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Chemosphere 235 (2019) 227e238

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Chemosphere

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Effects of nitrate on development and thyroid hormone signalingpathway during Bufo gargarizans embryogenesis

Lei Xie a, b, Yuhui Zhang a, Yanhua Qu c, Lihong Chai d, e, Xinyi Li a, Hongyuan Wang a, *

a College of Life Science, Shaanxi Normal University, Xi'an, 710119, Chinab College of Life and Environmental Science, Wenzhou University, Wenzhou, 325035, Chinac Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, Chinad School of Environmental Science and Engineering, Chang'an University, Xi'an, 710054, Chinae Key Laboratory of Subsurface Hydrology and Ecological Effect in Arid Region of Ministry of Education, Xi'an 710062, China

h i g h l i g h t s

� Nitrate could induce malformation and abnormal development in toad embryos.� Thyroid hormone signaling pathway were disrupted by nitrate.� Nitrate affected the expression levels of genes related to oxidative stress.� The spatial expression patterns of Dios and TRs were not changed by nitrate.

a r t i c l e i n f o

Article history:Received 28 April 2019Received in revised form22 June 2019Accepted 23 June 2019Available online 24 June 2019

Handling Editor: Jim Lazorchak

Keywords:NitrateEmbryoThyroid hormoneDevelopmentBufo gargarizans

* Corresponding author.E-mail address: hongyuanwang@snnu.edu.cn (H. W

https://doi.org/10.1016/j.chemosphere.2019.06.1770045-6535/© 2019 Elsevier Ltd. All rights reserved.

a b s t r a c t

Nitrate is known to disrupt the thyroid hormone, which is essential for the metamorphosis of am-phibians. However, few studies are focused on the effects of nitrate on the maternal thyroid hormone inearly amphibian embryos. We aimed to determine the impact of nitrate on maternal thyroid hormonesignaling pathway in Bufo gargarizans embryos. B. gargarizans embryos were exposed to different con-centrations of nitrate-nitrogen (NO3-N) for 7 days. High concentration of NO3-N (50, 100, and 200mg/L)could induce embryonic malformation and influence the development of embryos. In addition, maternalT4 and components of the thyroid hormone (TH) signaling pathway were detected by ELISA and RNA-seq, respectively. The expression levels of mRNA related to thyroid hormone and oxidative stress wereaffected in the early developing embryos in all NO3-N treatment groups. However, the T4 levels and thespatial expression patterns of type II iodothyronine deiodinase (D2), type III iodothyronine deiodinase (D3),thyroid hormone receptor a (TRa), and thyroid hormone receptor b (TRb) mRNA were not changed by ni-trate. In conclusion, the results of our study highlight the crucial role of the maternal thyroid hormonesignaling pathway in normal embryonic development, and the adverse effects of nitrate on theexpression levels of mRNA related to thyroid hormone signaling pathway and oxidative stress inB. gargarizans embryos.

© 2019 Elsevier Ltd. All rights reserved.

1. Introduction

In aquatic ecosystems, inorganic nitrogen occurs as three com-mon forms (ammonium, nitrite, and nitrate) (Rabalais, 2002). Ni-trate is the most stable and abundant form in waters, becauseammonia and nitrite are quickly oxidized to nitrate by bacteria andalgae (Rouse et al., 1999; Elisante and Muzuka, 2016). Natural

ang).

background concentrations of nitrate-nitrogen (NO3-N) in surfacewater and groundwater range from trace amounts to 0.6mg/L and3mg/L, respectively (Rouse et al., 1999; USGS, 2002; Panno et al.,2006). However, the man-made sources of nitrate, includingmunicipal and industrial wastewaters, runoff from feedlots, as wellas agricultural fertilization, have resulted in a significant increase ofnitrate in aquatic ecosystems (Camargo and Alonso, 2006; Wanget al., 2017). It has been reported that nitrate concentrations in avast area exceed the natural background concentrations and can beas high as with 35.7mg NO3-N/L in surface waters and 113mg NO3-

L. Xie et al. / Chemosphere 235 (2019) 227e238228

N/L in ground waters (Camargo et al., 2005; Ju et al., 2006; Xueet al., 2016). Thus, it is important to examine the effects of envi-ronmentally relevant concentrations of nitrate in species.

Many studies have focused on the thyroid effects of increasingnitrate in human and animals (Hecnar, 1995; Ward et al., 2010). Forexample, Drozd et al. (2016) found increased incidence of thyroidcancer in human was affected by high level of nitrate in thegroundwater. The presence of high concentrations of nitrate indrinking water had been considered as a goitrogenic factor in malerat (Zaki et al., 2004). In addition, Edwards et al. (2006) and Wanget al. (2015b) identified nitrate as an endocrine disruptor thataltered the thyroid hormone (TH) signaling pathway and delayedmetamorphosis, which was a TH dependent process, in amphibiantadpoles. Poulsen et al. (2018) suggested two main hypothesesconcerning mechanisms for thyroid hormone disruption by nitrate,namely (1) nitrate can affect iodide transport, which could impairthe thyroid hormone synthesis in thyroid gland; and (2) nitrate canbe converted to reactive nitric oxide, which could disturb theintegrity of essential proteins, resulting in the influence of genetranscription.

During the development of amphibians, TH can be providedeither maternally or synthesized by the thyroid glands. Thyroidglands form around the Nieuwkoop and Faber (NF) stage 43 inX. laevis (Nieuwkoop and Faber, 1956; Honda et al., 1993), and arefirst present at Gosner stage (Gs) 28 in B. gargarizans (Gosner, 1960;Liu et al., 2012). It has been previously shown that maternal TH isdetectable in eggs and embryos of Xenopus laevis before thyroidgland organogenesis begins (Morvan-Dubois et al., 2008; Fini et al.,2012), while endogenous TH can be detected as metamorphosisbegins (Brown and Cai, 2007). The two principle types of TH arethyroxine (T4) and triiodothyronine (T3). In vertebrates, type IIdeiodinase (D2) acts as an outer-ring deiodinase (ORD) byremoving iodine from the 5’ outer-ring site to convert T4 to themore active form T3, while type III deiodinase (D3) acts as an inner-ring deiodinase (IRD) catalyzes the removal of iodine atoms frominner-ring of T3 and T4 in order to generate the less active forms ofhormone diiodothyronine (T2) and reverse triiodothyronine (rT3),respectively (Power et al., 2001). In addition, previous studies findthat type I deiodinase (D1), which carries out both ORD and IRD,plays a minor role in deiodination during amphibian embryogen-esis (Morvan Dubois et al., 2006; Morvan-Dubois et al., 2008). Theactions of TH are mainly mediated by binding T3 to thyroid hor-mone receptors (TRa and TRb) (Moeller et al., 2006). Maternal THplays an important role in several metabolic and developmentalprocesses during early development, such as central nervous sys-tem (CNS) development (Darras, 2019), skin development (Ahmed,2018), and fetal skeleton development (Capelo et al., 2008). It istherefore necessary to use appropriate model organisms, such asamphibians, to assess and predict the potential impacts of nitrateon the maternal TH signaling pathway in humans.

TH plays particularly crucial role in antioxidant balance, sinceboth hyperthyroidism and hypothyroidism have been found to beassociated with oxidative stress in animals, including humans(Mancini et al., 2016). In addition, nitrates have the potential tointeract with mitochondrial function and modulate oxidative stress(Lundberg et al., 2011; Maguire et al., 2017). This means that thelevels of oxidative stress could be used as surrogate indicators forboth TH and nitrate pollution (Valavanidis et al., 2006; Herrera-Duenas et al., 2014). To avoid the damage from an overproductionof reactive oxygen species (ROS), which can lead to oxidative stress,cellular antioxidant defenses, such as superoxide dismutase (SOD)and glutathione peroxidase (GPx), have key functions (Carillonet al., 2013; Cordero-Herrera et al., 2015). The main role of SOD isto neutralize superoxide anion radicals (O2

�) into hydrogenperoxide (H2O2) (Zelko et al., 2002). Following this, the H2O2 is

converted to H2O by GPx and other antioxidant systems (Carillonet al., 2013). Additionally, heat shock proteins (HSPs) play acrucial role in maintaining protein homeostasis under sub-lethalstress conditions including oxidative stress (Kim et al., 2014).Therefore, the mRNA expression levels of SOD, GPx, and HSPs can beused as bioindicators of oxidative stress.

The Chinese toad (Bufo gargarizans) is widely distributed inmostareas of China and is not included in the International Union forConservation of Nature Red List. The embryos are spawned inponds, streams, and temporary pools that are often associated withagricultural areas receiving fertilizers. In addition, the free-livingembryos permit physiological experimentation independent ofmaternal factors (Fini et al., 2012). Due to these features,B. gargarizans could be considered a promising model organism forecotoxicological studies. In the present study, we evaluated theeffects of nitrate exposure on development in B. gargarizans em-bryos by examining malformation, body weight, total length, anddevelopmental stage of embryos. The effects of nitrate on theexpression levels of genes related to TH signaling pathway andoxidative stress in embryos were also evaluated. For a better un-derstanding the toxicology of nitrate on the embryos, the spatialexpression patterns of mRNA related to TH, including D2, D3, TRa,and TRbwere examined. The data should shed light on the effects ofnitrate on development and TH-related genes expression levelsduring amphibian embryonic development.

2. Materials and methods

2.1. Animal

Adult B. gargarizans of both sexes were obtained from a naturalpond in Qinling Mountains, Shaanxi Province, China (109�0700200E,33�5901800N) in February 2017. Males and females were then pairedand held into plastic aquariums with shallow dechlorinated tapwater. After spawning naturally, eggs were collected and raised in8 L tanks with shallow dechlorinated tap water. When all of theembryos reached Gosner stage (Gs) 3 (Gosner, 1960), they weretransferred to the experimental containers. The experiments werecarried out at approximately 18± 2 �C in a 12 h: 12 h light-darkphotoperiod. All studies were approved by the Laboratory AnimalCare and Use Committee of Shaanxi Normal University.

2.2. Chemicals and solutions

Sodium nitrate (NaNO3, molecular weight 84.9) was purchasedfrom Sigma-Aldrich Corporation (Sigma, St. Louis, MO). The stocksolution of nitratewas prepared by dissolving 6.07 g of NaNO3 in 1 Ldechlorinated tap water to final concentration of 1000mg/L NO3-N.Then the stock solutionwas dilutedwith dechlorinated tapwater totested concentration.

The pH, dissolved oxygen, and total hardness of the dechlori-nated tap water were measured using GDYS-201M multi param-eter water quality analyser (Little Swan, China). The conductivity,total chlorine, and total organic carbon (TOC) were measure byPC300 waterproof portable meter (Clean, USA), GDYS-101SN chlo-rine metre (Little Swan, China), and TOC-5000A TOC analyser(Shimadzu, Japan), respectively. In addition, the concentration ofNO3-N was measured using colorimetric aquarium nitrate test kit(Aquarium Pharmaceuticals Inc., USA).

A sample of the dechlorinated tap water was found to have thefollowing characteristics: pH 7.2± 0.2, dissolved oxygen6.8± 0.5mg/L, conductivity 241± 38mS/cm, total chlorine0.32± 0.06mg/L, TOC 3.69± 0.88mg/L and total hardness167± 27mg CaCO3/L.

L. Xie et al. / Chemosphere 235 (2019) 227e238 229

2.3. Experimental procedure and sampling

Based on the natural background concentration of NO3-N (5mg/L) in groundwater (Ministry of Health of the People's Republic ofChina, 2006) and the concentrations used in the previous studyof Wang et al. (2015b), six nominal NO3-N concentrations of testsolutions, three replicates for each, were set up: 0 (control), 5, 10,50, 100 and 200mg/L NO3-N, respectively. To ensure a consistentwater environmental conditions of experimental water, the watercharacteristics were checked at day 1, 3, 5, and 7. The resultsshowed that the measured values of NO3-N concentrations were1.97± 0.47, 6.14± 0.71, 10.52± 0.33, 50.78± 0.54, 102.74± 1.31, and199.28± 3.74 in control, 5, 10, 50, 100 and 200mg/L NO3-N groups,respectively.

Fifty embryos at Gs 3 for each concentration and replicate wereassigned to glass aquaria containing 4 L of tested solution (totaln¼ 150/treatment). The exposure solutions were renewed with a50% change on a daily basis, and the exposure time was 7 days(approximate to Gs 20e21). Embryos were observed daily fordetecting malformations, and the malformation rates were thencalculated. The malformed embryos were removed from theiraquaria immediately and documented using a dissecting micro-scope (Zeiss Discovery V12 stereoscope). After documented, mal-formed embryos were fixed for 24 h in phosphate buffered saline(PBS) containing 4% formaldehyde (pH 7.4) for scanning electronmicroscope (SEM) observations.

To analyze the effects of nitrate concentration on development,we sampled ten normal embryos randomly from each aquarium(n¼ 30/treatment) at day 5, 6 and 7, respectively. The developmentwas assayed by recording fresh body weight, total length anddevelopmental stage of individual sample. The body weight (mg)was weighted byMettler Toledo ME104E analytical balance (0.1mgprecision) after blotting water from the body. The total lengthmeasurements (mm)were recorded using Tesa-Cal Dura-Cal Digitalelectronic calipers (0.01 cm precision).

Developmental stages were determined using morphologycriteria described in Gosner (1960) and were recorded with aninteger. The Gosner stage (Gs) 19 is recognized by the heart beat. InGs 20, gill circulation is seen as a movement of corpuscles throughthe external gill filaments. In Gs 21, the cornea become transparentand the eyes are clearly discernible. In stage 22, the fins becometransparent and circulation with them begins. In stage 23, theexternal gills begin to disappear. In stage 24, there is only one sideof external gill left.

2.4. Scanning electron microscope

After 24 h fixing, the malformed embryos were dehydrated anddried according to the protocol from Ellinger and Murphy (1979).Finally, the samples were mounted on specimen stubs, sputtercoated with 10 nm Au/Pd. High-resolution SEM images werecaptured by Hitachi S-570 scanning electron microscope system.

2.5. Thyroid hormone measurement

The thyroid hormone levels in embryos at Gs 20 were measuredusing frog T4 ELISA kit (Jonln, cat no. JL47536). Approximately 0.3 gembryos from control and 200mg/L NO3-N treated groups werehomogenized in 1.2mL PBS with glass homogenizer on ice andwere sonicated with spasmodic ultrasonic disrupter. Then thesesamples were centrifugation at 5000�g for 10min at 4 �C, and thesupernatants were collected for T4 measurement. The measure-ment was carried out according to the manufacturer's instructions.Results were recorded on a microplate reader at 450 nm.

2.6. RNA extraction

For RNA-seq and qRT-PCR analysis, we randomly sampled fourembryos at Gs 20 from each treatment replicate. Embryos of eachtreatment replicate were pooled and snap frozen in liquid nitrogenfor 2 h and then stored at �80 �C for RNA extraction (n¼ 3/treat-ment). RNA extraction was carried out using E.Z.N.A.™ tissue RNAKit (Omega) according to the manufacturer's instructions. RNAquantity was tested using Thermo Scientific Nano Drop 2000spectrophotometer and RNA integrity was confirmed by 1% agarosegel electrophoresis.

2.7. RNA-seq analysis

The total RNA samples of control group and 200mg/L NO3-Nexposed group were sent to Guang zhou Gene Denovo Biotech-nology Co. Ltd for RNA-seq analysis. The process of RNA-seq anal-ysis were performed as manufacturer's protocol and the methoddescribed by Tan et al. (2013). More detailed procedures were listedin Supplemental Materials and Methods.

2.8. cDNA synthesis and quantification of gene expression by qRT-PCR

The total RNA of control group and all nitrate exposed groupswere reverse-transcribed to cDNA using the cDNA reverse tran-scription kit (BioRT Two Step RT-PCR Kit). Quantitative real-timePCR was conducted with CFX96™ Real-time Detection System(Bio-Rad) according to the manufacturer's protocol using the SYB-R®Pre-mix Ex Taq™ II (Tli RNaseH Plus) (TaKaRa, Code No. RR820A).Primer sequences of b-actin (internal control), SOD, GPx, HSP90, D2,D3, TRa, and TRb were designed by Primer 5.0 and listed inSupplementary Table S1. The expression level of each gene wascalculated using the 2�DDCT method Livak and Schmittgen (2001).More detailed procedures were listed in the Supplemental Mate-rials and Methods.

2.9. Whole-mount in situ hybridization

RNA sense and antisense probes for type II iodothyronine deio-dinase (D2), type III iodothyronine deiodinase (D3), thyroid hormonereceptor a (TRa), and thyroid hormone receptor b (TRb) were syn-thesized using the digoxigenin (DIG) RNA labeling kit (Roche, Cat.No.11175025910). For whole-mount ISH analysis, embryos at Gs 15,17 and 20 were randomly sampled (n¼ 12/treatment) and fixedwith 4% paraformaldehyde/0.1% Tween-20 in PBS (PBST) for 4 h atroom temperature. Then the whole-mount ISH were performed onthese embryos according to (Morvan Dubois et al., 2006). Embryoswere cleared in PBS and photographed under a dissecting micro-scope (Zeiss Discovery V12 stereoscope). More detailed procedureswere listed in the Supplemental Materials and Methods.

2.10. Statistical analysis

Statistical analyses were performed by IBM SPSS 23. The data ofmalformation rates, body weight, total length and developmentalstage were presented as mean± SD. Homogeneity of variances wasassessed using Levene's test, and Normality distributions wereexamined using KolmogoroveSmirnov normality test before con-ducting multiple comparison. One-way ANOVA with Fisher's leastsignificant difference (LSD) test was used to compare statisticallydifferences of malformation rates, body weight, total length,developmental stage, and genes expression. In addition, the mal-formation rates were analyzed by liner regression. Differences wereconsidered statistically significant at p� 0.05.

L. Xie et al. / Chemosphere 235 (2019) 227e238230

3. Results

3.1. Embryos malformations

Our results demonstrate that the malformation rates ofB. gargarizans embryos were 0.00± 0.00%, 0.67± 1.15%,1.33± 1.15%,3.33± 1.15%, 5.33± 3.06%, and 12.00± 3.46% for the control, 5, 10,50, 100, and 200mg/L NO3-N treatment groups after 7 days ofexposure (Fig. 1A). Exposure to 100 and 200mg/L NO3-N signifi-cantly increased the malformation rates of embryos (one-wayANOVA, F (5, 12)¼ 14.23, p< 0.05). Compared with the typical em-bryos (Fig. 1B1), the malformed embryos showed significant dif-ferences in appearance. Axial flexurewas a typical malformation, asseen in Fig. 1B2. In addition, the malformation of treatment em-bryos included pronounced prominence at the tail (Fig. 1B3), tumorand hyperplasia (Fig. 1B4), abdominal edema (Fig. 1B5) as well aswavy fin (Fig. 1B6). The percentages of each malformation in thedifferent treatments were listed in the Supplementary Table S2.Some malformed embryos in the 50, 100, and 200mg/L NO3-Ntreatment groups had two or more malformations.

The ultrastructure morphology of the embryos was observedusing scanning electron microscopy (Fig. 1C). The tail of a typicalembryo possessed of smooth, uniform surface with no pro-tuberances (Fig. 1C1). Several ciliated cells were sparsely distrib-uted on typical skin (Fig. 1C2). Additionally, the cilia of the ciliated

Fig. 1. The effect of nitrate on morphological malformations in B. gargarizans embryos. (B. gargarizans embryos. (B1) Normal embryo with yolk sac and tail in the control; Malformhyperplasia, (B5) abdominal edema, and (B6) wavy fin. bar¼ 1mm. Arrows indicate the mawf, wavy fin. (C) Morphological features of control and nitrate treated B. gargarizans embryocontrol embryos; (C4 and C7) gross morphology of the malformed embryo and (C5) magnsurface with hyperplasia.

cell were slender and neatly arranged (Fig. 1C3). The grossmorphology of the malformed embryo was characterized by axialflexure, fin hyperplasia and abdominal tumors (Fig. 1C4). Hyper-plastic fins were double-rowed, while the shape was distorted(Fig. 1C5). Abdominal tumors showed an outward protrusion, whiletheir epithelium was closely arranged (Fig. 1C6). Fig. 1C7 showsanother malformed embryo with tail edema and skin hyperplasia.Its edema was hollow and covered by a thin layer skin (Fig. 1C8).The cilia of the ciliated cell in the malformed embryo was disor-derly and unsystematic. In addition, the epithelium around theciliated cell were proliferated with different sizes (Fig. 1C9).

3.2. Effect of nitrate on growth and development of B. gargarizansembryos

Means of the fresh body weight, total length and developmentalstage were analyzed after 5, 6, and 7 days’ exposure. Results foundthat NO3-N exposure promoted growth and development ofB. gargarizans embryos (Table 1). On days 5 and 6, the embryos inthe 10, 50, 100, and 200mg/L NO3-N groups were significantlyheavier than embryos in the control group (one-way ANOVA, F (day

5), (5, 174)¼ 7.08, p< 0.05; one-way ANOVA, F (day 6), (5, 174)¼ 6.819,p< 0.05). On day 7, there were significant increase in the meanweight of the embryos in the 50, 100, and 200mg/L NO3-N treat-ment groups compared to the control group (one-way ANOVA, F (5,

A) Malformation rate. (B) Normal morphology and nitrate-induced malformations ofed embryo with (B2) axial flexion, (B3) pronounced prominence at tail, (B4) tumor,

lformed characters: ae, abdominal edema; hy, hyperplasia; pr, prominence; tu, tumor;s in Scanning Electron Microscope. (C1) Normal fin and (C2 and C3) normal surface ofification of fin hyperplasia, (C6) abdominal tumor, (C8) tail edema and (C9) abnormal

Table 1The body weight, total length and developmental stage of B. gargarizans embryos exposed to control and NO3-N treatment groups for 5,6 and 7 days.

exposure time groups (mg/L) body weight (mg) total length (mm) development stage

day 5 control 4.21± 0.84 5.38± 0.47 19.00± 0.005 4.42± 0.87 5.60± 0.33 19.00± 0.4110 5.38 ± 0.79* 5.91 ± 0.52* 19.21± 0.6150 5.63 ± 0.86* 5.98 ± 0.71* 19.53± 0.77100 5.90 ± 0.88* 6.26 ± 0.55* 19.90 ± 0.99*200 5.23 ± 0.92* 5.97 ± 0.72* 19.83 ± 0.93*

day 6 control 6.10± 0.79 6.71± 0.60 20.00± 0.005 6.29± 0.78 6.81± 0.58 20.53± 0.6810 6.68 ± 0.62* 6.99± 0.51 20.43± 0.5050 6.93 ± 0.65* 6.98± 0.64 21.33 ± 0.61*100 6.99 ± 0.83* 7.29 ± 0.70* 21.90 ± 0.32*200 6.83 ± 0.86* 7.29 ± 0.75* 21.70 ± 0.54*

day 7 control 7.70± 0.92 7.97± 0.46 20.60± 0.845 7.61± 0.73 8.15± 0.32 21.03± 0.3110 8.00± 0.63 8.02± 0.84 21.09± 0.6750 8.26 ± 0.70* 8.22± 0.55 21.05± 0.52100 8.29 ± 0.63* 8.59 ± 0.79* 22.00 ± 0.63*200 8.33 ± 0.81* 8.44 ± 0.47* 22.09 ± 0.29*

The data are presented as mean ± SD. Significant difference from the control group is indicated by *P � 0.05.

Fig. 2. Whole body T4 levels of Bufo gargarizans embryos in control and 200mg/Lgroup. The data are presented as mean± SD.

L. Xie et al. / Chemosphere 235 (2019) 227e238 231

174)¼ 9.53, p< 0.05).Similar to body weight, increased total lengths were observed at

10, 50, 100, and 200mg/L NO3-N treatment groups on day 5(one-way ANOVA, F (5, 174)¼ 8.90, p< 0.05). On days 6 and 7, only thetotal length of the 100 and 200mg/L NO3-N treatment groups weresignificantly longer than that of the control group (one-wayANOVA, F (day 6), (5, 174)¼ 4.34, p< 0.05; one-way ANOVA, F (day 7), (5,

174)¼ 2.81, p< 0.05).When compared to the control group, NO3-N also promoted the

mean developmental stage of B. gargarizans embryos. On day 5, thedevelopmental stage of the 100 and 200mg/L NO3-N groups weresignificantly increased compared with the control group (one-wayANOVA, F (5, 174)¼ 10.37, p< 0.05). On day 6, the developmentstages of the 50, 100, and 200mg/L NO3-N treatment groups were21.33± 0.61, 21.90± 0.32, and 21.70± 0.53, respectively, whichwere significantly higher than control group at 20.00± 0.00 (one-way ANOVA, F (5, 174)¼ 4.92, p< 0.05). On day 7, the condition wassimilar to that on the day 5, while the developmental stages of the100 and 200mg/L NO3-N groups were significantly increased whencompared to the control (one-way ANOVA, F (5, 174)¼ 3.48, p< 0.05).

3.3. Whole-body TH levels

The whole-body T4 levels of the embryos were detected byELISA. The T4 levels of control and 200mg/L NO3-N treatmentgroups were 8.91± 1.45 and 9.07± 0.41 pg/g, respectively (Fig. 2).There was no significant different between the two groups.

3.4. Transcriptome assembly and annotation

We sequenced the genome-wide transcriptomes of Gs20 ofB. gargarizans and obtained large amount of paired-end raw reads.After trimming and cleaning the rare adapter, poly-N or low-qualityreads, totals of 86,979,216 clean reads (two groups) wereconserved, with over 87% of sequences above Q30 quality, whichwere used to finish the de novo assembly. The resulting assemblycontained 137,264 unigenes. The length of unigenes ranged from200bp to 3 kb, with an N50 length of 1462 bp. The unigenes set wassubjected to BLAST similarity searches. Of 73,962 unigenes, 22829(30.87%), 7963 (10.77%), 15488 (20.94%) and 27097 (36.64%)showed significant similarities (BLAST, E value� 1.0� 10�5; orHMMER, E value� 1.0� 10�10) to protein-encoding genes in theCOG, GO, KEGG, and the Nr database, respectively (Supplemental

Table S3).Expression levels of genes involved in TH signaling pathway and

oxidative stress were identified in B. gargarizans embryos of boththe control group and the 200mg/L NO3-N treated group (Fig. 3;Supplemental Table S4). Compared with the control group, ex-pressions of transporter genes (SLC16A2 and SLCO1C) in the THsignaling pathway were found to be down-regulated in the NO3-Ntreated group. Expressions of deiodinase D1 and D2 were down-regulated, whereas D3 was up-regulated after treatment withNO3-N. TRs were disrupted by NO3-N compared to the controlgroup. Expressions of TRa and TRb were up-regulated followingtreatment with NO3-N. After TRs, expressions of corepressors(NCoR1 and Sin3A) and coactivators (p/CAF, SRC, and CBP) werefound to be down-regulated by NO3-N. The expression of RXR, themost frequent partner of TRs for binding as heterodimer, were alsodown-regulated after treated with NO3-N. As the target genes ofTRs, CCND1, GATA4, RCAN1, HIF1A, PLN, and NOTCH1were all down-regulated whileMYC,Wnt-4, and CTNNB1 were up-regulated in theNO3-N treated group. In addition, no significant differences in theexpression of SOD, GPx, and HSP90were found between the controland NO3-N treatment group.

Fig. 3. Proposed thyroid hormone signaling pathway from Wen and Shi (2016). T3 and T4 enter the cell through transporter proteins. D2 converts T4 to T3 whereas D1 and D3converts T3 and T4 into the inactive forms, T2 and rT3, respectively. TR/RXR heterodimers can both activate and repress the transcription of the target genes depending upon thepresence and absence of TH, respectively. T4, thyroxine; T3, triiodothyronine; rT3, reverse triiodothyronine; T2, diiodothyronine; SLCO1C, solute carrier organic anion transporterfamily, member 1C; SLC16A2, solute carrier family 16 (monocarboxylic acid transporters), member 2; D1, type I deiodinase; D2, type II deiodinase; D3, type III deiodinase; TR,thyroid hormone receptor; RXR, retinoid X receptor; TRE, thyroid hormone response elements; NCoR1, nuclear receptor co-repressor 1; Sin3A, paired amphipathic helix proteinSin3a; HDAC1, histone deacetylase 1; p/CAF, histone acetyltransferase; SRC, nuclear receptor coactivator 1; CBP, E1A/CREB-binding protein; CCND1, G1/S-specific cyclin-D1; GATA4,GATA-binding protein 4; RCAN1, calcipressin-1; HIF1A, hypoxia-inducible factor 1 alpha; MYC, Myc proto-oncogene protein; PLN, phospholamban; Wnt-4, wingless-type MMTVintegration site family, member 4; CTNNB1, catenin beta 1; Notch1, Notch homolog 1, translocation-associated.

L. Xie et al. / Chemosphere 235 (2019) 227e238232

3.5. Spatial expression of D2, D3, TRa, and TRb in B. gargarizansembryos

To better understand the function of TH signaling pathway, weidentified the mRNA expression pattern of D2, D3, TRa, and TRb inB. gargarizans embryos at 3 developmental stages by ISH. Resultsshowed that spatial expression patterns of D2, D3, TRa, and TRbwere not affected by nitratewhen compared with the control group(data not shown).

At the neural stage (Gs 14 and 15), expression of D2 mRNA wasfound in the branchial placode, cement gland, and along the neuralfolds (Fig. 4AeC). Expression of D3 mRNA could be seen in thebranchial placode, and cement glands at the neural stage(Fig. 4DeF). Expressions of TRa and TRb mRNA were observedaround the cement gland and neural tube posterior (Fig. 4Ge L).From the ventral view, the cement gland demonstrated anapproximate V-shaped structure (Fig. 4I and L).

At the tailbud stage (Gs17), expression of D2 was found in themidbrain, cement gland, somite and tailbud (Fig. 5AeC) whileexpression of D3 were detected in branchial arches, cement gland,otic vesicle, blood-forming region, somite and tailbud inB. gargarizans (Fig. 5DeF). In addition, our results demonstrated

that TRa and TRb were clearly expressed in the cement gland andtailbud at the tailbud stage (Fig. 5GeK). We also observed theexpression signal of TRb in both branchial arches and oral apparatus(Fig. 5L).

After hatching (Gs 20), expression regions of D2 included thecement gland, nose, branchial arches, oral apparatus, somite andtail (Fig. 6AeC). Expression of D3 were found in the cement gland,forebrain, branchial arches, oral apparatus, somite and tail(Fig. 6DeF). Strong signals of TRa could be seen in cement gland, tailbud and branchial arches region (Fig. 6G). Additionally, expressionsof TRa were found in the oral apparatus and nose (Fig. 6H and I).Expression of TRb mRNA were found in the cement gland, oralapparatus, branchial arch areas, and tailbud (Fig. 6J-L).

3.6. Effects of nitrate on D2, D3, TRa and TRb mRNA expression inembryos

Expression levels of the thyroid hormone-related genes (D2, D3,TRa, and TRb) are shown in Fig. 7A. At stage 20, expressions of D2and D3 were significantly decreased in all NO3-N groups whencompared to the control group (one-way ANOVA, F (D2) (5,

12)¼ 13.74, P< 0.05; one-way ANOVA, F (D3) (5, 12)¼ 3.74, P< 0.05)

Fig. 4. Expression of antisense D2, D3, TRa and TRb mRNA probe of B. gargarizans at neural stage. Arrows indicate the location of expression: bp, branchial placode; cg, cementgland; nf, neural fold; nt, neural tube. bar¼ 1mm.

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(Fig. 7A). In addition, when compared to the control group,expression of TRa were significantly increased in all NO3-N groups(one-way ANOVA, F (5, 12)¼ 17.38, P< 0.05). However, expressionsof TRb were not altered by NO3-N in any of the treatment groups(one-way ANOVA, F (5, 12)¼ 2.94, P> 0.05) (Fig. 7A).

3.7. Effects of nitrate on SOD, GPx, and HSP90 mRNA expression inembryos

At stage 20, expression levels of SOD and GPx were significantlydecreased in all NO3-N exposed embryos when compared to thecontrol group (one-way ANOVA, F (SOD) (5, 12)¼ 18.26, p< 0.05; one-way ANOVA, F (GPx) (5, 12)¼ 5.60, p< 0.05) (Fig. 7B). However, nosignificant difference of HSP90 mRNA level was found between thecontrol group and the NO3-N treated groups (one-way ANOVA, F (5,

12)¼ 2.82, p> 0.05).

4. Discussion

In the present study, exposure to 100 and 200mg/L NO3-Nsignificantly increased the malformation rates in B. gargarizansembryos (Fig. 1A). The dose-dependent tendency is consistent withthe study of Schuytema and Nebeker (1999), which found themalformation rates of X. laeviswere 4%, 29% and 50% for 111.0, 230.4and 470.4mg/L NO3-N after 5 days of exposure. It was suggestedthat the malformation induced by nitrate showed dose-dependentmanner in amphibians. However, it is difficult to compare thetolerance to nitrate between B. gargarizans and X. laevis since thesensitivity of various species to the same chemical substance maychange due to the differences in species characteristics. Forexample, B. gargarizans embryos hatched at Gs 19 (5 days of age),while X. laevis embryos hatched at NF stage 35e36 (2 days of age).The B. gargarizans embryosmight suffer shorter exposure time thanthat of X. laevis embryos without the protection of jelly coat. Inaddition, the habitat of B. gargarizans is mainly distributed near

Fig. 5. Expression of antisense D2, D3, TRa and TRbmRNA probe of B. gargarizans at tailbud stage. Arrows indicate the location of expression: ba, branchial arches; bf, blood-formingregion; cg, cement gland; mb, mind brain; oa, oral apparatus; ov, otic vesicles; st, somite; tb, tail bud. bar¼ 1mm.

L. Xie et al. / Chemosphere 235 (2019) 227e238234

farmland, while X. laevis generally live in slowly flowing water. Theenvironment they inhabit and the range of geographic distributionmight be another reason for the different tolerance (Fei et al., 2009).

The malformations of B. gargarizans were mainly characterizedby axial flexion, tumor, hyperplasia, abdominal edema andwavy finin our study (Fig. 1B and C). Malformations induced by nitrateexposure were also observed in the studies of other species. Forexample, nitrate could induce swelling, fin erosion, incomplete/anomalous pigmentation, and lack of scales in zebrafish(Learmonth and Carvalho, 2015), and induce lordosis, cardiac andabdominal edema in Pseudacris regilla and X. laevis embryos(Schuytema and Nebeker, 1999). Although the specific mechanismof nitrate-induced malformations remains unclear, the ante-roposterior axis curvature could be caused by inhibited neural andmesodermal convergent extension (Wallingford and Harland,2001), and body edema may be due to impaired osmoregulationcaused by high nitrate concentration (Hecnar, 1995).

Our results imply that nitrate exposure could promote growthand development of B. gargarizans embryos (Table 1). This growthpromoting effect was consistent with previous studies of Smithet al. (2005) and Wang et al. (2015b), which showed that nitratecould increase the fresh weight of Rana catesbeiana tadpoles andB. gargarizans tadpoles. However, some studies had demonstratednitrate exposure significantly decreased the larval length in RanaPipiens (Allran and Karasov, 2000), Bufo bufo, Bufo calamita, Pelo-phylax perezi, and Pelodytes ibericus (García-Mu~noz et al., 2011). Thedifferent effects of nitrate on growth and development might bedue to the different sensitivity of species to nitrate, because theyhave evolved in environments of different nutrient status and haveinherently different hormonal systems (Poulsen et al., 2018).

In the present study, maternal T4 and components of the THsignaling pathway were detected by ELISA and RNA-seq, respec-tively (Figs. 2 and 3). The TH signaling pathway has also beenidentified in X. laevis eggs and embryos (Morvan Dubois et al.,

Fig. 6. Expression of antisense D2, D3, TRa and TRb mRNA probe of B. gargarizans after hatching. Arrows indicate the location of expression: ba, branchial arches; cg, cement gland;fb, fore brain; ns, nose; oa, oral apparatus; st, somite; ta, tail. bar¼ 1mm.

Fig. 7. Relative transcription levels of (A) D2, D3, TRa, TRb, (B) SOD, GPx, and HSP90 compared to non-exposed controls at Gs 20 exposed to different concentrations of NO3-N. Thedata are presented as mean ± SD. *significantly different from control (p < 0.05).

L. Xie et al. / Chemosphere 235 (2019) 227e238 235

L. Xie et al. / Chemosphere 235 (2019) 227e238236

2006; Fini et al., 2012; Le Blay et al., 2018). It is known that TR havedual functions in the regulation of T3-inducible genes in the pres-ence and absence of T3, respectively (Sachs et al., 2000). MaternalT4, deiodinase and TR are present in embryos, and it is hypothe-sized that deiodinase converts maternal T4 to T3 by ORD, whichthen binds TR, allowing T3-inducible genes to be expressed at basallevels during early embryogenesis (Yen, 2015). In contrast, unli-ganded TR exerts an essential role in eye development in X. laevisembryos (Havis et al., 2006; Le Blay et al., 2018). It is possible that incertain tissues, the role of deiodinase is to maintain low levels of T3by IRD, thereby ensuring that TR is unliganded (Fini et al., 2012).

To further understand the functions of the TH signalingpathway, we identified the mRNA expression patterns of D2, D3,TRa, and TRb in B. gargarizans embryos by ISH at 3 developmentalstages. These genes were mainly expressed around the neural foldand neural tube at the neural stage, when neural organogenesis isinitiated. D2 and D3 are critically involved in determining theavailability of T3 in specific tissues and cell types due to their role inmodulating T3 availability by IRD or ORD (Fini et al., 2012). Thyroidhormone is essential for neonatal neurodevelopment (Bernal et al.,2003; Williams, 2008). Therefore, the current results indicate thatthe expression of these genes in the neural fold or neural tube isinvolved in providing thyroid hormone to neural cells, subse-quently regulating neurodevelopment.

At the tailbud stage (Gs17), the expression of D2 was found inthemidbrain, cement gland, somite and tailbud, and the expressionof D3 was detected in the branchial arches, cement gland, oticvesicle, blood-forming region, somite and tailbud in B. gargarizans.Our results were similar to the study of Tindall et al. (2007), whichreported that D2 was expressed in the forebrain, hindbrain andmidbrain, and D3 was expressed in the branchial arches, oticvesicle, blood-forming region, retina and notochord during tailbudstage in Xenopus tropicalis embryos. In addition, our results indicatethat TRa and TRb are clearly expressed in the cement gland andtailbud during the tailbud stage. Compared with the neural stage,the expression signals shift from the neural tube to the somite andtailbud, which are adjacent to the developing neural tube. Duringthe tailbud stage, embryos begin to develop recognizable heads andtails, along with the formation of many organs, including thebranchial arches, cement gland and optic vesicle. The expressionpatterns of D2, D3, TRa and TRb were consistent with these organs,and these patterns indicate that there are differing requirementsfor TH activity during this period.

During embryonic developments, our results indicate that post-hatching (Gs 20) expression patterns of D2, D3, TRa and TRb mRNAmaintain the patterns of the tailbud stage, and expression wasobserved in the cement gland, branchial arches, oral apparatus,somite, nose, and tail. These expression patterns were consistentwith the developing organs, such as the oral apparatus, branchialarches and cement gland. After hatching, embryos require well-developed organs to survive alone without the protection of a jel-ly coat. For example, embryos must ingest food through a func-tional oral apparatus to obtain energy. In addition, the cementgland allows the embryos to be adhered to hard surfaces, to preventthemselves from being washed away (Jin and Weinstein, 2018).Taken together, the dynamic distribution of the D2, D3, TRa and TRbsuggests that the TH signaling pathway is active during develop-ment. The regulation of genes by TH is tissue and developmentalstage specific to facilitate the coordination of different trans-formations in various organs.

In the present study, exposure to nitrate disrupted the maternalTH signaling pathway by down-regulating the expression levels ofthe transporter genes (SLC16A2 and SLCO1C) and deiodinase (D1and D2). In addition, the expression of TRa was up-regulatedfollowing nitrate treatment. The expression of corepressors

(NCoR1 and Sin3A), coactivators (p/CAF, SRC, and CBP) and RXRweredown-regulated by nitrate. However, maternal T4 levels were notaffected by nitrate, which may be due to the fact that nitrate doesnot react directly with T4. These data presented herein demon-strate that nitrate could affect the expression levels of geneinvolved in the maternal TH signaling pathway. The response of theTH signaling pathway to nitrate was consistent with the earlierstudy by Fini et al. (2012), which indicated that exposure to a THdisruptor modulated the expression of several TH target genes inX. laevis. We thus speculates that the mechanism for nitrateinduced disruption of gene expression may involve the conversionof nitrate to reactive nitric oxide, which disturbs the integrity ofessential proteins, ultimately affecting mRNA transcription andstability (Poulsen et al., 2018).

To validate the results of RNA-seq, the mRNA expression levelsof D2, D3, TRa, and TRb of B. gargarizans in control and nitratetreatment groups at Gs 20 were examined by qRT-PCR. The resultsshowed exposure to nitrate significantly decreased the expressionsof D2 and D3. The decreased expressions of D2 and D3 corre-sponded to a down-regulation trend in transcriptome profiles. D2and D3 make vital contributions to control the levels of TH(Morvan-Dubois et al., 2008). For example, an increased serumlevels of T4 was observed in D2 knockout mice (Schneider et al.,2001), while D3 knockout mice demonstrated low serum levels ofT4 and T3 from postnatal day 15 to adulthood (Hernandez et al.,2006). In this study, the levels of maternal T4 were not affectedby nitrate. Therefore, exposure to nitrate might disrupt the con-version from T4 to T3 by down-regulating the mRNA expression ofD2 and D3.

TRs, which play important roles in the effects of TH on cellularproliferation and differentiation, are used as molecular biomarkersfor evaluating TH signaling disrupting effects in amphibians (Opitzet al., 2006; Pascual and Aranda, 2013). In the present study,compared with control group, the expression of TRa was signifi-cantly increased in all nitrate treated groups. However, theexpression of TRb were not altered by nitrate exposure in alltreatment groups. Hinther et al. (2012) also reported that exposureto 5 and 50mg/L NO3-N did not result in a change in TRb transcriptlevels in cultured R. catesbeiana tadpole tail fin tissue. Previousstudies had shown that TRawas involved in main growth programsand TRb mediates the latest event in metamorphosis including tailresorption and organ remodeling (Furlow et al., 2004; Brown andCai, 2007). Thus, the increased TRa expression in nitrate treat-ment groups may promote the growth and development ofB. gargarizans embryos.

Nitrate can be reduced to nitrite, which is transformed into ni-tric oxide (NO), thus leading to the formation of free radicals(Pacher et al., 2007; Ortiz-Santaliestra et al., 2010). In addition,hyperthyroidism increase ROS production and hypothyroidismcause low availability of antioxidants (Mancini et al., 2016). SODand GPx are associated with eliminating ROS, while HSP90 isrelated to maintaining protein homeostasis (Lushchak, 2011). Inthis study, exposure to nitrate significantly decreased the expres-sions of SOD and GPx, but not the expression of HSP90. It has beenpublished that the increase or decrease in mRNA expressions ofSOD and GPx are consistent with the changes in enzymatic activityof SOD and GPx (Furukawa et al., 2004; Wang et al., 2015a). Thedown-regulated SOD and GPxmRNAmay reflect the low availabilityof antioxidants and lead to the accumulation of O2

� and H2O2.Oxidative stress occurs if the production of ROS is abnormallyincreased or antioxidant concentration decreases (Droge et al.,2006). Additionally, early studies have indicated that theincreased embryonic oxidative stress induced several teratogens inthe developing embryos (Damasceno et al., 2002; Ornoy, 2007).Thus, we could hypothesize that nitrate induced the accumulation

L. Xie et al. / Chemosphere 235 (2019) 227e238 237

of ROS and then resulted in oxidative stress, which might be apossible factor leading to embryonic malformations in nitratetreated B. gargarizans embryos.

5. Conclusion

In the present study, we found that exposure to 100 and 200mg/L NO3-N significantly increased the malformation rates ofB. gargarizans embryos. The decreased expression levels of SOD andGPx in 5, 10, 50, 100 and 200mg/L NO3-N groups showed that ni-trate could induce the accumulation of ROS and then result inoxidative stress. Thus, the morphological malformations may becaused by the nitrate induced oxidative stress. In addition, expo-sure to 50, 100, and 200mg/L NO3-N could promote the growth anddevelopment in B. gargarizans embryos. This growth promotingeffect might be associated with the abnormal expression levels ofD2,D3, and TRamRNA in 5,10, 50,100, and 200mg/L NO3-N groups.However, the spatial expression patterns of D2, D3, TRa and TRbduring embryonic development were not altered by 200mg/L NO3-N.

Based on the published surface water nitrate data in China, theconcentration of NO3-N exceeded 10mg/L (maximum at 35.7mg/L)inmany areas (Zhang et al., 2014; Xue et al., 2016). The NO3-N levelsin surface waters in these areas are high enough to probably causeadverse effects on B. gargarizans. The results of the present paperenhance the necessity of paying more attention to risk manage-ment, ecological security and aquatic ecotoxicological study ofnitrate.

Acknowledgments

The work was financially supported by the National NaturalScience Foundation of China (grant number 31572222), the NaturalScience Foundation of Shaanxi Province (grant number 2019JM-331).

Appendix A. Supplementary data

Supplementary data to this article can be found online athttps://doi.org/10.1016/j.chemosphere.2019.06.177.

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