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Ecotoxicology and Human Environmental Health

Iodoacetic acid disrupts the thyroid endocrine system in vitro and in vivo Ying Xia, Yan Mo, Qiyuan Yang, Yang Yu, Meiyu Jiang, Shumao Wei, Du Lu, Huan Wu, Guodong Lu, Yunfeng Zou, Zhiyong Zhang, and Xiao Wei Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b01802 • Publication Date (Web): 29 May 2018 Downloaded from http://pubs.acs.org on May 29, 2018

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Environmental Science & Technology

Iodoacetic acid disrupts the thyroid endocrine system in vitro and in vivo

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Ying Xia†, ||, Yan Mo†, ||, Qiyuan Yang†, ||, Yang Yu†, Meiyu Jiang†, Shumao Wei†, Du Lu†, Huan

4

Wu†, Guodong Lu∆, Yunfeng Zou∆, Zhiyong Zhang †,*, Xiao Wei †,*

5 6

†

Department of Occupational and Environmental Health, School of Public Health, Guangxi Medical University, Nanning 530021, China

7 8 9 10

∆

Guangxi Colleges and Universities Key Laboratory of Prevention and Control of Highly Prevalent Diseases, Department of Toxicology, School of Public Health, Guangxi Medical University, Nanning 530021, China

11 12 13

||

These authors contributed equally to this work.

14 15

*Correspondence to: Xiao Wei, Shuang Yong Road 22, Nanning 530021, China. Tel:

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86-771-5312371. Fax: 86-771-5312371. E-mail: [email protected]; [email protected]

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Key words: Iodoacetic acid, thyroid endocrine disruption, disinfection byproducts, drinking

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water

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Competing financial interests declaration: The authors declare they have no actual or

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potential competing financial interests.

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ACS Paragon Plus Environment


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Abstract

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Exposure to drinking water disinfection byproducts (DBPs) is potentially associated with

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adverse developmental effects. Iodoacetic acid (IAA), an unregulated DBP, has been shown to

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be cytotoxic, mutagenic, genotoxic, and tumorigenic. However, its endocrine-disrupting

26

effects remain unknown. This study evaluated the IAA-induced disruption of the thyroid

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endocrine system using in vitro and in vivo assays. Rat pituitary tumor GH3 cells were treated

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with IAA in the presence and absence of triiodothyronine (T3). IAA exposure significantly

29

reduced T3-activated GH3 cell proliferation, indicating the antagonistic activity of IAA in

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vitro. Sprague-Dawley rats were also subjected to IAA treatment through oral gavage for 28

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consecutive days. IAA exposure significantly down-regulated the mRNA expression levels of

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the thyrotropin receptor (TSHR), the sodium/iodide symporter (NIS), and type I deiodinase

33

and simultaneously reduced the protein expression levels of TSHR and NIS. IAA exposure

34

decreased T3 levels but increased the weights of hypothalamus and the levels of thyrotropin

35

releasing hormone and thyrotropin. In addition, IAA induced the formation of smaller and

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more depleted follicles or even vacuolization in the thyroid. These results suggested that IAA

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potentially disrupts the thyroid endocrine system both in vitro and in vivo.

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Introduction

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Iodoacetic acid (IAA) was first identified as a disinfection byproduct (DBP) in drinking

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water in the United States Nationwide DBP Occurrence Study.1, 2 High iodide levels in source

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water and chloramination are important factors in the formation of IAA.3, 4 IAA is generally

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detected in drinking water at µg/L levels, but the cytotoxicity and genotoxicity of IAA are

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higher than those of many regulated chloro/bromo-DBPs.1, 3, 5 A molecular mechanism of

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IAA-induced cytotoxicity and genotoxicity is the generation of oxidative stress. IAA causes

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toxicity in mammalian cells by inhibiting glyceraldehyde-3-phosphate dehydrogenase

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(GAPDH) and reducing the levels of pyruvate and ATP.6-8 IAA even induces the malignant

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transformation of NIH3T3 cells and the production of aggressive fibrosarcomas, showing that

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IAA is also a potential carcinogen.9 However, IAA is still an unregulated DBP and does not

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have a maximum contaminant level in regulations and guidelines.10

50

Previous studies mostly focused on the cytotoxicity, genotoxicity, and carcinogenicity of

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IAA.1, 3, 9, 11 Furthermore, contaminants in drinking water have attracted much interest in

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terms of their developmental toxicity. Because of their endocrine-disrupting effects, some

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DBPs have become regarded as potential inducers in drinking water. Epidemiological studies

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have revealed the association between longtime exposure to DBPs and increased risk of

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adverse pregnancy outcomes such as miscarriage, intrauterine growth retardation, still birth,

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low birth weight, and birth defects.12-17 A mouse whole embryo culture assay showing the

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developmental toxicity of a series of haloacetic acids demonstrated their ability to affect

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neural tube, eye, and heart development and to produce malpositioned and hypoplastic

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pharyngeal arches.18 Quantitative structure-activity relationship (QSAR) analysis of the

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developmental toxicity of monohaloacids have shown that IAA might have more

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developmental toxicity than other monohaloacids.19

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Thyroid hormone (TH), an essential endocrine hormone, is involved in the regulation of

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metabolism and development, in mammals. TH abnormality may result in the hypogenesis of

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bones, muscles, and the central nervous system in embryos.20, 21 Thyroid system function is

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regulated by the hypothalamus-pituitary-thyroid (HPT) axis.22 Thus, its function can be 3



66

disrupted by chemicals via multiple physiological steps within the HPT axis: (1) TH synthesis

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[e.g., iodine uptake, the sodium/iodide symporter (NIS), thyroperoxidase (TPO),

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thyroglobulin (TG), or the thyrotropin receptor (TSHR)], (2) TH transport and action [e.g.,

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thyroxine-binding globulin, transthyretin, albumin, hepatic phase II enzyme, or TH receptors

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(THR)], (3) TH metabolism [e.g., uridine diphosphoglucuronosyl transferases and deiodinases

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(DIOs)], and (4) the HPT feedback mechanism.23 However, the effect of IAA on TH is not yet

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fully understood.

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The purposes of this study were to determine the ability of IAA to disturb thyroid system

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functions in vitro and in vivo. A T-screen assay was initially performed to investigate whether

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IAA disrupts TH-induced GH3 cell proliferation. A 28-day repeated-dose toxicity study in

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Sprague-Dawley (SD) rats was subsequently conducted to confirm the effects of IAA on TH

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levels and thyroid functions and to examine the possible pathways involved in the disruption

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of the HPT axis.

79 80

Materials and methods

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Reagents. IAA was procured from Sigma-Aldrich (MO, USA). Dulbecco’s modified Eagle’s

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medium/nutrient mixture F-12 (DMEM/F-12), horse serum (HS), fetal bovine serum (FBS),

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trypsin-EDTA, and TRIzol were purchased from Invitrogen (NY, USA). Antibodies for TSHR

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(sc13936) and TPO (sc58432) were procured from Santa Cruz, Inc. (CA, USA). Antibodies

85

for NIS (ab17795) and TG (ab156008) were obtained from Abcam (MA, USA).

86 87

Cell culture. Rat pituitary tumor cell line GH3 was purchased from the Cell Center of the

88

Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China).

89

The cells were cultured in DMEM/F-12 medium containing 15% HS and 2.5% FBS at 37 °C

90

in a humidified 5% CO2 atmosphere.

91 92

Animals. SD rats were procured from the Laboratory Animal Center of Guangxi Medical

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University and housed under the following conditions: 23 °C ± 2 °C, 60% ± 5% humidity, 4


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and a light/dark (12 h/12 h) cycle. Animal ethics approval was obtained from Guangxi

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Medical University of Animal Ethics Committee.

96 97

T-screen assay. The T-screen assay was performed as published previously.24 GH3 cells were

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transferred to serum-free medium (DMEM/F-12 medium supplemented with 10 ng/mL

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sodium selenite, 10 µg/mL bovine insulin, 500 µg/mL bovine serum albumin, 10 µM

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ethanolamine, and 10 µg/mL human apotransferrin) and maintained for 48 h. Then, GH3 cells

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were detached and washed three times with serum-free medium. Approximately 2.5×103 cells

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in 50 µL of serum-free medium per well were plated in 96-well microplates. After 24 h,

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different concentrations of test compounds in 50 µL of serum-free medium were added to the

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respective well for 96 h. Cell proliferation was analyzed using the crystal violet method.11, 25

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Different concentrations of T3 were firstly tested to reach the optimal concentration. Then,

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the desired concentrations of IAA (without cytotoxicity) were analyzed in the presence of the

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optimal concentration of T3.

108 109

Animal treatment. The study was conducted as the Organization for Economic Cooperation

110

and Development (OECD) Test Guideline 407.26 Five-week-old SD rats with a weight of 100

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± 10 g were randomly grouped (7 males and 7 females) at the beginning of the experiments.

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They were kept in their cages for 1 week to allow them to acclimatize to the laboratory

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conditions prior to the onset of the treatment study. The rats were then exposed to 0, 6, 12,

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and 24 mg/kg bw of IAA in ultrapure water through oral gavage for 28 consecutive days. The

115

highest dose was determined based on an acute toxicity study of IAA in rats. Our preliminary

116

study indicated that 126 and 147 mg/kg bw were the acute oral LD50 values of IAA in male

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and female rats, respectively. Clinical manifestations, food consumption, and body weight

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were observed every day. All of the rats were sacrificed through exsanguination with 10%

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chloral hydrate anesthesia after 28 days. Blood samples were collected for clinical

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biochemical and hormonal analysis. Organs such as the hypothalamus, pituitary, thyroid,

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kidney, liver, thymus, heart, testes, epididymides, prostate, ovaries, and uterus were weighed. 5



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The relative weight of organs was calculated as follows: relative organ weight = organ weight

123

(g) / body weight (g).

124 125

Clinical biochemical and hormonal analysis. Clinical biochemical parameters included

126

alanine

127

aminotransferase-alanine aminotransferase ratio (AST/ALT), alkaline phosphatase (ALP),

128

total bilirubin (TB), total protein (TP), globulin (GLB), albumin (ALB), albumin-globulin

129

ratio (A/G), blood urea nitrogen (BUN), uric acid (UA), creatinine (CREA), sodium (Na),

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potassium (K), and chlorine (Cl). Total triiodothyronine (TT3), free triiodothyronine (FT3),

131

total thyroxin (TT4), and free thyroxin (FT4) were determined using radioimmunoassay kits

132

from Cisbio Bioassays (Codolet, France). Thyrotropin (TSH), thyrotropin-releasing hormone

133

(TRH), TG, and TPO were measured using enzyme-linked immunosorbent assay (ELISA)

134

kits from EIAab (Hubei, China). These commercially available kits were widely employed to

135

measure TH, TSH, and TRH before and have been proved to be effective in identifying

136

hyperthyroidism or hypothyroidism.27-31

aminotransferase

(ALT),

aspartate

aminotransferase

(AST),

aspartate

137 138

Histopathology and immunohistochemistry. The obtained organs were fixed, dehydrated,

139

embedded, sectioned, and stained according to standard procedures for histopathological

140

examinations. Immunohistochemical analysis was developed in 4.0 µm sections of tissues

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fixed with paraformaldehyde. After dewaxing and antigen retrieval, the sections were blocked

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and incubated with anti-NIS, anti-TSHR, anti-TG, and anti-TPO antibody at 4 °C overnight,

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combined with biotinylated secondary antibody, stained with diaminobenzidine (DAB), and

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lightly counterstained with hematoxylin. Immunopositive reactions were randomly selected

145

from three different areas in each section and analyzed with Image-Pro Plus 6.0 (MD, USA).

146 147

RNA isolation and quantitative real-time reverse transcriptase-polymerase chain

148

reaction (RT-qPCR). The total RNA of the hepatic genes (THRα and THRβ1) and thyroid

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genes (TSHR, NIS, DIO1, TPO, and TG) was isolated with TRIzol reagent and assessed 6


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through A260/A280 ratio measurement and agarose-formaldehyde denaturing gel electrophoresis

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as previously described.32, 33 RNA was reverse-transcribed to cDNA by using a PrimeScript

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RT reagent kit from Takara (Shiga, Japan). qPCR analysis was conducted with a SYBR

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Premix Ex Tap II kit from Takara (Shiga, Japan). PCR primers were designed using Oligo 6.0

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(CO, USA) (Table S1) and synthesized by Takara Biotech Co., Ltd. (Liaoning, China). Each

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sample was analyzed in triplicate, and the average cycle threshold (CT) was calculated. These

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data were quantified by the 2

157

expression relative to the control.

－∆∆CT

method and are presented as the fold change of gene

158 159

Statistical analysis. Data were analyzed using SPSS 16.0 (IL, USA) and GraphPad Prism 5.0

160

(CA, USA). Significant differences among treatment groups were evaluated through a

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one-way analysis of variance (ANOVA) test followed by Dunnett’s multiple comparison test

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versus the control group. The incidence of histopathological changes was analyzed using

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Fisher’s exact test. Two-tailed P< 0.05 indicated statistically significant differences. Data

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were expressed as the mean ± standard deviation (SD).

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Results

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IAA reduced GH3 cell proliferation in the presence of T3.

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The dose-dependent manner of GH3 cells stimulated by T3 is shown in Fig. 1A. The

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lowest T3 concentration that induced significant cell proliferation was 0.1 nM. The highest

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cell proliferation was found at 1 nM T3, and this observation was about 2-fold higher than

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that of the control group. The GH3 cells were then exposed to 0.125 - 8 µM IAA without T3.

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Fig. 1B shows that IAA was not significantly cytotoxic to GH3 cell lines within a range of

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0.125–2 µM. After the GH3 cells were treated with IAA in the presence of 1 nM T3, 2 µM

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IAA significantly reduced the GH3 cell growth compared with 0 µM IAA (Fig. 1C).

175 176

IAA altered the organ weights of SD rats but not their general observations, body weight

7



177

or food consumption.

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Male rats differed from female rats in their relative organ weights after IAA treatment

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(Table 1). There was a significant dose-dependent decrease in the relative organ weight of the

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heart and liver of males. However, in females, there was a decrease only in the ovaries. By

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contrast, the relative weight of the hypothalamus increased significantly in female rats.

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Additionally, there was an increasing trend in the thyroid relative weight of the female rats

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but not of the male rats.

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No abnormal clinical manifestations were observed in any group. As shown in Fig. S1

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and S2, IAA did not induce significant changes in the body weight or food consumption of

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SD rats.

187 188

IAA mediated the clinical biochemistry in SD rats.

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In the blood biochemical findings after IAA treatment (Table S2), the male rats also

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responded differently from the female rats in most clinical biochemical parameters, except a

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common decrease in GLB and a common increase in A/G ratio in the 24 mg/kg bw IAA

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groups and a common decrease in AST in the 6 mg/kg bw groups. The highest IAA dose (24

193

mg/kg bw) decreased the levels of ALP, TP, and BUN in the male rats but did not affect the

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female rats. ALP tended to decrease in the female rats, but the differences failed to reach

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statistical significance. Both AST and ALT decreased in the 6, 12, and 24 mg/kg bw groups

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of female rats, leaving the AST/ALT ratio unchanged. Na and Cl increased only in the 12 and

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24 mg/kg bw groups of the female rats.

198 199

IAA increased TRH, TSH, TG, and TPO but decreased TT3 in the serum of SD rats.

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A common significant decrease of TT3 was observed at the dose of 24 mg/kg bw IAA in

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both male and female rats (Table 2). This change was accompanied by increases in TG and

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TPO in the 12 and 24 mg/kg bw IAA groups of both genders. In male rats, TSH was

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increased in the 12 and 24 mg/kg bw groups, whereas in female rats, TRH was elevated in the

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12 mg/kg bw group. 8


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IAA induced histopathological changes in the thyroid of SD rats.

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The histopathological examination of the thyroid showed that the number of smaller and

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depleted thyroid follicles in the IAA-treated male or female rats increased compared with that

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of the control group but failed to reach statistical significance (Fig. 2B, 2C, and Table 3).

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However, IAA induced a significant increase in total number of smaller thyroid follicles in the

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male and female rats. The intrafollicular colloid was stained more faintly in these smaller or

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depleted follicles. The cellular vacuolization of the thyroid was observed in one male rat in

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the 24 mg/kg bw group (Fig. 2D). Slight edemas were observed in liver cells of most male

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and female rats in the treatment groups (Fig. S3). No other changes appeared in periportal

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zones after the rats were exposed to IAA.

216 217

IAA regulated the target gene expression in the thyroid and liver of SD rats.

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Expression of the NIS gene was down-regulated in all IAA treatment groups of male and

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female rats (Fig. 3). TSHR mRNA expression was also decreased at the doses of 12 mg/kg bw

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in both genders and at 6 mg/kg bw in female rats. Meanwhile, expression of the DIO1 gene

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was down-regulated in the 12 and 24 mg/kg bw groups of both genders. IAA enhanced the

222

expression of the TG and TPO genes and reduced the expression of the THRα gene at doses of

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12 mg/kg bw in male rats. Both the THRβ1 gene in the 24 mg/kg bw group of male rats and

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the THRα gene in the 6 mg/kg bw group of female rats were up-regulated.

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IAA reduced NIS and TSHR protein expression in the thyroid of SD rats.

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Consistent with the decrease in NIS and TSHR mRNA expression, the protein levels of

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NIS and TSHR were down-regulated in all IAA treatment groups of male and female rats (Fig.

229

4, S4). However, there were no evident expression changes in the TG and TPO proteins

230

compared with the control.

231 232

Discussion 9



233

Many epidemiological studies have shown the association between chronic DBPs

234

exposure and adverse developmental effects, but few DBPs have been tested for their direct

235

role in endocrine-disrupting effects. For all we know, this is the first study evaluating the

236

TH-disrupting effects of IAA in vitro and in vivo. The present study demonstrates that IAA is

237

a thyroid toxicant.

238

The T-screen assay is an in vitro model for chemicals that shows their potential

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TH-disrupting effects. GH3 cell is suitable for T-screen assay because of its special growth

240

characteristics. In the ‘70 s and ‘80 s, Samuels34 and Hinkle35 et al. reported that T3 can

241

stimulate rat pituitary tumor cell proliferation and induce growth hormone secretion. Thus,

242

this assay was developed to evaluate the agonistic and antagonistic potency of chemicals in

243

the absence and presence of T3 by Hohenwarter et al. in 1996, the so-called T-screen assay.24

244

This assay has been successfully employed to detect the effects of many TH-disrupting

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chemicals, such as pentachlorophenol, phosphorus-containing flame retardants, pesticides,

246

and hydroxylated polybrominated diphenyl ethers.36-40 In our study, IAA reduced GH3 cell

247

proliferation in the absence of T3, indicating potential non-agonistic activity. However, IAA

248

also inhibited cell proliferation when co-administered with T3. Thus, the assay revealed that

249

IAA exerted antagonistic effects on TH action. Previous studies have not yet displayed the

250

TH-disrupting effect of IAA in vitro.

251

For the in vivo assay, OECD Test Guideline 407, a repeated dose 28-day oral toxicity

252

study in rodents, is a screening assay conducted to detect endocrine disrupters acting through

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various toxicity mechanisms. Several chemicals have been evaluated with this assay.41-43 In

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our study, IAA treatment increased the weights of the hypothalamus and reduced the weights

255

of the ovaries of the female rats. The previous in vitro study, which reported that treatment

256

with IAA inhibits antral follicle growth and decreases estradiol levels in mouse ovarian

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follicles.44 These results indicate that IAA may be an ovarian toxicant in vivo and in vitro. In

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addition, as the dose of IAA increases, the relative thyroid weight has an upward trend in

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female rats but not in male rats. Therefore, IAA might have thyroid-mediated effects.

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Under normal physiological conditions, TRH secreted from the hypothalamus stimulates 10


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the pituitary to release TSH, which binds to TSHR on the surface of thyroid follicle cells.

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Several thyroid genes and proteins, such as NIS, TPO, and TG, are triggered to participate in

263

the synthesis of TH.45 The uptake of iodide into thyroid follicular cells is the initial step in TH

264

synthesis. NIS can transport and concentrate iodide into the thyroid via Na+/K+-ATPase.46

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NIS functioning is inhibited by many environmental toxicants, such as nitrate, iodide, and

266

bromide.47, 48 In the present study, the decrease in the mRNA and protein expression of TSHR

267

and NIS in the IAA-dosed group indicated that IAA is probably an inhibitor of TSHR and NIS.

268

In addition, Na+/K+-ATPase provides the driving force for the uptake of iodide by generating

269

a large concentration gradient in sodium. Na+/K+-ATPase pumps 3 Na+ out and takes 2 K+ in,

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which would requires one ATP. However, IAA-induced GAPDH inhibition causes a severe

271

reduction of cellular ATP levels.6, 7 The reduction of ATP may inhibit Na+/K+-ATPase activity.

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These studies indicate that IAA may affect metabolic processes at this level and inhibit TH

273

synthesis.

274

In thyroid follicular cells, iodide is activated by TPO. Then, the tyrosine residues of TG

275

are iodinated to produce monoiodinated and diiodinated residues, which coupled to form TH

276

(T4 and T3).22, 45 In this study, IAA treatment induced a significant change in the TPO and TG

277

transcript levels in the 12 mg/kg bw group of male rats, but no significant differences were

278

observed in the TPO and TG protein expression levels in the thyroid tissue. On the other hand,

279

the serum TPO and TG values increased in the 12 and 24 mg/kg bw groups of male and

280

female rats. Elevation of serum TG was proved to be an indicator of an abnormal or damaged

281

thyroid follicular structure.49, 50 Thus, the high TPO and TG levels in serum were possibly

282

attributable to the release of TPO and TG from the damaged thyroid follicular cells into the

283

serum under the dosing conditions of this study.

284

T4 is the main form of released TH. The biological activity of T3, however, is 4-fold

285

higher than that of serum T4. Serum T4 and T3 mostly bind to carrier proteins and are

286

transported to target tissues. The free (unbound) serum T4 and T3 levels were only 0.03% and

287

0.3%, respectively. Free THs, mainly T3, bind to THR in the nuclei of target cells to generate

288

their biological effects.45 Our study showed that THRα and THRβ1 mRNA expression levels 11



289

were altered, but such changes were independent of dosage. Further studies on other target

290

tissues should be conducted to help elucidate the THR-disrupting effects of IAA. Another

291

major pathway of T3 production is the conversion of T4 to T3 by type I and type II

292

deiodinases.51 Thus, the reduction in DIO1 mRNA expression in the present study might be a

293

response to the decrease in T3 in the 24 mg/kg bw groups of both genders. These observations

294

were further supported by the inhibition of TSHR and NIS in T3 synthesis and the results of

295

the T-screen assay in vitro.

296

The reduction in TH (T4 and T3) will provoke an increase in TSH and TRH release

297

through the negative HPT feedback mechanism.22 In the present study, the weights of the

298

hypothalamus and TRH level of female rats increased in the IAA-dosed groups following the

299

reduction in T3. Similarly, the TSH level of male rats was stimulated by the reduction in T3

300

after IAA treatment. The release of TRH and TSH can stimulate the hyperplasia and

301

hypertrophy of the thyroid, which synthesizes and secretes more TH to satisfy physiological

302

requirements.22 The increase in TPO and TG mRNA expression levels in IAA-dosed groups

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likely compensated for the reduction in T3. In the OECD Test Guideline 407, the

304

characteristic patterns of thyroid toxicants are to induce changes in the weight and/or

305

histopathology of the thyroid.26 Although IAA-treatment did not induce a statistically

306

significant difference in thyroid weights, the formation of small follicles was increasingly

307

observed in the exposed groups. These findings provided direct evidence supporting the

308

potential of IAA as a thyroid toxicant.

309

Decreased liver weight, abnormal AST, ALT, AST/ALT, ALP, TP, GLB, A/G, and BUN

310

values, and hepatocytic edema revealed the adverse effects of IAA on liver function. IAA has

311

been reported to significantly increase reactive oxygen species (ROS) levels5, 8 and to induce

312

cytotoxicity in primary rat hepatocytes in vivo.25 These results are in agreement with our

313

study. In general, decreased ALP in the serum, as observed in both genders, can be regarded

314

as a marker of reduced liver activity.52-54 ALP expression, which is TH dependent,55-60 may be

315

suppressed by IAA through the activation of TH-mediated effects.

316

In addition to in vitro assay, IAA had not been shown to exhibit T3-agonist activity in in 12


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vivo assay. In general, compounds that resemble the structure of TH can bind to nuclear THRs

318

in cells and then stimulate GH3 cell growth or regulate gene transcription and expression in

319

target organs. For example, Triac and Tetrac, which resemble the structures of T3 and T4,

320

respectively, induced GH3 cell growth.36 In contrast, this study indicates that IAA presents

321

antagonistic activity. In the T-screen assay, IAA inhibited GH3 cell growth in the presence of

322

T3. The possible mechanism of this antagonistic action was considered to be associated with

323

decreasing the rate of T3 entry into cells and inhibiting binding by nuclear receptors.61, 62

324

However, in vitro assay do not reflect the process of the absorption, distribution, metabolism,

325

or excretion of thyroid disruptors in vivo. Thyroid disruptors may interfere with TH functions

326

through multiple pathways and complicated feedback mechanisms in vivo. The present study

327

showed that IAA decreased T3 synthesis and metabolism by inhibiting the mRNA expression

328

of TSHR, NIS, and DIO1 in rats. Although IAA did not disrupt THR in the liver, consistent

329

with the findings of the T-screen assay, the expression of THR in other target organs still

330

needs to be evaluated in the future.

331

Overall, our results indicated that IAA reduced T3-mediated GH3 cell proliferation and

332

decreased T3 by down-regulating TSHR, NIS, and DIO1 mRNA expression in rats. Combined

333

with histopathological changes in the thyroid, this study suggests that IAA exposure disrupts

334

thyroid function and possibly induces potential health risks. Of particular note is that

335

exposure to IAA reduced the weights of the ovaries of the female rats. The finding revealed

336

that IAA may have reproductive toxicity. Hence, T-screen and OECD Test Guideline 407

337

assays are effective screening tests for evaluating the endocrine-mediated effects and

338

identifying thyroid toxicants. However, the effects of IAA on the physiological characteristics

339

of TH are more complex than previously assumed. Further mechanistic studies are therefore

340

required to elucidate IAA-induced anti-thyroid effects.

341 342

Associated Content

343

Supporting Information Available

344

Table S1. Primer sequences for RT-qPCR. 13



Page 14 of 32

345

Table S2. Clinical biochemistry values in SD rats exposed to IAA for 28 days.

346

Fig. S1. Body weight changes in male and female SD rats treated with IAA for 28 days. Data

347

are shown as the mean ± SD, n=7.

348

Fig. S2. Food consumption of males and females SD rats treated with IAA for 28 days. Data

349

are shown as the mean of each group, n=7.

350

Fig. S3. Histological micrographs of the liver of SD rats treated with IAA for 28 days. (A)

351

Normal liver cells. (B) Slight edemas of liver cells were frequently observed in IAA-treated

352

rats.

353

Fig. S4. Immunohistochemical micrographs of NIS (A), TSHR (B), TG (C), and TPO (D)

354

protein expression in the thyroid of SD rats treated with IAA for 28 days.

355 356 357

Acknowledgements

358

This research was supported by grants from National Natural Science Foundation of

359

China (No. 81360421, 81560524), the China Postdoctoral Science Foundation (No.

360

2014T70839,

361

2012GXNSFBA053109), the Outstanding Young and Middle-aged Excellent Teachers'

362

Training in Higher Education Institutions of Guangxi (No. GXQG022014019), and the

363

Guangxi Medical University Training Program for Distinguished Young Scholars (No. 2017).

2013M540686),

the

Guangxi

Natural

Science

Foundation

(No.

364

We thank Professor Gang Chen and Dr. Jingjing Zeng (Department of Pathology,

365

Guangxi Medical University) for their technical support in the histological analyses. The

366

authors would like to thank the anonymous reviewers and the editor for their comments and

367

suggestions.

14


Page 15 of 32


368

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561

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21



562

Table captions

563

Table 1. Relative organ weights in SD rats exposed to IAA for 28 days.

564

Table 2. Serum levels of hormones, TG and TPO in SD rats exposed to IAA for 28 days.

565

Table 3. Histopathological changes in the thyroid of SD rats exposed to IAA for 28 days at

566

different doses (mg/kg bw).

567 568

Figure captions

569

Fig. 1. (A) Dose-response relationship of the T3-activated GH3 cell response. (B)

570

Cytotoxicity of IAA to GH3 cell. (C) Antagonistic effects of different concentrations of IAA

571

in the T-screen assay. Data represent the mean ± SE from triplicate experiment. *P< 0.05,

572

Treatment groups compared with the control. #P< 0.05, 2 µM IAA treatment groups

573

compared with the 0 µM IAA treatment group in the presence of T3.

574 575

Fig. 2. Histological micrographs of the thyroid of SD rats treated with IAA for 28 days. (A)

576

Normal thyroid follicles. (B, C) Follicles are smaller (B) or even depleted (C). (D) Follicles

577

are more vacuolated.

578 579

Fig. 3. Gene expressions levels in the thyroid and liver of SD rats treated with IAA for 28

580

days. Data are the mean ± SD in each group, n=7. *P< 0.05, IAA treatment groups compared

581

with the negative control.

582 583

Fig. 4. Expression of NIS, TSHR, TG, and TPO protein in the thyroid of SD rats treated with

584

IAA for 28 days. Data are the mean ± SD in each group, n=7. *P< 0.05, IAA treatment groups

585

compared with the negative control.

22


Page 22 of 32

Page 23 of 32


586

Table 1. Relative organ weights in SD rats exposed to IAA for 28 days. Relative organ weights

Control

IAA (mg/kg bw) 6

12

24

Male Hypothalamus (‰)

0.148±0.042

0.157±0.065

0.163±0.045

0.176±0.047

Pituitary (‰)

0.034±0.004

0.033±0.002

0.032±0.003

0.031±0.003

Thyroid (‰)

0.047±0.006

0.044±0.005

0.047±0.009

0.048±0.010

Kidney (%)

0.686±0.046

0.714±0.042

0.706±0.024

0.702±0.042

Liver (%)

4.220±0.212

4.022±0.214

3.851±0.294*

3.793±0.268*

Thymus (%)

0.183±0.022

0.192±0.021

0.176±0.022

0.174±0.039

Heart (%)

0.329±0.023

0.302±0.017*

0.307±0.017

0.296±0.016*

Testes (%)

1.019±0.091

0.997±0.084

1.081±0.081

1.119±0.168

Epididymides (%)

0.246±0.024

0.268±0.031

0.271±0.016

0.265±0.028

Prostate (%)

0.123±0.023

0.145±0.009

0.134±0.020

0.120±0.018

Hypothalamus (‰)

0.187±0.043

0.311±0.070*

0.344±0.071*

0.281±0.044*

Pituitary (‰)

0.062±0.006

0.060±0.008

0.057±0.011

0.059±0.007

Thyroid (‰)

0.049±0.009

0.056±0.006

0.058±0.007

0.055±0.010

Kidney (%)

0.719±0.054

0.784±0.041

0.724±0.058

0.756±0.063

Liver (%)

3.707±0.172

3.694±0.162

3.683±0.132

3.764±0.290

Thymus (%)

0.217±0.025

0.201±0.032

0.231±0.041

0.236±0.034

Female

23



Page 24 of 32

Heart (%)

0.324±0.026

0.364±0.038

0.338±0.043

0.328±0.018

Ovaries (%)

0.091±0.020

0.063±0.005*

0.066±0.012*

0.062±0.012*

Uterus (%)

0.047±0.016

0.040±0.017

0.033±0.004

0.041±0.018

587

Data are expressed as the mean ± SD, n=7.

588

* Significantly different from the control at P < 0.05.

24


Page 25 of 32


589

Table 2. Serum levels of hormones, TG, and TPO in SD rats exposed to IAA for 28 days. Parameters

Control

IAA (mg/kg bw) 6

12

24

Male TT3 (nM /L)

0.66±0.15

0.76±0.12

0.70±0.18

0.48±0.14*

TT4 (nM /L)

65.67±16.9

68.20±11.79

58.19±14.68

50.63±8.86

FT3 (pM /L)

4.51±0.40

4.79±1.03

4.55±0.37

4.50±0.56

FT4 (pM /L)

38.01±3.84

40.64±7.18

41.16±9.13

36.29±9.07

TSH (IU/L)

0.76±0.16

1.03±0.22

2.05±0.79*

2.50±0.50*

TRH (mIU/L)

6.72±2.02

6.90±2.50

5.11±0.28

4.66±1.97

TG (ng/L)

18.03±2.61

16.88±1.39

27.63±11.90*

36.43±3.39*

TPO (µg/L)

0.06±0.01

0.15±0.07

0.35±0.06*

0.32±0.06*

TT3 (nM /L)

0.90±0.25

0.77±0.17

0.75±0.10

0.61±0.06*

TT4 (nM /L)

36.64±7.21

38.23±7.81

45.78±4.54

45.53±8.79

FT3 (pM /L)

4.02±0.78

4.27±0.82

4.64±1.04

3.90±0.70

FT4 (pM /L)

38.87±3.38

39.47±4.76

40.70±13.66

37.16±8.82

TSH (IU/L)

2.67±0.85

1.98±0.88

2.44±0.81

2.63±0.99

TRH (mIU/L)

3.40±1.26

5.38±1.44

5.90±1.61*

5.19±1.43

TG (ng/L)

32.54±7.51

40.31±6.14

62.58±18.96*

73.69±0.87*

TPO (µg/L)

0.05±0.03

0.15±0.08

0.26±0.11*

0.33±0.05*

Female

25



590

Data are expressed as the mean ± SD, n=7.

591


26


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592

Table 3. Histopathological changes in the thyroid of SD rats exposed to IAA for 28 days at

593

different doses (mg/kg bw).

Organ findings

Male Control

6

Female 12

24

Control

6

Male + Female 12

24

Control

6

12

24

Vacuolization

0/7a

0/7 0/7 1/7

0/7

0/7 0/7 0/7

0/14b

0/14 0/14 1/14

Smaller follicles

1/7

2/7 3/7 4/7

1/7

1/7 2/7 4/7

2/14

3/14 5/14 8/14*

Depleted follicles

2/7

3/7 2/7 3/7

1/7

2/7 2/7 2/7

3/14

5/14 4/14 5/14

594

a Incidence: Affected rats/total examined (n=7).

595

b Incidence: Affected rats/total examined (n=14).

596


27



Fig. 1 A

B

C


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Fig. 2 A

B

C

D



Fig. 3


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Fig. 4



TOC Art 46x26mm (600 x 600 DPI)


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Iodoacetic acid disrupts the thyroid endocrine ... - ACS Publications

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