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Disruptive effects of two organotin pesticides on the thyroid signaling pathway in Xenopus laevis during metamorphosis
Science of the Total Environment 697 (2019) 134140
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Disruptive effects of two organotin pesticides on the thyroid signaling pathway in Xenopus laevis during metamorphosis Shuying Li a, Kun Qiao a, Yao Jiang a, Qiong Wu a, Scott Coffin b, Wenjun Gui a,⁎, Guonian Zhu a a b
Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310058, PR China Department of Environmental Sciences, University of California, Riverside, CA 92521, United States
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Fenbutatin oxide and fentin hydroxide showed thyroid disrupting effects on Xenopus laevis. • SPR indicated organotin pesticides could bind to thyroid receptor with different binding ability. • Reporter gene assay indicated antagonist activity of fentin hydroxide but not fenbutatin oxide.
a r t i c l e
i n f o
Article history: Received 27 May 2019 Received in revised form 25 August 2019 Accepted 26 August 2019 Available online 27 August 2019 Editor: Yolanda Picó Keywords: Organotin pesticides Xenopus laevis Thyroid endocrine disruption Surface plasmon resonance Reporter gene assay
a b s t r a c t Organotin compounds are the ubiquitous environmental pollutants due to their wide industrial and agricultural applications and unexpected releasing into the environment, which show characteristic of endocrine disruptors to interfere with the synthesis, receptor binding or action of endogenous-hormones. Organotin pesticides (OTPs) are used in agriculture and may impact endocrine functions on organisms. Thyroid hormones (THs) play fundamental roles in regulating the basal metabolism and energy balance, while thyroid function can be impaired by environmental contaminants. Therefore, it is crucial to clarify the effects and mechanisms of OTPs on hypothalamus-pituitary-thyroid (HPT) axis. In this study, Xenopus laevis tadpoles at stage 51 were exposed to fentin hydroxide and fenbutatin oxide (0.04, 0.20 and 1.00 μg·L−1) for 21 days. It was found that both compounds caused inhibitory effects on metamorphic development of tadpoles (e.g., significant decrease in hindlimb length and retarding development). Triiodothyronine (T3) significantly decreased in tadpoles exposed to 0.20 μg/L and 1.00 μg/L of the two OTPs for 14 days or 21 days. The expressions of TH responsive genes trβ, bteb and dio2 were down-regulated, while tshβ and slc5a5 were up-regulated. Surface plasmon resonance (SPR) binding assays showed that fentin hydroxide had a moderate affinity to recombinant human thyroid hormone receptor β but fenbutatin oxide did not have. Result of the SPR assay was highly consistent with the luciferase reporter gene assays that fentin hydroxide suppressed the relative luciferase activity in the presence of T3 while fenbutatin oxide did not, demonstrating fentin hydroxide but not fenbutatin oxide displayed an antagonistic activity against T3-TR complex mediated transcriptional activation. Overall, the findings elucidated the mechanisms induced by OTPs along HPT axis. These results highlighted the adverse influences of organotin pesticides on thyroid
⁎ Corresponding author at: Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310058, PR China. E-mail address: [email protected] (W. Gui).
https://doi.org/10.1016/j.scitotenv.2019.134140 0048-9697/© 2019 Elsevier B.V. All rights reserved.
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S. Li et al. / Science of the Total Environment 697 (2019) 134140
hormone- dependent development in vertebrates and the need for more comprehensive investigations of their potential ecological risks. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Organotins (OTs) are organometallic compounds used for various industrial purposes including disinfectants, pesticides, and most frequently as biocides in antifouling paints (Gui et al., 2016; Li et al., 2016a; Marques et al., 2018; Yan et al., 2018). Aquatic pollution attributed to the use of OTs is of great concern due to the adverse biological effects on nontarget biota, even at Sn concentration as low as ng·L−1 (Alzieu, 2000). Although many countries worldwide had banned on OT-based paints from the 1980s (Furdek et al., 2012), OTs (such as tributyltin and triphenyltin) are still detected in water, sediments and biota at concentrations ranging from ng Sn·L−1 (or g−1) to μg Sn·L−1 (or g−1) (Antizar-Ladislao, 2008; Furdek et al., 2012; Radke et al., 2013). These OTs can be easily accumulated by living organisms and further transferred to humans through food chain (Furdek et al., 2012), consequently increasing health and environmental risks. However, OTs such as fentin hydroxide, fenbutatin oxide, azocyclotin and fentin acetate are still used extensively as pesticides in agricultural applications (Gui et al., 2016; Li et al., 2016a). Few studies focused on the toxicity and mechanism induced by these organotin pesticides (OTPs). Previous studies showed that OTs could cause a range of toxic outcomes, including genetic, hepatic, renal, neural and immune toxicity (Bertuloso et al., 2015; Coutinho et al., 2016). For example, Merlo et al. (2016) reported that tributyltin exerted its toxicity on the nervous system of rat after treated with 100 ng kg−1 for 15 days. It is confirmed that some of OTs (such as tributyltin and triphenyltin) are capable of altering functions of tissues (e.g. hypothalamus, pituitary, gonad, adrenal, and thyroid gland) on some non-target organisms, playing as endocrine disruptors mainly through interacting with transcriptional regulators including nuclear and steroid receptors (e.g. estrogen receptor (ER), retinoid X receptor (RXR), and peroxisome proliferator activated receptor (PPAR)) (Bertuloso et al., 2015; Coutinho et al., 2016). For instance, Marques et al. (2018) described that OTs might induce endocrine disorders due to the capability of regulation on the enzyme P450arom. In fish, tributyltin or azocyclotin exposure not only reduced 17βestradiol serum levels but also changed ER expression in different sites along hypothalamus-pituitary-gonadal (HPG) axis (Ma et al., 2016; Marques et al., 2018). Moreover, a few studies showed the effects of OTs on hypothalamus-pituitary-thyroid (HPT) axis. Adeeko et al. (2003) reported that tributyltin exposure would lead to reduction in serum thryoid hormones (THs) triiodothyronine (T3) and thyroxine (T4) levels in pregnancy rat. Sharan et al. (2014) demonstrated that decreases in the activities of thryoid hormone receptors α and β (TRα, and TRβ) as well as lower protein expression of TRβ were found in hepatocarcinoma cells after tributyltin exposure. Furthermore, in amphibian Xenopus laevis, the colloid depletion and follicle malformation were observed in thyroid gland after 19 days of tributyltin exposure (Shi et al., 2012). These studies showed a potential thyroid disruption induced by OTs. Our previous study also provided the evidences of azocyclotin-induced metamorphosis disruption in Xenopus laevis (Li et al., 2016a). However, effects induced by fentin hydroxide and fenbutatin oxide on TH-dependent development at the morphological and functional level are still poorly understood. The molecular structures of fentin hydroxide and fenbutatin oxide are similar to those of other OTs, but the thyroid endocrine disrupting effects of the two chemicals on environmental organisms remains unknown. Amphibians share multiple similarities with vertebrates in development, and as such, amphibian metamorphosis is regarded as an ideal model to study TH-dependent development and the mechanisms of TH action in vertebrates (Zhang et al., 2014; Freitas et al., 2016).
Considering pesticide exposure has been regarded as one of the main causes of the decline in amphibian populations worldwide (Hayes et al., 2006; Freitas et al., 2016), the evaluation of effects and mechanisms caused by OTPs could elucidate their potential risks to the populations of amphibian and even vertebrates. In the present study, in order to explore the thyroid endocrine disrupting effect of fentin hydroxide and fenbutatin oxide, tadpoles (X. laevis) were exposed to fentin hydroxide or fenbutatin oxide. Then, the developmental retarding effects, the THs levels and the expressions of genes involved in X. laevis metamorphic pathways such as basic transcription element binding protein (bteb), deiodinases (dio2), thyroid hormone receptors (trβ), transthyretin (ttr), thyriod stimulating hormone (tshβ), sodium/iodide cotransporter (slc5a5) were evaluated at different time point. In vitro, we employed the firefly luciferase assay to investigate the THdisrupting effects of the two OTPs. What’ more, the molecular interactions between thyroid receptor (TR) and the OTPs were determined by using a surface plasmon resonance (SPR) biosensor to reveal the underlying mechanisms of thyroid disruption. 2. Materials and methods 2.1. Chemicals and reagents Two OTPs standards of fenbutatin oxide (CAS: 13356–08-6, purity N95%) and fentin hydroxide (CAS: 76–87-9, purity N95%) were obtained from Zhejiang Heben Pesticide & Chemicals Co., Ltd. Dimethyl sulfoxide (DMSO, cell culture reagent), T3 and tricaine methanesulfonate (MS222) were purchased from Sigma (St. Louis, USA). OTPs stock solutions (100 mg·L−1) were dissolved in DMSO and stored at −20 °C for no more than one month. TRIzol™, RNase-free water, PrimeScript RT reagent Kit and SYBR Premix Ex Tap™ II were purchased from Takara (Dalian, China). Purified recombinant human thyroid hormone receptor β (hTRβ) protein was purchased from CusaBio (Wuhan, China). 2.2. Metamorphosis assay X. laevis (Nasco, Fort Atkins, WI, USA) were maintained according to our previous study (Li et al., 2017). Development stages of tadpoles were determined according to Nieuwkoop and Faber (NF) (Nieukoop and Faber, 1967). Detailed information was provided in Supporting Information (SI-1). X. laevis tadpoles of NF stage 51 were individually exposed to the two OTPs (with the same concentration sets of 0.04, 0.20 and 1.00 μg·L−1 for each OTP, exposure concentrations were set based on the published study (Li et al., 2016a) for 21 days (20 tadpoles per glass tank (50 cm × 25 cm × 30 cm) filled with 18 L exposure solution. Each exposure solution contained 0.01% (by volume) DMSO), during which time the tadpoles were undergoing for metamorphic development (Nieukoop and Faber, 1967). Tadpoles in pesticide-free exposure solution (containing 0.01% DMSO, v/v) were set as vehicle control (VC). Each treatment was conducted in triplicate. Dead tadpoles were removed twice a day and exposure solutions were renewed at an interval of 24 h. Water samples were collected before and after renewal and stored in darkness at −80 °C until analysis. Tadpoles were randomly sampled from each tank at 7th, 14th and 21th d after exposure (10 tadpoles per tank per time) for the survival rate (%), malformation rate (%), hindlimb length (mm), and developmental stage check. At each sampling time point, 3 tadpoles were randomly collected and anesthetized using MS-222 (0.01 g·L−1), and then undergone for mRNA (hindlimb) and THs (head) analysis.
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2.3. Quantification of chemicals in exposure solutions The fentin hydroxide and fenbutatin oxide in exposure solutions were determined by high performance liquid chromatographyinductively coupled plasma-mass spectrometry (HPLC-ICP-MS) which were described in a previous study (Gui et al., 2016) with slightly modified. Detailed information was provided in Supporting Information (SI2).
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DMSO and 0.05% (by volume) surfactant P20. Buffer samples containing 4.5–5.8% (by volume) DMSO were injected to construct a DMSO calibration plot to correct for bulk refractive index shift (Rich et al., 2002). Running buffer was set at a flow rate of 30 μl·min−1. For each test chemical, the association phase of the SPR measurement was set to 120 s, and the buffer flow was continued to allow a dissociation time of 300 s. The kinetic and affinity constants were obtained by fitting the experimental data to a reversible 1:1 bimolecular interaction model in Biacore Evaluation Software.
2.4. Thyroid hormones assay 2.8. Statistical analysis The measurement of THs was performed using a commercial enzyme-linked immunosorbent assay (ELISA) kit (Uscnlife, Wuhan, China). Briefly, three tadpoles (head) from each tank were weighed and homogenized in 4 mL of assay buffer. After centrifugation (7, 500 g) at 4 °C for 15 min, the supernatant was separated and undertaken for T3 and T4 measurement. The ELISA was conducted in 2 wells technical replicates and 3 biological replicates. The detection limits for T4 and T3 of the method were 1.46 ng·mL−1 and 47.1 pg·mL−1, respectively. The kits were certified for use with X. laevis sample by validating the correlation between a series of diluted and spiked samples in reference to both T3 and T4 standard curves.
Statistical analysis was performed using SPSS® version 20.0 (SPSS, USA). All data were expressed as means ± standard deviation (SD). The Kruskal-Wallis test was used for measurement of endpoints (hind limb length, developmental stage). Statistical differences in gene expression and hormone concentrations were evaluated by one-way analysis of variance (ANOVA) following Tukey's post-hoc test. A p-value b0.05 was considered statistically significant. 3. Results 3.1. Quantification of chemicals in solution
2.5. Quantitative real-time PCR Total RNA was extracted using TRIzol® reagent as described by our previous report (Li et al., 2016b). Primer sequences of genes were provided in Table S1 which were developed in a previous study (Zhang et al., 2014). The 2-△△Ct method was used to calculate the target mRNA expression (Livak and Schmittgen, 2001). Detailed information was described in the Supporting Information (SI-3).
The real concentrations of fentin hydroxide and fenbutatin oxide in the exposure solutions were monitored in the metamorphosis test at one renewal period. Results showed that the degradation rates of fentin hydroxide and fenbutatin oxide in water phase (24 h) were all b20% during 24 h of refreshing interval (Table 1), which conformed to the requirement of OECD Guideline 203 (OECD, 1992). It indicated that the fluctuations of the OTPs in exposure solutions were acceptable and the refreshing interval was reasonable.
2.6. Cell-based bioassays 3.2. Developmental toxicity endpoints Thyroid hormone receptor (TR) activity was assessed by using established in vitro GH3-TRE cell (cell provided by Dr. Qiangwei Wang) assay. Bioassay protocols were based on previous report (Xiang et al., 2017). Briefly, cells were plated in a 96-well microplate at a density of 1.5 × 104 cells per well in DMEM/F-12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12) medium (Thermo fisher, USA) with penicillin/streptomycin (Thermo fisher, USA) and 100 U/mL hygromycin B (APExBIO, USA) followed by a 24 h incubation. For agonistic/antagonistic activity tests, cells were exposed to T3 ranging from 10−12 to 10−7 M (set as positive control), and pesticides ranging from 10−10 to 10−7 M. After 24 h, light produced was measured with Spectra Max i3 (Molecular Devices, USA) using the Luciferase Reporter Assay Kit (Promega, USA). All tests were carried out three independent times, each with six replicates per test concentration. Cytotoxicity induced by tested chemical was performed using a 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4sulfophenyl)-2H-tetrazolium (MTS) assay on cells. 2.7. Surface plasmon resonance (SPR) binding assay SPR assays were performed on a Biacore™ T200 system (GE Healthcare, Sweden) with hTRβ protein. In both Homo sapiens and Xenopus laevis, TRs are nuclear receptors which control transcription, and thereby have effects in all cells within the body. Thyroid hormone receptors are encoded by two TR genes. There is some specie/tissue specificity in the distribution of these isoforms (Mackenzie, 2018). The hTRβ protein was covalently immobilized to a CM7 sensor chip (GE Healthcare, Sweden) using an amine-coupling procedure, in which HBS-N (0.01 M HEPES (4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid) and 0.15 M NaCl, pH 7.4) containing 0.05% surfactant P20 (Polysorbate 20) (GE Healthcare, Sweden) was used as immobilized running buffer. Chemicals were dissolved in HBS-N buffer containing 5% (by volume)
Tadpoles at NF stage 51 were exposed to either fentin hydroxide or fenbutatin oxide in order to investigate their effects on development of frog via TH signaling disruption. Exposed to fentin hydroxide or fenbutatin oxide for 21 days did not significantly affect survival rates of tadpoles (Table S2). However, tadpoles exhibited pronounced morphological inhibitions including hindlimb growth and lower developmental stage (Fig. 1). All tadpoles developed to stages 52–54 from stage 51 on day 7. Compared to VC (29.6%), reductions in proportion of stage 54 were observed in the groups of fentin hydroxide at 0.04 μg·L−1 (14.8%, p = 0.047) and 1.00 μg·L−1 (7.4%, p = 0.013). Similar results were also found in fenbutatin oxide exposure groups. The percentages of stage 54 were 14.8% (p = 0.047) and 11.1% (p = 0.038) in 0.20 and 1.00 μg/L groups, respectively. In all exposure groups, hindlimb length tended to decrease, while significantly decreases only observed at 1.00 μg·L−1 fenbutatin oxide (p = 0.013). Table 1 Concentrations of organotin pesticides in exposure solutions. Nominal concentrations (μg·L−1)
Vehicle control Fentin hydroxide
Fenbutatin oxide
0.04 0.20 1.00 0.04 0.20 1.00
Measured concentrationsa (μg·L−1) 0h
24 h
ND 0.046 ± 0.001 0.212 ± 0.009 1.114 ± 0.031 0.042 ± 0.003 0.209 ± 0.012 1.094 ± 0.025
ND 0.036 ± 0.003 0.167 ± 0.004 0.944 ± 0.018 0.031 ± 0.004 0.159 ± 0.008 0.817 ± 0.037
ND, not detected. a Values represent mean ± SD (n = 3).
Degradation rate (%)
– 19.3 14.2 15.3 16.1 13.3 15.7
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Fig. 1. Developmental retarding effect of fentin hydroxide and fenbutatin oxide on hind limb length (mm) of tadpoles (A), as well as developmental stage ratios of tadpoles exposed to fentin hydroxide (B) and fenbutatin oxide (C). Asterisk indicated significant differences (*, p b 0.05) between exposure groups and the corresponding vehicle control.
On day 14, all tadpoles developed to stages 54–55 in VC group while parts of tadpoles remained in stage 53 in all exposure groups. The proportion of stage 55 significantly decreased in 1.00 μg·L−1 fentin hydroxide group (3.7%, p = 0.047) and 1.00 μg·L−1 fenbutatin oxide group (3.7%, p = 0.047), respectively (18.5% in VC group). Increased percentages of stage 53 were observed in fentin hydroxide (0.04 μg·L−1, 18.5%, p = 0.038; 0.20 μg·L−1, 18.5%, p = 0.038; 1.00 μg·L−1, 29.6%, p = 0.015) and fenbutatin oxide (1.00 μg·L−1, 40.7%, p = 0.032) compared with VC. Significant decreases in hindlimb length were found in all 0.20 μg·L−1 (p b 0.05) and 1.00 μg/L (p b 0.05) treated groups. Following 21d exposure, the inhibiting effects on development became pronounced. Tadpoles developed to stages 54–56 in VC, but most of the tadpoles remained in stage 54 in fentin hydroxide and fenbutatin oxide exposure groups. Moreover, significant greater inhibitions to hindlimb length were observed in 0.20 and 1.00 μg·L−1 treated groups (p b 0.05). 3.3. Thyroid hormone levels TH levels in tadpoles were examined after exposure to the two OTPs. The total T4 levels were not significantly altered in any exposure group at any sampling point (Fig. 2). On day 7, no significant changes in T3 content were observed. While on day 14, T3 levels were significantly
decreased in 0.04 μg/L (8.08 ng·g−1, p = 0.014) and 1.00 μg/L (8.80 ng·g−1, p = 0.038) fentin hydroxide exposure groups, relative to VC. Additionally, decreased T3 levels were observed in 1.00 μg/L (7.26 ng·g−1, p = 0.037) fenbutatin oxide on day 14. On day 21, significantly decreased T3 levels were measured in both fentin hydroxide (0.20 μg/L: 15.01 ng·g−1, p = 0.027; 1.00 μg/L: 11.46 ng·g−1, p = 0.006) and fenbutatin oxide (0.20 μg/L: 18.27 ng·g−1, p = 0.043; 1.00 μg/L: 16.62 ng·g−1, p = 0.018) treatments compared with VC. 3.4. Gene transcription Transcriptional profiles of genes involved in TH-signaling pathway and TH-response (bteb, dio2, trβ, ttr, tshβ and slc5a5) were further examined in tadpoles after exposure to fentin hydroxide and fenbutatin oxide (Fig. 3). Exposed to fentin hydroxide or fenbutatin oxide failed to alter the expression of the target genes on day 7 except that in 1.00 μg/L fentin hydroxide exposure group, the expression of trβ in tadpole hindlimb was reduced by 0.46-fold (p = 0.037). On day 14, significant down-regulations (p b 0.05) of bteb, dio2 and trβ mRNA expression were observed in all treatments of the two tin-pesticide. On the contrary, significant concentration-associated increases (p b 0.05) of tshβ and slc5a5 expression were observed in all treatments of the two chemicals compared to VC. Similar alterations were found in the
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expressions of the selected genes after 21 days exposures (significant down-regulations in bteb, dio2 and trβ, significant up-regulations in tshβ and slc5a5 (all compared to VC (p b 0.05)). 3.5. Binding kinetics of chemicals to hTRβ Binding assays were performed in a quantification screening mode and kinetic analyses for agonists and antagonists were performed to determine the rate constants associated with Human TRβ (hTRβ). As a positive control, T3 displayed a concentration-dependent response which could reach equilibrium and return to baseline within seconds, indicating a rapid association and a fast disassociation between T3 and TR (Fig. 4A). Fenbutatin oxide and fentin hydroxide both showed direct binding interaction with hTRβ (Fig. 4B, C), but the association and dissociation curves of fentin hydroxide and fenbutatin oxide trended to differ from those of T3. The association (ka) and dissociation (kd) rate constants and the equilibrium dissociation constants (KD) for the tested compounds were represented in Table 2. Compared with T3, up to 13fold and 1000-fold lower association rate constants were observed in fentin hydroxide and fenbutatin oxide, respectively. Results indicated that fentin hydroxide had a moderate binding ability while fenbutatin oxide had a very weak binding ability to hTRβ. 3.6. Luciferase reporter gene assay The cytotoxic concentrations of tested chemicals were determined by MTS assay before performing receptor assay (data not shown), and results showed that the tested concentrations of chemicals did not affect the viability and proliferation of GH3-TRE cells. The relative luciferase activity (RLA) induced by thyroid hormone T3 in TRβ-mediated luciferase reporter gene assay was shown in Fig. 5A. T3 induced RLA in a concentration-dependent manner ranging from 10−12 to 10−6 M, and the EC50 (the term half maximal effective concentration) of T3 was 2.8 × 10−9 M. Agonistic and antagonistic effects of the pesticides mediated via TR were estimated. For evaluation of the agonistic effect, GH3 cells were treated with the pesticides both ranging from 1 × 10−10 to 1 × 10−7 M in the absence of T3. It was found that only fentin hydroxide showed weak TR agonistic effects (Fig. 5A) with the REC20 (20% relative effective concentration (Xiang et al., 2017), i.e., concentration of the
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tested chemicals showing 20% of the RLA of 1 × 10−7 M T3) of fentin hydroxide was N10−7 M. The antagonistic activity was conducted in the presence of 1 × 10−9 M T3 (Fig. 5B). Results showed that fentin hydroxide displayed a weak antagonistic effect against T3 while fenbutatin oxide had no significant influence on T3. The 20% relative inhibitory concentration (RIC20), concentration of the tested chemicals showing a 20% reduction in the RLA of 1 × 10−9 M T3) of fentin hydroxide was 2.2 × 10−8 M. 4. Discussion Great concerns have been raised in recent years over the potential adverse effects of OTs on the endocrine system (Ma et al., 2016; Li et al., 2017; Finnegan et al., 2018; Yan et al., 2018). The actions of OTs span many endocrine axes, including HPG and HPT axes (Santos-Silva et al., 2018). OTs may impact developmental steps controlled by the HPT axis (Li et al., 2016c) such as metamorphosis in the amphibian Xenopus laevis. However, effects induced by OTPs on TH-dependent development at the morphological and functional levels are poorly understood. In this study, significantly inhibitory effects were observed on tadpole metamorphosis which was characterized by multiple morphological changes including shortened hindlimb protrusion and delayed development stages, particularly after 14 and 21 days of fentin hydroxide and fenbutatin oxide exposure. Our results demonstrated that fentin hydroxide and fenbutatin oxide inhibited metamorphic development of tadpoles. These findings agreed with previous studies in which it had been demonstrated that organotin compounds could delay metamorphosis and inhibit the growth of tadpole (Shi et al., 2012; Li et al., 2016a). Our findings further supported the hypothesis that the metamorphosis of X. laevis could be inhibited by OTPs. Moreover, inhibitory effects of OTPs on thyroid actions were further supported by decreased T3 levels and altered expression of TH-related genes including bteb, trβ, dio2, tshβ and slc5a5 in previous researches (Zhang et al., 2014; Li et al., 2016c). TH is a key variable affecting the biological developmental such as amphibian metamorphosis (Kloas, 2002). It is already known that some endocrine disrupting chemicals targeting thyroid gland have the ability to change circulating levels of TH, altering the relationship between TH biosynthesis and elimination, which results in acceleration or retardation of metamorphosis (Kloas,
Fig. 2. T3 and T4 levels in Xenopus laevis tadpoles after exposed to different concentrations of fentin hydroxide (A and B) and fenbutatin oxide (C and D) for 7, 14 and 21 d. Data were shown as mean ± SD. *p b 0.05 indicated significant differences between exposure groups and the corresponding vehicle control.
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Fig. 3. The mRNA expression of genes in Xenopus laevis tadpoles that were exposed to different concentrations of fentin hydroxide (A) and fenbutatin oxide (B) for 7, 14 and 21 days. Data were shown as mean ± SD. *p b 0.05 indicates significant differences between exposure groups and the corresponding vehicle control.
2002). For examples, decreased levels of T4 and T3 were observed in X. laevis after exposed to triadimefon (Li et al., 2016b). Azocyclotin led to a decreased level of T3 in X. laevis tadpole (Li et al., 2016a). In the present study, significant decreases in T3 levels (which were associated with inhibition of metamorphic development) were observed after 14 days and 21 days of exposure, indicating the effects of fenbutatin oxide and fentin hydroxide on metamorphic development are mainly mediated by TH signaling disruption. T3 levels in plasma are regulated by deiodinases, in which iodothyronine deiodinase II (Dio2) is responsible for converting T4 to T3 (Bianco and Kim, 2006). In X. laevis, the increased mRNA expression of dio2 has shown to be concomitant with the beginning of metamorphosis (Arrojo et al., 2013). The disruption of dio2 expression resulted in a state of localized hypothyroidism (Arrojo et al., 2013). Our results demonstrated that dio2 mRNA expression was down-regulated in
X. laevis exposed to the highest concentration of organotin pesticides on day 14, and this effect was enhanced in tadpoles following 21 days of exposure. Decreases in dio2 mRNA expression should lead to reduced T3 levels, consistent with the decrease of dio2 expression, the reducing levels of T3 were also observed. Therefore, the down-regulation of dio2 might be responsible for the decreased T3 levels. In addition, with the prolongation of exposure time, the relative levels of T4 (compared to corresponding VC) generally showed a slight upward trend after exposure to fentin hydroxide. While exposed to fenbutatin oxide, the relative levels of T4 remained basically unchanged. However, how fentin hydroxide or fenbutatin oxide affects dio2 and why T4 displays differential alternation after exposure to the two chemicals remain unknown and further studies are needed to confirm the mechanisms. Two important genes related to TH synthesis pathway, slc5a5 and tshβ, were examined in this study. As reported, the up-regulation of
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Fig. 4. Representative sensorgrams for kinetic analysis of hTRβ interactions. The T3 (A) was injected at concentrations of 0.0125, 0.025, 0.05 and 0.1 μM (from bottom to top). Fenbutatin oxide (B) was injected at concentrations of 0.039, 0.078, 0.312, 1.25 and 2.5 mM (from bottom to top). Fentin hydroxide (C) was injected at concentrations of 0.625, 1.25, 2.5 and 5.0 mM (from bottom to top). All chemicals were injected for 120 s, and disassociation was monitored for 300 s. The data of each interaction were fitted to a 1:1 bimolecular interaction model.
slc5a5 and tshβ might promote the TH production in tadpoles (Wang et al., 2013), while TH can be regulated by thyroid stimulating hormone (TSH) via feedback mechanisms (Wang et al., 2013). Similar results have demonstrated that the decrease of TH was accompanied with the increases in mRNA expression of tshβ and slc5a5 after thyroid disruptor exposure (Wang et al., 2013; Li et al., 2016c), which is consistent with our findings. Significant down-regulation of T3 but up-regulation of the two
genes were observed in the present study. Therefore, the up-regulation of slc5a5 and tshβ could be regarded as a compensatory response to decreased T3. But more measurements of thyroid development should be further applied to verify this compensatory mechanism. Concurrently, decreased transcription levels of bteb were observed following exposure to both fentin hydroxide and fenbutatin oxide. BTEB (basic transcription element binding protein) could bind the GC-rich basic transcription
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Table 2 Kinetic parameters of the binding of TR. Interaction
ka (1/Ms)
kd (1/s)
KD (M)
T3 Fenbutatin oxide Fentin hydroxide
3.33E+06 3.22E+03 2.49E+05
6.69E−03 1.28E−02 1.55E−02
2.01E−09 3.97E−06 6.21E−08
*ka, association rate constants. kd, dissociation rate constants. KD, equilibrium dissociation constants.
element and functions as a transcriptional activator on TH-response genes promoters (Hoopfer et al., 2010; Zhang et al., 2014). So, the decreased tendency of bteb was consistent with the reduction of T3. The antagonistic effects of the two OTPs on frog development were further supported by expression of the decrease of bteb. TR function as transcription factors whose activity is regulated by the binding of T3 (Hayes et al., 2010) and mediated TH-regulated gene expression by binding to response elements (Morvan-Dubois et al., 2008). In the present study, with 7 days of development there were retarding effects of the organotins, though thyroid hormones were unaltered. The metamorphosis of tadpole was mainly regulated by THs, as well as the transcription of specific sets of genes, including the thyroid hormone receptors and associated coactivators (Vitt and Caldwell, 2014). Hence, the decreased mRNA expression of trβ might explain this interesting finding. Previous in vitro studies also demonstrated that tributyltin disrupted TR transcriptional regulation whatever in absence or presence of T3 (Sharan et al., 2014). The present luciferase reporter gene assay showed that fentin hydroxide weakly stimulated the transcriptional activity of TR in absence of T3 but conversely slightly suppressed the transcriptional activity of TR in presence of T3. However, fenbutatin oxide displayed no influence on transcriptional regulation of TR. Although tributyltin, fentin hydroxide and fenbutatin oxide all belong to members of the OTs family, there are still obvious differences in their chemical structures. This difference in chemical structure might explain the aforementioned their different effects on transcriptional regulation of TR, also reminding that different organotin may have different molecular mechanisms although they may have the same or similar toxicological characterizations. But further studies are still needed to clarify the detailed molecular mechanism. Recent studies indicated that environmental chemicals could bind to TRs and might activate or inhibit the action of T3, or change the affinity of TRs for response elements in specific target genes, these impacts might alter the normal ability of TRs to bind to TH, resulting in
interference with thyroid function and TH signaling (Freitas et al., 2016). In the present study, SPR was applied for the evaluation of molecular interaction between chemicals and TR protein. SPR is a high sensitivity and reliable detection method which allows for the analysis of biomolecular interactions and molecular mechanisms (Nguyen et al., 2015). A wide range of applications have been developed for affinity binding analysis using SPR biosensors, including ligand-receptor kinetics (Nguyen et al., 2015; Li et al., 2017; Xiang et al., 2017). The dynamic interaction curve of chemicals binding to biomacromolecule given by SPR can clearly reveal the binding strength of chemicalsbiomacromolecule and the differences in binding mode between different compounds and the same biomacromolecule. Commonly, agonists display relatively fast association and dissociation rate constants from the surface of biomacromolecule. In contrast, antagonists show slower association and dissociation rates (Rich et al., 2002). In the present study, compared to T3 (an endogenous TRβ agonist)-hTRβ interaction, lower association and dissociation rate constants which shared the features of antagonistic interaction were observed in fentin hydroxidehTRβ interaction, indicating a potential inhibiting action on T3 binding to TR. For fenbutatin oxide, its interaction with hTRβ could almost be ignored (the ka value almost 1000-fold lower than that of T3). The evidence of differential interaction of fentin hydroxide and fenbutatin oxide binding to hTRβ in this SPR-based affinity binding assay was highly consistent with the result of the luciferase reporter gene assay. In summary, previous studies have reported thyroid endocrine disrupting effects induced by OTs (Brtko and Dvorak, 2015; Li et al., 2016a; Finnegan et al., 2018; Santos-Silva et al., 2018). In the present study, we found that fentin hydroxide and fenbutatin oxide significantly altered thyroid hormone levels and expression of thyroid hormonerelated genes, and induced pronounced morphological inhibitions in tadpoles of X. laevis. It is deduced that both fentin hydroxide and fenbutatin oxide repress dio2, thereby reducing T3. Moreover, fentin hydroxide but not fenbutatin oxide disrupts the normal binding interaction between TR and T3, therefore suppressing TH-TR complexmediated transcriptional activation, and further affecting physiological function of X. laevis such as the retarding of metamorphosis development. The overall results clearly reveal the disruption of fentin hydroxide and fenbutatin oxide along thyroid signaling pathway in Xenopus laevis during metamorphosis. Given that thyroid hormone signaling is conserved across vertebrate species, our findings from X. laevis highlight potential adverse effects of OTPs on vertebrates. Further studies are warranted to better understand the potential environmental risks of OTPs to aquatic ecosystems and human health.
Fig. 5. Agonistic and antagonistic activities of tested compounds in the TR reporter (α and β) gene assay using stably transfected GH3-TRE cells. A, Cells were treated with increasing concentrations of T3 (1 × 10−12 to 1 × 10−7 M), fentin hydroxide and fenbutatin oxide (1 × 10−10 to 1 × 10−7 M) to detect the activities. Data were presented as mean fold induction compared with vehicle control. B, Cells were treated with 1 × 10−10 to 1 × 10−7 M of fenbutatin oxide with 1 × 10−9 M T3. Values were presented as percent induction, with 100% activity defined as the activity achieved with 1 × 10−9 M of T3.
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Declaration of competing interest The authors have declared no conflict of interest. Acknowledgments We would like to thank Dr. Qiangwei Wang (Zhejiang University) for the donation of GH3-TRE cells. This work was financially supported by the National Natural Science Foundation of China (No. 31572026), the China Postdoctoral Science Foundation (2017M621947) and the Zhejiang Provincial Natural Science Foundation of China (No. LZ18C030001 and No. LZ12C14001). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2019.134140. References Adeeko, A., Li, D., Forsyth, D.S., Casey, V., Cooke, G.M., Barthelemy, J., Cyr, D.G., Trasler, J.M., Robaire, B., Hales, B.F., 2003. Effects of in utero tributyltin chloride exposure in the rat on pregnancy outcome. Toxicol. Sci. 74, 407–415. Alzieu, C., 2000. Environmental impact of TBT: the French experience. Sci. Total Environ. 258, 99–102. Antizar-Ladislao, B., 2008. Environmental levels, toxicity and human exposure to tributyltin (TBT)-contaminated marine environment. A review. Environ. Int. 34, 292–308. Arrojo, E.D.R., Fonseca, T.L., Werneckdecastro, J.P., Bianco, A.C., 2013. Role of the type 2 iodothyronine deiodinase (D2) in the control of thyroid hormone signaling. BBAGen. Subjects 1830, 3956–3964. Bertuloso, B.D., Podratz, P.L., Merlo, E., Araújo, J.F.P.D., Lima, L.C.F., Miguel, E.C.D., Souza, L.N.D., Gava, A.L., Oliveira, M.D., Miranda-Alves, L., 2015. Tributyltin chloride leads to adiposity and impairs metabolic functions in the rat liver and pancreas. Toxicol. Lett. 235, 45–59. Bianco, A.C., Kim, B.W., 2006. Deiodinases: implications of the local control of thyroid hormone action. J. Clin. Invest. 116, 2571–2579. Brtko, J., Dvorak, Z., 2015. Triorganotin compounds—ligands for "rexinoid" inducible transcription factors: biological effects. Toxicol. Lett. 234, 45–59. Coutinho, J.V., Freitaslima, L.C., Freitas, F.F., Freitas, F.P., Podratz, P.L., Magnago, R.P., Porto, M.L., Meyrelles, S.S., Vasquez, E.C., Brandão, P.A., 2016. Tributyltin chloride induces renal dysfunction by inflammation and oxidative stress in female rats. Toxicol. Lett. 260, 52–69. Finnegan, C., Ryan, D., Enright, A.M., Garcia-Cabellos, G., 2018. A review of strategies for the detection and remediation of organotin pollution. Crit. Rev. Env. Sci. Tec. 1, 1–42. Freitas, J.S., Kupsco, A., Diamante, G., Felicio, A.A., Almeida, E.A., Schlenk, D., 2016. Influence of temperature on the thyroidogenic effects of diuron and its metabolite 3,4DCA in tadpoles of the American bullfrog (Lithobates catesbeianus). Environ. Sci. Technol. 50, 13095–13104. Furdek, M., Vahčič, M., Ščančar, J., Milačič, R., Kniewald, G., Mikac, N., 2012. Organotin compounds in seawater and Mytilus galloprovincialis mussels along the Croatian Adriatic Coast. Mar. Pollut. Bull. 64, 189–199. Gui, W.J., Tian, C.X., Sun, Q.Q., Li, S.Y., Zhang, W., Tang, J., Zhu, G.N., 2016. Simultaneous determination of organotin pesticides by HPLC-ICP-MS and their sorption, desorption, and transformation in freshwater sediments. Water Res. 95, 185–194. Hayes, T.B., Case, P., Chui, S., Chung, D., Haeffele, C., Haston, K., Lee, M., Mai, V.P., Marjuoa, Y., Parker, J., 2006. Pesticide mixtures, endocrine disruption, and amphibian declines: are we underestimating the impact? Environ. Health Persp. 114, 40–50. Hayes, T.B., Khoury, V., Narayan, A., Nazir, M., Park, A., Brown, T., Adame, L., Chan, E., Buchholz, D., Stueve, T., Gallipeau, S., 2010. Atrazine induces complete feminization and chemical castration in male African clawed frogs (Xenopus laevis). P. Natl. Acad. Sci. Usa. 107, 4612–4617. Hoopfer, E.D., Huang, L., Denver, R.J., 2010. Basic transcription element binding protein is a thyroid hormone-regulated transcription factor expressed during metamorphosis in Xenopus laevis. Develop. Growth Differ. 44, 365–381.
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Contents lists available at ScienceDirect
Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Disruptive effects of two organotin pesticides on the thyroid signaling pathway in Xenopus laevis during metamorphosis Shuying Li a, Kun Qiao a, Yao Jiang a, Qiong Wu a, Scott Coffin b, Wenjun Gui a,⁎, Guonian Zhu a a b
Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310058, PR China Department of Environmental Sciences, University of California, Riverside, CA 92521, United States
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Fenbutatin oxide and fentin hydroxide showed thyroid disrupting effects on Xenopus laevis. • SPR indicated organotin pesticides could bind to thyroid receptor with different binding ability. • Reporter gene assay indicated antagonist activity of fentin hydroxide but not fenbutatin oxide.
a r t i c l e
i n f o
Article history: Received 27 May 2019 Received in revised form 25 August 2019 Accepted 26 August 2019 Available online 27 August 2019 Editor: Yolanda Picó Keywords: Organotin pesticides Xenopus laevis Thyroid endocrine disruption Surface plasmon resonance Reporter gene assay
a b s t r a c t Organotin compounds are the ubiquitous environmental pollutants due to their wide industrial and agricultural applications and unexpected releasing into the environment, which show characteristic of endocrine disruptors to interfere with the synthesis, receptor binding or action of endogenous-hormones. Organotin pesticides (OTPs) are used in agriculture and may impact endocrine functions on organisms. Thyroid hormones (THs) play fundamental roles in regulating the basal metabolism and energy balance, while thyroid function can be impaired by environmental contaminants. Therefore, it is crucial to clarify the effects and mechanisms of OTPs on hypothalamus-pituitary-thyroid (HPT) axis. In this study, Xenopus laevis tadpoles at stage 51 were exposed to fentin hydroxide and fenbutatin oxide (0.04, 0.20 and 1.00 μg·L−1) for 21 days. It was found that both compounds caused inhibitory effects on metamorphic development of tadpoles (e.g., significant decrease in hindlimb length and retarding development). Triiodothyronine (T3) significantly decreased in tadpoles exposed to 0.20 μg/L and 1.00 μg/L of the two OTPs for 14 days or 21 days. The expressions of TH responsive genes trβ, bteb and dio2 were down-regulated, while tshβ and slc5a5 were up-regulated. Surface plasmon resonance (SPR) binding assays showed that fentin hydroxide had a moderate affinity to recombinant human thyroid hormone receptor β but fenbutatin oxide did not have. Result of the SPR assay was highly consistent with the luciferase reporter gene assays that fentin hydroxide suppressed the relative luciferase activity in the presence of T3 while fenbutatin oxide did not, demonstrating fentin hydroxide but not fenbutatin oxide displayed an antagonistic activity against T3-TR complex mediated transcriptional activation. Overall, the findings elucidated the mechanisms induced by OTPs along HPT axis. These results highlighted the adverse influences of organotin pesticides on thyroid
⁎ Corresponding author at: Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310058, PR China. E-mail address: [email protected] (W. Gui).
https://doi.org/10.1016/j.scitotenv.2019.134140 0048-9697/© 2019 Elsevier B.V. All rights reserved.
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S. Li et al. / Science of the Total Environment 697 (2019) 134140
hormone- dependent development in vertebrates and the need for more comprehensive investigations of their potential ecological risks. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Organotins (OTs) are organometallic compounds used for various industrial purposes including disinfectants, pesticides, and most frequently as biocides in antifouling paints (Gui et al., 2016; Li et al., 2016a; Marques et al., 2018; Yan et al., 2018). Aquatic pollution attributed to the use of OTs is of great concern due to the adverse biological effects on nontarget biota, even at Sn concentration as low as ng·L−1 (Alzieu, 2000). Although many countries worldwide had banned on OT-based paints from the 1980s (Furdek et al., 2012), OTs (such as tributyltin and triphenyltin) are still detected in water, sediments and biota at concentrations ranging from ng Sn·L−1 (or g−1) to μg Sn·L−1 (or g−1) (Antizar-Ladislao, 2008; Furdek et al., 2012; Radke et al., 2013). These OTs can be easily accumulated by living organisms and further transferred to humans through food chain (Furdek et al., 2012), consequently increasing health and environmental risks. However, OTs such as fentin hydroxide, fenbutatin oxide, azocyclotin and fentin acetate are still used extensively as pesticides in agricultural applications (Gui et al., 2016; Li et al., 2016a). Few studies focused on the toxicity and mechanism induced by these organotin pesticides (OTPs). Previous studies showed that OTs could cause a range of toxic outcomes, including genetic, hepatic, renal, neural and immune toxicity (Bertuloso et al., 2015; Coutinho et al., 2016). For example, Merlo et al. (2016) reported that tributyltin exerted its toxicity on the nervous system of rat after treated with 100 ng kg−1 for 15 days. It is confirmed that some of OTs (such as tributyltin and triphenyltin) are capable of altering functions of tissues (e.g. hypothalamus, pituitary, gonad, adrenal, and thyroid gland) on some non-target organisms, playing as endocrine disruptors mainly through interacting with transcriptional regulators including nuclear and steroid receptors (e.g. estrogen receptor (ER), retinoid X receptor (RXR), and peroxisome proliferator activated receptor (PPAR)) (Bertuloso et al., 2015; Coutinho et al., 2016). For instance, Marques et al. (2018) described that OTs might induce endocrine disorders due to the capability of regulation on the enzyme P450arom. In fish, tributyltin or azocyclotin exposure not only reduced 17βestradiol serum levels but also changed ER expression in different sites along hypothalamus-pituitary-gonadal (HPG) axis (Ma et al., 2016; Marques et al., 2018). Moreover, a few studies showed the effects of OTs on hypothalamus-pituitary-thyroid (HPT) axis. Adeeko et al. (2003) reported that tributyltin exposure would lead to reduction in serum thryoid hormones (THs) triiodothyronine (T3) and thyroxine (T4) levels in pregnancy rat. Sharan et al. (2014) demonstrated that decreases in the activities of thryoid hormone receptors α and β (TRα, and TRβ) as well as lower protein expression of TRβ were found in hepatocarcinoma cells after tributyltin exposure. Furthermore, in amphibian Xenopus laevis, the colloid depletion and follicle malformation were observed in thyroid gland after 19 days of tributyltin exposure (Shi et al., 2012). These studies showed a potential thyroid disruption induced by OTs. Our previous study also provided the evidences of azocyclotin-induced metamorphosis disruption in Xenopus laevis (Li et al., 2016a). However, effects induced by fentin hydroxide and fenbutatin oxide on TH-dependent development at the morphological and functional level are still poorly understood. The molecular structures of fentin hydroxide and fenbutatin oxide are similar to those of other OTs, but the thyroid endocrine disrupting effects of the two chemicals on environmental organisms remains unknown. Amphibians share multiple similarities with vertebrates in development, and as such, amphibian metamorphosis is regarded as an ideal model to study TH-dependent development and the mechanisms of TH action in vertebrates (Zhang et al., 2014; Freitas et al., 2016).
Considering pesticide exposure has been regarded as one of the main causes of the decline in amphibian populations worldwide (Hayes et al., 2006; Freitas et al., 2016), the evaluation of effects and mechanisms caused by OTPs could elucidate their potential risks to the populations of amphibian and even vertebrates. In the present study, in order to explore the thyroid endocrine disrupting effect of fentin hydroxide and fenbutatin oxide, tadpoles (X. laevis) were exposed to fentin hydroxide or fenbutatin oxide. Then, the developmental retarding effects, the THs levels and the expressions of genes involved in X. laevis metamorphic pathways such as basic transcription element binding protein (bteb), deiodinases (dio2), thyroid hormone receptors (trβ), transthyretin (ttr), thyriod stimulating hormone (tshβ), sodium/iodide cotransporter (slc5a5) were evaluated at different time point. In vitro, we employed the firefly luciferase assay to investigate the THdisrupting effects of the two OTPs. What’ more, the molecular interactions between thyroid receptor (TR) and the OTPs were determined by using a surface plasmon resonance (SPR) biosensor to reveal the underlying mechanisms of thyroid disruption. 2. Materials and methods 2.1. Chemicals and reagents Two OTPs standards of fenbutatin oxide (CAS: 13356–08-6, purity N95%) and fentin hydroxide (CAS: 76–87-9, purity N95%) were obtained from Zhejiang Heben Pesticide & Chemicals Co., Ltd. Dimethyl sulfoxide (DMSO, cell culture reagent), T3 and tricaine methanesulfonate (MS222) were purchased from Sigma (St. Louis, USA). OTPs stock solutions (100 mg·L−1) were dissolved in DMSO and stored at −20 °C for no more than one month. TRIzol™, RNase-free water, PrimeScript RT reagent Kit and SYBR Premix Ex Tap™ II were purchased from Takara (Dalian, China). Purified recombinant human thyroid hormone receptor β (hTRβ) protein was purchased from CusaBio (Wuhan, China). 2.2. Metamorphosis assay X. laevis (Nasco, Fort Atkins, WI, USA) were maintained according to our previous study (Li et al., 2017). Development stages of tadpoles were determined according to Nieuwkoop and Faber (NF) (Nieukoop and Faber, 1967). Detailed information was provided in Supporting Information (SI-1). X. laevis tadpoles of NF stage 51 were individually exposed to the two OTPs (with the same concentration sets of 0.04, 0.20 and 1.00 μg·L−1 for each OTP, exposure concentrations were set based on the published study (Li et al., 2016a) for 21 days (20 tadpoles per glass tank (50 cm × 25 cm × 30 cm) filled with 18 L exposure solution. Each exposure solution contained 0.01% (by volume) DMSO), during which time the tadpoles were undergoing for metamorphic development (Nieukoop and Faber, 1967). Tadpoles in pesticide-free exposure solution (containing 0.01% DMSO, v/v) were set as vehicle control (VC). Each treatment was conducted in triplicate. Dead tadpoles were removed twice a day and exposure solutions were renewed at an interval of 24 h. Water samples were collected before and after renewal and stored in darkness at −80 °C until analysis. Tadpoles were randomly sampled from each tank at 7th, 14th and 21th d after exposure (10 tadpoles per tank per time) for the survival rate (%), malformation rate (%), hindlimb length (mm), and developmental stage check. At each sampling time point, 3 tadpoles were randomly collected and anesthetized using MS-222 (0.01 g·L−1), and then undergone for mRNA (hindlimb) and THs (head) analysis.
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2.3. Quantification of chemicals in exposure solutions The fentin hydroxide and fenbutatin oxide in exposure solutions were determined by high performance liquid chromatographyinductively coupled plasma-mass spectrometry (HPLC-ICP-MS) which were described in a previous study (Gui et al., 2016) with slightly modified. Detailed information was provided in Supporting Information (SI2).
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DMSO and 0.05% (by volume) surfactant P20. Buffer samples containing 4.5–5.8% (by volume) DMSO were injected to construct a DMSO calibration plot to correct for bulk refractive index shift (Rich et al., 2002). Running buffer was set at a flow rate of 30 μl·min−1. For each test chemical, the association phase of the SPR measurement was set to 120 s, and the buffer flow was continued to allow a dissociation time of 300 s. The kinetic and affinity constants were obtained by fitting the experimental data to a reversible 1:1 bimolecular interaction model in Biacore Evaluation Software.
2.4. Thyroid hormones assay 2.8. Statistical analysis The measurement of THs was performed using a commercial enzyme-linked immunosorbent assay (ELISA) kit (Uscnlife, Wuhan, China). Briefly, three tadpoles (head) from each tank were weighed and homogenized in 4 mL of assay buffer. After centrifugation (7, 500 g) at 4 °C for 15 min, the supernatant was separated and undertaken for T3 and T4 measurement. The ELISA was conducted in 2 wells technical replicates and 3 biological replicates. The detection limits for T4 and T3 of the method were 1.46 ng·mL−1 and 47.1 pg·mL−1, respectively. The kits were certified for use with X. laevis sample by validating the correlation between a series of diluted and spiked samples in reference to both T3 and T4 standard curves.
Statistical analysis was performed using SPSS® version 20.0 (SPSS, USA). All data were expressed as means ± standard deviation (SD). The Kruskal-Wallis test was used for measurement of endpoints (hind limb length, developmental stage). Statistical differences in gene expression and hormone concentrations were evaluated by one-way analysis of variance (ANOVA) following Tukey's post-hoc test. A p-value b0.05 was considered statistically significant. 3. Results 3.1. Quantification of chemicals in solution
2.5. Quantitative real-time PCR Total RNA was extracted using TRIzol® reagent as described by our previous report (Li et al., 2016b). Primer sequences of genes were provided in Table S1 which were developed in a previous study (Zhang et al., 2014). The 2-△△Ct method was used to calculate the target mRNA expression (Livak and Schmittgen, 2001). Detailed information was described in the Supporting Information (SI-3).
The real concentrations of fentin hydroxide and fenbutatin oxide in the exposure solutions were monitored in the metamorphosis test at one renewal period. Results showed that the degradation rates of fentin hydroxide and fenbutatin oxide in water phase (24 h) were all b20% during 24 h of refreshing interval (Table 1), which conformed to the requirement of OECD Guideline 203 (OECD, 1992). It indicated that the fluctuations of the OTPs in exposure solutions were acceptable and the refreshing interval was reasonable.
2.6. Cell-based bioassays 3.2. Developmental toxicity endpoints Thyroid hormone receptor (TR) activity was assessed by using established in vitro GH3-TRE cell (cell provided by Dr. Qiangwei Wang) assay. Bioassay protocols were based on previous report (Xiang et al., 2017). Briefly, cells were plated in a 96-well microplate at a density of 1.5 × 104 cells per well in DMEM/F-12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12) medium (Thermo fisher, USA) with penicillin/streptomycin (Thermo fisher, USA) and 100 U/mL hygromycin B (APExBIO, USA) followed by a 24 h incubation. For agonistic/antagonistic activity tests, cells were exposed to T3 ranging from 10−12 to 10−7 M (set as positive control), and pesticides ranging from 10−10 to 10−7 M. After 24 h, light produced was measured with Spectra Max i3 (Molecular Devices, USA) using the Luciferase Reporter Assay Kit (Promega, USA). All tests were carried out three independent times, each with six replicates per test concentration. Cytotoxicity induced by tested chemical was performed using a 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4sulfophenyl)-2H-tetrazolium (MTS) assay on cells. 2.7. Surface plasmon resonance (SPR) binding assay SPR assays were performed on a Biacore™ T200 system (GE Healthcare, Sweden) with hTRβ protein. In both Homo sapiens and Xenopus laevis, TRs are nuclear receptors which control transcription, and thereby have effects in all cells within the body. Thyroid hormone receptors are encoded by two TR genes. There is some specie/tissue specificity in the distribution of these isoforms (Mackenzie, 2018). The hTRβ protein was covalently immobilized to a CM7 sensor chip (GE Healthcare, Sweden) using an amine-coupling procedure, in which HBS-N (0.01 M HEPES (4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid) and 0.15 M NaCl, pH 7.4) containing 0.05% surfactant P20 (Polysorbate 20) (GE Healthcare, Sweden) was used as immobilized running buffer. Chemicals were dissolved in HBS-N buffer containing 5% (by volume)
Tadpoles at NF stage 51 were exposed to either fentin hydroxide or fenbutatin oxide in order to investigate their effects on development of frog via TH signaling disruption. Exposed to fentin hydroxide or fenbutatin oxide for 21 days did not significantly affect survival rates of tadpoles (Table S2). However, tadpoles exhibited pronounced morphological inhibitions including hindlimb growth and lower developmental stage (Fig. 1). All tadpoles developed to stages 52–54 from stage 51 on day 7. Compared to VC (29.6%), reductions in proportion of stage 54 were observed in the groups of fentin hydroxide at 0.04 μg·L−1 (14.8%, p = 0.047) and 1.00 μg·L−1 (7.4%, p = 0.013). Similar results were also found in fenbutatin oxide exposure groups. The percentages of stage 54 were 14.8% (p = 0.047) and 11.1% (p = 0.038) in 0.20 and 1.00 μg/L groups, respectively. In all exposure groups, hindlimb length tended to decrease, while significantly decreases only observed at 1.00 μg·L−1 fenbutatin oxide (p = 0.013). Table 1 Concentrations of organotin pesticides in exposure solutions. Nominal concentrations (μg·L−1)
Vehicle control Fentin hydroxide
Fenbutatin oxide
0.04 0.20 1.00 0.04 0.20 1.00
Measured concentrationsa (μg·L−1) 0h
24 h
ND 0.046 ± 0.001 0.212 ± 0.009 1.114 ± 0.031 0.042 ± 0.003 0.209 ± 0.012 1.094 ± 0.025
ND 0.036 ± 0.003 0.167 ± 0.004 0.944 ± 0.018 0.031 ± 0.004 0.159 ± 0.008 0.817 ± 0.037
ND, not detected. a Values represent mean ± SD (n = 3).
Degradation rate (%)
– 19.3 14.2 15.3 16.1 13.3 15.7
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Fig. 1. Developmental retarding effect of fentin hydroxide and fenbutatin oxide on hind limb length (mm) of tadpoles (A), as well as developmental stage ratios of tadpoles exposed to fentin hydroxide (B) and fenbutatin oxide (C). Asterisk indicated significant differences (*, p b 0.05) between exposure groups and the corresponding vehicle control.
On day 14, all tadpoles developed to stages 54–55 in VC group while parts of tadpoles remained in stage 53 in all exposure groups. The proportion of stage 55 significantly decreased in 1.00 μg·L−1 fentin hydroxide group (3.7%, p = 0.047) and 1.00 μg·L−1 fenbutatin oxide group (3.7%, p = 0.047), respectively (18.5% in VC group). Increased percentages of stage 53 were observed in fentin hydroxide (0.04 μg·L−1, 18.5%, p = 0.038; 0.20 μg·L−1, 18.5%, p = 0.038; 1.00 μg·L−1, 29.6%, p = 0.015) and fenbutatin oxide (1.00 μg·L−1, 40.7%, p = 0.032) compared with VC. Significant decreases in hindlimb length were found in all 0.20 μg·L−1 (p b 0.05) and 1.00 μg/L (p b 0.05) treated groups. Following 21d exposure, the inhibiting effects on development became pronounced. Tadpoles developed to stages 54–56 in VC, but most of the tadpoles remained in stage 54 in fentin hydroxide and fenbutatin oxide exposure groups. Moreover, significant greater inhibitions to hindlimb length were observed in 0.20 and 1.00 μg·L−1 treated groups (p b 0.05). 3.3. Thyroid hormone levels TH levels in tadpoles were examined after exposure to the two OTPs. The total T4 levels were not significantly altered in any exposure group at any sampling point (Fig. 2). On day 7, no significant changes in T3 content were observed. While on day 14, T3 levels were significantly
decreased in 0.04 μg/L (8.08 ng·g−1, p = 0.014) and 1.00 μg/L (8.80 ng·g−1, p = 0.038) fentin hydroxide exposure groups, relative to VC. Additionally, decreased T3 levels were observed in 1.00 μg/L (7.26 ng·g−1, p = 0.037) fenbutatin oxide on day 14. On day 21, significantly decreased T3 levels were measured in both fentin hydroxide (0.20 μg/L: 15.01 ng·g−1, p = 0.027; 1.00 μg/L: 11.46 ng·g−1, p = 0.006) and fenbutatin oxide (0.20 μg/L: 18.27 ng·g−1, p = 0.043; 1.00 μg/L: 16.62 ng·g−1, p = 0.018) treatments compared with VC. 3.4. Gene transcription Transcriptional profiles of genes involved in TH-signaling pathway and TH-response (bteb, dio2, trβ, ttr, tshβ and slc5a5) were further examined in tadpoles after exposure to fentin hydroxide and fenbutatin oxide (Fig. 3). Exposed to fentin hydroxide or fenbutatin oxide failed to alter the expression of the target genes on day 7 except that in 1.00 μg/L fentin hydroxide exposure group, the expression of trβ in tadpole hindlimb was reduced by 0.46-fold (p = 0.037). On day 14, significant down-regulations (p b 0.05) of bteb, dio2 and trβ mRNA expression were observed in all treatments of the two tin-pesticide. On the contrary, significant concentration-associated increases (p b 0.05) of tshβ and slc5a5 expression were observed in all treatments of the two chemicals compared to VC. Similar alterations were found in the
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expressions of the selected genes after 21 days exposures (significant down-regulations in bteb, dio2 and trβ, significant up-regulations in tshβ and slc5a5 (all compared to VC (p b 0.05)). 3.5. Binding kinetics of chemicals to hTRβ Binding assays were performed in a quantification screening mode and kinetic analyses for agonists and antagonists were performed to determine the rate constants associated with Human TRβ (hTRβ). As a positive control, T3 displayed a concentration-dependent response which could reach equilibrium and return to baseline within seconds, indicating a rapid association and a fast disassociation between T3 and TR (Fig. 4A). Fenbutatin oxide and fentin hydroxide both showed direct binding interaction with hTRβ (Fig. 4B, C), but the association and dissociation curves of fentin hydroxide and fenbutatin oxide trended to differ from those of T3. The association (ka) and dissociation (kd) rate constants and the equilibrium dissociation constants (KD) for the tested compounds were represented in Table 2. Compared with T3, up to 13fold and 1000-fold lower association rate constants were observed in fentin hydroxide and fenbutatin oxide, respectively. Results indicated that fentin hydroxide had a moderate binding ability while fenbutatin oxide had a very weak binding ability to hTRβ. 3.6. Luciferase reporter gene assay The cytotoxic concentrations of tested chemicals were determined by MTS assay before performing receptor assay (data not shown), and results showed that the tested concentrations of chemicals did not affect the viability and proliferation of GH3-TRE cells. The relative luciferase activity (RLA) induced by thyroid hormone T3 in TRβ-mediated luciferase reporter gene assay was shown in Fig. 5A. T3 induced RLA in a concentration-dependent manner ranging from 10−12 to 10−6 M, and the EC50 (the term half maximal effective concentration) of T3 was 2.8 × 10−9 M. Agonistic and antagonistic effects of the pesticides mediated via TR were estimated. For evaluation of the agonistic effect, GH3 cells were treated with the pesticides both ranging from 1 × 10−10 to 1 × 10−7 M in the absence of T3. It was found that only fentin hydroxide showed weak TR agonistic effects (Fig. 5A) with the REC20 (20% relative effective concentration (Xiang et al., 2017), i.e., concentration of the
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tested chemicals showing 20% of the RLA of 1 × 10−7 M T3) of fentin hydroxide was N10−7 M. The antagonistic activity was conducted in the presence of 1 × 10−9 M T3 (Fig. 5B). Results showed that fentin hydroxide displayed a weak antagonistic effect against T3 while fenbutatin oxide had no significant influence on T3. The 20% relative inhibitory concentration (RIC20), concentration of the tested chemicals showing a 20% reduction in the RLA of 1 × 10−9 M T3) of fentin hydroxide was 2.2 × 10−8 M. 4. Discussion Great concerns have been raised in recent years over the potential adverse effects of OTs on the endocrine system (Ma et al., 2016; Li et al., 2017; Finnegan et al., 2018; Yan et al., 2018). The actions of OTs span many endocrine axes, including HPG and HPT axes (Santos-Silva et al., 2018). OTs may impact developmental steps controlled by the HPT axis (Li et al., 2016c) such as metamorphosis in the amphibian Xenopus laevis. However, effects induced by OTPs on TH-dependent development at the morphological and functional levels are poorly understood. In this study, significantly inhibitory effects were observed on tadpole metamorphosis which was characterized by multiple morphological changes including shortened hindlimb protrusion and delayed development stages, particularly after 14 and 21 days of fentin hydroxide and fenbutatin oxide exposure. Our results demonstrated that fentin hydroxide and fenbutatin oxide inhibited metamorphic development of tadpoles. These findings agreed with previous studies in which it had been demonstrated that organotin compounds could delay metamorphosis and inhibit the growth of tadpole (Shi et al., 2012; Li et al., 2016a). Our findings further supported the hypothesis that the metamorphosis of X. laevis could be inhibited by OTPs. Moreover, inhibitory effects of OTPs on thyroid actions were further supported by decreased T3 levels and altered expression of TH-related genes including bteb, trβ, dio2, tshβ and slc5a5 in previous researches (Zhang et al., 2014; Li et al., 2016c). TH is a key variable affecting the biological developmental such as amphibian metamorphosis (Kloas, 2002). It is already known that some endocrine disrupting chemicals targeting thyroid gland have the ability to change circulating levels of TH, altering the relationship between TH biosynthesis and elimination, which results in acceleration or retardation of metamorphosis (Kloas,
Fig. 2. T3 and T4 levels in Xenopus laevis tadpoles after exposed to different concentrations of fentin hydroxide (A and B) and fenbutatin oxide (C and D) for 7, 14 and 21 d. Data were shown as mean ± SD. *p b 0.05 indicated significant differences between exposure groups and the corresponding vehicle control.
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Fig. 3. The mRNA expression of genes in Xenopus laevis tadpoles that were exposed to different concentrations of fentin hydroxide (A) and fenbutatin oxide (B) for 7, 14 and 21 days. Data were shown as mean ± SD. *p b 0.05 indicates significant differences between exposure groups and the corresponding vehicle control.
2002). For examples, decreased levels of T4 and T3 were observed in X. laevis after exposed to triadimefon (Li et al., 2016b). Azocyclotin led to a decreased level of T3 in X. laevis tadpole (Li et al., 2016a). In the present study, significant decreases in T3 levels (which were associated with inhibition of metamorphic development) were observed after 14 days and 21 days of exposure, indicating the effects of fenbutatin oxide and fentin hydroxide on metamorphic development are mainly mediated by TH signaling disruption. T3 levels in plasma are regulated by deiodinases, in which iodothyronine deiodinase II (Dio2) is responsible for converting T4 to T3 (Bianco and Kim, 2006). In X. laevis, the increased mRNA expression of dio2 has shown to be concomitant with the beginning of metamorphosis (Arrojo et al., 2013). The disruption of dio2 expression resulted in a state of localized hypothyroidism (Arrojo et al., 2013). Our results demonstrated that dio2 mRNA expression was down-regulated in
X. laevis exposed to the highest concentration of organotin pesticides on day 14, and this effect was enhanced in tadpoles following 21 days of exposure. Decreases in dio2 mRNA expression should lead to reduced T3 levels, consistent with the decrease of dio2 expression, the reducing levels of T3 were also observed. Therefore, the down-regulation of dio2 might be responsible for the decreased T3 levels. In addition, with the prolongation of exposure time, the relative levels of T4 (compared to corresponding VC) generally showed a slight upward trend after exposure to fentin hydroxide. While exposed to fenbutatin oxide, the relative levels of T4 remained basically unchanged. However, how fentin hydroxide or fenbutatin oxide affects dio2 and why T4 displays differential alternation after exposure to the two chemicals remain unknown and further studies are needed to confirm the mechanisms. Two important genes related to TH synthesis pathway, slc5a5 and tshβ, were examined in this study. As reported, the up-regulation of
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Fig. 4. Representative sensorgrams for kinetic analysis of hTRβ interactions. The T3 (A) was injected at concentrations of 0.0125, 0.025, 0.05 and 0.1 μM (from bottom to top). Fenbutatin oxide (B) was injected at concentrations of 0.039, 0.078, 0.312, 1.25 and 2.5 mM (from bottom to top). Fentin hydroxide (C) was injected at concentrations of 0.625, 1.25, 2.5 and 5.0 mM (from bottom to top). All chemicals were injected for 120 s, and disassociation was monitored for 300 s. The data of each interaction were fitted to a 1:1 bimolecular interaction model.
slc5a5 and tshβ might promote the TH production in tadpoles (Wang et al., 2013), while TH can be regulated by thyroid stimulating hormone (TSH) via feedback mechanisms (Wang et al., 2013). Similar results have demonstrated that the decrease of TH was accompanied with the increases in mRNA expression of tshβ and slc5a5 after thyroid disruptor exposure (Wang et al., 2013; Li et al., 2016c), which is consistent with our findings. Significant down-regulation of T3 but up-regulation of the two
genes were observed in the present study. Therefore, the up-regulation of slc5a5 and tshβ could be regarded as a compensatory response to decreased T3. But more measurements of thyroid development should be further applied to verify this compensatory mechanism. Concurrently, decreased transcription levels of bteb were observed following exposure to both fentin hydroxide and fenbutatin oxide. BTEB (basic transcription element binding protein) could bind the GC-rich basic transcription
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Table 2 Kinetic parameters of the binding of TR. Interaction
ka (1/Ms)
kd (1/s)
KD (M)
T3 Fenbutatin oxide Fentin hydroxide
3.33E+06 3.22E+03 2.49E+05
6.69E−03 1.28E−02 1.55E−02
2.01E−09 3.97E−06 6.21E−08
*ka, association rate constants. kd, dissociation rate constants. KD, equilibrium dissociation constants.
element and functions as a transcriptional activator on TH-response genes promoters (Hoopfer et al., 2010; Zhang et al., 2014). So, the decreased tendency of bteb was consistent with the reduction of T3. The antagonistic effects of the two OTPs on frog development were further supported by expression of the decrease of bteb. TR function as transcription factors whose activity is regulated by the binding of T3 (Hayes et al., 2010) and mediated TH-regulated gene expression by binding to response elements (Morvan-Dubois et al., 2008). In the present study, with 7 days of development there were retarding effects of the organotins, though thyroid hormones were unaltered. The metamorphosis of tadpole was mainly regulated by THs, as well as the transcription of specific sets of genes, including the thyroid hormone receptors and associated coactivators (Vitt and Caldwell, 2014). Hence, the decreased mRNA expression of trβ might explain this interesting finding. Previous in vitro studies also demonstrated that tributyltin disrupted TR transcriptional regulation whatever in absence or presence of T3 (Sharan et al., 2014). The present luciferase reporter gene assay showed that fentin hydroxide weakly stimulated the transcriptional activity of TR in absence of T3 but conversely slightly suppressed the transcriptional activity of TR in presence of T3. However, fenbutatin oxide displayed no influence on transcriptional regulation of TR. Although tributyltin, fentin hydroxide and fenbutatin oxide all belong to members of the OTs family, there are still obvious differences in their chemical structures. This difference in chemical structure might explain the aforementioned their different effects on transcriptional regulation of TR, also reminding that different organotin may have different molecular mechanisms although they may have the same or similar toxicological characterizations. But further studies are still needed to clarify the detailed molecular mechanism. Recent studies indicated that environmental chemicals could bind to TRs and might activate or inhibit the action of T3, or change the affinity of TRs for response elements in specific target genes, these impacts might alter the normal ability of TRs to bind to TH, resulting in
interference with thyroid function and TH signaling (Freitas et al., 2016). In the present study, SPR was applied for the evaluation of molecular interaction between chemicals and TR protein. SPR is a high sensitivity and reliable detection method which allows for the analysis of biomolecular interactions and molecular mechanisms (Nguyen et al., 2015). A wide range of applications have been developed for affinity binding analysis using SPR biosensors, including ligand-receptor kinetics (Nguyen et al., 2015; Li et al., 2017; Xiang et al., 2017). The dynamic interaction curve of chemicals binding to biomacromolecule given by SPR can clearly reveal the binding strength of chemicalsbiomacromolecule and the differences in binding mode between different compounds and the same biomacromolecule. Commonly, agonists display relatively fast association and dissociation rate constants from the surface of biomacromolecule. In contrast, antagonists show slower association and dissociation rates (Rich et al., 2002). In the present study, compared to T3 (an endogenous TRβ agonist)-hTRβ interaction, lower association and dissociation rate constants which shared the features of antagonistic interaction were observed in fentin hydroxidehTRβ interaction, indicating a potential inhibiting action on T3 binding to TR. For fenbutatin oxide, its interaction with hTRβ could almost be ignored (the ka value almost 1000-fold lower than that of T3). The evidence of differential interaction of fentin hydroxide and fenbutatin oxide binding to hTRβ in this SPR-based affinity binding assay was highly consistent with the result of the luciferase reporter gene assay. In summary, previous studies have reported thyroid endocrine disrupting effects induced by OTs (Brtko and Dvorak, 2015; Li et al., 2016a; Finnegan et al., 2018; Santos-Silva et al., 2018). In the present study, we found that fentin hydroxide and fenbutatin oxide significantly altered thyroid hormone levels and expression of thyroid hormonerelated genes, and induced pronounced morphological inhibitions in tadpoles of X. laevis. It is deduced that both fentin hydroxide and fenbutatin oxide repress dio2, thereby reducing T3. Moreover, fentin hydroxide but not fenbutatin oxide disrupts the normal binding interaction between TR and T3, therefore suppressing TH-TR complexmediated transcriptional activation, and further affecting physiological function of X. laevis such as the retarding of metamorphosis development. The overall results clearly reveal the disruption of fentin hydroxide and fenbutatin oxide along thyroid signaling pathway in Xenopus laevis during metamorphosis. Given that thyroid hormone signaling is conserved across vertebrate species, our findings from X. laevis highlight potential adverse effects of OTPs on vertebrates. Further studies are warranted to better understand the potential environmental risks of OTPs to aquatic ecosystems and human health.
Fig. 5. Agonistic and antagonistic activities of tested compounds in the TR reporter (α and β) gene assay using stably transfected GH3-TRE cells. A, Cells were treated with increasing concentrations of T3 (1 × 10−12 to 1 × 10−7 M), fentin hydroxide and fenbutatin oxide (1 × 10−10 to 1 × 10−7 M) to detect the activities. Data were presented as mean fold induction compared with vehicle control. B, Cells were treated with 1 × 10−10 to 1 × 10−7 M of fenbutatin oxide with 1 × 10−9 M T3. Values were presented as percent induction, with 100% activity defined as the activity achieved with 1 × 10−9 M of T3.
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Declaration of competing interest The authors have declared no conflict of interest. Acknowledgments We would like to thank Dr. Qiangwei Wang (Zhejiang University) for the donation of GH3-TRE cells. This work was financially supported by the National Natural Science Foundation of China (No. 31572026), the China Postdoctoral Science Foundation (2017M621947) and the Zhejiang Provincial Natural Science Foundation of China (No. LZ18C030001 and No. LZ12C14001). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2019.134140. References Adeeko, A., Li, D., Forsyth, D.S., Casey, V., Cooke, G.M., Barthelemy, J., Cyr, D.G., Trasler, J.M., Robaire, B., Hales, B.F., 2003. Effects of in utero tributyltin chloride exposure in the rat on pregnancy outcome. Toxicol. Sci. 74, 407–415. Alzieu, C., 2000. Environmental impact of TBT: the French experience. Sci. Total Environ. 258, 99–102. Antizar-Ladislao, B., 2008. Environmental levels, toxicity and human exposure to tributyltin (TBT)-contaminated marine environment. A review. Environ. Int. 34, 292–308. Arrojo, E.D.R., Fonseca, T.L., Werneckdecastro, J.P., Bianco, A.C., 2013. Role of the type 2 iodothyronine deiodinase (D2) in the control of thyroid hormone signaling. BBAGen. Subjects 1830, 3956–3964. Bertuloso, B.D., Podratz, P.L., Merlo, E., Araújo, J.F.P.D., Lima, L.C.F., Miguel, E.C.D., Souza, L.N.D., Gava, A.L., Oliveira, M.D., Miranda-Alves, L., 2015. Tributyltin chloride leads to adiposity and impairs metabolic functions in the rat liver and pancreas. Toxicol. Lett. 235, 45–59. Bianco, A.C., Kim, B.W., 2006. Deiodinases: implications of the local control of thyroid hormone action. J. Clin. Invest. 116, 2571–2579. Brtko, J., Dvorak, Z., 2015. Triorganotin compounds—ligands for "rexinoid" inducible transcription factors: biological effects. Toxicol. Lett. 234, 45–59. Coutinho, J.V., Freitaslima, L.C., Freitas, F.F., Freitas, F.P., Podratz, P.L., Magnago, R.P., Porto, M.L., Meyrelles, S.S., Vasquez, E.C., Brandão, P.A., 2016. Tributyltin chloride induces renal dysfunction by inflammation and oxidative stress in female rats. Toxicol. Lett. 260, 52–69. Finnegan, C., Ryan, D., Enright, A.M., Garcia-Cabellos, G., 2018. A review of strategies for the detection and remediation of organotin pollution. Crit. Rev. Env. Sci. Tec. 1, 1–42. Freitas, J.S., Kupsco, A., Diamante, G., Felicio, A.A., Almeida, E.A., Schlenk, D., 2016. Influence of temperature on the thyroidogenic effects of diuron and its metabolite 3,4DCA in tadpoles of the American bullfrog (Lithobates catesbeianus). Environ. Sci. Technol. 50, 13095–13104. Furdek, M., Vahčič, M., Ščančar, J., Milačič, R., Kniewald, G., Mikac, N., 2012. Organotin compounds in seawater and Mytilus galloprovincialis mussels along the Croatian Adriatic Coast. Mar. Pollut. Bull. 64, 189–199. Gui, W.J., Tian, C.X., Sun, Q.Q., Li, S.Y., Zhang, W., Tang, J., Zhu, G.N., 2016. Simultaneous determination of organotin pesticides by HPLC-ICP-MS and their sorption, desorption, and transformation in freshwater sediments. Water Res. 95, 185–194. Hayes, T.B., Case, P., Chui, S., Chung, D., Haeffele, C., Haston, K., Lee, M., Mai, V.P., Marjuoa, Y., Parker, J., 2006. Pesticide mixtures, endocrine disruption, and amphibian declines: are we underestimating the impact? Environ. Health Persp. 114, 40–50. Hayes, T.B., Khoury, V., Narayan, A., Nazir, M., Park, A., Brown, T., Adame, L., Chan, E., Buchholz, D., Stueve, T., Gallipeau, S., 2010. Atrazine induces complete feminization and chemical castration in male African clawed frogs (Xenopus laevis). P. Natl. Acad. Sci. Usa. 107, 4612–4617. Hoopfer, E.D., Huang, L., Denver, R.J., 2010. Basic transcription element binding protein is a thyroid hormone-regulated transcription factor expressed during metamorphosis in Xenopus laevis. Develop. Growth Differ. 44, 365–381.
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