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Unraveling the toxic effects of neonicotinoid insecticides on the thyroid endocrine system of lizards
Environmental Pollution 258 (2020) 113731
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Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
Unraveling the toxic effects of neonicotinoid insecticides on the thyroid endocrine system of lizards* Yinghuan Wang a, Peng Xu a, Jing Chang a, Wei Li a, Lu Yang a, Haoting Tian b, * a
Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, 18 Shuangqing Road, Haidian District, Beijing, 100085, PR China Shandong Provincial Key Laboratory of Water and Soil Conservation and Environmental Protection, College of Resource and Environment, Linyi University, Linyi 276005, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 September 2019 Received in revised form 1 December 2019 Accepted 3 December 2019 Available online 17 December 2019
The widespread use of neonicotinoids has resulted in large residues in the soil, which has a major impact on the lizards that inhabit the soil. Thyroid hormones play an important role in the growth and development of lizards. In this report, we assessed the disrupting effects of thyroid system on lizards after 28 days of continuous exposure to dinotefuran, thiamethoxam, and imidacloprid, respectively. Neonicotinoid insecticides could seriously affect the concentration of T4 in lizard plasma and the conversion of T4 to T3 in the thyroid gland. Specifically, exposure to dinotefuran affected the intake and utilization of iodine in the thyroid gland, resulting in insufficient thyroid function, which in turn lead to thyroid epithelial hyperplasia and follicular volume enlargement by negative feedback. Exposure to thiamethoxam could activate thyroid function, significantly increasing plasma T3 and T4 concentrations and promoting the binding of T3 and thyroid hormone receptors. Imidacloprid exposure could inhibit the secretion of thyroid hormones, leading to down-regulation of thyroid hormone receptors and related phase II metabolic enzyme genes. This study verified that the continuous exposure of neonicotinoids could affect the lizard thyroid endocrine system. The harm of neonicotinoids to reptiles deserved more attention. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Neonicotinoids Lizards Thyroid Disrupting effects
1. Introduction Neonicotinoids are widely used in crop protection and pest control because of their broad-spectrum, high-efficiency insecticidal activity and low toxicity to mammals (Morrissey et al., 2015; Zhang et al., 2018). Neonicotinoids are currently the most widely used insecticides in the world, accounting for a quarter of the world’s pesticide share (Jeschke et al., 2011). The environmental friendliness of neonicotinoids is mainly reflected in the fact that their affinity for acetylcholinesterase in insects is much higher than that in mammals (Tomizawa and Casida, 2005). However, in recent years, many reports have pointed out that neonicotinoid insecticides may cause harm to non-target organisms in ecosystems (Bass et al., 2015; Bonmatin et al., 2015). Due to the non-volatile, high water solubility and long degradation period in the soil, neonicotinoids have been detected in soils and surface waters
* This paper has been recommended for acceptance by Christian Sonne. * Corresponding author. E-mail addresses: [email protected], [email protected] (H. Tian).
https://doi.org/10.1016/j.envpol.2019.113731 0269-7491/© 2019 Elsevier Ltd. All rights reserved.
around the world (Yi et al., 2019). The high residues of neonicotinoids in the soil pose a huge threat to the animals that inhabit the soil. Reptiles are the least studied species in toxicology (Amaral et al., 2012). The striking fact is that since 2000, reptiles have experienced global decline due to habitat loss, global temperature changes and pollutant threats (Bohm et al., 2013; Gibbon et al., 2000). Among reptiles, lizards (refers to Lacertilia) have the largest variety and are very sensitive to contaminants (Mingo et al., 2016). The use of pesticides is considered to be one of the important reasons for the decline in the population of lizards (Randhawa et al., 2014). Eremias argus, a small lizard mainly distributed in the north of Yangzi River. E. argus mainly inhabits grasslands, sand dunes or riparian areas close to the mountains (Park et al., 2014). Their habitats are highly overlapping with pesticide application areas. In recent years, the population of E. argus has been significantly reduced (Park et al., 2014). China is a big agricultural country that produces and consumes neonicotinoids (Shao et al., 2013). The harm of neonicotinoid residues in the soil to lizards should not be ignored. Thyroid hormone has important physiological functions in the growth and development of vertebrates (Dunlap, 1995), which can
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regulate metabolism, growth and protein synthesis (Langer, 2010). For example, thyroid hormones dominate the metabolism of pollutants in the liver of mice (Martin et al., 2007) and play a crucial role in the metamorphosis of amphibians (Fort et al., 2011; Hersikorn and Smits, 2011). Thyroid hormones are closely related to body metabolic rate, tail regeneration and molting frequency in reptiles (Kanaho et al., 2006). Moreover, thyroid hormones are directly involved in physiological processes such as thermoregulation (Dunlap, 1995), reproduction (Bicho et al., 2013), metabolism (Johnalder, 1990), and molting (Chiu et al., 1967) in lizards. Field studies have shown that the thyroid system of lizards living in agricultural areas can be disrupted, which ultimately affects the reproductive function of lizards (Bicho et al., 2013). In recent years, neonicotinoids have been reported to cause endocrine disrupting effects in vertebrates (Kapoor et al., 2011; Wang et al., 2019a). However, there is no report on the effects of neonicotinoids on the thyroid system. Therefore, more attention should be paid to the effects of pesticides on thyroid system in lizards. To verify the effects of neonicotinoids on the lizard thyroid system, we selected three typical neonicotinoids (dinotefuran, thiamethoxam, and imidacloprid) for 28 days of continuous exposure to E. argus. The effects of neonicotinoids on endocrine disruption in thyroid system of lizards were evaluated comprehensively from histopathology of thyroid gland, plasma thyroid hormones level change and expression level change of thyroid system related genes in liver. Meanwhile, principal component analysis was used to further analyze the differences in the effects of three neonicotinoids on the lizard thyroid system. This study verified the interference effect of neonicotinoids on the function of the thyroid system during continuous exposure. It provided basic data support for the effect of neonicotinoids on terrestrial vertebrates.
2. Materials and methods 2.1. Chemicals Imidacloprid (IMI, 99.4% purity), thiamethoxam (TMX, 98.2% purity) and dinotefuran (DIN, 95.0% purity) were kindly supplied by Institute for the Control of Agrochemicals, Ministry of Agriculture, China. The solvents (analytical grade) were bought from Dikma (Beijing, China).
2.2. Animals husbandry The E. argus were collected from the breeding colony in Changping District, Beijing. They are housed in a manually controlled pesticide-free micro-ecological system. Lizard breeding was carried out in a 5 1.2 0.4 m solid bottom aquarium. A mixture of mollisol and deciduous leaves, about 10e15 cm in length, was applied at the bottom of the aquarium for lizard habitat. UV lamps, humidifiers and air conditioners were used to maintain proper feeding conditions. The relevant parameters of the feed zone were set as follows. Temperature: 25e30 C, humidity: 30e60%, fluorescent lamp: 100 W, light: dark photoperiod: 14:10h. The mealworm (Tenebrio molitor) and water were used for lizard rearing. Prior to the formal experiment, sexually mature lizards were transferred to glass cages (60 50 50 cm) one week in advance to accommodate experimental conditions and artificial contact. In the formal experiment, the environmental parameters were controlled in accordance with the feeding conditions.
2.3. Dosing and exposure experiment Exposure dose (20 mg/kg bw) and dosing method was set in line with previous study (Wang et al., 2019b). The selected neonicotinoid insecticides DIN, TMX and IMI were first dissolved in ethanol and then dispersed into corn oil. The mixture was administrated to each group through oral gavage. The experiment contained four groups (control group, DIN exposure group, TMX exposure group and IMI exposure group). Based on our past experience (Wang et al., 2019a), twenty lizards (10 males and 10 females) were used in each group. The lizards were administrated with DIN, TMX and IMI every 3 days for 28 days in the DIN, TMX and IMI exposure group, respectively. The adverse reactions were observed during exposure. The lizards were euthanized with carbon dioxide at the end of the exposure. Thyroid tissues in each group were stored in a paraformaldehyde solution for histopathological evaluation. The plasmas of lizards in each group were collected to analyze the concentrations of triiodothyronine (T3) and tetraiodothyronine (T4). Livers in each group were stored at 80 C in 1 mL RNA storage reagent for analysis of mRNA expression. 2.4. Thyroid gland histopathology The thyroid gland was fixed with paraformaldehyde solution, sectioned and routinely stained with hematoxylin and eosin. The sections were then encoded and analyzed using an optical microscope (Olympus, BX35). Cell height and follicular area were determined using the instrument’s own software cellSens. 2.5. Thyroid hormone concentration analysis ELISA kits for measuring T3 and T4 concentrations were obtained from Elabscience Biotechnology Co., Ltd. (Wuhan, China). The assays were performed in accordance with the manufacturer’s instructions. 2.6. RNA extraction, cDNA reverse transcription and real-time PCR The isolation of RNA, reverse transcription synthesis of cDNA and real-time PCR were carried out based on our previous developed method (Wang et al., 2019a). Total RNA was isolated from lizard livers using Trizol reagent (Beijing life technology, China). DNA was incubated with DNase-1 (Ambion) to remove trace DNA. RNA was dissolved in RNase-free water and stored at 80 C. The reverse transcription mixture contained 4 mL of DNTP, 2 mL of Oligo (dT) 15 primer and 22 mL of total RNA. Heat the mixture at 70 C for 5 min and cool quickly on ice. After cooling, 8 mL of 5 6 buffer, 2 mL of M-mlv and 40 units of RNAsin (RNase inhibitor) were added at a total volume of 41 mL. The mixture was incubated at 42 C for 50 min and then heated to 95 C for 5 min to inactivate the reverse transcription reaction. SYBR GREEN PCR Kit (Tiangen biotechnology co., LTD., Beijing, China) for real-time PCR in the MX3005P realtime quantitative polymerase chain reaction system (Stratagene, USA). The thermal cycles were set to 40 cycles at 95 C for 5 min, 30 s at 95 C, 40 s at 54 C and 40 s at 72 C. MxPro software is used for sample analysis. The deiodinases (dio1, dio2), thyroid hormone receptors (tra, trb), sulfotransferase (sult), urine diphosphate glucuronyltransferase (udp) and transthyretin (ttr) were selected. The designed primers were listed in Table S1. According to our previous experimental results (Chang et al., 2017), b-actin is the most stable housekeeping gene in lizards. The mRNA expressions of selected genes were normalized by the expression of b-actin.
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2.7. Data analysis Software SPSS 16.0 was used to carry out the statistical analysis. Two sample t-tests were used to determine the difference in mean values of thyroid hormone levels and gene expression between the control and exposed groups. Principal component analysis (PCA) was carried out using thyroid hormone concentrations and related gene expressions as initial factors. Data mapping in lower dimensional spaces was performed to observe the data distribution of the control and exposure groups. Meanwhile, the basic features in the principal component structure were studied by detecting the contribution of the initial factors to the principal component.
3. Results
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Table 1 The cell height of follicular epithelia, follicular area and follicle aspect ratio in all groups. Cell height of follicular epithelial/mm CK group DIN exposure group TMX exposure group IMI exposure group
4.07 3.48 4.75 4.49
± ± ± ±
1.09 0.89 0.88 0.95
Follicular area/mm2 142.27 335.05 220.64 200.36
± ± ± ±
Follicle aspect ratio 57.41 122.95 83.31 111.35
1.25 1.28 1.65 1.38
± ± ± ±
0.15 0.24 0.69 0.20
In the IMI exposure group, an increase in regenerative follicles was observed around the follicles (Fig. 1, red arrows). 3.2. Analysis of thyroid hormone concentration
3.1. Thyroid gland histopathology The thyroid histopathological observation of the lizard at the end of continuous exposure was shown in Fig. 1. In the control group, the thyroid gland exhibited a normal histological appearance. Colloidal-filled follicles were observed surrounded by round or cubic epithelial cells. The follicles were mainly elliptical. The height of follicular epithelial cells, follicular area and follicle aspect ratio values in control and exposure groups were displayed in Table 1. Compared with the control group, the height of follicular epithelial cells in the DIN, TMX and IMI exposure groups varied by 14.5%, 16.7% and 10.3%, respectively. Compared with the control group, the follicular area of the DIN, TMX and IMI exposure groups increased by 135%, 54.9% and 40.8%, respectively. Among them, the increase of follicular area in the DIN exposure group was significant compared with the control group (p < 0.05). The aspect ratio of follicles in the DIN and IMI exposure groups did not change significantly compared with the control group. However, in TMX exposure group, the aspect ratio of follicles in the TMX exposure group increased by 32%. Meanwhile, the decrease of colloids in follicles was observed in all exposure groups (Fig. 1, black arrows).
At the end of continuous exposure, the plasma concentrations of T3 and T4 in the control and exposure groups were exhibited in Fig. 2. Two sample t-tests were used to analyze the difference in mean values of thyroid hormone levels between exposure and control groups (results shown in Table S2). Compared with the control group, the T3 concentration in the plasma of the TMX exposure group increased significantly, and the concentration of T3 in the plasma of the IMI exposure group decreased significantly, while the concentration of T3 in the plasma of the DIN exposed group did not change significantly. There was no significant change in plasma T4 concentration in the TMX exposure group compared with that of the control group, but the plasma T4 concentration was significantly reduced in the DIN and IMI exposure group. 3.3. The expression of thyroid system relative genes in the liver At the end of continuous exposure, the results of gene expression associated with the thyroid system were shown in Fig. 3. Two sample t-tests were used to analyze the difference in mean values of thyroid system relative gene expression levels between exposure
Fig. 1. Representative histology of the thyroid. A: Control group; B: DIN exposure group; C: TMX exposure group; D: IMI exposure group.
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Fig. 2. The plasma concentrations of T3 and T4 in control group, DIN, TMX and IMI exposure group at the end of continues exposure. þ means significant increase compared with the control group, * means significant decrease compared with the control group (p < 0.05, two sample t-test). CK means control check.
Fig. 3. Relative gene levels of tra, trb, dio1, dio2, udp, sult and ttr in liver at end of continues exposure. The results were expressed as the relative ratio of the expression level of each mRNA to the expression level of b-actin. þ means significant increase compared with the control group, * means significant decrease compared with the control group (p < 0.05, two sample t-test). CK means control check.
and control groups (results shown in Table S2). In the DIN exposure group, the expression level of dio1 was significantly downregulated, and the expression levels of dio2 and ttr were significantly up-regulated compared with that of the control group. In the TMX exposure group, the expression levels of dio1 and ttr decreased significantly, and the expression levels of tra, trb and dio2 increased significantly compared with the control group. In the IMI exposure group, except for the significantly increase of dio2 expression and no significant change of tra expression, the expression of other genes decreased significantly. 3.4. Principal component analysis PCA was carried out using thyroid hormone concentrations and related gene expressions in all groups as initial factors to obtain principal components 1 and 2 (55.4% for PC1 and 27.4% for PC2). PC1 and PC2 were taken as x and y axes, re-map the data distinguishing by group. The results were shown in Fig. 4. The average coordinates of PCA scores in the control group, DIN, TMX, and IMI
exposure groups were (0.45, 1.20), (0.41, 0.41), (1.62, 0.21) and (0.76, 1.40), respectively. 4. Discussion Like other lizards, the thyroid gland of the E. argus is a discrete band structure that traverses the middle of the trachea horizontally (Sciarrillo et al., 2008). Under the microscope, follicles and follicular epithelial cells could be clearly observed. Changes in thyroid structure of lizards could reflect changes in activity of thyroid (Virgilio et al., 2003). Compared with the control group, the height of follicular epithelial cells in DIN exposed group decreased by 14.5%, but the follicular area significantly increased by 135%. Meanwhile, the follicles were filled with gelatin. This was a typical symptom in the prophase of goiter. Goiter is usually caused by insufficient thyroid function, and then negative feedback stimulated the secretion of thyroid-stimulating hormone from the pituitary gland. Eventually caused thyroid epithelial hyperplasia, glial accumulation, increased follicular area, and squeezing of follicular
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Fig. 4. Score plot of PCA on thyroid-related hormones and genes in control and three exposure groups.
epithelial cells (Centanni et al., 2006). Therefore, exposure to DIN might affect the pre-synthesis of thyroid hormones, which finally lead to hypothyroidism in lizards. In the TMX exposure group, follicular epithelial cells showed a high columnar shape, and their height increased by 16.7% compared with the control group. This indicated that follicular epithelial cells were in active state. This result was consistent with a significant increase in T3 concentration in lizard plasma in the TMX exposed group. Meanwhile, it was worth noting that in the TMX exposed group, the aspect ratio of follicles increased significantly compared with the control group. This result showed that TMX exposure not only caused hyperthyroidism, but also caused morphological damage to the lizard thyroid gland. Under the exposure of IMI, there was no significant change in follicular epithelial cell height and follicular area compared with the control group. However, we observed reabsorbed vacuoles in the follicles (Fig. 1, red arrows). The generation of resorbed vacuoles was related to the release of T3 and T4 (Bicho et al., 2013). This might be a negative feedback regulation caused by a decrease in the concentration of T3 and T4 in lizard plasma under IMI exposure. Results of thyroid-related hormone concentrations and genes could verify histopathological results. The comprehensive effect of three neonicotinoids selected in this study on the thyroid-related hormones and genes of the E. argus were shown in Fig. 5. In lizards, thyroid hormones exert physiological functions mainly by binding to thyroid hormone receptors and specific nuclear proteins (Umemura et al., 2005). In the TMX exposure group, a significant increase in the expression of tra and trb in the liver was consistent with a significant increase in plasma T3 levels. In the IMI exposure group, a significant decrease in plasma T3 and T4 concentrations led to a down-regulation of trb expression in the liver. This result indicated that in the lizard liver, the expression of the thyroid receptor protein gene was directly regulated by the thyroid concentration in the plasma. Deiodinase is an important regulatory enzyme that catalyzes the conversion of T4 to T3 (Robert et al., 2006). Compared with the control group, the expression of dio1 in the liver was significantly inhibited in all treated groups, while
the expression of dio2 was significantly increased. The dio1 gene dominates the deiodination of the outer and inner rings of iodothyronine derivatives. The dio2 gene is responsible for regulating the transformation from T4 to T3 (Brown, 2005; Orozco and Valverde-R, 2005). The concentration ratio of T3 to T4 in plasma is considered to be an indicator of the change in deiodinase activity (Campos and Freire, 2016). Compared with the control group, the ratio of T3 to T4 in the three exposed groups showed a significant increase (Table S3). These results showed that the up-regulation effect of dio2 was higher than that of dio1 down-regulation, which promoted the transformation of T4 to T3. Meanwhile, thyroid hormones could undergo sulfation and glucuronidation, and these phase II reactions promote the metabolism of thyroid hormones (Schnitzler et al., 2012). Sulfotransferase (sult) inactivates T3 and promotes its excretion in bile and urine (Schuur et al., 1999). Urine diphosphate glucuronyltransferase (udp) enhances glucuronidation of T4 and its excretion in bile (Mcnabb, 2007). In the IMI exposure group, both sult and udp expression were significantly inhibited, which was consistent with a significant decrease in T3 and T4 concentrations in the IMI exposed group. This result indicated that the decrease in thyroid hormone concentration affected its subsequent phase II metabolism in IMI exposure group. T4 could be catalyzed to T3 by dio2 or degraded by udp. In the IMI exposed group, dio2 expression was significantly increased while udp expression was significantly inhibited. This suggested that there was a competitive negative feedback regulation relationship between the conversion of T4 to T3 and the phase II metabolism of T4. The inhibition of sult and udp expression alleviated the decrease in thyroid hormone concentration. Transthyretin (ttr) is generally produced in the choroid plexus and liver of animals and is involved in regulating the transport of thyroid hormones to the central nervous system (Schreiber, 2002a; Schreiber, 2002b). Compared with the control group, the genes of ttr showed significant up-regulation in the DIN exposed group, while the T4 concentration in plasma was significant inhibited. This implied that there was negative feedback regulation between the expression of ttr and thyroid hormone concentration. Compared
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Fig. 5. Overview of the effects of three neonicotinoids selected on the thyroid-related hormones and genes of the E. argus.
with the control group, plasma T3 concentration did not change significantly, but T4 concentration decreased significantly in DIN exposure group. This phenomenon indicated that DIN exposure affected the pre-synthesis of thyroid hormone, but the conversion of T4 to T3 mitigated this effect. Thyroid peroxidase promotes iodination of tyrosine residues in thyroglobulin, and then couples two iodotyrosine residues in the chain to form thyroid hormones (Godlewska and Banga, 2019). DIN exposure might seriously interfere with this process and adversely affected the intake and utilization of iodine. In the IMI exposure group, the significant decrease in the expression of ttr relative to the control group was associated with decreased levels of thyroid hormone in plasma. After principal component analysis using thyroid hormone concentrations and related gene expression as initial factors (Fig. 4), we investigated the contribution of each factor to the principal component (Table S4). The three main contributing factors of PC1 were T3, tra and trb. The contribution values of T3, tra, and trb to PC1 were 0.958, 0.968, and 0.962, respectively. Therefore, PC1 could be considered as a T3 related term, which was mainly related to the concentration of T3 and the binding of T3 and thyroid hormone receptors. The three main contributing factors of PC2 were T4, dio1 and dio2. The contribution values of T4, dio1 and dio2 to PC2 were 0.905, 0.837 and 0.876, respectively. Therefore, PC2 could be considered as a T4 related term, which was mainly related to the concentration of T4 and conversion of T4 to T3. On the PC1 axis, the average coordinate distances of DIN, TMX and IMI points deviating from those of the control group were 0.05, 2.07 and 0.30, respectively. This result showed that TMX promoted the generation of T3 and the binding of T3 to thyroid hormone receptors while IMI inhibited this process. TMX exposure had the most severe effect on the concentration of T3 and the binding of T3 to thyroid hormone receptors in terms of the influence of three neonicotinoids. On the PC2 axis, the average coordinate distances of DIN, TMX and IMI points deviating from those of the control group were 0.79, 1.41 and 2.60 respectively. This result showed that all exposed groups would seriously affect the T4 related items, especially promoted the conversion of T4 to T3. This was the same effect of three neonicotinoids on the thyroid system. The degree of effect was IMI > TMX > DIN. In summary, neonicotinoids mainly affect the concentration of T4 in the plasma and the transformation of T4 to T3 in the thyroid gland. Specifically, DIN exposure could affect iodine intake and utilization in thyroid gland, resulting in thyroid insufficiency, negative feedback leading to thyroid epithelial hyperplasia and follicular volume enlargement. TMX exposure not only caused
hyperthyroidism, but also caused morphological damage to the lizard thyroid gland. Exposure to TMX stimulated excitation of the thyroid gland, increased plasma T3 and T4 concentrations and promoted the binding of T3 and thyroid hormone receptors. Although IMI exposure did not cause significant histological damage to the lizard thyroid, IMI exposure inhibited the synthesis of thyroid hormones, resulting in down-regulation of thyroid hormone receptors and related phase II metabolic enzyme genes. CRediT authorship contribution statement Yinghuan Wang: Project administration. Peng Xu: Investigation. Jing Chang: Data curation. Wei Li: Validation. Lu Yang: Software. Haoting Tian: Funding acquisition, Methodology. Acknowledgements This study was supported by the Shandong Provincial Key Laboratory of Water and Soil Conservation and Environmental Protection (Contract Authorization No. STKF201932) and National Natural Science Foundation of China (Contract Authorization No. 41807478). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.envpol.2019.113731. References Amaral, M.J., Bicho, R.C., Carretero, M.A., Sanchezhernandez, J.C., Faustino, A.M., Soares, A.M., Mann, R.M., 2012. The use of a lacertid lizard as a model for reptile ecotoxicology studies: part 2-biomarkers of exposure and toxicity among pesticide exposed lizards. Chemosphere 87, 765e774. Bass, C., Denholm, I., Williamson, M.S., Nauen, R., 2015. The global status of insect resistance to neonicotinoid insecticides. Pestic. Biochem. Physiol. 121, 78e87. Bicho, R.C., Amaral, M.J., Faustino, A.M., Power, D.M., Rema, A., Carretero, M.A., Soares, A.M., Mann, R.M., 2013. Thyroid disruption in the lizard Podarcis bocagei exposed to a mixture of herbicides: a field study. Ecotoxicology 22, 156e165. Bohm, M., Collen, B., Baillie, J.E., Bowles, P., Chanson, J., Cox, N.A., Hammerson, G., Hoffmann, M., Livingstone, S.R., Ram, M., 2013. The conservation status of the world’s reptiles. Biol. Conserv. 157, 372e385. Bonmatin, J., Giorio, C., Girolami, V., Goulson, D., Kreutzweiser, D.P., Krupke, C.H., Liess, M., Long, E.Y., Marzaro, M., Mitchell, E.A., 2015. Environmental fate and exposure; neonicotinoids and fipronil. Environ. Sci. Pollut. Control Ser. 22, 35e67. Brown, D.D., 2005. The role of deiodinases in amphibian metamorphosis. Thyroid 15, 815e821.
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Orozco, A., Valverde-R, C., 2005. Thyroid hormone deiodination in fish. Thyr. Off. J. Am. Thyr. Assoc. 15, 799e813. Park, H., Suk, H.Y., Jeong, E., Park, D., Lee, H., Min, M., 2014. Population genetic structure of endangered Mongolian racerunner (Eremias argus) from the Korean Peninsula. Mol. Biol. Rep. 41, 7339e7347. Randhawa, M.A., Anjum, M.N., Butt, M.S., Yasin, M., Imran, M., 2014. Minimization of imidacloprid residues in cucumber and bell pepper through washing with citric acid and acetic acid solutions and their dietary intake assessment. Int. J. Food Prop. 17, 978e986. Robert, O., Achim, T., Claudia, L., Ilka, L., Sabine, H., Tobias, B., Thomas, B., Werner, K., 2006. Expression of sodium-iodide symporter mRNA in the thyroid gland of Xenopus laevis tadpoles: developmental expression, effects of antithyroidal compounds, and regulation by TSH. J. Endocrinol. 190, 157e170. Schnitzler, J., Klaren, P.H.M., Bouquegneau, J., Das, K., 2012. Environmental factors affecting thyroid function of wild sea bass (Dicentrarchus labrax) from European coasts. Chemosphere 87, 1009e1017. Schreiber, G., 2002a. The evolution of transthyretin synthesis in the choroid plexus. Clin. Chem. Lab. Med. 40, 1200e1210. Schreiber, G., 2002b. The evolutionary and integrative roles of transthyretin in thyroid hormone homeostasis. J. Endocrinol. 175, 61e73. Schuur, A.G., Bergman, A., Brouwer, A., Visser, T.J., 1999. Effects of pentachlorophenol and hydroxylated polychlorinated biphenyls on thyroid hormone conjugation in a rat and a human hepatoma cell line. Toxicol. In Vitro 13, 417e425. Sciarrillo, R., De Falco, M., Virgilio, F., Laforgia, V., Capaldo, A., Valiante, S., Varano, L., 2008. Morphological and functional changes in the thyroid gland of methyl thiophanate-injected lizards, Podarcis sicula. Arch. Environ. Contam. Toxicol. 55, 254e261. Shao, X., Liu, Z., Xu, X., Li, Z., Qian, X., 2013. Overall status of neonicotinoid insecticides in China: production, application and innovation. J. Pestic. Sci. 38, 1e9. Tomizawa, M., Casida, J.E., 2005. Neonicotinoid insecticide toxicology: mechanisms of selective action. Annu. Rev. Pharmacol. Toxicol. 45, 247e268. Umemura, T., Kurahashi, N., Kondo, T., Katakura, Y., Sata, F., Kawai, T., Kishi, R., 2005. Acute effects of styrene inhalation on the neuroendocrinological system of rats and the different effects in male and female rats. Arch. Toxicol. 79, 653e659. Virgilio, F., Sciarrillo, R., Laforgia, V., Varano, L., 2003. Response of the thyroid gland of the lizard Podarcis sicula to endothelin-1. J. Exp. Zool. A Comp. Exp. Biol. 296, 137e142. Wang, Y.H., Zhang, Y., Zeng, T., Li, W., Yang, L., Guo, B.Y., 2019a. Accumulation and toxicity of thiamethoxam and its metabolite clothianidin to the gonads of Eremias argus. Sci. Total Environ. 667, 586e593. Wang, Y.H., Zhang, Y., Li, W., Han, Y.T., Guo, B.Y., 2019b. Study on neurotoxicity of dinotefuran, thiamethoxam and imidacloprid against Chinese lizards (Eremias argus). Chemosphere 217, 150e157. Yi, X., Zhang, C., Liu, H., Wu, R., Tian, D., Ruan, J., Zhang, T., Huang, M., Ying, G., 2019. Occurrence and distribution of neonicotinoid insecticides in surface water and sediment of the Guangzhou section of the Pearl River, South China. Environ. Pollut. 251, 892e900. Zhang, P., Ren, C., Sun, H., Min, L., 2018. Sorption, desorption and degradation of neonicotinoids in four agricultural soils and their effects on soil microorganisms. Sci. Total Environ. 615, 59e69.
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Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
Unraveling the toxic effects of neonicotinoid insecticides on the thyroid endocrine system of lizards* Yinghuan Wang a, Peng Xu a, Jing Chang a, Wei Li a, Lu Yang a, Haoting Tian b, * a
Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, 18 Shuangqing Road, Haidian District, Beijing, 100085, PR China Shandong Provincial Key Laboratory of Water and Soil Conservation and Environmental Protection, College of Resource and Environment, Linyi University, Linyi 276005, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 September 2019 Received in revised form 1 December 2019 Accepted 3 December 2019 Available online 17 December 2019
The widespread use of neonicotinoids has resulted in large residues in the soil, which has a major impact on the lizards that inhabit the soil. Thyroid hormones play an important role in the growth and development of lizards. In this report, we assessed the disrupting effects of thyroid system on lizards after 28 days of continuous exposure to dinotefuran, thiamethoxam, and imidacloprid, respectively. Neonicotinoid insecticides could seriously affect the concentration of T4 in lizard plasma and the conversion of T4 to T3 in the thyroid gland. Specifically, exposure to dinotefuran affected the intake and utilization of iodine in the thyroid gland, resulting in insufficient thyroid function, which in turn lead to thyroid epithelial hyperplasia and follicular volume enlargement by negative feedback. Exposure to thiamethoxam could activate thyroid function, significantly increasing plasma T3 and T4 concentrations and promoting the binding of T3 and thyroid hormone receptors. Imidacloprid exposure could inhibit the secretion of thyroid hormones, leading to down-regulation of thyroid hormone receptors and related phase II metabolic enzyme genes. This study verified that the continuous exposure of neonicotinoids could affect the lizard thyroid endocrine system. The harm of neonicotinoids to reptiles deserved more attention. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Neonicotinoids Lizards Thyroid Disrupting effects
1. Introduction Neonicotinoids are widely used in crop protection and pest control because of their broad-spectrum, high-efficiency insecticidal activity and low toxicity to mammals (Morrissey et al., 2015; Zhang et al., 2018). Neonicotinoids are currently the most widely used insecticides in the world, accounting for a quarter of the world’s pesticide share (Jeschke et al., 2011). The environmental friendliness of neonicotinoids is mainly reflected in the fact that their affinity for acetylcholinesterase in insects is much higher than that in mammals (Tomizawa and Casida, 2005). However, in recent years, many reports have pointed out that neonicotinoid insecticides may cause harm to non-target organisms in ecosystems (Bass et al., 2015; Bonmatin et al., 2015). Due to the non-volatile, high water solubility and long degradation period in the soil, neonicotinoids have been detected in soils and surface waters
* This paper has been recommended for acceptance by Christian Sonne. * Corresponding author. E-mail addresses: [email protected], [email protected] (H. Tian).
https://doi.org/10.1016/j.envpol.2019.113731 0269-7491/© 2019 Elsevier Ltd. All rights reserved.
around the world (Yi et al., 2019). The high residues of neonicotinoids in the soil pose a huge threat to the animals that inhabit the soil. Reptiles are the least studied species in toxicology (Amaral et al., 2012). The striking fact is that since 2000, reptiles have experienced global decline due to habitat loss, global temperature changes and pollutant threats (Bohm et al., 2013; Gibbon et al., 2000). Among reptiles, lizards (refers to Lacertilia) have the largest variety and are very sensitive to contaminants (Mingo et al., 2016). The use of pesticides is considered to be one of the important reasons for the decline in the population of lizards (Randhawa et al., 2014). Eremias argus, a small lizard mainly distributed in the north of Yangzi River. E. argus mainly inhabits grasslands, sand dunes or riparian areas close to the mountains (Park et al., 2014). Their habitats are highly overlapping with pesticide application areas. In recent years, the population of E. argus has been significantly reduced (Park et al., 2014). China is a big agricultural country that produces and consumes neonicotinoids (Shao et al., 2013). The harm of neonicotinoid residues in the soil to lizards should not be ignored. Thyroid hormone has important physiological functions in the growth and development of vertebrates (Dunlap, 1995), which can
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regulate metabolism, growth and protein synthesis (Langer, 2010). For example, thyroid hormones dominate the metabolism of pollutants in the liver of mice (Martin et al., 2007) and play a crucial role in the metamorphosis of amphibians (Fort et al., 2011; Hersikorn and Smits, 2011). Thyroid hormones are closely related to body metabolic rate, tail regeneration and molting frequency in reptiles (Kanaho et al., 2006). Moreover, thyroid hormones are directly involved in physiological processes such as thermoregulation (Dunlap, 1995), reproduction (Bicho et al., 2013), metabolism (Johnalder, 1990), and molting (Chiu et al., 1967) in lizards. Field studies have shown that the thyroid system of lizards living in agricultural areas can be disrupted, which ultimately affects the reproductive function of lizards (Bicho et al., 2013). In recent years, neonicotinoids have been reported to cause endocrine disrupting effects in vertebrates (Kapoor et al., 2011; Wang et al., 2019a). However, there is no report on the effects of neonicotinoids on the thyroid system. Therefore, more attention should be paid to the effects of pesticides on thyroid system in lizards. To verify the effects of neonicotinoids on the lizard thyroid system, we selected three typical neonicotinoids (dinotefuran, thiamethoxam, and imidacloprid) for 28 days of continuous exposure to E. argus. The effects of neonicotinoids on endocrine disruption in thyroid system of lizards were evaluated comprehensively from histopathology of thyroid gland, plasma thyroid hormones level change and expression level change of thyroid system related genes in liver. Meanwhile, principal component analysis was used to further analyze the differences in the effects of three neonicotinoids on the lizard thyroid system. This study verified the interference effect of neonicotinoids on the function of the thyroid system during continuous exposure. It provided basic data support for the effect of neonicotinoids on terrestrial vertebrates.
2. Materials and methods 2.1. Chemicals Imidacloprid (IMI, 99.4% purity), thiamethoxam (TMX, 98.2% purity) and dinotefuran (DIN, 95.0% purity) were kindly supplied by Institute for the Control of Agrochemicals, Ministry of Agriculture, China. The solvents (analytical grade) were bought from Dikma (Beijing, China).
2.2. Animals husbandry The E. argus were collected from the breeding colony in Changping District, Beijing. They are housed in a manually controlled pesticide-free micro-ecological system. Lizard breeding was carried out in a 5 1.2 0.4 m solid bottom aquarium. A mixture of mollisol and deciduous leaves, about 10e15 cm in length, was applied at the bottom of the aquarium for lizard habitat. UV lamps, humidifiers and air conditioners were used to maintain proper feeding conditions. The relevant parameters of the feed zone were set as follows. Temperature: 25e30 C, humidity: 30e60%, fluorescent lamp: 100 W, light: dark photoperiod: 14:10h. The mealworm (Tenebrio molitor) and water were used for lizard rearing. Prior to the formal experiment, sexually mature lizards were transferred to glass cages (60 50 50 cm) one week in advance to accommodate experimental conditions and artificial contact. In the formal experiment, the environmental parameters were controlled in accordance with the feeding conditions.
2.3. Dosing and exposure experiment Exposure dose (20 mg/kg bw) and dosing method was set in line with previous study (Wang et al., 2019b). The selected neonicotinoid insecticides DIN, TMX and IMI were first dissolved in ethanol and then dispersed into corn oil. The mixture was administrated to each group through oral gavage. The experiment contained four groups (control group, DIN exposure group, TMX exposure group and IMI exposure group). Based on our past experience (Wang et al., 2019a), twenty lizards (10 males and 10 females) were used in each group. The lizards were administrated with DIN, TMX and IMI every 3 days for 28 days in the DIN, TMX and IMI exposure group, respectively. The adverse reactions were observed during exposure. The lizards were euthanized with carbon dioxide at the end of the exposure. Thyroid tissues in each group were stored in a paraformaldehyde solution for histopathological evaluation. The plasmas of lizards in each group were collected to analyze the concentrations of triiodothyronine (T3) and tetraiodothyronine (T4). Livers in each group were stored at 80 C in 1 mL RNA storage reagent for analysis of mRNA expression. 2.4. Thyroid gland histopathology The thyroid gland was fixed with paraformaldehyde solution, sectioned and routinely stained with hematoxylin and eosin. The sections were then encoded and analyzed using an optical microscope (Olympus, BX35). Cell height and follicular area were determined using the instrument’s own software cellSens. 2.5. Thyroid hormone concentration analysis ELISA kits for measuring T3 and T4 concentrations were obtained from Elabscience Biotechnology Co., Ltd. (Wuhan, China). The assays were performed in accordance with the manufacturer’s instructions. 2.6. RNA extraction, cDNA reverse transcription and real-time PCR The isolation of RNA, reverse transcription synthesis of cDNA and real-time PCR were carried out based on our previous developed method (Wang et al., 2019a). Total RNA was isolated from lizard livers using Trizol reagent (Beijing life technology, China). DNA was incubated with DNase-1 (Ambion) to remove trace DNA. RNA was dissolved in RNase-free water and stored at 80 C. The reverse transcription mixture contained 4 mL of DNTP, 2 mL of Oligo (dT) 15 primer and 22 mL of total RNA. Heat the mixture at 70 C for 5 min and cool quickly on ice. After cooling, 8 mL of 5 6 buffer, 2 mL of M-mlv and 40 units of RNAsin (RNase inhibitor) were added at a total volume of 41 mL. The mixture was incubated at 42 C for 50 min and then heated to 95 C for 5 min to inactivate the reverse transcription reaction. SYBR GREEN PCR Kit (Tiangen biotechnology co., LTD., Beijing, China) for real-time PCR in the MX3005P realtime quantitative polymerase chain reaction system (Stratagene, USA). The thermal cycles were set to 40 cycles at 95 C for 5 min, 30 s at 95 C, 40 s at 54 C and 40 s at 72 C. MxPro software is used for sample analysis. The deiodinases (dio1, dio2), thyroid hormone receptors (tra, trb), sulfotransferase (sult), urine diphosphate glucuronyltransferase (udp) and transthyretin (ttr) were selected. The designed primers were listed in Table S1. According to our previous experimental results (Chang et al., 2017), b-actin is the most stable housekeeping gene in lizards. The mRNA expressions of selected genes were normalized by the expression of b-actin.
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2.7. Data analysis Software SPSS 16.0 was used to carry out the statistical analysis. Two sample t-tests were used to determine the difference in mean values of thyroid hormone levels and gene expression between the control and exposed groups. Principal component analysis (PCA) was carried out using thyroid hormone concentrations and related gene expressions as initial factors. Data mapping in lower dimensional spaces was performed to observe the data distribution of the control and exposure groups. Meanwhile, the basic features in the principal component structure were studied by detecting the contribution of the initial factors to the principal component.
3. Results
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Table 1 The cell height of follicular epithelia, follicular area and follicle aspect ratio in all groups. Cell height of follicular epithelial/mm CK group DIN exposure group TMX exposure group IMI exposure group
4.07 3.48 4.75 4.49
± ± ± ±
1.09 0.89 0.88 0.95
Follicular area/mm2 142.27 335.05 220.64 200.36
± ± ± ±
Follicle aspect ratio 57.41 122.95 83.31 111.35
1.25 1.28 1.65 1.38
± ± ± ±
0.15 0.24 0.69 0.20
In the IMI exposure group, an increase in regenerative follicles was observed around the follicles (Fig. 1, red arrows). 3.2. Analysis of thyroid hormone concentration
3.1. Thyroid gland histopathology The thyroid histopathological observation of the lizard at the end of continuous exposure was shown in Fig. 1. In the control group, the thyroid gland exhibited a normal histological appearance. Colloidal-filled follicles were observed surrounded by round or cubic epithelial cells. The follicles were mainly elliptical. The height of follicular epithelial cells, follicular area and follicle aspect ratio values in control and exposure groups were displayed in Table 1. Compared with the control group, the height of follicular epithelial cells in the DIN, TMX and IMI exposure groups varied by 14.5%, 16.7% and 10.3%, respectively. Compared with the control group, the follicular area of the DIN, TMX and IMI exposure groups increased by 135%, 54.9% and 40.8%, respectively. Among them, the increase of follicular area in the DIN exposure group was significant compared with the control group (p < 0.05). The aspect ratio of follicles in the DIN and IMI exposure groups did not change significantly compared with the control group. However, in TMX exposure group, the aspect ratio of follicles in the TMX exposure group increased by 32%. Meanwhile, the decrease of colloids in follicles was observed in all exposure groups (Fig. 1, black arrows).
At the end of continuous exposure, the plasma concentrations of T3 and T4 in the control and exposure groups were exhibited in Fig. 2. Two sample t-tests were used to analyze the difference in mean values of thyroid hormone levels between exposure and control groups (results shown in Table S2). Compared with the control group, the T3 concentration in the plasma of the TMX exposure group increased significantly, and the concentration of T3 in the plasma of the IMI exposure group decreased significantly, while the concentration of T3 in the plasma of the DIN exposed group did not change significantly. There was no significant change in plasma T4 concentration in the TMX exposure group compared with that of the control group, but the plasma T4 concentration was significantly reduced in the DIN and IMI exposure group. 3.3. The expression of thyroid system relative genes in the liver At the end of continuous exposure, the results of gene expression associated with the thyroid system were shown in Fig. 3. Two sample t-tests were used to analyze the difference in mean values of thyroid system relative gene expression levels between exposure
Fig. 1. Representative histology of the thyroid. A: Control group; B: DIN exposure group; C: TMX exposure group; D: IMI exposure group.
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Fig. 2. The plasma concentrations of T3 and T4 in control group, DIN, TMX and IMI exposure group at the end of continues exposure. þ means significant increase compared with the control group, * means significant decrease compared with the control group (p < 0.05, two sample t-test). CK means control check.
Fig. 3. Relative gene levels of tra, trb, dio1, dio2, udp, sult and ttr in liver at end of continues exposure. The results were expressed as the relative ratio of the expression level of each mRNA to the expression level of b-actin. þ means significant increase compared with the control group, * means significant decrease compared with the control group (p < 0.05, two sample t-test). CK means control check.
and control groups (results shown in Table S2). In the DIN exposure group, the expression level of dio1 was significantly downregulated, and the expression levels of dio2 and ttr were significantly up-regulated compared with that of the control group. In the TMX exposure group, the expression levels of dio1 and ttr decreased significantly, and the expression levels of tra, trb and dio2 increased significantly compared with the control group. In the IMI exposure group, except for the significantly increase of dio2 expression and no significant change of tra expression, the expression of other genes decreased significantly. 3.4. Principal component analysis PCA was carried out using thyroid hormone concentrations and related gene expressions in all groups as initial factors to obtain principal components 1 and 2 (55.4% for PC1 and 27.4% for PC2). PC1 and PC2 were taken as x and y axes, re-map the data distinguishing by group. The results were shown in Fig. 4. The average coordinates of PCA scores in the control group, DIN, TMX, and IMI
exposure groups were (0.45, 1.20), (0.41, 0.41), (1.62, 0.21) and (0.76, 1.40), respectively. 4. Discussion Like other lizards, the thyroid gland of the E. argus is a discrete band structure that traverses the middle of the trachea horizontally (Sciarrillo et al., 2008). Under the microscope, follicles and follicular epithelial cells could be clearly observed. Changes in thyroid structure of lizards could reflect changes in activity of thyroid (Virgilio et al., 2003). Compared with the control group, the height of follicular epithelial cells in DIN exposed group decreased by 14.5%, but the follicular area significantly increased by 135%. Meanwhile, the follicles were filled with gelatin. This was a typical symptom in the prophase of goiter. Goiter is usually caused by insufficient thyroid function, and then negative feedback stimulated the secretion of thyroid-stimulating hormone from the pituitary gland. Eventually caused thyroid epithelial hyperplasia, glial accumulation, increased follicular area, and squeezing of follicular
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Fig. 4. Score plot of PCA on thyroid-related hormones and genes in control and three exposure groups.
epithelial cells (Centanni et al., 2006). Therefore, exposure to DIN might affect the pre-synthesis of thyroid hormones, which finally lead to hypothyroidism in lizards. In the TMX exposure group, follicular epithelial cells showed a high columnar shape, and their height increased by 16.7% compared with the control group. This indicated that follicular epithelial cells were in active state. This result was consistent with a significant increase in T3 concentration in lizard plasma in the TMX exposed group. Meanwhile, it was worth noting that in the TMX exposed group, the aspect ratio of follicles increased significantly compared with the control group. This result showed that TMX exposure not only caused hyperthyroidism, but also caused morphological damage to the lizard thyroid gland. Under the exposure of IMI, there was no significant change in follicular epithelial cell height and follicular area compared with the control group. However, we observed reabsorbed vacuoles in the follicles (Fig. 1, red arrows). The generation of resorbed vacuoles was related to the release of T3 and T4 (Bicho et al., 2013). This might be a negative feedback regulation caused by a decrease in the concentration of T3 and T4 in lizard plasma under IMI exposure. Results of thyroid-related hormone concentrations and genes could verify histopathological results. The comprehensive effect of three neonicotinoids selected in this study on the thyroid-related hormones and genes of the E. argus were shown in Fig. 5. In lizards, thyroid hormones exert physiological functions mainly by binding to thyroid hormone receptors and specific nuclear proteins (Umemura et al., 2005). In the TMX exposure group, a significant increase in the expression of tra and trb in the liver was consistent with a significant increase in plasma T3 levels. In the IMI exposure group, a significant decrease in plasma T3 and T4 concentrations led to a down-regulation of trb expression in the liver. This result indicated that in the lizard liver, the expression of the thyroid receptor protein gene was directly regulated by the thyroid concentration in the plasma. Deiodinase is an important regulatory enzyme that catalyzes the conversion of T4 to T3 (Robert et al., 2006). Compared with the control group, the expression of dio1 in the liver was significantly inhibited in all treated groups, while
the expression of dio2 was significantly increased. The dio1 gene dominates the deiodination of the outer and inner rings of iodothyronine derivatives. The dio2 gene is responsible for regulating the transformation from T4 to T3 (Brown, 2005; Orozco and Valverde-R, 2005). The concentration ratio of T3 to T4 in plasma is considered to be an indicator of the change in deiodinase activity (Campos and Freire, 2016). Compared with the control group, the ratio of T3 to T4 in the three exposed groups showed a significant increase (Table S3). These results showed that the up-regulation effect of dio2 was higher than that of dio1 down-regulation, which promoted the transformation of T4 to T3. Meanwhile, thyroid hormones could undergo sulfation and glucuronidation, and these phase II reactions promote the metabolism of thyroid hormones (Schnitzler et al., 2012). Sulfotransferase (sult) inactivates T3 and promotes its excretion in bile and urine (Schuur et al., 1999). Urine diphosphate glucuronyltransferase (udp) enhances glucuronidation of T4 and its excretion in bile (Mcnabb, 2007). In the IMI exposure group, both sult and udp expression were significantly inhibited, which was consistent with a significant decrease in T3 and T4 concentrations in the IMI exposed group. This result indicated that the decrease in thyroid hormone concentration affected its subsequent phase II metabolism in IMI exposure group. T4 could be catalyzed to T3 by dio2 or degraded by udp. In the IMI exposed group, dio2 expression was significantly increased while udp expression was significantly inhibited. This suggested that there was a competitive negative feedback regulation relationship between the conversion of T4 to T3 and the phase II metabolism of T4. The inhibition of sult and udp expression alleviated the decrease in thyroid hormone concentration. Transthyretin (ttr) is generally produced in the choroid plexus and liver of animals and is involved in regulating the transport of thyroid hormones to the central nervous system (Schreiber, 2002a; Schreiber, 2002b). Compared with the control group, the genes of ttr showed significant up-regulation in the DIN exposed group, while the T4 concentration in plasma was significant inhibited. This implied that there was negative feedback regulation between the expression of ttr and thyroid hormone concentration. Compared
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Y. Wang et al. / Environmental Pollution 258 (2020) 113731
Fig. 5. Overview of the effects of three neonicotinoids selected on the thyroid-related hormones and genes of the E. argus.
with the control group, plasma T3 concentration did not change significantly, but T4 concentration decreased significantly in DIN exposure group. This phenomenon indicated that DIN exposure affected the pre-synthesis of thyroid hormone, but the conversion of T4 to T3 mitigated this effect. Thyroid peroxidase promotes iodination of tyrosine residues in thyroglobulin, and then couples two iodotyrosine residues in the chain to form thyroid hormones (Godlewska and Banga, 2019). DIN exposure might seriously interfere with this process and adversely affected the intake and utilization of iodine. In the IMI exposure group, the significant decrease in the expression of ttr relative to the control group was associated with decreased levels of thyroid hormone in plasma. After principal component analysis using thyroid hormone concentrations and related gene expression as initial factors (Fig. 4), we investigated the contribution of each factor to the principal component (Table S4). The three main contributing factors of PC1 were T3, tra and trb. The contribution values of T3, tra, and trb to PC1 were 0.958, 0.968, and 0.962, respectively. Therefore, PC1 could be considered as a T3 related term, which was mainly related to the concentration of T3 and the binding of T3 and thyroid hormone receptors. The three main contributing factors of PC2 were T4, dio1 and dio2. The contribution values of T4, dio1 and dio2 to PC2 were 0.905, 0.837 and 0.876, respectively. Therefore, PC2 could be considered as a T4 related term, which was mainly related to the concentration of T4 and conversion of T4 to T3. On the PC1 axis, the average coordinate distances of DIN, TMX and IMI points deviating from those of the control group were 0.05, 2.07 and 0.30, respectively. This result showed that TMX promoted the generation of T3 and the binding of T3 to thyroid hormone receptors while IMI inhibited this process. TMX exposure had the most severe effect on the concentration of T3 and the binding of T3 to thyroid hormone receptors in terms of the influence of three neonicotinoids. On the PC2 axis, the average coordinate distances of DIN, TMX and IMI points deviating from those of the control group were 0.79, 1.41 and 2.60 respectively. This result showed that all exposed groups would seriously affect the T4 related items, especially promoted the conversion of T4 to T3. This was the same effect of three neonicotinoids on the thyroid system. The degree of effect was IMI > TMX > DIN. In summary, neonicotinoids mainly affect the concentration of T4 in the plasma and the transformation of T4 to T3 in the thyroid gland. Specifically, DIN exposure could affect iodine intake and utilization in thyroid gland, resulting in thyroid insufficiency, negative feedback leading to thyroid epithelial hyperplasia and follicular volume enlargement. TMX exposure not only caused
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