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Environmental Pollution 243 (2018) 394e403

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Environmental Pollution

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Differences in reproductive toxicity of TBBPA and TCBPA exposure inmale Rana nigromaculata*

Hangjun Zhang a, b, c, Wenli Liu a, Bin Chen a, Jianbo He a, Feifei Chen a, Xiaodong Shan a,Qiongxia Du a, Ning Li a, Xiuying Jia a, Juan Tang a, *

a College of Life and Environmental Sciences, Hangzhou Normal University, Xuelin Road 16#, Xiasha Gaojiao Dongqu, Hangzhou, Zhejiang Province, 310036,Chinab Guangzhou Key Laboratory of Environmental Exposure and Health, School of Environment, Jinan University, Guangzhou, 510632, Chinac Key Laboratory of Hangzhou City for Ecosystem Protection and Restoration, Hangzhou Normal University, Hangzhou, 310036, China

a r t i c l e i n f o

Article history:Received 12 April 2018Received in revised form4 August 2018Accepted 25 August 2018Available online 30 August 2018

Keywords:Tetrabromobisphenol ATetrachlorobisphenol AReproductive toxicityAndrogen receptorRana nigromaculata

* This paper has been recommended for acceptMichele.* Corresponding author. College of Life and Enviro

Normal University, Hangzhou, Zhejiang, 310036, ChinE-mail address: tangjuanttjj@163.com (J. Tang).

https://doi.org/10.1016/j.envpol.2018.08.0860269-7491/© 2018 Elsevier Ltd. All rights reserved.

a b s t r a c t

Tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA) are persistent toxic environmentalpollutants that cause severe reproductive toxicity in animals. The goal of this study was to compare thereproductive toxic effects of TBBPA and TCBPA on male Rana nigromaculata and to expound on themechanisms leading to these effects. Healthy adult frogs were exposed to 0, 0.001, 0.01, 0.1, and 1mg/L ofTBBPA and TCBPA for 14 days. Sperm numbers were counted by erythrometry. Sperm mobility anddeformities were observed under a light microscope (400� ). We used commercial ELISA kits todetermine the serum content of testosterone (T), estradiol (E2), luteinizing hormone (LH) and folliclestimulating hormone (FSH). Expression of androgen receptor (AR) mRNA was detected using real-timeqPCR. Sperm numbers and sperm mobility were significantly decreased and sperm deformity wassignificantly increased in a concentration dependent manner following exposure to TBBPA and TCBPA.Sperm deformity was significantly greater in the 1mg/L TCBPA (0.549) treatment group than in the 1mg/L TBBPA (0.397) treatment group. Serum T content was significantly greater in the 0.01, 0.1 and 1mg/LTBBPA and TCBPA experimental groups compared with controls, while E2 content was significantlygreater in only the 1mg/L TBBPA and TCBPA experimental groups. Expression levels of LH and FSHsignificantly decreased in the 1mg/L TBBPA and TCBPA treatment groups. AR mRNA expressiondecreased markedly in all the treated groups. Our results indicated that TBBPA and TCBPA inducedreproductive toxicity in a dose-dependent manner, with TCBPA having greater toxicity than TBBPA.Furthermore, changes in T, E2, LH, and FSH levels induced by TBBPA and TCBPA exposure, which led toendocrine disorders, also caused disturbance of spermatogenesis through abnormal gene expressions ofAR in the testes.

© 2018 Elsevier Ltd. All rights reserved.

1. Introduction

In recent decades, with the rapid development of technologyand industry, a variety of chemical products has been developed.However, widespread use of chemical products with persistenttoxic pollutants may lead to many environmental problems. Tet-rabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA)

ance by Dr. Harmon Sarah

nmental Sciences, Hangzhoua.

are two types of persistent toxic environmental pollutants, pri-marily used in flame retardants, that have become common in theenvironment around the world (de Wit et al., 2010; Saint-Louis andPelletier, 2004; Berger et al., 2004; Morris et al., 2004). TBBPA hasbeen found in drinking water stored in polyethylene-carbonatecontainers (Peterman et al., 2000), and up to 300 ng/g TBBPA hasbeen detected in the estuarine sediment of the Saint LawrenceRiver in Quebec, Canada. In addition, high levels of TBBPA havebeen found in the sludge sediment of a sewage treatment plant inSwitzerland and in marine sediment near the Osaka Bay Estuarinein Japan (Letcher and Chu, 2010). TBBPA can enter the atmospherethrough various channels and can exist as an in atmospheric par-ticulates (Zweidinger et al., 1979). The average concentration of

H. Zhang et al. / Environmental Pollution 243 (2018) 394e403 395

TBBPA in the workshop air was 0.20 ng/m3, and the average con-centration of TBBPA in the computer maintenance plant was0.035 ng/m3 (Tollb€ack et al., 2006). TCBPA also has been detected inriver sediments (Letcher and Chu, 2010). One study found that theconcentration of TCBPA in a river sediment sample was 542.6 ng/g(Yuan et al., 2010). TCBPA has also been found in human breast milk(Bastos et al., 2008) and in urine (Rodríguez-G�omez et al., 2014).Therefore, it is essential to further investigate the potentiallyharmful impacts of TBBPA and TCBPA on both wildlife and humans.

Reproductive toxicity effects of TBBPA and TCBPA have beenreported in some organisms (Chapin et al., 2008). Studies haveshown that TBBPA has weak estrogenic activity and reproductivetoxicity (Thomsen et al., 2001). In one study, TBBPA exposureresulted in reduced sperm counts in Sterlet as well as an increase inthe amount of abnormal sperm (Linhartova et al., 2015). Moreover,TBBPA may induce testicular cell apoptosis (Kuiper et al., 2007;Zatecka et al., 2013) and reductions in sperm quality (Ogunbayoet al., 2008). Linhartova et al. (2015) suggested that TBBPA expo-sure caused epididymal sperm DNA damage and protein distribu-tion anomalies. TCBPA has been shown to alter animal behaviorthat may lead to potential developmental toxicity and/or endocrinedisorders (Zatecka et al., 2014). However, despite research on theeffects of these toxins, it is imperative to compare differences inreproductive toxicity and to clarify the reproductive toxicitymechanism of TBBPA and TCBPA. One study has compared thedevelopmental toxicity and estrogenic activity of TBBPA and TCBPAin zebrafish (Song et al., 2014). In this study, the half lethal con-centration values of the chemicals suggests that TCBPA has greatertoxicity than TBBPA, and this result is particularly beneficial indetermining the water quality guidelines for test chemicals in riverand lakes. Further comparison of the reproductive toxicity of TBBPAand TCBPA can provide a scientific basis for choosing suitable flameretardants and for setting water quality guidelines for thesechemicals in river and lakes.

Sperm count, motility, and deformity are the basic indicators ofsperm quality in male reproductive toxicology; these parameterscan be used to evaluate the fertility of a species (Ankley et al., 1998).The male sex steroid testosterone (T) acts as a significant factor inspermatogenesis and ensures the normal phenotype of malereproductive system (Scott et al., 2009). One study showed thatTBBPA interfered with the secretion of T (Dankers et al., 2013).Estradiol (E2) is also essential for the formation of sperm. Studieshad reported that TBBPA has some properties similar to estrogen(Kitamura et al., 2002; Samuelsen et al., 2001) and confirmed thatTBBPA exhibits anti-estrogenic activity (Kitamura et al., 2005).However, the estrogenic activity of TCBPA may be higher than BPA(Vandenberg et al., 2009). Luteinizing hormone (LH) and folliclestimulating hormone (FSH) influenced sperm formation, spermmaturation, testis development, and other reproductive functions(Shiraishi and Matsuyama, 2017). Furthermore, LH and FSH canaffect the development of spermatocyte and regulate androgenreceptors (AR) in the testis. AR also affects sperm formation andmale fertility (Wang et al., 2009a). Damage to androgen receptorshas been shown to cause infertility in male mice (Beck et al., 2016).Thus, the study of T, E2, LH, FSH and the AR gene helps to clarify themechanisms by which TBBPA and TCBPA affect the sperm index.While numerous studies have reported on the reproductive toxicityof TBBPA and TCBPA in mammals (Szychowski and Wojtowicz,2016; Zatecka et al., 2014; Zatecka et al., 2013), little work hasexamined reproductive toxicity of these chemicals in amphibians.

Stuart et al. (2004) found that the number and variety of am-phibians around the world has been rapidly decreasing. Theattenuation problem of a variety of amphibian species has been thetopic of many studies (Houlahan et al., 2000; Kiesecker et al., 2001;Palen and Schindler, 2010). At least 2468 amphibians are

experiencing a decline in numbers (Stuart et al., 2004). There are100 species of amphibians in China (27.3% of all species) that areconsidered extinct, critically endangered, or endangered (Xie et al.,2007). It was confirmed that environmental pollution was one ofthe main causes of amphibians declines (Hayes et al., 2002) andthat amphibians are vulnerable to the toxicity of these compounds(Kerby et al., 2010). Amphibians can be used as a yardstick tomeasure the environmental health of a global ecosystem. Among allamphibians, Rana nigromaculata has a high sensitivity to pollutionand environmental impacts, which can be a good biological indi-cator of environmental pollution and ecosystem health (Hopkins,2007). R. nigromaculata has been listed as an endangered speciesby the International Union for the Conservation of Nature (IUCN)since 2004. Therefore, R. nigromaculata is a suitable study species toclarify the reproductive toxicity of TBBPA and TCBPA.

Here, we focused on TBBPA and TCBPA-induced reproductivetoxicity, with the hypothesis that PNP-induced reproductiveendocrine disturbance may be associated with changes in T, E2, LHand FSH content changes and AR gene expression in maleR. nigromaculata. We also compared the reproductive toxicity ofdifferences between TBBPA and TCBPA. Those results of this studywill provide a new scientific basis and theoretical basis for the toxiceffects of both TBBPA and TCBPA on amphibian population.

2. Materials and methods

2.1. Chemicals

TBBPAwas purchased fromAladdin Industrial Corporation (FengXian, Shanghai, China). TCBPA was purchased from TCI IndustrialDevelopment Corporation (Shanghai, China). Dimethyl sulfoxide(DMSO) was obtained from Lingfeng Chemical Reagent Corporation(Shanghai, China). FSH, LH, T and estradiol assay ELISA kits wereobtained from Nanjing Jiancheng Bioengineering, Inc., China.Chemical information for TBBPA, TCBPA and DMOS is given inTable 1.

2.2. Animals acquisition and treatment

Three year old male R. nigromaculata frogs were purchased fromthe outskirts of Huzhou (Zhejiang, China). Frogs were acclimatizedin aquariums (30 cm� 30 cm� 60 cm) for 14 days. Subsequently,robust male frogs were chosen and randomly assigned to twelvegroups of 20 frogs each. Treatment groups were subjected to toxinexposure of either TBBPA or TCBPA at concentrations of 0, 0.001,0.01, 0.1, 1mg/L. The control group was kept in a 2 L volume ofdechlorinated tap water. A 0.5% DMSO treated group served as areagent blank. TBBPA and TCBPA were dissolved in 0.5% DMSO toget stock solutions. Four groups were exposed to either 0.001, 0.01,0.1, or 1mg/L TBBPA and four groups were exposed to either 0.001,0.01, 0.1, or 1mg/L TCBPA for 14 days. All control and treatmentgroups were maintained under standard experimental conditions(temperature range of 18�Ce20 �C; pH range of 6.0e7.0; dissolvedoxygen range of 6e8mg/L). A static displacement method was usedto change aerated water every day by 6:00 p.m. Frogs were fedEisenia fetida twice a day (8:00 a.m., 6:00 p.m.) and maintained in anatural light/dark cycle.

After the 14 day exposure to either TBBPA or TCBPA, we sacri-ficed the frogs by pithing. No other individual deaths wereobserved throughout the experiment. Blood was collected by car-diac puncture and then transferred to centrifuge tubes for serumseparation and precipitation (centrifugation at 4000 rpm for10min, 4 �C). Serum samples were stored at - 20 �C prior to getready for T, E2, LH, and FSH assays. After the appurtenant waseliminated, tissue from the testis was cut and weighed. Tissues

Table 1Data on TBBPA, TCBPA and DMSO.

Chemicals Abbreviation Suppliers CAS Purity (%)

3,3,5,5-Tetrabromobisphenol A TBBPA Aladdin Industrial Corporation 79-94-7 >98.02,20 ,6,6-Tetrachlorobisphenol A TCBPA TCI 79-95-8 >98.0Dimethyl sulfoxide DMSO Shanghai Lingfeng Chemical Reagent Corporation 67-68-5 >99.0

H. Zhang et al. / Environmental Pollution 243 (2018) 394e403396

were washed twice in a phosphate-buffered solution (PBS) andstored at - 80 �C with liquid nitrogen prior to chemical treatment.Animal care and experimental treatments followed the guidelinesof the Experimentation Ethics Review Committee.

2.3. Determination of TBBPA and TCBPA concentrations in water

Water samples containing TBBPA, TCBPA and 0.5% DMSO werecollected and chemical concentrations were determined based onpreviously reported methods (Chu et al., 2005; Lu et al., 2018).Solid-phase extraction (SPE) was performed using an Oasis HLBcartridge (6mL, 200mg, Waters, Massachusetts, USA). Watersamples were injected into an ultrahigh-performance liquid chro-matography tandem mass spectrometer (UPLC�MS/MS) (AcquityUPLC, Waters). A 3/1 signal-to-noise ratio (S/N) and method-detection limits (MDLs) were used to determine the detectionlimits in the water samples (n¼ 3). Specific details are described inthe Supplemental Material (S1). The measured actual exposureconcentrations, qualitative assessment, and quantification of TBBPAand TCBPA are provided in the Supplemental Material (Table S1-3).

2.4. Sperm assays

Sperm suspensions were collected for sperm index de-terminations. Phosphate-buffered solutionwas added to centrifugetubes (1.5 mL) with a ratio of 1:9 according to the weight of thecollected testicles. Sperm counts were conducted by placing a 20 mLsample of each sperm suspension on a slide in a hemocytometerchamber. The number of sperm in four squares (A1-A4) werecounted under a microscope (400 � ), and the sperm count wasdetermined as follows: sperm count ¼ (A1þA2þA3þA4)/100 � 4 � 106 (Jia et al., 2014).

Sperm motility was observed under an optical400 � microscope. Sperm motility was expressed as a percentage(h), which was calculated as: h¼ (aþ bþ c)/(aþ bþ cþ d), where ais the number of sperm with high mobility; b is the number withnormal mobility; c is the number with weak mobility; and d is thenumber of dead sperm (Jia et al., 2009).

To determine sperm deformity, an 800 mL sperm suspensionwascollected by filtering through 4 layers of lens paper. We centrifugedthe filtrate (centrifugation at 400� g, 5min), removed the super-natant, gently agitated the remaining liquid and then addedmethanol to the precipitate. After incubation, the solution wasstained by 1% eosin Y. Then, 10 mL of the dyeing liquid mixture wasput on a hemocytometer slide to determine spermmorphology.Werecorded the percentage of sperm cell abnormalities observedunder a light microscope at 400�magnification.

2.5. Morphology observation by electron-transmission microscope

Testes samples were fixed in 2.5% glutaraldehyde and stored at4 �C overnight. Samples were post-fixed in 1.5% osmic acid for 1 h,washed with 0.1M phosphate buffered saline, and then dehydratedin a graded series of 50%e100% alcohol. Samples were then fixed topure acetone and sliced. Sections were stained with uranyl acetateand lead citrate, and imaged using an electron-transmission mi-croscope (H-7650, Hitachi, Japan).

2.6. Histomorphometric analysis

Fresh testes tissue was first stained with hematoxylin and eosinand then observed under a BX20 fluorescence microscope(OLYMPUS, Japan). Specific details are described in the Supple-mental Material (S2).

2.7. Hormone analysis

Commercial ELISA kits were used to determine the content of T,E2, LH and FSH in the serum. For each sample, 40 mL of serum wasadded to a well of a 96-well plate, followed by 10 mL of the corre-sponding standard biotin labeled antibody and 50 mL ofstreptavidin-HRP. Plates were incubated for 60min at 37 �C. Afterplates were washed with scrubbing solution 5 times, a 50 mL vol-ume of both staining fluid A and B were placed in every well andgently mixed. Plates were incubated at 37 �C for 10min in the dark.Finally, a 50-mL volume of stop-solution was added into every well,and the assay was visualized at 450 nm wavelength for 10min.Testosterone, E2, LH, and FSH hormone analyses were repeatedthree times.

2.8. Androgen receptor gene expression level

Total RNA was extracted using Trizol Reagent (Invitrogen, USA)and purified using an RNase-free DNase Set kit (Qiagen, USA). RNAquality was determined by gel electrophoresis and comparison of260/280 nm absorbance ratios. RNA was reverse transcribed intocDNA by using SuperScript™ III First-Strand Synthesis SuperMix kit(Invitrogen Life Technologies, Germany), according to the manu-facturer's instructions. Primers are shown in Table 2. Subsequently,a CFX384 Touth™ Real-Time PCR Detection System was used toperform the qRT-PCR (Bio-Rad, USA). The reaction mixturesincluded 10.0 mL power SYBR® Green Master Mix (Applied Bio-systems, USA), 8.0 mL SDW, 1.0 mL cDNA, 0.5 mL forward primers and0.5 mL reverse primers. Reactions were carried out in a thermo-cycler under the following conditions: initial denaturation at 95 �Cfor 1min, followed by 40 cycles of denaturation at 95 �C andannealing at 63�Cfor 25 s. The gene was used to normalize geneexpression. Changes in gene expression were quantified using theCt (2- △△Ct) method (Livak and Schmittgen, 2001).

2.9. Statistical analysis

The data was analyzed using Origin 8.5 software; themean ± standard deviation (SD) are given. Statistical analyses wereperformed using GraphPad Prism software (San Diego, CA, USA).*P < 0.05, **P < 0.01, #P< 0.05, ##P< 0.01, and $ all indicate sig-nificant differences.

3. Results

3.1. Sperm analysis

Sperm indices are shown in Fig. 1. Sperm numbers weresignificantly lower in the groups exposed to 0.01, 0.1 and 1mg/LTBBPA (P< 0.01, Fig. 1 A) compared with controls; a significant

Table 2Real-time PCR primers and conditions.

Gene Genbank Accession Primer Sequences (50to 30) Size/bp Annealing/oC

AR KY427935 F:50-GGAGGCACTGGAGCATCTGAG-30 150 60R:50eCCTTCACGGTACTGGGAGTTTGA-30

H. Zhang et al. / Environmental Pollution 243 (2018) 394e403 397

decrease was also detected in frogs exposed to 0.1 and 1mg/LTCBPA (P< 0.01). Additionally, sperm counts were significantlylower in frogs exposed to 1mg/L TBBPA (1.61� 106) as compared to1mg/L TCBPA (2.45� 106).

A significant decrease in sperm motility was observed in the1mg/L TCBPA exposure group (P< 0.01, Fig. 1 B). After 14 days ofexposure to 0.01, 0.1 and 1mg/L TBBPA, a significant decrease inmotility was also observed in treatment groups compared with thecontrol group (P< 0.05). Furthermore, the decrease in spermmotility was more significant in the 0.01, 0.1, and 1mg/L TCBPAgroups (0.47, 0.443, and 0.4, respectively) than in the correspondingTBBPA groups (0.330, 0.353, and 0.267, respectively).

Sperm deformity in the testes of male frogs exposed to 0.001,0.01, 0.1 and 1mg/L TBBPA and TCBPA are shown in Fig. 1 C.Compared with the control group of male frogs, significant spermdeformity was detected in the 0.001, 0.1 and 1mg/L TBBPA treat-ment group, as well as in the 0.01, 0.1 and 1mg/L TCBPA treatmentgroups (P< 0.01). However, sperm deformity increased moresignificantly in the 1mg/L TCBPA group (0.549) than in the corre-sponding TBBPA group (0.397).

3.2. Ultrastructural observations of sperm in the testes

Morphological observations of the sperm of frogs exposed to0.001, 0.01, 0.1 and 1mg/L TBBPA for 14 days are shown in Fig. 2.Sperm from the control groupwas normal (Fig.1 A and B). However,the 0.001mg/L TBBPA treatment group exhibited vacuoles in thetesticular sperm (Fig. 2 C). Compared with the control group,abnormal condensations were found in the end of sperm tails afterfrogs were exposed to 0.01mg/L TBBPA (Fig. 2 D). Spermatozoawith swollen necks and abnormal condensations in the sperm tailwere observed in the group treatedwith 0.1mg/L TBBPA (Fig. 2 E). Alarge number of vacuoles, abnormal condensation of sperm tails,excessive lysosome in the end of the tail, or absence of sperm tailswere detected in the testes of frogs exposed to 1mg/L TBBPA (Fig. 2F).

Morphological observations of the sperm from frogs exposed to0.001, 0.01, 0.1 and 1mg/L TCBPA for 14 days are shown in Fig. 2. Thecontrol group showed no abnormal sperm (Fig. 2 G and H). Incontrast, abnormal condensation in the sperm tail was noted in the

Fig. 1. Effects of TBBPA and TCBPA on the number of sperm cells, sperm motility and sperm dL) for 14 days. Bars represent mean ± S.D. Asterisks and dollar signs denote significant dif$P< 0.05, $$P< 0.01).

0.001mg/L TCBPA treatment group (Fig. 2 I). Abnormal condensa-tion in the sperm tail and spermatozoa with swollen necks werefound in frogs exposed to 0.01mg/L TCBPA (Fig. 2 J). Numerousvacuoles, spermatozoa with swollen necks and abnormal conden-sation in sperm tails were observed in the 0.1mg/L TCBPA treat-ment group (Fig. 2 K). An absence of sperm tails was noted in frogsexposed to 1mg/L TCBPA (Fig. 2 L).

3.3. Histopathological evaluation

HE-stained testes sections from male frogs exposed to 0.001,0.01, 0.1 and 1mg/L TBBPA for 14 days are shown in Fig. 3. Testeswith normal histomorphology were found in the control group(Fig. 3 A). Histopathological observation revealed no differencesafter frogs were exposed to 0.5% DMSO (Fig. 3 B). However, vacuoleswere observed after exposure to 0.001mg/L TBBPA (Fig. 3 C). After14 days of 0.01mg/L TBBPA exposure, an increase in the disorder ofthe spermatogenic cell array was observed (Fig. 3 D). Spermato-genic cells were dispersed and shed to different degrees in frogsfrom the 0.1mg/L TBBPA treatment group (Fig. 3 E). Aberrant vac-uoles and a significant increase in the disorder of the spermato-genic cell array were noted after male frogs were exposed to 1mg/LTBBPA (Fig. 3 F).

HE-stained testes sections from male frogs exposed to 0.001,0.01, 0.1 and 1mg/L TCBPA for 14 days are shown in Fig. 3. Fig. 3 Gshows the normal histomorphology of testes without TCBPAexposure. No differences in histomorphology were observed in thegroup exposed to 0.5% DMSO as compare with the control group. Incontrast, vacuoles were detected in frogs exposed to 0.001mg/LTCBPA for 14 days (Fig. 3 I). An increase in the disorder of thespermatogenic cell array was noted in the 0.01mg/L TCBPA treat-ment group (Fig. 3 J). Detachment was observed to varying extentsin frogs exposed to 0.1mg/L TCBPA (Fig. 3 K), and increased vacu-oles were detected in the 1mg/L TCBPA-treated group (Fig. 3 L).

3.4. Testosterone and estradiol analysis

The effects of 0.001, 0.01, 0.1 and 1mg/L TBBPA and TCBPAexposure on serum T and E2 content are shown in Fig. 4. Comparedwith the control group, T content increased significantly after male

eformity in testes. Male frogs were exposed to TBBPA and TCBPA (0.001, 0.01, 0.1, 1mg/ferences between TBBPA and TCBPA compared with the control (*P< 0.05, **P< 0.01,

Fig. 2. Morphology of sperm from male frogs exposed to 0, 0.001, 0.01, 0.1 or 1mg/L TBBPA and TCBPA for 14 days. (A) Sperm from the testes of the control group (3000� ). (B)Sperm from the testes of frogs exposed to 0.5% DMSO. (C) Vacuoles in the sperm tails after exposure to 0.001mg/L TBBPA for 14 d (3000� ) (white arrow). (D) Abnormalcondensation of sperm tails after exposure to 0.01 g/L TBBPA for 14 d (3000� ) (black arrow). (E) Spermatozoa with a swollen neck of the sperm tail after exposure to 0.1mg/L TBBPAfor 14 d (3000� ) (short black arrow). (F) Absence of sperm tails after exposure to 1mg/L TBBPA for 14 d (3000� ) (white triangle). (G) Normal sperm from the testes of the controlgroup (3000� ). (H) Sperm from the testes after frogs were exposed to 0.5% DMSO. (I) Abnormal condensation of sperm tails after frogs were exposed to 0.001 g/L TCBPA for 14 d(3000� ; black arrow). (J) Abnormal condensation and spermatozoa with swollen necks of sperm tails after frogs were exposed to 0.01mg/L TCBPA for 14 d (3000� ) (short blackarrow). (K) Vacuoles and swollen necks of the sperm tails were observed after exposure to 0.1mg/L TCBPA for 14 d (3000� ) (white arrow). (L) Absence of sperm tails after exposureto 1mg/L TCBPA for 14 d (3000� ) (white triangle).

H. Zhang et al. / Environmental Pollution 243 (2018) 394e403398

frogs were exposed to 0.01mg/L TBBPA (P< 0.05, Fig. 4 A). Signifi-cant increases were observed in the 0.1 and 1mg/L TBBPA treat-ment groups and the 0.01, 0.1 and 1mg/L TCBPA treatment groups(P< 0.01) compared with the control group. The increase in Tcontent was more significant in male frogs exposed to 1mg/LTBBPA than 1mg/L TCBPA.

Compared with the male frogs in the control group, E2 serumcontent increased significantly in both the TBBPA and TCBPA 1mg/Ltreatment groups (P< 0.01, Fig. 4 B). E2 content also increasedsignificantly in the group exposed to 0.01mg/L TCBPA (P< 0.01).However, the increase in E2 content was more significant in the0.01mg/L TCBPA treatment group than in the corresponding TBBPAgroup, whereas the increase was more significant in the 1mg/LTBBPA group than in the corresponding TCBPA group.

3.5. LH and FSH analysis

The levels of LH and FSH after TBBPA and TCBPA exposure areshown in Fig. 5. LH content decreased significantly after the malefrogs were exposed to 0.001e0.1mg/L of TBBPA compared with thecontrol group (P< 0.01, Fig. 5 A). LH content decreased significantlyin the groups exposed to 0.1 and 1mg/L TCBPA compared with thecontrol group (P< 0.01). These results demonstrated a dosage effect

for both TBBPA and TCBPA exposure on LH expression. Further-more, the decrease in LH content was more significant in the0.001mg/L TBBPA group than in the corresponding TCBPA group,whereas the decrease was more significant in the 1mg/L TCBPAgroup than in the corresponding TBBPA group.

After a 14-day exposure to 0.1 and 1mg/L TBBPA, FSH contentdecreased significantly compared with the control group (P< 0.05,Fig. 5 B). However, compared with the control group, FSH contentshowed no significant change after the male frogs were exposed toTCBPA. In addition, the decrease in FSH content was more signifi-cantly following exposure to TBBPA as compared with TCBPA in the0.1mg/L groups.

3.6. AR gene expression level analysis

The relative levels of AR mRNA increased significantly after the14 day exposure to 0.001, 0.01, 0.1, and 1mg/L TBBPA and TCBPAcompared with that in the corresponding control group (P< 0.01,Fig. 6). These results demonstrate that TBBPA and TCBPA have adose-effect relationship on AR gene expression level. The decreasein AR gene expression was more significant following exposure toTBBPA than to TCBPA in the 0.001e1mg/L groups (see Fig. 7).

Fig. 3. HE staining of testes tissue from male frogs exposed to 0, 0.001, 0.01, 0.1, 1mg/L TBBPA and TCBPA. A to F are TBBPA exposure and G to L are TCBPA exposure. (A and G)Normal control groups. (B and H) No difference was observed in 0.5% DMSO exposure group. (C) Vacuoles were observed in the group exposed to 0.001mg/L TBBPA (short blackarrow). (D) Disorder of the spermatogenic cell array was observed in the 0.01mg/L TBBPA treatment group (black arrow). (E) Detachment was noted in the 0.1mg/L TBBPA treatmentgroup (black triangle). (F) A more significant disorder of the spermatogenic cell array was observed in the 1mg/L TBBPA treatment group (black arrow). (I) Vacuoles were observedin the group exposed to 0.001mg/L TCBPA (short black arrow). (J) Disorder of the spermatogenic cell array was noted in the 0.01mg/L TCBPA treatment group (black arrow). (K)Detachment was observed in the 0.1mg/L TCBPA treatment group (black triangle). (L) Aberrant vacuoles were observed in the group exposed to 1mg/L TCBPA (short black arrow).

H. Zhang et al. / Environmental Pollution 243 (2018) 394e403 399

4. Discussion

We investigated the reproductive toxicity of TBBPA and TCBPAexposure in male R. nigromaculata and compared the reproductivetoxicity differences between TBBPA and TCBPA treatment groups.Our results demonstrated that TBBPA and TCBPA caused a decreasein sperm number and activity, and caused an increase in spermdeformities. TBBPA and TCBPA exposure also caused changes in T,E2, LH, and FSH. Thus, TBBPA and TCBPA caused reproductivedamage, and this damage was more serious in groups exposed toTCBPA. In addition, TBBPA and TCBPA induced changes in T, E2, LHand FSH levels, which may lead to endocrinopathy and cause thedisturbance of spermatogenesis by abnormal gene expressions ofAR in the testes.

The main factor leading to decreases in amphibian populationsis environmental pollutants (Hayes et al., 2010). TBBPA and TCBPAare used in food packaging materials, such as cans and metal jarlids, and in high-impact windows, automobile parts, finishes,coatings, and other items (Summerfield et al., 1998). In recent years,TBBPA and TCBPA have been detected in sludge, sediment, andenvironmental water, as well as in drinking water and food

products (Patrolecco et al., 2004). TBBPA and TCBPA have also beenfound in human serum and breast milk (Kuiper et al., 2007). TBBPAand TCBPA exposure may cause different toxic effects in differentspecies (Table 3). For instance, one study indicated that TCBPA andTBBPA can cause lipid accumulation in zebrafish, and TCBPA wasfound to be more toxic than TBBPA at 1 mM (Riu et al., 2014).However, studies also showed that exposure to TBBPA and TCBPAcould lead to epithelial cancer and affinity and activity in the hu-man body; in this study, toxicity was more significant upon TBBPAexposure (Hoffmann et al., 2017; Riu et al., 2011). Another studyindicated that TCBPA causes more serious antiandrogenic activitythan TBBPA at 0.45 mM (Sun et al., 2006); we also found that thereproductive toxicity induced by TCBPA was higher than TBBPA.Recent studies have demonstrated that TBBPA can reduce spawningactivity in zebrafish and increase the number of immature oocytes(Kuiper et al., 2007). Furthermore, previous work demonstratedthat TBBPA had a toxic effect on reproduction in maleR. nigromaculata (Ogunbayo et al., 2008). Analysis of animal spermis crucial for determining reproductive toxicology in males, andsperm deformity is the most direct evidence of the influence ofpollutants in the testes of animals (Ankley et al., 1998). Our results

Fig. 4. Effects of TBBPA and TCBPA on testosterone and estradiol levels in testes. Male frogs were exposed to TBBPA and TCBPA (0.001, 0.01, 0.1, 1mg/L) for 14 days. Bars representmean± S.D. Asterisks and dollar signs denote significant differences between TBBPA and TCBPA compared with the control (*P< 0.05, **P< 0.01, $P< 0.05, $$P< 0.01).

Fig. 5. Effects of TBBPA and TCBPA on luteinizing hormone and follicle stimulating hormone in the serum of frog testes. Male frogs were exposed to TBBPA and TCBPA (0.001, 0.01,0.1, 1mg/L) for 14 days. Bars represent mean± S.D. Asterisks and dollar signs denote significant differences between TBBPA and TCBPA compared with the control (*P< 0.05,**P< 0.01, $P< 0.05, $$P< 0.01).

H. Zhang et al. / Environmental Pollution 243 (2018) 394e403400

demonstrated that after R. nigromaculata were exposed to TBBPAand TCBPA, sperm count and sperm motility decreased signifi-cantly, while sperm deformity increased significantly; these find-ings are consistent with recent research that showed the twoindices above in frogs exposed to TBBPA and TCBPA were signifi-cantly lower compared to controls (Linhartova et al., 2015). We alsofound that TBBPA and TCBPA exposure caused testes damage,vacuoles, disorder of the spermatogenic cell array, and dispersionand shedding of spermatogenic cells to different degrees. We alsoobserved examples of abnormal condensation of sperm tails,spermatozoa with swollen sperm tail necks, and even the absenceof sperm tails entirely. Therefore, these results suggest that TBBPAand TCBPA induced reproductive harm in the testes ofR. nigromaculata and that the reproductive toxicity of TCBPA washigher than TBBPA in male frogs. In addition, TBBPA and TCBPAtoxicity could account for the decrease in the populations ofamphibians.

Testosterone and E2 can have an endocrine-disrupting influencein the reproductive system of male amphibians (Akhtar et al., 2011).Elevated T content could theoretically lead to a more masculinephenotype because of its function in promoting the developmentand differentiation of the testes (Scott et al., 2009). Estrogen is a keyfactor in the formation of many tissues within the nervous systemand reproductive tract of both males and females. However, su-perabundant E2may cause sex reversal in amphibians (Piprek et al.,2012) and lead to interference of spermatogenesis. Here, T and E2serum content in male R. nigromaculata was significantly upregu-lated after exposure toTBBPA and TCBPA. The increase of Tand E2 inthe serum of male frogs in TBBPA-treated group was more signifi-cant than that in the TCBPA-treated groups at a high concentration.A previous study indicated that exposure of MA-10 mesenchymalcells to TBBPA in vitro promotes the synthesis of T (Roelofs et al.,2015). Because T and E2 levels play a role in the induction of aro-matases, T can transform into E2 (Hecker et al., 2005). Song et al.

Fig. 6. Effects of TBBPA and TCBPA on androgen receptor expression levels in frogtestes. Male frogs were exposed to TBBPA and TCBPA (0.001, 0.01, 0.1, 1mg/L) for 14days. Bars represent mean ± S.D. Asterisks and dollar signs denote significant differ-ences between TBBPA and TCBPA compared with the control (*P< 0.05, **P< 0.01,$P< 0.05, $$P< 0.01).

Fig. 7. Proposed signaling pathway of reproductive toxicity related to TBBPA- andTCBPA-induced hormonal changes.

H. Zhang et al. / Environmental Pollution 243 (2018) 394e403 401

(2014) suggested that TBBPA and TCBPA were potent toxicants forestrogenic activity, which is consistent with our results. This studysuggests that TBBPA and TCBPA can affect the endocrine system,

Table 3Comparison of TBBPA and TCBPA-induced toxic effects on different species.

Species Toxicity categories TBBPA and

Zebrafish Lipid accumulation 1 mMHuman Epithelial cancer 10 nMZebrafish Developmental toxicity 1.0mg/LZebrafish Estrogenic activity 1.0mg/LHuman Antiandrogenic activity 10.45 mMHuman Affinity and activity 1 mMMale Rana nigromaculata Reproductive toxicity 0.001, 0.01,

leading to obstacles to sperm formation and reduced sperm qualityin male R. nigromaculata.

FSH and LH play very important roles in the formation andfunction of the gonads (Gharib et al., 1990). Similarly, studies haveshown that FSH and LH are the major hormones that control thegonads (Dickey and Swanson,1998). However, concentrations of LHand FSH from pituitary cells of adult rats decreased after exposureto a high dose of BPA (Fernandez et al., 2009), and BPA also causeddamage to the process stimulating the formation of FSH in humancells (Kwintkiewicz et al., 2010). Moreover, a recent study indicatedthat BPA exposure could reduce concentrations of LH and FSH in theserum (Wisniewski et al., 2015). Here, we found that there was anegative relationship between levels of LH and TBBPA and TCBPAconcentrations, which was consistent with another study demon-strating that BPA can cause decreases of LH and FSH (Zhang et al.,2013). LH levels had a greater decrease at a low doses of TBBPAcompared with TCBPA, but decreased more significantly at highdoses of TCBPA as compared with TBBPA. The increase of LH con-centration in the 1mg/L TBBPA group and the increase of FSHconcentration in the 0.01mg/L TCBPA groupmight be related to thepositive feedback effects caused by increased T and increased E2 intreatment groups. A previous study confirmed that the secretionregulation of FSH requires aromatization to E2 in the pituitarygland, while T and E2 are equally effective in regulating LH secre-tion (Pitteloud et al., 2008). Another recent study found that LHlevels were associated with the offset in negative feedback effectscaused by decreased T and increased E2 (Jia et al., 2018). Thus,TBBPA and TCBPA may affect T and E2 content, which causeschanges to LH and FSH hormone levels resulting in reproductivehormone abnormalities in amphibians.

Collins et al. (2003) suggested that androgen receptors controlthe combination with androgens to regulate gene expression. Oneprevious study showed that TBBPA and TCBPA can cause damage toreceptors (Rosenmai et al., 2014). TBBPA exhibited antiandrogenicactivity in a yeast AR-dependent reporter gene assay, but was amore effective antagonist in vitro for AR compared with otherbisphenols that have similar structure (Roelofs et al., 2015). Expo-sure to TBBPA significantly increased expression levels of ARbmRNA in the testes of males and decreased expression levels of ARamRNA in the testes of juveniles (Huang et al., 2013). Our resultsshowed that mRNA levels of AR decreased significantly in malefrogs exposed to TCBPA and exhibited even greater decreases infrogs exposed to TBBPA. The knockout of AR in different mousetesticular cell types can cause spermatogenesis and male repro-ductive dysfunction (Wang et al., 2009b). Thus, TBBPA and TCBPAmay interfere with transcription changes involved in the regulationof spermatogenesis and cause decreased AR gene expression,which causes reproductive toxicity in amphibians.

In conclusion, our findings in R. nigromaculata indicated thatTBBPA and TCBPA both induce reproductive toxicity damage andthat frogs are more sensitive to TCBPA than to TBBPA. This repro-ductive toxicity damage might be mediated by T, E2, LH, and FSHlevels and AR gene expression.

TCBPA doses Toxicity comparison Reference

TCBPA> TBBPA Riu et al., 2014TBBPA> TCBPA Hoffmann et al., 2017TCBPA> TBBPA Song et al., 2014TCBPA> TBBPA Song et al., 2014TCBPA> TBBPA Sun et al., 2006TBBPA> TCBPA Riu et al., 2011

0.1, 1mg/L TCBPA> TBBPA This study

H. Zhang et al. / Environmental Pollution 243 (2018) 394e403402

5. Conclusions

We researched the toxic effects and toxicity mechanisms ofTBBPA and TCBPA in the reproductive system of male frogs andcompared TBBPA and TCBPA. Sperm deformity values were 0.549and 0.397 following exposure to 1mg/L of TBBPA and TCBPA for 14days, respectively. Our study revealed that even a low dose ofTBBPA or TCBPA could cause endocrine-disrupting and hormone-disrupting effects in male frogs. The comparison analysis resultsbetween TBBPA- and TCBPA-treated groups suggested that TCBPAhas stronger toxicity to frogs than TBBPA. The decrease in LH con-tent was more significant in frogs exposed to TBBPA at a low con-centration and in frogs exposed to TCBPA at a high concentration.FSH content decreased significantly in the 0.1 and 1mg/L TBBPAgroups, but we did not observe a dose-dependent relationship withTCBPA. Decreases in FSH content was more significant in the0.1mg/L TBBPA groups than in the 0.1mg/L TCBPA groups. ARmRNA expression decreasedmarkedly in all the TBBPA- and TCBPA-treatment groups compared with controls and decreased signifi-cantly in the TBBPA treatment groups compared with the TCBPAtreatment groups. In conclusion, TBBPA- and TCBPA-inducedchanges in T, E2, LH and FSH levels can cause endocrine disordersand influence the spermatogenesis in the testes of R. nigromaculataby AR gene expression anomaly.

Acknowledgments

This work was financially supported by the National NatureScience Foundation of China (No. 21876037), the Excellent TalentInnovation Experiment Program for University Students in China(201710346009), the Program for College Students Science andTechnology Innovation Activity in Zhejiang Province(4105C5151710241), the Program for 151 Talents in ZhejiangProvince (No. 4108Z061700303) and the Program for 131 Talentsin Hangzhou City (No. 4105F5061700104).

Appendix A. Supplementary data

Supplementary data related to this article can be found athttps://doi.org/10.1016/j.envpol.2018.08.086.

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