Early life Exposure to Triclosan Impacted Thyroid Follicular Structure and Decreased Thyroid Hormone Levels in Zebrafish

Background: Triclosan (TCS) is an antimicrobial chemical widely used in personal care products. Most of TCS is discharged into aquatic ecosystem after usage. While TCS has potential thyroid-disrupting effect, it is unknown whether early exposure to TCS affects thyroid grand and thyroid hormone levels. We used zebrafish model to examine this topic. Methods: The fertilized zebrafish eggs were exposed to TCS. We inspected hatching and mortality of larvae. The total triiodothyronine (TT 3 ), total thyroxine (TT 4 ), free triiodothyronine (FT 3 ), and free thyroxine (FT 4 ) levels were measured at 7, 14 and 120 days post fertilization (dpf). Thyroid histopathological analysis was conducted at 120 dpf of zebrafish. Results: The hatching rate of embryos decreased from 95.8% in control group to 18.3% in TCS highest exposure in 3 days. In 900 ng/mL TCS exposure all larvae died within one day. With increasing concentration of TCS exposure, TT 3 and FT 3 level decreased after 14 days. FT 4 decreased, and the height and nuclear area of thyroid follicular cell increased with TCS exposure after 120 days. Conclusions: High TCS exposure decreased the hatching rate of zebrafish embryos, and also decreased thyroid hormone levels owing to structural change of thyroid follicles. Our study raised the concern that TCS exposure in early life may profoundly affect neurodevelopment.

showed that TCS exposure resulted in hyperplasia of thyroid follicles [11] . There were only two related studies on potential thyroid-disrupting effect of TCS in fish [11,12] , it is unknown whether early life exposure to TCS will affect offspring thyroid grand and thyroid hormone (TH) levels. In this study, we aimed to use zebrafish to examine this topic.
Zebrafish is a proved model to study thyroid gland and hormones because the development of its thyroid system is generally comparable to human [13] . While thyroid hormones are critical for fetal neuro-development, up to our knowledge, no study has examined early exposure to TCS (from fertilized eggs, embryo to adult stage) and zebrafish thyroid hormone levels. In this study, we used zebrafish model to examine the impact of early exposure to TCS at environmentally relevant levels and the TCS levels in human bio-samples on the hatching rate, mortality, and thyroid hormones levels, as well as the histopathology of thyroid follicles.

Zebrafish strains and maintenance
In this study, we used the zebrafish strains of AB wild-type line. First, 5-month-old male and female zebrafish were acclimated in tanks containing dechlorinated tap water for 4 weeks and under a photoperiod of 14:10 hour light/dark cycle. The fish were fed with brine shrimp twice per day. The remaining food and feces were removed at thirty minutes after feeding.
Next, zebrafish fertilized eggs were obtained/collected within 30 min after natural mating, rinsed in water, and the unfertilized eggs were discarded. Zebrafish larvae were fed with paramecium twice per day from 5 days post fertilization (dpf) till 14 dpf. After that, brine shrimp was fed from 15 dpf. The pH (maintained at 7.5 ± 0.5), conductivity (maintained at 550 ± 50 μS), temperature (maintained at 28 ± 0.5 ℃), and dissolved oxygen of the exposure media were monitored.

TCS exposure
We randomly assigned and exposed fertilized zebrafish eggs (600 eggs per TCS group) to 6 levels TCS exposures from 0 (Dimethyl Sulphoxide, DMSO as blank control), 3, 30, 100, 300 to 900 ng/mL up to 7 and 14 days, and we put 200 fertilized zebrafish in each culture dish of 100 ml medium. We changed half of the medium each time for twice per day. The fertilized zebrafish eggs were cultured at 28.0±0.5 °C under 14:10 light/dark photoperiod cycle in a climate chamber during the duration of the experiment.
After TCS treatment for 7 and 14 days post fertilization (dpf) respectively, larvae TH levels were measured. Specifically, at 7 dpf, fish larvae were collected and put into three groups for each TCS treatment level as three replicates (50 larvae each replicate) for THs measurement. Then, the larvae left were continuingly exposed to TCS/or control until 14 dpf, fish larvae were collected and put into three replicates per each TCS treatment level (100 larvae in each replicate) for THs measures.
In addition, we exposed fertilized zebrafish eggs to 6 TCS exposures levels (30 eggs per each exposure level) for 120 days. At 120 dpf, the fish were deceased by immersing in ice-cold water. The heads were used for thyroid histological study and the bodies of fish were collected for thyroid hormone measurement. All fish samples (larvae at 7 and 14 dpf, heads and bodies of fish at 120 dpf) were immediately frozen and stored at −80°C for measurement of thyroid hormones.
These exposure doses of TCS were set based on the exposure levels of human and wildlife [9,14] . 3-100 ng/mL was close to the levels of urinary TCS concentrations in Chinese pregnant women [9] as well as TCS levels in some rivers/lakes [3] . We used DMSO as a solvent to enhance its solubility because TCS has low water solubility [15] , and the concentration of DMSO in both the experimental and control groups was 0.01%. Zebrafish embryos are tolerant to low concentrations (0.01%) of DMSO [16] .

Hatching rate
The hatching of fertilized eggs was inspected and recorded twice per day at the first 3 days (i.e. 72 hours). Hatching rate was calculated as the number of larvae hatched during the first 3 days divided by the total number of fertilized eggs in each TCS exposure level. The death of larvae was also inspected and recorded twice per day, and dead larvae were immediately removed from dishes/tanks. Mortality was calculated as the number of larvae death divided by the total number of hatched larvae.

Thyroid hormone measurements
We measured thyroid hormone levels of zebrafish at 7, 14 and 120 dpf. The samples of larvae at 7 and 14 dpf, and the body (without head, viscera or gut) of fish at 120 dpf stored at −80°C were taken out from freezer and immediately sonicated in 0.01 M PBS (0.01 M) at 1mg/5μL (w/v, w: wet weight of 50 or 100 larvae or every fish body, v: volume of 0.01 M PBS, mg/μL; pH 7.2) and homogenized at 4°C . Next, to disrupt the structures, we put samples on intermittent sonic oscillation for 5 minutes on ice, and then vortexed vigorously for 10 min. Then, all the samples were centrifuged at 12,000 × g for 5 min at 4 °C. The supernatant was collected for thyroid hormones and total protein concentration measurement. Total protein concentration was measured with the Pierce © BCA Protein Assay Kit (Thermo Fisher Scientific Inc., Rockford, IL, USA) to normalize thyroid hormone concentration [17] . Free trithyroxine (FT 3 ), free thyroxine (FT 4 ), total trithyroxine (TT 3 ) and total thyroxine (TT 4 ) levels were measured with ELISA (enzyme-linked immunosorbent assay) (Labor Diagnostika Nord commercial kit, Germany). The limit of detection is 0.1 ng/mL for TT 3 , 8 nmol/L for TT 4 , 0.3 pg/mL for FT 3 and 1pg/mL for FT 4 . Values below the limit of detection (LOD) were replaced with values equal to the LOD divided 2.

Hematoxylin and eosin (H&E) staining
The heads of zebrafish at 120 dpf frozen at −80°C were taken out from freezer and fixed in 10% neutral formalin (Zhongshan Beijing Biotechnology Co., Ltd., Beijing, China) at temperature 25˚C for at least 12 h, and transferred to 70% ethanol. Each zebrafish head was placed in processing cassettes, dehydrated through a serial alcohol gradient, and embedded in wax blocks. Serial transverse cross-sections were cut using a microtome 5 μm and dewaxed in xylene, rehydrated through decreasing concentrations of ethanol, and washed in PBS. The sections were then stained with hematoxylin for 4-5 min and with eosin for 1-2 min at 25˚C (Zhongshan Beijing Biotechnology Co., Ltd.). Stained thyroid follicle sections were observed and photographed under light microscopy (BX53-p; Olympus Corporation, Tokyo, Japan). We located thyroid follicles according to their being dispersed among the afferent branchial arterioles (such as ventral aorta) in the throat region [18] .

The nuclear size and height of follicular cells
Image Pro-Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA) was used to quantitatively analyze. The nuclear size of the thyroid follicular cells was assessed quantitatively by measuring the long and short diameters of the cell nuclei. The size of at least 50 thyroid follicular cell nuclei from each fish at a total magnification of ×1000, at least three different follicles or from three separate areas of the section in cases where follicular structure was unclear or absent. Photograph of each follicle was taken at the largest follicle diameter (determined by observing serial sections) with an Olympus digital camera (BX53-p; Olympus Corporation, Tokyo, Japan). The nuclear size was calculated based on the formula for an ellipse (long diameter × short diameter × π/4) [18] . The height of follicular cell was calculated by five measurements along the follicle perimeter at regular intervals, 40-80 follicular cell heights for each fish [19] .

Statistical analysis
The ANOVA F-test was used to compare the differences in hormone levels, the height and the nuclear size of the thyroid follicle cells, the chi square test was used to compare the difference in hatching rate and mortality of larvae among TCS exposure groups. The level of significance was two-sided p value < 0.05. All analyses were performed using the SAS 9.3 software (SAS Institute, Inc., Cary, NC, USA).

The effect of TCS exposure on hatching rate
In this study, the hatching rate of zebrafish embryos decreased with increasing TCS exposure at 2 dpf. In control group, hatching rate was 42% in the first 48 hours and 95.8% at 72 hpf, and the larvae presented a well-developed head, body and tail. Following the higher concentrations of TCS exposure, hatching rate decreased especially in the first 48 hours. The lowest was 18.3% in the highest (900 ng/mL) TCS exposure group (p < 0.01) ( Table 1) at 72 hpf.

The effect of TCS exposure on mortality of zebrafish larvae
In TCS exposure level of 900 ng/mL, larvae died shortly after its hatching. In larvae exposed to the highest level (900 ng/mL) of TCS, the mortality reached 100% at 3 dpf. In the other TCS exposure levels, the mortalities were similar at 7 dpf ( Table 2).
After 7 days exposure, we randomly collected fish larvae (50 larvae each for triplicate) from each TCS exposure group to measure thyroid hormone levels. We then continued to observe mortality in the remaining larvae during 8 and 14 dpf, and the mortality was similar among TCS exposure levels from 0 to 300 ng/mL (p=0.16, Table 2).

The thyroid hormone levels in Zebrafish larvae after 7 days of TCS exposure
The TT 3 , TT 4 , FT 3 and FT 4 levels did not differ significant among 5 TCS exposure groups from 0 to 300 ng/mL (Figure 1 and Table S1). For the TCS exposure level of 900 ng/mL, since the larvae all died by the day three soon after they were hatched, thyroid hormone levels were not measured in this group.
The thyroid hormone levels in larvae after 14 days of TCS exposure TT 3 and FT 3 levels in TCS exposure groups were all lower than the control group after 14 days of exposure. Following exposure to TCS from 3 ng/mL to 300 ng/mL for 14 days, TT 3 were on average 0.188 to 0.194 ng/mg lower than that of control group (all p < 0.01, Figure 1 and Table S2). Also, FT 3 levels were 9.49 -10.74 ng/mg lower (p < 0.01) in larvae exposed to TCS level at 30, 100, 300 ng/mL than the control group. For FT 4 level, overall it tended to decrease with higher level of TCS exposure (p trend < 0.05 , Table S2). Similarly, TT 4 levels were lower in larvae exposed to TCS than that in control group, but the differences were not statistically significant.

The thyroid hormone levels in zebrafish after 120 days of TCS exposure
The effects of TCS exposure for 120 days on thyroid hormone levels are presented in Figure 2 and Table S3. Following exposure to TCS at 30, 100, and 300 ng/mL for 120 days, FT 4 levels were on average 0.06, 0.08, 0.09 pg/mg lower than that of control group (all p < 0.05) (Figure 2 and Table   S3). The TT 3 , TT 4 , and FT 3 levels were similar among 5 TCS groups.

Thyroid Histopathology in zebrafish after 120 days of TCS exposure
With the increasing exposure levels to TCS, the thyroid follicular cell of zebrafish at 120 dpf became hypertrophy (enlarged follicular cell size and nuclear size) and hyperplasia (increased number of follicular cell), and the nuclear area and the epithelial cell height of thyroid follicles was significant increased (Figures 3 and 4, Table S4). The oval thyroid follicles of zebrafish in control group consisted of an outer thyroid epithelial layer which surrounded into an inner lumen; the inner lumen of thyroid follicles was filled with colloid (Figure 4, Panel A). After exposure to TCS for 120 days, colloid in follicle decreased obviously (Panel B, C, D, E and F in Figure 4 and  Figure 4; B, C, D and E in Figure 5), and diffuse hyperplasia (F in Figure 4) in thyroid follicles.
Colloid depletion of follicles was observed in all zebrafish exposed to TCS (B, C, D, E and F in Figure   4). In the zebrafish exposed to high TCS levels at 100 ng/mL and 300 ng/mL, the thyroid follicles had obvious morphological alterations, for example, the epithelial cells became edema, thicker and increased significantly, follicular interstitial hyperplasia and inflammatory cells increased (D, E and F in Figure 4).

Discussion
In this study, we examined the effects of early TCS exposure on the thyroid hormone (TH) levels at two growth phases (larvae and adult) and found thyroid morphological change after 120 days of TCS exposure. High TCS exposure decreased the hatching rate of zebrafish embryos. Furthermore, TCS exposure also decreased TT 3 , FT 3 and FT 4 levels after 14 days and decreased FT 4 level after 120 days owing to histopathological change of thyroid follicular cell. With increasing concentration of TCS exposure, the height and nuclear size (area) of thyroid follicular cell increased with TCS exposure after 120 days. Considering the importance of thyroid hormone to nervous system development, our study implicates that TCS exposure in early life may profoundly affect neurodevelopment.
Zebrafish is a useful animal model for the study on thyroid hormones and the chemical pollutions in water, and its high (71%) genetic is similar to human [20] . We found that with the increase of TCS exposure concentration zebrafish hatching rate decreased and the mortality rate increased of zebrafish larvae. This result was consistent with previous finding in zebrafish [14] , after being exposed to 500 ng/mL of TCS for 6 days, a significantly delayed hatchability and increased mortality were observed in zebrafish. After larvae stage, zebrafish step into sexual mature phase at 120 dpf [21] . We observed adverse effect of early exposure to TCS (from fertilized eggs, embryo to adult stage) on thyroid hormone levels, and this might be due to the cumulative effect of TCS exposure on the physiological characteristics of the development of zebrafish thyroid glands. The first follicular thyroid of zebrafish larvae emerges at about 55 hpf and weak signals of T 4 can be detected in submandibular about 72 hpf [22] . There are about 6-7 thyroid follicles at 7 dpf [13] . The thyroid hormone level of zebrafish became stable after 7 days [23] . In our experiments, TT 4 , FT 4 and FT 3 levels in TCS exposed groups tended to decrease, although not significant, after 7 days of exposure. The thyroid disruption effect of TCS was more obvious after 14 days of exposure. FT 4 level decreased significantly with the increasing TCS exposure concentration both at 14 and 120 dpf. The impact of TCS exposure on thyroid hormone levels (TT 3 , FT 3 and FT 4 ) in zebrafish larvae at 14 dpf provides causal support to our previous findings in population study [9] . Our previous study observed an inverse association between maternal urinary TCS and cord blood FT 3 level in Chinese newborns [9] .
THs are secreted and released by the thyroid gland. FT 3 is the most active form of TH, and it comes from FT 4 deiodination. FT 3 and FT 4 , TT 3 and TT 4 can transform into each other and maintain dynamic balance in blood. Most TT 4 and few TT 3 are synthesized by thyroid follicular epithelial cells directly and then are transported to blood and be function [24,25] . TT 4 and TT 3 are usually used as an indicator of the reserve capacity of the thyroid gland [26] . Once the thyroid function is disrupted, the FT 3 and FT 4 levels are the first to be affected, lead to TT 3 and TT 4 level and histopathological change of thyroid [27] . In general, T 4 is more sensitive compared with the changes of T 3 [26,28,29] , however, in this study, with higher TCS exposure levels, TT 3 and FT 3 concentrations decreased statistically in larvae at 14 dpf. Exposure to 120 dpf, TT 4 , FT 3 and FT 4 levels were all lower than that at 14 dpf, and the height and nuclear area of thyroid follicular cell at 120 dpf were obviously increased with the increasing TCS exposure concentration. The histopathology results are consistent with the changed levels of thyroid hormone in this study. The changes in the epithelial cell height and follicle nuclear size are likely the result of negative feedback of decreased thyroid hormone.
To study the impact of environmental chemicals exposure on health effects, if zebrafish larvae were used as models, generally the whole fish is used as biological samples [12,30] , and in our study, we measured THs levels in hole body of larvae at 7 and 14 dpf. However, if adult zebrafish were used as models, plasma or body are generally used as biological samples [31] , but we measured TH levels in body (without head, viscera or gut) of adult fish at 120 dpf. In zebra fish, THs both expressed in hepatic and muscle [32] . THs levels in muscle can reflect the growth and development of the body, and in our study, we found that TCS exposure level was related to TH levels in the muscle and therefore presumably affects the body growth of the zebrafish.
TCS was found in wastewater treatment plants worldwide [3] . One study found that TCS has been detected in more than half of the rivers and lakes in Savannah, Georgia, USA, with a median level of 0.14 ng/mL and the maximum level of 2.3 ng/mL [3] . In some stream, the concentration even reached up to 86.2 ng/mL [3] . This study selected the TCS exposure levels of 3, 30, 100, 300 and 900 ng/mL based on our population exposure level, wildlife zebrafish exposure level and the TCS level in environmental data. The lowest concentration (3 ng/mL) was selected based on the median urinary TCS concentration (2.52 ng/mL), and 100 ng/mL was close to the highest urinary TCS concentrations from population of pregnant women in China [9] . In fish, the LC50 for TCS ranges from 180 to 602 μg/L [33][34][35] . In zebrafish, TCS LC50 values was 420μg/L(95% confidence interval, 400 ng/mL-470 ng/mL) for embryos in exposure to TCS for 96 hours (starting from fertilized eggs), and 340μg/L for zebrafish adults also with an exposure of 96 hours [14] . TCS exposure level of 100 ng/mL in this study was 25% of the concentration for LC50. We also use relatively higher concentrations of TCS (300 and 900 ng/mL) to study possible toxicity during short-term laboratory exposures. The exposure concentrations 900 ng/mL of TCS was a 10-fold of the highest urinary TCS concentration in our previous population study in China. While the toxic effects of TCS were well recognized on bacterial resistance, reproductive toxicity and cytotoxicity in aquatic organisms [36][37][38] , the results on thyroid hormone disruption were inconsistent [39,40] .Previous studies found that exposure of TCS decreased thyroid hormone levels and inhibited metamorphosis [39,41] , or did not change the thyroid hormone level nor alter the metamorphosis in anuran [40] . Most animal studies mainly focused on rodents [42,43] . In adult rat, TCS treatment reduced the levels of blood TT 4 and FT 4 , while no change was found in the level of blood TSH [44,45] [46,47] . Another study in female mice found that exposed to TCS (300 mg/kg/day) during pregnancy decreased approximately 30% of TT 4 in dams at the postpartum day 22 [47] . Only two recent studies have examined the influence of TCS exposure on thyroid disruption in fish [11,12] . One study found that TCS exposure caused hatching delay of 6-13 hours in medaka fish [12] . The other study found that sub-chronic (21 days) exposure to 100 μg/g TCS caused hyperplasia of thyroid follicle in adult zebrafish [11] . A recent study [48] found that offspring of zebrafish that exposed to TCS decreased survival rate and delayed maturation. This can be explained by decreased thyroid hormone resulting from TCS exposure.
We speculate that TCS may inhibit thyroid hormones secretion by acting as a disruptor of the hypothalamic-pituitary-thyroid axis or due to the increased thyroid hormones clearance. Mechanisms have been studied but there is controversial. TCS exposure may promote [11] or inhibit [49] the sodiumiodide symporter (NIS)-mediated iodide uptake in animals. TCS increased TSH gene transcription and decrease thyroid hormones in circulation of zebrafish [11] . In rat, TCS inhibited the activity of thyroid peroxidase (TPO) at concentration of 50 ng/mL to inhibit thyroid hormone synthesis [49] . In addition, evidence suggests that TCS inhibited liver phase I and II enzyme mRNA expression in zebrafish liver cells [50] . TCS may interfere with thyroid function by increasing the hepatic enzymes activity of glucuronyltransferase and pentoxyresorufin-O-deethylase (PROD) in livers [51,52] or by activating of the nuclear receptor, pregnane X receptor (PXR) [43] , as a result, the metabolism of T 4 causing decrease in T 4 bioavailability [42] . In 2018, it was also found that TCS caused the hypothyroidism in thyroid cells of rats through p38/TRHr-dependent pathway [37] . Still, the mechanism that TCS inhibit the thyroid hormones need further investigation.
Our study found that zebrafish exposed with TCS for 120 days (from embryo to adult phase) caused thyroid histopathology change and disturbed thyroid hormone levels. We are the first study to explore the thyroid disturbed effect of long-time TCS exposure on zebrafish from fertilized eggs, larvae to adult phases, and to focus on effect of TCS exposure on thyroid hormones of zebrafish from early life.
We did not measure TSH level in zebrafish, although TSH receptor is responsible for thyroid gland differentiation in zebrafish [53] , Alt et al. [54] reported that in zebrafish the formation, growth and differentiation of thyroid follicles appears to be independent of TSH. The limitation of our study is that we did not measure the concentration of TH levels in hole body of adult zebrafish, the trend of TH levels at 120 dpf were not fully consistent with that at 14 dpf.

Conclusions
In summary, we found that exposure to TCS delayed the hatching of zebrafish embryos and decreased thyroid hormone (FT 3 , FT 4 and TT 3 ) levels in zebrafish larvae, also decreased FT 4 level in zebrafish adult. Furthermore, TCS exposure also affect the structure of thyroid follicles by increasing nuclear area and height of zebrafish thyroid follicle cell. Considering that the thyroid hormones are crucial for neurodevelopment in early life, our study raises the concern that early TCS exposure (from conception) may profoundly influence child neurobehavioral development at such highly sensitive window.

Funding Information
This study was supported by grants from the National Natural Science Foundation of China

Competing financial interests
The authors declare they have no actual or potential competing financial interests.

Ethics approval and consent to participate
All animal in this study underwent ethics approval by Ethics Committee of Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine.

Data availability
The authors declare that all data supporting the findings of this study are available within the article and its supplementary information files, or from the corresponding authors on reasonable request.     Thyroid hormone values in zebrafish exposed to control and TCS for120 days after fertilizing. Thyroid hormones concentrations in fish at 120 dpf were measured for each exposure group, with each sample homogenized from every single fish at 120 dpf. p trend for FT4 = 0.01. *p < 0.05, ** p <0.01.