similar thyroid-disrupting potency in terms of T3 decrease was observed for BP-8 in

391

zebrafish larvae in the present study; however, its detection levels have been relatively lower

392

than those of BP-3. The highest detection level of BP-8 in a water environment was 117 ng/L

393

in seawater collected from Hong Kong.3

394

Considering the importance of thyroid hormone regulation in early development and normal


Page 18 of 31

Page 19 of 31


395

physiological functions, and the fact that our observations in fish were based on rather short-

396

term exposure of 144 h, consequences of long-term exposure to BPs on thyroid functions and

397

performances of later life stages in the fish warrant further investigation.

398 399

Acknowledgements

400

This study was funded by the Korea Ministry of Environment (MOE) as ‘‘the Environmental

401

Health Action Program (1485014458)".

402 403

Supporting Information Available

404

Details of preliminary range-finding tests, quality assurance information, housekeeping gene

405

selection, and chemical analysis of BPs in the exposure media can be found in supporting

406

information. In addition, the results of qPCR and hormone analysis are presented in figures.

407

This information is available free of charge via the Internet at http://pubs.acs.org.

408 409

Author Information

410

Corresponding Author

411

*Tel: 82-2-880-2738. Fax: 82-2-745-9104. E-mail: [email protected] (K. Choi).

412

ORCIDs

413

Jungeun Lee: 0000-0001-8336-2952

414

Sujin Kim: 0000-0001-5932-6249

415

Young Joo Park: 0000-0002-3671-6364

416

Hyo-Bang Moon: 0000-0001-6618-2036

417

Kyungho Choi: 0000-0001-7460-792X

418

Notes

419

The authors declare no competing ﬁnancial interest.



420


Page 20 of 31

Page 21 of 31

421


6. References

422

(1) Montes-Grajales, D.; Fennix-Agudelo, M.; Miranda-Castro, W. Occurrence of personal

423

care products as emerging chemicals of concern in water resources: A review. Sci. Total

424

Environ. 2017, 595, 601−614.

425 426

(2) Ramos, S.; Homem, V.; Alves, A.; Santos, L. A review of organic UV-filters in wastewater treatment plants. Environ. Int. 2016, 86, 24−44.

427

(3) Tsui, M. M.; Leung, H. W.; Wai, T. C.; Yamashita, N.; Taniyasu, S.; Liu, W.; Murphy,

428

M. B. Occurrence, distribution and ecological risk assessment of multiple classes of UV

429

filters in surface waters from different countries. Water Res. 2014, 67, 55−65.

430

(4) Gao, C. J.; Liu, L. Y.; Ma, W. L.; Zhu, N. Z.; Jiang, L.; Li, Y. F.; Kannan, K.

431

Benzophenone-type UV filters in urine of Chinese young adults: concentration, source and

432

exposure. Environ. Pollut. 2015, 203, 1−6.

433

(5) Hines, E. P.; Mendola, P.; von Ehrenstein, O. S.; Ye, X.; Calafat, A. M.; Fenton, S. E.

434

Concentrations of environmental phenols and parabens in milk, urine and serum of lactating

435

North Carolina women. Reprod. Toxicol. 2015, 54, 120−128.

436

(6) Coronado, M.; De Haro, H.; Deng, X.; Rempel, M. A.; Lavado, R.; Schlenk, D.

437

Estrogenic activity and reproductive effects of the UV-filter oxybenzone (2-hydroxy-4-

438

methoxyphenyl-methanone) in fish. Aquat. Toxicol. 2008, 90 (3), 182−187.

439

(7) Kim, S.; Jung, D.; Kho, Y.; Choi, K. Effects of benzophenone-3 exposure on endocrine

440

disruption and reproduction of Japanese medaka (Oryzias latipes)—A two generation

441

exposure study. Aquat. Toxicol. 2014, 155, 244−252.

442

(8) Schlumpf, M.; Cotton, B.; Conscience, M.; Haller, V.; Steinmann, B.; Lichtensteiger,

443

W. In vitro and in vivo estrogenicity of UV screens. Environ. Health Perspect. 2001, 109,

444

239−244.

445

(9) Kunz, P. Y.; Galicia, H. F.; Fent, K. Comparison of in vitro and in vivo estrogenic



446

activity of UV filters in fish. Toxicol. Sci. 2006, 90 (2), 349−361.

447

(10) Molina-Molina, J. M.; Escande, A.; Pillon, A.; Gomez, E.; Pakdel, F.; Cavaillès, V.;

448

Olea, N.; Aït-Aïssa, S.; Balaguer, P. Profiling of benzophenone derivatives using fish and

449

human estrogen receptor-specific in vitro bioassays. Toxicol. Appl. Pharmacol. 2008, 232 (3),

450

384−395.

451

(11) Suzuki, T.; Kitamura, S.; Khota, R.; Sugihara, K.; Fujimoto, N.; Ohta, S. Estrogenic

452

and antiandrogenic activities of 17 benzophenone derivatives used as UV stabilizers and

453

sunscreens. Toxicol. Appl. Pharmacol. 2005, 203, 9−17.

454 455 456 457 458 459

(12) Patrick, L. Thyroid disruption: mechanisms and clinical implications in human health. Altern. Med. Rev. 2009, 14 (4), 326−347. (13) Yen, P. M. Physiological and molecular basis of thyroid hormone action. Physiol. Rev. 2001, 81 (3), 1097−1142. (14) Boas, M.; Feldt-Rasmussen, U.; Skakkebæk, N. E.; Main, K. M. Environmental chemicals and thyroid function. Eur. J. Endocrinol. 2006, 154 (5), 599−611.

460

(15) Miller, M. D.; Crofton, K. M.; Rice, D. C.; Zoeller, R. T. Thyroid-disrupting

461

chemicals: interpreting upstream biomarkers of adverse outcomes. Environ. Health Perspect.

462

2009, 117 (7), 1033.

463

(16) Jarry, H.; Christoffel, J.; Rimoldi, G.; Koch, L.; Wuttke, W. Multi-organic endocrine

464

disrupting activity of the UV screen benzophenone 2 (BP2) in ovariectomized adult rats after

465

5 days treatment. Toxicology 2004, 205 (1), 87−93.

466

(17) Schmutzler, C.; Bacinski, A.; Gotthardt, I.; Huhne, K.; Ambrugger, P.; Klammer, H.;

467

Jarry, H. The ultraviolet filter benzophenone 2 interferes with the thyroid hormone axis in rats

468

and is a potent in vitro inhibitor of human recombinant thyroid peroxidase. Endocrinology

469

2007, 148 (6), 2835−2844.

470

(18) Schmutzler, C.; Gotthardt, I.; Hofmann, P. J.; Radovic, B.; Kovacs, G.; Stemmler, L.;


Page 22 of 31

Page 23 of 31


471

Nobis, I.; Bacinski, A.; Mentrup, B.; Ambrugger, P.; Gruters, A.; Malendowicz, L. K.;

472

Christoffel, J.; Jarry, H.; Seidlova-Wuttke, D.; Wuttke, W.; Kohrle, J. Endocrine disruptors

473

and the thyroid gland−a combined in vitro and in vivo analysis of potential new biomarkers.

474

Environ. Health Perspect. 2007, 115, 77−83.

475

(19) Krause, M.; Klit, A.; Blomberg Jensen, M.; Søeborg, T.; Frederiksen, H.; Schlumpf,

476

M.; Drzewiecki, K. T. Sunscreens: are they beneficial for health? An overview of endocrine

477

disrupting properties of UV filters. Int. J. Androl. 2012, 35 (3), 424−436.

478

(20) Kim, S.; Kim, S.; Won, S.; Choi, K. Considering common sources of exposure in

479

association studies-Urinary benzophenone-3 and DEHP metabolites are associated with

480

altered thyroid hormone balance in the NHANES 2007–2008. Environ. Int. 2017, 107, 25−32.

481

(21) Aker, A. M.; Watkins, D. J.; Johns, L. E.; Ferguson, K. K.; Soldin, O. P.; Anzalota Del

482

Toro, L. V.; Alshawabkeh, A. N.; Cordero, J. F.; Meeker, J. D. Phenols and parabens in

483

relation to reproductive and thyroid hormones in pregnant women. Environ. Res. 2016, 151,

484

30−37.

485

(22) Ghisari, M.; Bonefeld-Jorgensen, E. C. Impact of environmental chemicals on the

486

thyroid hormone function in pituitary rat GH3 cells. Mol. Cell. Endocrinol. 2005, 244 (1),

487

31−41.

488

(23) Gutleb, A. C.; Meerts, I. A.; Bergsma, J. H.; Schriks, M.; Murk, A. J. T-screen as a

489

tool to identify thyroid hormone receptor active compounds. Environ. Toxicol. Pharmacol.

490

2005, 19, 231−238.

491

(24) Arturi, F.; Presta, I.; Scarpelli, D.; Bidart, J. M.; Schlumberger, M.; Filetti, S.; Russo,

492

D. Stimulation of iodide uptake by human chorionic gonadotropin in FRTL-5 cells: effects on

493

sodium/iodide symporter gene and protein expression. Eur. J. Endocrinol. 2002, 147,

494

655−661.

495

(25) Lecat-Guillet, N.; Ambroise, Y. Enhanced iodide sequestration by 3-biphenyl-5, 6-



496

dihydroimidazo[2,1-b]thiazole

497

ChemMedChem 2008, 3, 1211−1216.

in

sodium/iodide

symporter

Page 24 of 31

(NIS)-expressing

cells.

498

(26) Weiss, S. J.; Philp, N. J.; Ambesi-Impiombato, F. S.; Grollman, E. F. Thyrotropin-

499

stimulated iodide transport mediated by adenosine 3' 5'-monophosphate and dependent on

500

protein synthesis. Endocrinology 1984, 114, 1099−1107.

501 502

(27) Guo, Y.; Zhou, B. Thyroid endocrine system disruption by pentachlorophenol: an in vitro and in vivo assay. Aquat. Toxicol. 2013, 142, 138−145.

503

(28) Kim, S.; Jung, J.; Lee, I.; Jung, D.; Youn, H.; Choi, K. Thyroid disruption by triphenyl

504

phosphate, an organophosphate flame retardant, in zebrafish (Danio rerio) embryos/larvae,

505

and in GH3 and FRTL-5 cell lines. Aquat. Toxicol. 2015, 160, 188−196.

506

(29) Lee, S.; Kim, C.; Youn, H.; Choi, K. Thyroid hormone disrupting potentials of

507

bisphenol A and its analogues-in vitro comparison study employing rat pituitary (GH3) and

508

thyroid follicular (FRTL-5) cells. Toxicol. In Vitro 2017, 40, 297−304.

509

(30) Gentilcore, D.; Porreca, I.; Rizzo, F.; Ganbaatar, E.; Carchia, E.; Mallardo, M.; De

510

Felice, M.; Ambrosino, C. Bisphenol A interferes with thyroid specific gene expression.

511

Toxicology 2013, 304, 21−31.

512

(31) Yu, L.; Deng, J.; Shi, X.; Liu, C.; Yu, K.; Zhou, B. Exposure to DE-71 alters thyroid

513

hormone levels and gene transcription in the hypothalamic–pituitary–thyroid axis of zebrafish

514

larvae. Aquat. Toxicol. 2010, 97 (3), 226−233.

515

(32) Wang, Q.; Liang, K.; Liu, J.; Yang, L.; Guo, Y.; Liu, C.; Zhou, B. Exposure of

516

zebrafish embryos/larvae to TDCPP alters concentrations of thyroid hormones and

517

transcriptions of genes involved in the hypothalamic–pituitary–thyroid axis. Aquat. Toxicol.

518

2013, 126, 207−213.

519 520

(33) Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2∆∆CT method. Methods 2001, 25, 402−408.


Page 25 of 31

521 522


(34) Zoeller, R. T.; Tan, S. W.; Tyl, R. W. General background on the hypothalamicpituitary-thyroid (HPT) axis. Crit. Rev. Toxicol. 2007, 37 (1−2), 11−53.

523

(35) Liu, C.; Ha, M.; Cui, Y.; Wang, C.; Yan, M.; Fu, W.; Quan, C.; Zhou, J.; Yang, K. JNK

524

pathway decreases thyroid hormones via TRH receptor: a novel mechanism for disturbance

525

of thyroid hormone homeostasis by PCB153. Toxicology 2012, 302 (1), 68−76.

526

(36) Paul, K. B.; Hedge, J. M.; Rotroff, D. M.; Hornung, M. W.; Crofton, K. M.; Simmons,

527

S. O. Development of a thyroperoxidase inhibition assay for high-throughput screening.

528

Chem. Res. Toxicol. 2014, 27 (3), 387−399.

529 530 531 532 533 534 535 536

(37) Divi, R. L.; Doerge, D. R. Mechanism-based inactivation of lactoperoxidase and thyroid peroxidase by resorcinol derivatives. Biochemistry 1994, 33 (32), 9668−9674. (38) Divi, R. L.; Doerge, D. R. Inhibition of thyroid peroxidase by dietary flavonoids. Chem. Res. Toxicol. 1996, 9 (1), 16−23. (39) Song, M.; Kim, Y. J.; Park, Y. K.; Ryu, J. C. Changes in thyroid peroxidase activity in response to various chemicals. J. Environ. Monit. 2012, 14 (8), 2121−2126. (40) Riedel, C.; Levy, O.; Carrasco, N. Post-transcriptional regulation of the sodium/ iodide symporter by thyrotropin. J. Biol. Chem. 2001, 276 (24), 21458−21463.

537

(41) Serrano-Nascimento, C.; Calil-Silveira, J.; Nunes, M. T. Posttranscriptional regulation

538

of sodium-iodide symporter mRNA expression in the rat thyroid gland by acute iodide

539

administration. Am. J. Physiol. Cell Physiol. 2010, 298 (4), C893−C899.

540

(42) Thienpont, B.; Tingaud-Sequeira, A.; Prats, E.; Barata, C.; Babin, P. J.; Raldúa, D.

541

Zebrafish eleutheroembryos provide a suitable vertebrate model for screening chemicals that

542

impair thyroid hormone synthesis. Environ. Sci. Technol. 2011, 45 (17), 7525−7532.

543 544 545

(43) Porazzi, P.; Calebiro, D.; Benato, F.; Tiso, N.; Persani, L. Thyroid gland development and function in the zebrafish model. Mol. Cell. Endocrinol. 2009, 312 (1−2), 14−23. (44) Vergauwen, L.; Cavallin, J. E.; Ankley, G. T.; Bars, C.; Gabriëls, I. J.; Michiels, E. D.;



Page 26 of 31

546

Fitzpatrick, K. R.; Periz-Stanacev, J.; Randolph, E. C.; Robinson, S. L.; Saari, T. W.;

547

Schroeder, A. L.; Stinckens, E.; Swintek, J.; Van Cruchten, S. J.; Verbueken, E.; Villeneuve,

548

D. L.; Knapen, D. Gene transcription ontogeny of hypothalamic-pituitary-thyroid axis

549

development in early-life stage fathead minnow and zebrafish. Gen. Comp. Endocrinol. 2018.

550

(45) Vogel, C.; Marcotte, E. M. Insights into the regulation of protein abundance from

551

proteomic and transcriptomic analyses. Nat. Rev. Genet. 2012, 13 (4), 227.

552

(46) Murk, A. J.; Rijntjes, E.; Blaauboer, B. J.; Clewell, R.; Crofton, K. M.; Dingemans,

553

M. M.; Furlow, J. D.; Kavlock, R.; Köhrle, J.; Opitz, R.; Traas, T.; Visser, T. J.; Xia, M.;

554

Gutleb, A. C. Mechanism-based testing strategy using in vitro approaches for identification of

555

thyroid hormone disrupting chemicals. Toxicol. In Vitro 2013, 27 (4), 1320−1346.

556

(47) Van der Geyten, S.; Byamungu, N.; Reyns, G.; Kühn, E.; Darras, V. Iodothyronine

557

deiodinases and the control of plasma and tissue thyroid hormone levels in hyperthyroid

558

tilapia (Oreochromis niloticus). J. Endocrinol. 2005, 184, 467−479.

559

(48) Van der Geyten, S.; Toguyeni, A.; Baroiller, J. F.; Fauconneau, B.; Fostier, A.;

560

Sanders, J. P.; Visser, T. J.; Kühn, E. R.; Darras, V. M. Hypothyroidism induces type I

561

iodothyronine deiodinase expression in tilapia liver. Gen. Comp. Endocrinol. 2001, 124 (3),

562

333−342.

563

(49) Barter, R. A.; Klaassen, C. D. UDP-glucuronosyltransferase inducers reduce thyroid

564

hormone levels in rats by an extrathyroidal mechanism. Toxicol. Appl. Pharmacol. 1992, 113

565

(1), 36−42.

566 567 568

(50) Bastomsky, C. H. Enhanced thyroxine metabolism and high uptake goiters in rats after a single dose of 2,3,7,8-tetrachlorodibenzop-dioxin. Endocrinology 1977, 101 (1), 292−296. (51) Jemnitz, K.; Veres, Z.; Monostory, K.; Vereczkey, L. Glucuronidation of thyroxine in

569

primary

570

glucuronosyltranferases by methylcholanthrene, clofibrate, and dexamethasone alone and in

monolayer

cultures

of

rat

hepatocytes:

in


vitro

induction

of

UDP-

Page 27 of 31

571


combination. Drug Metab. Dispos. 2000, 28 (1), 34−37.

572

(52) Kerdivel, G.; Le Guevel, R.; Habauzit, D.; Brion, F.; Ait-Aissa, S.; Pakdel, F.

573

Estrogenic potency of benzophenone UV filters in breast cancer cells: proliferative and

574

transcriptional activity substantiated by docking analysis. PLoS One 2013, 8 (4), e60567.

575

(53) Morohoshi, K.; Yamamoto, H.; Kamata, R.; Shiraishi, F.; Koda, T.; Morita, M.

576

Estrogenic activity of 37 components of commercial sunscreen lotions evaluated by in vitro

577

assays. Toxicol. In Vitro 2005, 19 (4), 457−469.

578

(54) Cyr, D. G.; MacLatchy, D. L.; Eales, J. G. The influence of short-term 17β-estradiol

579

treatment on plasma T3 levels and in vitro hepatic T4 5′-monodeiodinase activity in immature

580

rainbow trout, Salmo gairdneri. Gen. Comp. Endocrinol. 1988, 69 (3), 431−438.

581

(55) Leatherland, J. F. Effects of 17β-estradiol and methyl testosterone on the activity of

582

the thyroid gland in rainbow trout, Salmo gairdneri Richardson. Gen. Comp. Endocrinol.

583

1985, 60 (3), 343−352.

584

(56) Mercure, F.; Holloway, A. C.; Tocher, D. R.; Sheridan, M. A.; Kraak, G.; Leatherland,

585

J. F. Influence of plasma lipid changes in response to 17β oestradiol stimulation on plasma

586

growth hormone, somatostatin, and thyroid hormone levels in immature rainbow trout. J.

587

Fish Biol. 2001, 59 (3), 605−615.

588

(57) Ichikawa, K.; Hashizume, K.; Miyamoto, T.; Sakurai, A.; Yamauchi, K.; Nishii, Y.;

589

Yamada, T. Differences in nuclear thyroid hormone receptors among species. Gen. Comp.

590

Endocrinol. 1989, 74 (1), 68−76.

591

(58) Loraine, G. A.; Pettigrove, M. E. Seasonal variations in concentrations of

592

pharmaceuticals and personal care products in drinking water and reclaimed wastewater in

593

southern California. Environ. Sci. Technol. 2006, 40, 687−695.

594

(59) Kim, S.; Choi, K. Occurrences, toxicities, and ecological risks of benzophenone-3, a



595

common component of organic sunscreen products: a mini-review. Environ. Int. 2014, 70,

596

143−157.


Page 28 of 31

Page 29 of 31


597 598

Abstract Art

599



600 601

Figure 1. Transcriptional changes of (A) the Trhr, Tshβ, and Trβ genes in GH3 cells

602

following exposure to BP (3.2-100 µM), BP-1 (1-32 µM), BP-2 (0.32-10 µM), BP-3 (3.2-100

603

µM), BP-4 (10-320 µM), and BP-8 (3.2-100 µM), as well as the (B) Nis, Tg, and Tpo genes in

604

FRTL-5 cells after exposure to BP, BP-1, BP-2, BP-3, BP-4 (10-320 µM), and BP-8 (3.2-100

605

µM) in a half-log scale dilution. Colors represent the direction and extent of transcriptional

606

changes of the genes relative to that of the solvent control, 0.1% DMSO. The results are

607

based on three biological replicates.

608


Page 30 of 31

Page 31 of 31


609 610

Figure 2. Relative changes in (A) thyroid hormone levels and (B) expression of genes related

611

to the stimulation of thyroid hormone synthesis (tshβ, slc5a5, tg, tpo, pax8) and its

612

metabolism (dio1, dio2, ugt1ab) in whole-body of 6 dpf zebrafish larvae after exposure to

613

BP-1 (100, 320, and 1000 µg/L), BP-3 (32, 100, and 320 µg/L), and BP-8 (32, 100, and 320

614

µg/L). Colors represent the direction and extent of changes relative to solvent control,

615

0.005% DMSO (n=8 for solvent control; n=4 for BPs).


Thyroid hormone-disrupting potentials of major benzophenones in two

Recommend Documents