Fluoride caused thyroid endocrine disruption in male zebrafish (Danio rerio)

Aquatic Toxicology 171 (2016) 48–58

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Fluoride caused thyroid endocrine disruption in male zebrafish (Danio rerio) Chen Jianjie 1 , Xue Wenjuan 1 , Cao Jinling ∗ , Song Jie, Jia Ruhui, Li Meiyan State Key Laboratory of Ecological Animal Husbandry and Environmental Veterinary Medicine, College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, Shanxi, China

a r t i c l e

i n f o

Article history: Received 26 October 2015 Received in revised form 14 December 2015 Accepted 15 December 2015 Available online 24 December 2015 Keywords: Fluoride Male zebrafish Thyroid Structure Endocrine-related gene Expressions

a b s t r a c t Excessive fluoride in natural water ecosystem has the potential to detrimentally affect thyroid endocrine system, but little is known of such effects or underlying mechanisms in fish. In the present study, we evaluated the effects of fluoride on growth performance, thyroid histopathology, thyroid hormone levels, and gene expressions in the HPT axis in male zebrafish (Danio rerio) exposed to different determined concentrations of 0.1, 0.9, 2.0 and 4.1 M of fluoride to investigate the effects of fluoride on thyroid endocrine system and the potential toxic mechanisms caused by fluoride. The results indicated that the growth of the male zebrafish used in the experiments was significantly inhibited, the thyroid microtrastructure was changed, and the levels of T3 and T4 were disturbed in fluoride-exposed male fish. In addition, the expressional profiles of genes in HPT axis displayed alteration. The expressions of all studied genes were significantly increased in all fluoride-exposed male fish after exposure for 45 days. The transcriptional levels of corticotrophin-releasing hormone (CRH), thyroid-stimulating hormone (TSH), thyroglobulin (TG), sodium iodide symporter (NIS), iodothyronine I (DIO1), and thyroid hormone receptor alpha (TR␣) were also elevated in all fluoride-exposed male fish after 90 days of exposure, while the inconsistent expressions were found in the mRNA of iodothyronineII (DIO2), UDP glucuronosyltransferase 1 family a, b (UGT1ab), transthyretin (TTR), and thyroid hormone receptor beta (TR␤). These results demonstrated that fluoride could notably inhibit the growth of zebrafish, and significantly affect thyroid endocrine system by changing the microtrastructure of thyroid, altering thyroid hormone levels and endocrine-related gene expressions in male zebrafish. All above indicated that fluoride could pose a great threat to thyroid endocrine system, thus detrimentally affected the normal function of thyroid of male zebrafish. © 2015 Published by Elsevier B.V.

1. Introduction Fluoride is widely distributed in the environment as inorganic or organic compounds as a result of its great reactivity. Fluoride plays an important role in the growth and development of humans and animals (Chachra et al., 2010; Chen et al., 2013; Shim et al., 2011). The fluoride levels in unpolluted fresh surface waters generally range from 0.01 to 0.3 mg/L (Camargo, 2003). However, natural and anthropogenic processes cause accumulation of flu-

Abbreviations: CRH, corticotrophin-releasing hormone; THS, thyroidstimulating hormone; TG, thyroglobulin; NIS, sodium iodide symporter; DIO1, iodothyronine I; DIO2, iodothyronineII; TR␣, thyroid hormone receptor alpha; TR␤, thyroid hormone receptor beta; UGT1ab, UDP glucuronosyltransferase 1 family a, b; TTR, transthyretin. ∗ Corresponding author. E-mail address: [email protected] (C. Jinling). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.aquatox.2015.12.010 0166-445X/© 2015 Published by Elsevier B.V.

oride compounds in surface waters and groundwater reserves, such as a variety of industrial and domestic emissions of easily dissolved fluoride ions, the improper use of agricultural pesticides and fertilizers, the differentiation of natural fluoride minerals, volcanic eruptions, etc., which are the major sources of fluoride pollution in freshwater ecosystems (Camargo, 2003). The fluoride content of groundwater at fluorosis areas in China was 2.3–8.0 mg/L (Zheng et al., 2006). The fluoride content in boiled water was found to be higher than 5 mg/L, even up to 45 mg/L (Ren and Jiao, 1988). Fluoride concentration in industrial wastewater was determined at 96.8 mg/L (Ding et al., 1998), and in extreme cases, up to 3000–5000 mg/L (Wu et al., 2006). Fish live in the water and can take up fluoride from the water (Tripathi et al., 2009). Excessive fluoride can lead to detrimental effects in fish, such as abundant accumulation in tissues (Moren et al., 2007; Shi et al., 2009a; Yoshitomi et al., 2007), growth inhibition (Chen et al., 2013; Shi et al., 2009b; Yoshitomi and Nagano, 2012), developmental disorder (Camargo, 2003), metabolic disor-

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der (Bajpai and Tripathi, 2010; Chen et al., 2012) behavioral changes (Camargo, 2003; Tripathi et al., 2004), pathological changes and apoptosis (Bhatnagar et al., 2007; Haque et al., 2012; Cao et al., 2013a,b), chromosome aberration and gene mutation (Tripathi et al., 2009). However, very few studies have looked at the toxic effects of fluoride on thyroid endocrine system in fish, such as thyroid hormone (THs), histopathological changes and endocrinerelated gene expressions, and the underlying mechanisms of fluoride on the thyroid system have not been clarified. Thyroid hormones (THs), secreted and released by the thyroid gland, play an important role in the regulation of growth, development, and metabolism in the vertebrates (Jugan et al., 2010). The thyroid homeostasis is regulated by hypothalamus–pituitary–thyroid axis (HPT), which is responsible for the thyroid hormone dynamics by coordinating their synthesis, ˜ 2011). In secretion, transport and metabolism (Carr and Patino, mammals, the HPT axis functions by stimulating thyrotropinreleasing hormone (TRH) from hypothalamus, which regulates the secretion of thyroid-stimulating hormone (TSH) from pituitary. TSH adjusts the synthesis of THs (T3 and T4). In amphibians and teleosts, corticotrophin-releasing hormone (CRH) stimulates the secretion of TSH (De Groef et al., 2006). THs in plasma of fish are bound to transthyretin (TTR), a specific THs transport protein in teleosts (Power et al., 2000), and only free hormones enter into target cells to launch a response. Previous studies showed that environmental pollutants could interfere with thyroid hormonal homeostasis and HPT axis, such as the hormone levels, enzyme activities, and the alterations of gene transcriptions, which have been used to evaluate the effects on thyroid endocrine disruption (Chen et al., 2012; Yu et al., 2010). Moreover, gene expression levels in HPT axis have been used as endpoints to estimate the detrimental effects of environmental pollutants (Hermsen et al., 2012) and provide further insight for clarifying the mechanisms of environmental pollutants. However, the underlying mechanisms of fluoride on thyroid endocrine disruption in male zebrafish have still not been clarified. Zebrafish are widely used as a predominant ecological toxicological model in the field of life science research because of its advantages, such as small size, high reproductive performance, rapid organogenesis, morphological and physiological similarities to mammals, high sensitivity to the harmful substances, etc., (Segner, 2009). Previous studies indicated that zebrafish are ideal models for endocrine disruption by chemicals in the laboratory (Chen et al., 2012; Kanungo et al., 2012; Liu et al., 2011; Tu et al., 2013). Therefore, in the present study, to remove sex as a factor/variable, the growth performance, histopathological changes of thyroid gland, the levels of THs (T3 and T4), and the gene mRNA profiles in the HPT axis were examined in male zebrafish exposed to different determined concentrations of 0.1, 0.9, 2.0 and 4.1 M fluoride to investigate the effects of fluoride on thyroid endocrine system of zebrafish and the underlying toxic mechanisms caused by fluoride.

2. Materials and methods

49

2.2. Fluoride exposure Approximately six hundred male zebrafish were randomly divide into four groups and exposed to different concentrations of 0 (control), 20, 40, 80 mg/L fluoride (in the form of NaF) dissolved in 120 L of dechlorinated-tap water in 150 L aquaria for 90 days, respectively. Each concentration had three replicates (n = 50 for each replicate). Exposure media were renewed every three days. The selected exposure concentrations were previously ascertained by range-finding study and did not result in any obvious deformation or mortality. The controlled conditions for the exposure were: water temperature, 28 ± 1 ◦ C; light period, 14 h light: 10 h dark; pH, 7.0.–7.4; dissolved oxygen, 7.0 ± 0.1 mg/L; total ammonia nitrogen, 1.05 ± 0.12 mg/L; nitrite nitrogen, 0.062 ± 0.012 mg/L; hardness, 20.0 ± 0.35 mg/L (as CaCO3 ). Heavy metal concentrations in the exposure media were below detection limits and the chemical components met the water quality standard of fisheries (National Environmental Protection Agency, 1990). Fluoride concentrations were monitored daily during the exposure using the fluoride ion selective electrode method (Inkielewicz et al., 2003). Determined fluoride concentrations in each tank remained relatively constant between the 3-days water renewals. The average measured concentrations of fluoride for 0 (control), 20, 40, 80 mg/L were 0.1 ± 0.02, 0.9 ± 0.26, 2.0 ± 1.90 and 4.1 ± 3.90 M, respectively. The fish were handled according to the National Institute of Health Guidelines for the handling and care of experimental animals. All studies were approved by the Laboratory Animal Care and Use Committee of Shanxi Agricultural University, Shanxi of China in 2014. After exposure for 45 and 90 days, fish were anesthetized in 80 mg/L of MS-222, and the whole body length and body weight of 30 male zebrafish from each treatment group were measured. Then, eighteen fish from each group were sacrificed and the heads were collected. Considering the possible diurnal fluctuations in hormone levels, the fish were sampled between 8:00 and 10:00 a.m. (Leiner et al., 2000). Blood of nine male fish from each group were collected from the caudal vein with chilled heparinized syringes and was kept on ice. Plasma samples were obtained after centrifugation at 2500 × g for 20 min and were stored at −20 ◦ C for hormone assay. Since thyroid follicles are almost invisible, heads including thyroid follicles, hypothalamus and pituitary were sampled for histopathological analysis and gene expression analysis in HPT axis. Nine heads containing thyroid follicles from each group were fixed in Bouin’s solution for histopathological analysis. Nine heads from each group were snap-frozen in liquid nitrogen and stored at −80 ◦ C for gene expression analysis. 2.3. Determination of growth parameters Growth performances were determined as follows: weight gain rate (%) = (final body weight−initial body weight)/initial body weight × 100; length gain rate (%) = (final body length− initial body length)/initial body length × 100; specific growth rate (%/d) = (ln final body weight−ln initial body weight)/d × 100; condition factor (K) (g/cm) = weight/length3 × 100. 2.4. Histopathological analysis of thyroid

2.1. Test animals Adult male zebrafish three months old (Danio rerio) (AB strain), with mean body weight and body length of 0.19 ± 0.03 g and 2.5 ± 0.3 cm, were obtained from Taiyuan fish hatchery in Shanxi Province, PR China. They were kept in a flow-through system with dechlorinated tap water (pH, 7.0–7.4; water temperature, 28 ± 1 ◦ C; light regime, 14-h light and 10-h dark) for 15 days to acclimate to laboratory conditions before exposure. Fish were fed with commercially available adult zebrafish compound feed.

After being fixed for 24 h, the head specimens were dehydrated in graded ethanol, embedded in paraffin blocks, and cut on Rotary microtome (Paraffin machine, Leica RM 2245, Germany) at 5 ␮m. The sections were stained with Delafield’s hematoxylin and alcoholic Eosin, dehydrated in graded alcohol, and then mounted in neutral balsam. Thyroid histopathology endpoints, such as follicle area, colloid area, and the height of epithelial cells from photomicrographs were quantified by Image Pro Plus (version 6.0, Media Cybemetics, USA). Thirty follicles were chosen for analysis in each

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Fig. 1. Effects of fluoride on growth parameters in male zebrafish exposed to different concentrations of fluoride for 45 days and 90 days. (A) Body weight (BW); (B) Weight gain rate (WGR); (C) Specific growth rate (SGR); (D) Body length (BL); (E) Length gain rate (LGR); (F) Condition factor. All date were represented as mean ± standarddeviation (SD) (n = 30).

Fig. 2. Histology of the thyroidal tissue in control male zebrafish. (A and B) The single thyroid follicles(→) are distributed adjacent to the ventral aorta (*) in the gill region. (C) Thyroid follicular along with the afferent gill arterial() into the gills.

group. Five measurements along the follicle circumference were made for the determination of epithelial cell height. 2.5. Hormone assay T3 and T4 levels in plasma samples were measured by enzymelinked immunosorbent assay (ELISA) using commercially available kits (Qiaodu biotechnology company, Shanghai, China) specific for these two hormones. These assays were performed according to the protocols provided by the manufacturer. Briefly, double antibody sandwich method was used to determine the T3 or T4 level. Purified fish T3 or T4 antibody was used to coat microtiter plate wells and solid-phase antibody was made. Then T3 or T4 antigens were

added to wells. Combined T3 or T4 antibody which was labeled with HRP, became antibody–antigen–enzyme–antibody complex. After being washed completely, TMB substrate solution was added. TMB substrate became blue color at HRP enzyme-catalysis. The reaction was terminated by the addition of a sulphuric acid solution and the color change was measured spectrophotometrically at a wavelength of 450 nm. The concentration of T3 or T4 in the samples was then determined by comparing O.D. of the samples to the standard curve. The ELISA for T3 and T4 was validated for use with zebrafish samples by demostrating parallelism between a series of diluted and spiked samples in relation to the standard curve attached to the ELISA kit. The detection limits were 1.0–20 pmol/L for T3, 3–7 ␮g/L

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Fig. 3. Effects of fluoride on the thyroid tissue in male zebrafish exposed to various concentrations of fluoride for 45 and 90 days, respectively. (A) Thyroid of control fish, a homogeneously stained colloid surrounded by the monolayer cuboid to flat epithelial cells; (B) Thyroid of fish exposed to 0.9 M of fluoride, the thyroids colloids were decreased; (C and D) Thyroid of fish exposed to 2.0 M of fluoride, the decreased thyroid colloid and the abnormal thickening of the follicle cell layer and decreased colloid density; (E and F) Thyroid of fish exposed to 4.1 M of fluoride, the decreased colloid density and the significantly increased hyperplasia and hypertrophy. a. Thyroid of control fish, a homogeneously stained colloid surrounded by the monolayer cuboid to flat epithelial cells; b and c. Thyroid of fish exposed to 0.9 M of fluoride, the number of abnormal follicles surrounded by thickened epithelial cells was significantly increased; d and f: Thyroid of fish exposed to 2.0 M of fluoride, most of the flat epithelial cells were arranged in disorder around the colloid; g–i. Thyroid of fish exposed to 4.1 M of fluoride, in-and out-foldings and the volume of follicles were significantly increased, increased enrichment, severe colloid depletion. c: Colloid; cd: Colloid decrease; f: Follicle; ft: Follicular epithelial thickening.

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Pencentage of control(%)

550

0.1 M

(A)

0.9 M

2.0 M

550

4.1 M

450 350 *

250 *

150

*

* *

* *

*

50 -50

Epithelial cell heigh

Follicle area

Colloid area

Pencentage of control(%)

52

0.1 M

(B)

0.9 M **

2.0 M

4.1 M

450 350

*

250 150

* *

* *

*

50 -50

Epithelial cell heigh

Follicle area

Colloid area

Fig. 4. Effects of fluoride on the thyroid parameters in male zebrafish exposed to various concentrations of fluoride for 45 days. (A) 45 days; (B) 90 days. Asterisks represent significantly different when compared with controls, respectively: * for p < 0.05 and ** for p < 0.01.

for T4. The inter- and intra-assay coefficients of variation for all of the hormones were both below 10%. 2.6. Gene expression analysis Total RNA was isolated from head samples using Trizol Reagent (Takara Biotechnology Company, Dalian, China) according to the manufacturer’s instructions. To remove genomic DNA contamination, total RNA was digested by RNase-free DNase I (Promega Madison, WI, USA) and then purified. The total RNA recovered from DNase I digestion was measured at 260 and 280 nm using a spectrophotometer (M2, Molecular Devices, Sunnyvale, CA, USA). The quality of RNA in each sample was verified by measuring the 260/280 nm ratios and by 1% agarose formaldehyde gel electrophoresis with ethidium bromide staining. Afterwards, equal amounts of RNA (500 ng) were reverse-transcribed into cDNA using the reverse transcriptase kit (Takara Biotechnology Company, Dalian, China) in a total volume of 10 ␮L according to the manufacturer’s instructions. Specific primers of CRH, TSH, TG, DIO1, DIO2, NIS, TTR, UGT1ab, TR␣, TR␤, and ␤-actin (Table 1) were designed with Beacon Designer 8. Before quantitative analyses, the amplification efficiencies between target genes and internal control genes were compared, and the results showed that the amplification efficiencies between target genes and internal control genes were between 100% and 105%, and the differences were less than 5%. So the comparative Ct method was used to determine mRNA levels by real-time fluorescent quantitative PCR (QRT-PCR) in the study. QRT-PCR was conducted by using the FTC2000 QRT-PCR system (Canada) and two-Step QRT-PCR kit (Takara Biotechnology Company, Dalian, China). The real-time PCR reactions were carried out in a final volume of 20 ␮L, containing 10 ␮L of 1 × SYBR Premix Ex Taq II, 0.4 ␮m of each primer, 0.4 ␮L of ROX Reference Dye II, and RT reaction solution. The real-time PCR reaction condition was as follows: initial denaturation at 94 ◦ C for 4 min, followed by 45 PCR cycles of 94 ◦ C for 20 s, 60 ◦ C for 30 s, and 72 ◦ C for 30 s, and then followed by the reaction melting curve analysis to verify the specificity of the amplified products. Negative control without template was used to eliminate contamination. Quantitative values were obtained from the threshold cycle (Ct) number, which is the increase in signal that is associated with an exponential growth of PCR product when detected. QRT-PCR was conducted in three replicate samples for each selected gene. The expression level of each target gene was normalized to the mRNA content of its reference gene (␤-actin). The existence of primer-dimers and secondary products was checked using melting curve analysis. Our data indicated that the amplification was specific. Only 1 PCR product was amplified for each individual primer set and verified by sequencing of the amplicon. Data were analyzed and were expressed as relative gene expression using the 2−Ct method (Livak and Schmittgen, 2001).

2.7. Statistical analysis The experimental data were presented as the means ± standard deviations (SD) and statistically analyzed using SPSS 20.0 software (IBM company). Before analysis for difference in gene expression levels, the normality and equal variance for the data were checked by Levene’s test. No violation of the assumptions for analysis of variance (ANOVA) was detected. There was no significant difference among the replicate tanks within each treatment. Therefore, the data from all the three replicates were combined. One-way analysis of variance (ANOVA) was used to detect the significant differences in gene expression levels among treatments followed by Tukey test for multiple comparisons between treatment group and control group. Values were considered to be statistically significant when p < 0.05. 3. Results 3.1. The effects of fluoride on growth performance in male zebrafish No mortality occurred during the 90-days exposure. There were no significant differences between the control and the treatment groups in their initial body weight and body length (p > 0.05 among all treatments). The growth of male zebrafish was significantly affected by fluoride. After 45 days of exposure, fish exposed to 0.9 and 2.0 M of fluoride had 4.02% and 8.04% increase in body weight relative to the control fish, whereas fish exposed to 4.1 M of fluoride had 8.54% inhibition (p < 0.05) (Fig. 1A). The body lengths of all fluorideexposed fish were lower than that of the control fish (p < 0.05) (Fig. 1C). After 90 days of exposure, control fish increased in body weight by approximately 29.28% whereas fish in all fluorideexposed fish lost approximately 1.20%, 5.98% and 13.15% of their body weight, respectively (p < 0.05) (Fig. 1A). Control fish had 12.68% increases in body length, while fish in all fluoride-exposed fish had 1.76%, 3.97% and 8.04% decrease, respectively (p < 0.05) (Fig. 1C). After exposure for 45 days, the weight gain rate, specific growth rate, and condition factor in fish exposed to 0.9 and 2.0 M of fluoride were significantly increased relative to the control group (p < 0.05), respectively. While the weight gain rate and specific growth rate in fish exposed to 4.1 M of fluoride were decreased significantly by 340.05% and 351.33% relative to the control group (p < 0.05). While the length gain rate in all fluoride-exposed fish were significantly decreased compared with the control group. After exposure for 90 days, the weight gain rate, length gain rate, specific growth rate were lower remarkably in all fluoride-exposed fish than that of the control group (p < 0.05). Generally, these inhibitions were most pronounced at higher fluoride concentration. Fish exposed to 4.1 M of

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Table 1 Nucleotide sequences of primers used for quantitative real-time RT-PCR and product sizes. Name

Name sequence of primers

␤-actin

Forward: CTTCTGGTCGTACTACTG Reverse: GGTCAGGATCTTCATCAG Forward: CCAATTACGCACAGATTC Reverse: TGGCTCTACATTCATACG Forward: CAGACAGACATCCTCATAC Reverse: GTAGATGGTGTAGTCAGTG Forward: GGAAGAGCTGACAGTAAG Reverse: GGACAGATGATGAAGAGG Forward: CCTACCTGTACTTTGGAG Reverse: CCTGGACTGATAGAATCC Forward: CAGTAACCAAGTCATTCG Reverse: CGCACATAAGTCACATTC Forward: GTGGTGGATGAGATGAAC Reverse: GCCTCCCTGATAGATAAC Forward: CTGCTCCAAACTCTAAGG Reverse: CCGAAGTTGACCACTAAC Forward: CCACTGGCTATCATTACC Reverse: GGAATAGGACGGATGAAG Forward: CTAGCACTACTGAACATC Reverse: GGTATCCAACACTATGAC Forward: CTATGGTGATGCTTCCTC Reverse: GACGGTCTCTATGAATGG

CRH TSH TG NIS TTR DIO1 DIO2 TR␣ TR␤ UGT1ab

0.1 M

0.9 M

2.0 M

2.0

109

NM 001007379

119

AY135147

201

DQ278875

103

NM 001089391

169

BC081488

99

BC076008

130

NM 212789

92

NM 131396

177

NM 131340

92

NM 213422

4.1 M *

2.5 *

*

*

*

*

1.5 1.0 0.5

Genbank accession No. AF057040.1

0.1 M

(A)

12.0 Total T4 concentration of male(ng/L)

Total T3 concentration of male(ng/L)

3.0

Size 152

0.9 M

2.0 M

4.1 M

(B)

10.0

*

*

8.0 6.0 4.0 2.0 0.0

0.0 45d

Exposure time

90d

45d

Exposure time

90d

Fig. 5. Effects of fluoride on thyroid hormone levels in male zebrafish exposed to various concentrations of fluoride for 45 and 90 days. (A) T3 levels; (B) T4 levels. Results are expressed as mean ± SD (n = 9). Asterisks represent significantly different when compared with controls, respectively: * for p < 0.05 and ** for p < 0.01.

fluoride had the lowest weight gain rate, length gain rate, and specific growth rate by −57.90%, −62.58%, and −54.55%, respectively (p < 0.05). While the condition factor increased by 8.13% (p < 0.05). 3.2. The effects of fluoride on the microstructure of the thyroid tissues Histopathological changes were determined in the thyroid tissue sections after exposure to fluoride for 45 and 90 days. The thyroid follicles were distributed around the ventral aorta in the basi branchial region. In the control fish, round or oval thyroid follicles of different size were filled with a homogeneously stained colloid surrounded by the monolayer cuboid to flat epithelial cells (Figs. Fig. 22 Fig. 33A, a, ). The representative histopathological abnormalities were observed after exposure to fluoride for 45 days and 90 days, including the follicle size and shape. After exposure for 45 days, the thyroid follicles in fish exposed to 0.9 and 2.0 M of fluoride was larger than the control and the thyroids colloids were decreased (Fig. 3B, C). More severe decrease and rupture of thyroids colloids were observed in fish exposed to 2.0 and 4.1 M of fluoride (Fig. 3E). The thickened follicle cell layer and decreased colloid density were also observed in fish exposed to 2.0 M (Fig. 3C, D). The decreased colloid density was also observed in fish exposed to 4.1 M (Fig. 3F). Significant hyperplasia and hypertrophy increased in the follicle epithelial cells (Fig. 3D–F). The epithelial cells were

changed from cuboid into columnar shape (Fig. 3D–F). Slight colloid depletion was observed by appearing the scalloped edges of the colloid (Fig. 3D, F). After exposure for 90 days, the number of abnormal follicles surrounded by thickened epithelial cells was significantly increased in fish exposed to 0.9 and 2.0 M of fluoride (Fig. 3b–d). More severe alterations were found in fish exposed to 2.0 and 4.1 M of fluoride. Most of the flat epithelial cells were arranged in disorder around the colloid (Fig. 3e–g). Moreover, the shape of the follicles was affected showing slight in-and out-foldings and the volume of follicles was significantly increased compared with the control group (Fig. 3h). Most of the colloids texture appeared abnormal, such as increased enrichment (Fig. 3f–h), severe colloid depletion (Fig. 4e, g) (Fig. 3i). The thyroid histopathology after exposure to fluoride is illustrated in Fig. 4. After exposure for 45 days and 90 days, the colloid area and epithelial cell height were significantly higher in all fluoride-exposed fish than that in the control (Fig. 4). There were positive correlations between fluoride dose and colloid area or epithelial cell height (R = 0.968, R = 0.937). However, follicle areas were significantly decreased in fish exposed to 2.0 and 4.1 M of fluoride for 45 days, and only in fish exposed to 4.1 M of fluoride for 90 days compared with the control fish.

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0.9 M

4.1 M

0.1 M 3.0

3.0

Fold change of male TSH mRNA level

** **

2.5 2.0

**

**

* 1.5

*

1.0 0.5 0.0

2.0 M

**

3.0

**

2.5 *

2.0

** *

1.5 1.0 0.5

*

1.5 1.0 0.5

3.0

90d

Exposure time

(D)

0.9 M

2.0 M

4.1 M

**

2.5 **

2.0 *

*

1.5

*

1.0 0.5

0.1 M

3.5

90d

Exposure time 0.9 M

2.0 M

4.1 M

(E) **

3.0 2.5 2.0

**

**

*

**

*

1.5

Exposure time

45d

1.0 0.5 0.0

Fold change of male DIO2 mRNA level

Fold change of male DIO1 mRNA level

**

0.0

45d

2.5

0.1 M

0.9 M

90d 2.0 M

4.1 M

(F) **

2.0 ** 1.5

* *

1.0

** 0.5

0.0

45d

90d

Exposure time

0.1 M

(G)

0.9 M

2.0 M

4.1 M

** 2.0 ** **

1.5

Exposure time

45d

* 1.0 ** 0.5

3.5

0.1 M

0.9 M

90d

2.0 M

4.1 M

(H) **

3.0 2.5

** *

2.0

*

1.5 1.0 ** 0.5 0.0

0.0 Exposure time

0.1 M (I)

0.9 M

45d

90 d

2.0 M

4.1 M

** **

4.0 **

3.5 **

**

2.5 2.0

*

1.5 1.0 0.5

3.0 Fold change of male TRβ mRNA level

4 5d

Fold change of male TRα mRNA level

**

**

0.1 M

0.0

3.0

4.1 M

2.0

4.1 M **

Fold change of male NIS mRNA level

Fold change of male TG mRNA level

0.9 M

(C)

3.5

4.5

**

45d

4.0

2.5

2.0 M

**

2.5

90d

Exposure time

0.1 M 4.5

0.9 M

(B)

0.0 45d

Fold change of male UGTlab mRNA level

2.0 M

(A)

Fold change of male TTR mRNA level

Fold change of male CRH mRNA level

0.1 M 3.5

(J)

Exposure time

90d

0.1 M

2.0 M

0.9 M

4.1 M

** 2.5 ** 2.0 ** 1.5 1.0 0.5 0.0

0.0 45d

Exposure time (days)

90d

45d

Exposure time

90d

Fig. 6. Effects of fluoride on the expression of genes associated with HPT axis in male zebrafish exposed to various concentrations of fluoride for 45 and 90 days. (A) CRH; (B) TSH; (C) TG; (D) NIS; (E) DIO1; (F) DIO2; (G) UGT1ab; (H) TTR; (I) TR␣; (J) TR␤. Results are expressed as mean ± SD (n = 9). Asterisks represent significantly different when compared with controls, respectively: * for p < 0.05 and ** for p < 0.01.

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3.3. Effects of fluoride on T3 and T4 levels in male zebrafish The effects of fluoride on T3 and T4 levels in male zebrafish are shown in Fig. 5. As shown in Fig. 5, T3 levels were significant increased in all fluoride-exposed male zebrafish compared with those in the control group (p < 0.05) during the whole exposure period (Fig. 5A). T4 levels were unchanged in all fluoride-exposed male fish compared with those in the control fish (p > 0.05) after 45 days of exposure (Fig. 5B). After exposure for 90 days, male fish exposed to 2.0 and 4.1 M of fluoride had prominently decreased T4 levels by 13.47% and 17.59% (p < 0.05) (Fig. 5B). 3.4. Effects of fluoride on endocrine-related gene expressions in male zebrafish After exposure for 45 days and 90 days, the levels of CRH and TSH mRNA were significantly up-regulated in all fluoride-exposed fish relative to the control fish. Especially the CRH expressions in fish exposed to 2.0 and 4.1 M of fluoride were extremely prominently elevated by 2.32-fold and 2.93-fold for 45 days, and 1.61-fold and 1.56-fold for 90 days, respectively (p < 0.01) (Fig. 6A). All fluorideexposed fish have highly significantly enhanced TSH levels by 1.63fold, 2.02-fold, 2.40-fold for 45 days, respectively (p < 0.01) (Fig. 6B). Meanwhile the TSH levels were significantly increased by 1.42-fold and 1.99-fold for 90 days, respectively. The gene transcriptions of TG were significantly increased in all fluoride-exposed fish during the whole exposure period (p < 0.05) (Fig. 6C), especially were remarkably elevated in fish exposed to 2.0 and 4.1 M of fluoride by 2.22-fold and 3.14-fold for 45 days, and 1.57-fold and 3.90-fold for 90 days, respectively (p < 0.01). After exposure for 45 days, significant elevation in NIS mRNA expression was observed in all fluoride-exposed fish (Fig. 6D), among which the fish in 2.0 and 4.1 M group had significantly increased NIS levels by 1.89-fold and 2.61-fold, respectively (p < 0.01). After exposure for 90 days, the NIS mRNA expressions were significantly increased by 1.20-fold and 1.24-fold in fish exposed to the 2.0 and 4.1 M of fluoride, respectively (p < 0.05) (Fig. 6D). After exposure for 45 days, the mRNA levels of DIO1 and DIO2 were significantly increased in all fluoride-exposed fish compared with those in the control fish (Fig. 6E, F), in which the expressions were highly significantly elevated by 1.62-fold and 2.76-fold for DIO1 mRNA, and by 1.39-fold and 1.86-fold for DIO2 mRNA in fish exposed to 2.0 and 4.1 M of fluoride, respectively (p < 0.01). After exposure for 90 days, similar expression results were obtained for the DIO1 mRNA, while, fluoride significantly decreased the DIO2 transcription levels in 2.0 and 4.1 M of fluoride groups relative to the control group (Fig. 6E, F). After exposure for 45 days, fluoride exposure remarkably elevated the UGT1ab mRNA levels in all treatment groups by 1.64-fold, 1.97-fold, and 1.27-fold, respectively (p < 0.01) (Fig. 6G). TTR transcription levels were significantly increased in fish exposed to 2.0 and 4.1 M of fluoride (p < 0.05) (Fig. 6H). After 90 days of exposure, UGT1ab mRNA levels were significantly increased in fish exposed to 2.0 M of fluoride (p < 0.05) and were highly significantly decreased in fish exposed to 4.1 M of fluoride compared with those in the control fish (p < 0.01). Fluoride exposure significantly increased the TTR mRNA levels in fish exposed to 2.0 and 4.1 M of fluoride, while highly significantly decreased the TTR expression in 4.1 M of fluoride-exposed fish ((p < 0.01). After 45 days of exposure, the mRNA levels of TR␣ and TR␤ were prominently raised in all fluoride-exposed fish relative to the control group (p < 0.01) (Fig. 6I, J). After exposure for 90 days, the mRNA level of TR␣ was significantly increased in all fluoride-exposed fish, in which the TR␣ expressions were significantly increased by 2.19-fold and 3.74-fold in fish exposed to 2.0 and 4.1 M of fluoride (p < 0.01) compared with the control fish, respectively (Fig. 6I).

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However, the TR␤ mRNA levels were relatively unchanged in all fluoride exposed groups (Fig. 6J). 4. Discussion 4.1. Effects of fluoride on growth performance in male zebrafish Growth is the net result of many essential processes, such as assimilation, respiration, and excretion, which is one of the most important indices to evaluate the effects of environmental stressors on animals at the population level (Wendelaar Bonga, 1997). In the present study, exposure to fluoride significantly reduced the growth in male zebrafish. Similar results were obtained in Siberian sturgeon (Acipenser baerii), which indicated that the growth was significantly inhibited at waterborne fluoride concentration greater than 7.8 mg/L after exposure for 12 weeks or after exposure to 10, 25, and 62.5 mg/L fluoride for 90 days (Shi et al., 2009a,b). A study on common carp (Cyprinus carpio) indicated that weight gain rate and specific growth rate in all fluoride-exposed fish were prominently decreased after 30, 60, and 90 days of exposure, respectively (Chen et al., 2013). Growth inhibition due to fluoride exposure has also been found in other fish species, such as climbing perch (Anabas testudineus), snakehead (Corydoras punctatus), walking catfish (Clarias batrachus), catfish (Heteropneustes fossilis), and clown featherback (Chitala ornata) (Samal, 1994). Their body weight were decreased by 68% on average after exposure to waterborne fluoride over a range of 6.9–52.5 mg/L (Samal, 1994). All above indicated that exposure to fluoride could significantly inhibit the growth in fish. The inhibition of growth as a response to fluoride exposure may be caused by the following reasons. Firstly, fluoride generally resulted in the reduction in both food intake and food assimilation efficiency, consistent with the anorexia observed in other fishes (Camargo, 2003; Kumar et al., 2007; Chen et al., 2013), thus, their growth were inhibited; Secondly, fluoride could highly accumulate in bones, which might distort the growth of fishes (Shi et al., 2009a); Thirdly, fluoride ions could act as enzymatic poisons to depressing the enzyme activity and ultimately suspend the metabolic processes, synthesis of proteins and glycolysis (Camargo, 2003). 4.2. Effects of fluoride on histopathological changes of thyroid in male zebrafish Exposure to fluoride caused obvious histopathological changes in the thyroid tissues. The observed histopathological changes were more obvious in fish exposed to high fluoride concentrations. The histopathological observations indicated an obvious thyroid activation evidenced by conspicuous alterations in the follicles, which was supported by the previous studies of potassium-perchlorate and monocrotophos pesticide on thyroid in zebrafish and goldfish, respectively (Schmidt et al., 2012; Zhang et al., 2013). Similar histological alterations have previously been connected to thyroid activation, epithelial cell height or follicle number as the indicator of thyroid activation (Liu et al., 2006; Schmidt et al., 2012). In the present study, the epithelial cell height, follicle area, and colloid area were all sensitive to fluoride. This was similar to the studies by Goleman et al. (2002) and Liu et al. (2006, 2008), which indicated epithelial cell height as the most sensitive endpoint in Xenopus laevis tadpoles and juvenile zebrafish, respectively. The changes in the epithelial cell height and follicle appearance are likely the result of negative feedback-induced secretion of TSH by the pituitary, which is considered to respond to exposure of thyroid-disrupting chemicals by intensified production of TSH. The activating effect of TSH is mediated by a G-protein-coupled TSH-receptor primarily expressed in thyroid and gonads (Farid and Szkudlinski, 2004; MacKenzie et al., 2009). Despite this knowledge, the reasons that

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cause the proliferation of follicle size instead of the proliferation of thyroid follicles are unclear and need further investigation. 4.3. Effects of fluoride on hormone levels in male zebrafish THs are important for the growth and development of fish (Brown, 1997). A large number of studies have shown that environmental toxins affected dynamic balance of thyroid hormones in homeostasis, and different materials had different impact of THs levels (Brown et al., 2004; Zhang et al., 2013). In the present study, THs concentrations were also significantly affected by fluoride. T3 levels were significantly increased in all fluoride-exposed fish during the exposure period. T4 levels were only prominently decreased in fish exposed to 2.0 and 4.1 M of fluoride for 90 days. All above results suggested that fluoride could interfere with the THs levels. These results were consistent with significant increases in T3 and remarkable decrease in T4 levels in zebrafish larvae after exposure to HEX and TEB (Yu et al., 2013). On the contrary, the THs levels were sharply reduced in bufo gargarizans tadpoles after exposed to different concentration of fluoride (Zhao et al., 2013). These results indicated that fluoride might have the ability to act as endocrine disruptors via a range of different mechanisms, such as disruption of thyroid hormone homeostasis and failed adaptation and auto-regulation of thyroid hormone through HPT axis. It should be noted that compared to studies from mammals, in which thyroid hormones levels (T3 and T4) were significantly decreased after exposure to fluoride (Basha et al., 2011). The different effects of fluoride on thyroid hormones in fish and mammals remained to be further investigated. 4.4. Effects of fluoride on gene expression of thyroid in male zebrafish Secretion functions of CRH, stimulating the secretion of adrenocorticotropin hormone from the pituitary, and TSH, the main regulator of TSH secretion, are triggered by the changes of circulating THs concentrations (De Groef et al., 2006). The mRNA levels of CRH and TSH mRNA levels can be used as endpoints to determine whether environmental chemicals disturb thyroid function (Yu et al., 2011). This study showed that after exposure for 45 days, the CRH and TSH mRNA expressions were significantly upregulated in fluoride-exposed fish for 45 and 90 days. Previous studies found that T4 regulated the transcription levels of CRH and TSH in fish by a negative feedback mechanism, namely, T4 content was reduced with the increase of CRH and TSH gene transcriptions, which have been indicated in zebrafish larvae when exposure to PBDE mixture DE-71 (Yu et al., 2010), BDE-209 (Chen et al., 2012), and MCLR (Yan et al., 2012). On the other hand, T4 content was increased with the decrease of CRH and TSH mRNA levels (EllisHutchings et al., 2006; Manchado et al., 2008). Similar results were obtained in DE-71-exposed zebrafish which found that prolonged exposure to DE-71 significantly decreased the mRNA levels of CRH and TSH in the brain, which were associated with elevated levels of plasma T4 in zebrafish (Yu et al., 2011). Combine with these results, the results of the present study suggested that the up-regulations of CRH and TSH mRNA levels might be correlated to the hypothalamus and pituitary negative feedback adjustment mechanism for the regulation of the decreased T4 levels. Sodium iodide symporter is an integral membrane protein on the basement membrane of thyroid follicular epithelial cells that transports sodium and iodide across the basolateral plasma into follicular cells of the thyroid gland, which is the first step in the synthesis of thyroid hormone (Dohán and Carrasco, 2003). Thyroglobulin, as a protein precursor of thyroid hormone, can be used to produce and storage T4 and T3 in thyroid gland. Therefore, changes of NIS and TG mRNA levels are very important to the pro-

duction of THs. Moreover, the levels of NIS and TG mRNA can be helpful markers for monitoring the thyroid activity during development (Shi et al., 2009c). The present study indicated that the mRNA expression of NIS and TG were significantly elevated in fish exposed to fluoride during the whole exposure period, which indicated that fluoride could induce thyroid endocrine disruption in male zebrafish. Moreover, fluoride increased the T3 levels during the exposure and only decreased the T4 in the higher dose and the latter period. Therefore, we speculate that the upregulation of NIS and TG mRNA could possible be related to the changes of plasma THs concentrations. Deiodinases are crucial regulators during the circulation and metabolism of THs in vertebrates, and are sensitive to variation in THs levels. Three types of deiodinases were described in fish: DIO1, DIO2, and DIO3. DIO1 and DIO2 are able to convert T4 into T3 while DIO3 is an absolutely inactive enzyme (Orozco and Valverde, 2005). In zebrafish, it has been found that DIO1 plays a vital role in iodine recovery and THs degradation but has a minimal effect on THs homeostasis of plasma (Van der Geyten et al., 2005). However, DIO2 plays an important role in the production of active T3 and has the adequate availability of local and systemic T3 in zebrafish (Walpita et al., 2007). In the present study, DIO1 levels were significantly increased during the whole exposure period. DIO2 mRNA expressions were significantly increased after exposure for 45 days. However, DIO2 expressions were remarkably decreased in fish exposed to 2.0 and 4.1 M of fluoride after exposure for 90 days. Similar result was obtained in study on hypothyroidism, which found that hypothyroidism increased the activities and mRNA expressions of DIO1 and DIO2 (Orozco and Valverde, 2005). Our results indicated that DIO1 was more sensitive to the alteration of THs levels than DIO2. This may probably be that after fluoride exposure, the DIO1 may be enhanced to try to degrade the elevated T3 levels. These results have important effects on TH homeostasis in tissues, which further proved that fluoride affected the development and growth in male zebrafish by impairing the THs signaling pathway. TTR binds THs and transports them to various target tissues, which plays an important role in the thyroid axis in fish (Kawakami et al., 2006; Power et al., 2000). Thus, TTR gene was regarded as a biomarker for thyroid system disruption (Yu et al., 2011). Previous studies found that the reduction of TTR were consistent with the decreased THs (He et al., 2012; Yu et al., 2010), which indicated that THs might regulate plasma TTR directly or indirectly. However, the TTR level was elevated in lampreys when THs concentrations in serum were at the lowest (Manzon et al., 2007). Moreover, Shi et al. (2009a,b,c) found that the significantly decrease of TTR mRNA transcription inversely correlated to the remarkably increase of T3 levels. Therefore, it is unlikely for plasma THs concentrations to be responsible for the difference in TTR expression (Li et al., 2009). In the present study, the TTR mRNA expressions were significantly increased in fish exposed to 0.9 and 2.0 M of fluoride during the experiment period while decreased in fish exposed to 4.1 M of fluoride after 90 days of exposure, which might affect the binding and transport of THs and hence posed a potentially threat to the thyroid function. Further investigations are needed to understand the underlying mechanism in the changes of TTR gene levels. It has been indicated that the induction of uridine diphosphate glucoronosyltransferases (UDPGT) decreased the levels of circulating THs (Hood and Klaassen, 2000). UDPGT catalyses the glucuronidation of T4 and thereby increases the biliary excretion of conjugated hormone, which could partly explain why T4 levels were decreased. Previous studies in rodents indicated that the decreased T4 levels and increased UGT activities or gene transcription were observed (Hester et al., 2006; Wolf et al., 2006; Szabo et al., 2009). Study on zebrafish larvae showed that the T4 levels were significantly reduced, while the UGT gene expressions were significantly increased after exposure to DE-71 for 14

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days or exposed to hexaconazole and tebuconazole for 120 h postfertilization (Yu et al., 2010, 2013). Nevertheless, study by Szabo et al. (2009) found that UGT and T4 were not the most sensitive markers for DE-71 and the regulation of UGT to THs was thought to be nuclear receptor specific. In the present study, the UGT1ab mRNA expressions were significantly increased after exposure for 45 days and in fish exposed to 2.0 M of fluoride for 90 days in the present study, which had no direct correlation with the T4 level. Since no studies indicated that fluoride had the potential to activate the aryl hydrocarbon receptor (AhR), the decrease of T4 levels may not be directly related to the regulation of UGT. Hence, further study is needed to clarify the mechanistic correlations between metabolic genes and THs levels. THs activities were mainly regulated by different isoforms of thyroid hormone receptor (TRs), which plays important roles in embryogenesis and larval development and are encoded by TR␣ and TR␤ gene, respectively (Liu and Chan, 2002). Previous studies have showed that the expression of TR subtype varied relying on tissue-specific and development state-specific functions in fish and other vertebrates (Forrest and Vennstrom, 2000; Nelson and Habibi, 2009). In the present study, TR␣ mRNA expressions were significantly elevated in male zebrafish with the increase of fluoride concentration and exposure time, while TR␤ mRNA expressions was up-regulated after 45 days of exposure and relatively unchanged after exposure for 90 days. The different effects of environmental pollutants on TR␣ and TR␤ gene transcriptional levels were also found in the study of PFOS on zebrafish larvae, which showed that TR␣ was increased while TR␤ was decreased (Shi et al., 2009c). Thus, our results combined with previous studies indicated that TR␣ and TR␤ had different functions. Crump et al. (2008) found that the T3 administration significantly enhanced the levels of TRs, which therefore affected the transcription of other genes in thyroid function. Therefore, it was speculated that fluoride increased the T3 levels, which significantly up-regulated the TRs gene transcription. This may be a feedback mechanism responded to the disturbance of HPT axis homeostasis. The abnormal expressions of TR␣ and TR␤ mRNA could result in the failure of THs to bind and activate the appropriate post-receptor response cascades (Liu et al., 2011). Therefore, In the present study, we further speculate that the change of TRs gene expressions might play a crucial role in the thyroid disruption induced by fluoride. In summary, this study explored the effects of fluoride on growth performance, histopathology, thyroid hormone and the expression of thyroid-related genes in endocrine regulation system. Our results showed that fluoride exposure could significantly inhibit the growth of male zebrafish. More importantly, fluoride could disturb circulating THs homeostasis in male zebrafish and damage the TH-dependent physiological functions by changing the normal structure of thyroid, disturbing the levels of T3 and T4, and a series of genes transcriptions involved in HPT axis in male zebrafish. The results indicated that fluoride could pose a great threat to thyroid endocrine system, thus detrimentally affected the normal function of thyroid of male zebrafish. Further studies are needed to focus on the molecular mechanisms for the effects of fluoride on the thyroid of male zebrafish. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments This work was supported by funding from the National Natural Science Foundation of China (31440087; 31502141); the China Postdoctoral Science Foundation (2012M520601; 2013T60267);

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the Doctor Initial Funding of Shanxi Agricultural University (XB2009003); the Postdoctoral Science Foundation of Shanxi Agricultural University (92462).

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