Metabolism of Diflubenzuron in Lizard (Eremias Argus) And

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Cite This: J. Agric. Food Chem. 2018, 66, 11640−11646 pubs.acs.org/JAFC

Metabolism of Diﬂubenzuron in Lizard (Eremias argus) and Comparative Toxicity of Diﬂubenzuron and Its Metabolite † † ‡ † ‡ † ‡ † † † Huili Wang, Yun Xie, , Meng Jiao, , Xiao Hu, , Jitong Li, Peng Xu, Yanfeng Zhang, † and Jing Chang*, † Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Shuangqing RD 18, Beijing 100085, China ‡ University of Chinese Academy of Sciences, Yuquan RD 19 a, Beijing 100049, China

*S Supporting Information

ABSTRACT: The metabolic process of diflubenzuron in rat or fish has been well studied, but little is known about its elimination pathway in lizard. The current study predicted the metabolic route of diflubenzuron in lizard feces and compared the toxicity of diflubenzuron and 4-chloroaniline on lizard thyroid system. The amido bond cleavage was the major route for diflubenzuron elimination in lizard feces. 4-Chloroaniline as the most toxic diflubenzuron metabolite was also abundant in feces. According to liver slices, 4-chloroaniline exposure induced significant changes of nuclear shape, while diflubenzuron exposure caused significant hepatocytes clustering. On the basis of thyroid hormone and thyroid-related gene levels, triiodothyronine (T3) level in lizard liver was regulated by thyroid hormone receptors, while thyroxine (T4) concentration was modulated by dio2 and udp genes after diflubenzuron or 4-chloroaniline exposure. These results showed that both diflubenzuron and 4- chloroaniline could disrupt lizard thyroid system, which could provide evidence for lizard population decline. KEYWORDS: diflubenzuron, 4-chloroaniline, elimination, histopathology, thyroid

− ■ INTRODUCTION molting.11 13 Because of its properties, diflubenzuron has been widely used to control grasshoppers in grassland. The half-lives From 2008 to 2018, the found lizard species have been fl 14,15 increasing (http://www.reptile-database.org/db-info/ of di ubenzuron in soil ranged from 80 to 119 days. SpeciesStat.html). However, the populations have been Thereby, it can place an enormous strain on higher organisms declining with the years. The Mongolian racerunner (Eremias after bioaccumulation through the trophic chain. Eremias argus, argus) is a small lacertid lizard species, which is distributed which inhabits in grassland and takes grasshoppers as their 1,2 main food, may accumulate diflubenzuron inevitably. Little is widely in China, Korea, Mongolia, and Russia. However, its fl fl populations in Korea are small and decreasing, and Eremias known about the in uences of di ubenzurononlizard population decline. argus has been listed under the Protection of Wild Fauna and fl Flora Act in South Korea.3 As compared to a previous survey Although di ubenzuron can be accumulated through the food chain, it is considered to show very low toxicity in in 1991, six species of reptiles including Eremias argus were not fl found in Songshan Nature Reserve, Beijing, from 2009 to mammals. However, when metabolized or degraded, di uben- 4 zuron can produce an extremely toxic compound 4-chloroani- 2010. The reasons for Eremias argus populations decline are 16 still unclear, and few research has focused on this issue. line. 25 mg/L 4-chloroaniline alone or 15 mg/L of a mixture 5 (75% 4-chloroaniline and 25% diflubenzuron) induced 50% Lizards are commonly found in agriculture areas. Pesticides 17 See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. application has been regarded as one of the possible reasons to mortality of tilapia (Oreochromis niloticus). In rat and mice,

Downloaded via RESEARCH CTR ECO-ENVIRONMENTAL SCI on April 19, 2019 at 07:09:01 (UTC). fl induce lizard population decline.6 More than 100 pesticides the major route of unabsorbed di ubenzuron elimination is via fi feces and from the liver via bile to feces or urine after have been listed as con rmed or possible endocrine disruption 18 chemicals (ECDs) by an official source.7 Previous studies on absorption. 4-Chloroaniline is not a major metabolite in both fi 19 18 endocrine disrupting effects of pesticides have focused on their sh and rat. However, the metabolic process of fl reproductive toxicity to nontarget organisms, such as estrogen, di ubenzuron in lizard has not been studied. Moreover, the ff androgens, antiestrogen, or antiandrogen effects.8 In recent toxic e ects of 4-chloroaniline on lizard as compared to fl years, studies have found that the thyroid axis is also one of the di ubenzuron are still unclear. target locations of ECDs.9 The hypothalamus−pituitary− In this study, we predicted the elimination pathway of fl thyroid (HPT) axis plays an important role in regulation of di ubenzuron in lizard by investigating the metabolites in 10 fl growth, metabolic, and reproductive systems. In that case, feces. The disruption of di ubenzuron and 4-chloroaniline on the disruption of lizard thyroid system may contribute to the the lizard thyroid system was also compared. This study may population decline. However, we still do not know much about the mechanisms of interaction between the pesticides and Received: July 15, 2018 lizard thyroid system. Revised: October 7, 2018 Diflubenzuron acts as a chitin synthesis inhibitor, and chitin- Accepted: October 22, 2018 synthesizing organisms are sensitive to this chemical during Published: October 22, 2018

© 2018 American Chemical Society 11640 DOI: 10.1021/acs.jafc.8b03713 J. Agric. Food Chem. 2018, 66, 11640−11646 Journal of Agricultural and Food Chemistry Article provide some evidence to assess the risk of diflubenzuron and follows: − (no lesions); mild, + (1−4 lesions); moderate, ++ (5−8 its metabolites to lizards. lesions); and severe, +++ (≥9 lesions). Thyroid Hormones Analysis. The thyroxine (T4) and triiodothyronine (T3) levels were measured by an enzyme-linked ■ MATERIALS AND METHODS immunosorbent assay (ELISA) kit specified for lizards (T3 kit was Reagents. Diflubenzuron and 4-chloroaniline (purity > 98%) were from Elabscience Biotechnology Co., Ltd.; T4 kit was from Cloud- purchased from J&K Chemical Technology (Beijing, China). The Clone Corp.) according to the manuals. The competing ELISA solvents including acetonitrile, methanol, and n-haxane (HPLC principle was applied in both kits. Samples diluted 5-fold were used in fi grade) were obtained from Dikma (Beijing, China). TRNzol A+, the T3 and T4 assay to t the calibration curves. reverse transcription (RT) Kit, and qPCR SYBR Green Kit were Quantitative Real-Time PCR. The isolation of RNA, cDNA fl synthesis method has been described in our previous paper.23 Briefly, purchased from TIANGEN Biotech (Beijing, China). The di uben- + zuron or 4-chloroaniline stock solution was prepared with carrier total RNA in lizard liver was extracted using TRNzol-A reagent. The solvent (methanol) and corn oil. concentration and purity of extracted RNA were evaluated using Exposure Experiment. The 2−3 year-old male lizards (3.5−4.0 NanoDrop 2000 microvolume spectrophotometers. The reverse g) were collected in our breeding colony in Changping District, transcription was achieved using RT kit with gDNase. The hypothalamus−pituitary−thyroid axis-related genes including Beijing, China. Lizards were allowed to acclimate in experiment cages α β (60 cm × 60 cm × 40 cm) for 7 days before experiment. The thyroid hormone receptors (tr , tr ), deiodinases (dio1, dio2), temperature and humidity were set at 25−30 °C and 30−50%, uridinediphosphate glucuronosyl-transferase (udp), and sulfotransfer- respectively. Lizards were fed with mealworms (Tenebrio molitor) ase (sult) and their respective primers were listed in Table S1. Quantitative real-time PCR was performed in a MX3005P real-time twice a day. The animal care and use procedure was approved by the quantitative polymerase chain reaction system (Stratagene, U.S.) Research Center for Eco-Environmental Sciences, Chinese Academy using the SYBR Green PCR Kit. The PCR system was operated of Sciences. Animal welfare and experimental procedures followed the according to the following protocols: denaturation at 95 °C for 5 min, Guide for the Care and Use of Laboratory Animals (Ministry of followed by 40 cycles of amplification at 95 °C for 30 s, at 54 °C for Science and Technology of China, 2006). 40 s, and at 72 °C for 40 s. The dissociation curve was obtained at 95 For 7-day diflubenzuron exposure, male lizards were randomly °C for 40 s, at 54 °C for 40 s, and a gradual heating to 95 °C. Relative separated into control and exposure groups (n = 6 for each group), quantification of each gene expression level was normalized according which were orally administrated with corn oil and 20 mg/kg to the β-actin gene expression. diflubenzuron, respectively. After 7 days, the feces were collected − ° Data Analysis. The body weight to body length ratio (BWL) was and stored at 20 C before analysis. calculated as follows: For 21-day diflubenzuron and 4-chloroaniline exposure, male lizards were randomly separated into three groups, including control, BWL= body weight/body length diflubenzuron, and 4-chloroaniline exposure groups (n = 6 for each group). In the exposure groups, lizards were orally administrated with The mass spectrum of predicted metabolites was compared to 20 mg/kg diflubenzuron or 4-chloroaniline per week. In the control compounds in the NIST library. group, only carrier solvent-methanol and corn oil were exposed. The statistical analysis was performed using SPSS software ff Lizards at both control and exposure groups were fed normally. After (Version 13.0, U.S.). Di erences of BWL, thyroid hormone levels, 3 weeks of exposure, lizards were killed through decollation. Blood and gene expressions between control and exposure group were was collected and centrifuged (2500g, 10 min) to collect the serum. analyzed according to analysis of variance (ANOVA). A probability of fi Lizard liver was also collected and stored in RNA store. For p < 0.05 was considered to be statistically signi cant. The correlation fi between thyroid hormone and related genes was analyzed with histopathology analysis, a part of the liver was xed with 4% ’ ff paraformaldehyde. Samples including serum and liver were stored at Pearson s test. The proportion of individuals exhibiting the di erent − ° liver lesions among exposed and control groups was compared using 20 C before analysis. ’ Chemical Analysis. The metabolic process of diflubenzuron was Fisher s Exact Tests. mainly studied through lizard excreted feces. It is an effective method The heat map and clustering analysis were performed on R to investigate the metabolites of diflubenzuron. In our study, lizard software. feces (1.5−2.0 g) were collected from 0 to 7 days after diflubenzuron exposure. The metabolites were extracted with 15 mL of acetonitrile ■ RESULTS in a 50 mL polypropylene centrifuge tube. The tube was mixed on a Proposed Metabolic Pathways of Diflubenzuron. The vortex mixer for 2 min, ultrasonicated for 10 min, and then metabolic process of diflubenzuron was shown in Figure 1. The centrifuged at 8000 rpm for 5 min. The upper phase was collected, major route of metabolism in lizard is via the cleavage of the and then the steps above were repeated. The upper phases were benzoyl−ureido bridge. Another pathway is through hydrox- combined and dehydrated with 2 g of anhydrous magnesium sulfate. fl The extracts were dried on a vacuum rotary at 35 °C and redissolved ylation and methylation of di ubenzuron. In lizard feces, four in 1 mL of n-haxane. major metabolites were detected by GC/MS/MS and The analysis of diflubenzuron metabolites was performed on a identified through comparison with compounds in the NIST Thermo-TSQ 8000 GC/MS/MS equipped with an electron-impact library. Hydroxylated and methylated diflubenzuron was eluted ionization (EI) source and Thermo column TR-35MS (0.25 mmΦ × at 5.98 min (Figure 2). From the mass spectrum (Figure 2), 30 mID × 0.25 mm). The ramped temperature program was held at there were three special fragment ions, 311, 325, and 341 m/z, 50 °C for 1 min, ramped at 20 °C/min to 200 °C for 1 min, ramped which represented diflubenzuron, methylated diflubenzuron, at 5 °C/min to 260 °C for 10 min, and ramped at 20 °C/min to 280 and further hydroxylated diflubenzuron. Yet we were not sure ° C for 2 min. The mass spectrum was operated at full scan mode. The about the hydroxylation and methylation sites. 4-Chlorophe- scan range was from 50 to 550 m/z. nylurea was eluted at 6.60 min, which showed 153, 125, 90 m/ Histopathology. Lizard liver sections were trimmed, placed in cassettes, embedded in paraffin, sectioned at 6 μm, stained with z as characterized fragment ions. The metabolite of 4- hematoxylin and eosin, and observed under light microscope (ZEISS, chlorophenylurea, 4-chloroaniline was eluted at 7.55 min. AXIO) according to the method described by MacFarland et al.20 The The special fragment ions from 127 to 92 m/z indicated the liver lesions were quantified using semiquantitative scoring cleavage of the carbon−chloride bond, which were highly method.21,22 Twelve tissue sections were randomly chosen under predicted to be 4-chloroaniline. At 8.61 min, 2,6-difluor- the light microscope in each group. The scoring system was defined as obenzamide was eluted, which was also the product of

11641 DOI: 10.1021/acs.jafc.8b03713 J. Agric. Food Chem. 2018, 66, 11640−11646 Journal of Agricultural and Food Chemistry Article

length ratio (BWL) was calculated in both control and exposure groups (Table 1). No significant differences were found in BWL between control and diflubenzuron (or 4- chloroaniline) exposure groups during 21 days of exposure (one-way ANOVA, p < 0.05). Liver Histopathology. The liver sections in both control and exposure groups were shown in Figure 3. In the control group, the central vein was very obvious, and hepatocytes were distributed evenly with clear round cell nucleus. As compared to the control group, 4-chloroaniline exposure caused nuclear changes of hepatocytes (Figure 3b and Table S2, Fisher’s Exact Test, p < 0.001), which converted from globular to rod-like. Significant hepatocytes clustering was observed in the diflubenzuron exposure group (Figure 3c, Fisher’sExact Test, p = 0.009). More kupffer cells were found in the control and diflubenzuron exposure group as compared to 4- chloroaniline exposure (Figure 3A,C). At the same time, the Figure 1. Proposed metabolic pathways of diflubenzuron in lizard sinusoid became narrowed and congested especially in the 4- feces. chloroaniline exposure group (Table S2). Thyroid Hormone Levels in Lizard Serum. After hydrolysis. The fragment ion from 157 m/z to 141 m/z exposure, the thyroid hormone levels in lizard serum were − fl shown in Table 1. The T4 level was significantly increased indicated the cleavage of the C N bond in 2,6-di uorobenza- fl mide, and then 113 m/z demonstrated its further decarbon- (1.7-fold) in the di ubenzuron exposure group as compared to ylation. The concentrations of these detected metabolites were that in the control group (one-way ANOVA, p < 0.05). In fl semiquantified by peak areas. The concentrations from high to contrast, both di ubenzuron and 4-chloroaniline exposure fi low are 4-chlorophenylurea, 4-chloroaniline, hydroxylated and induced signi cantly up-regulation of T3 levels. The T3 levels methylated diflubenzuron, and 2,6-difluorobenzamide. increased 3.0- and 4.5-fold by 4-chloroaniline and diflubenzur- Toxicity Comparison between Diflubenzuron and 4- on, respectively. Chloroaniline. As one of the major metabolites in lizard feces, Quantification of Thyroid System-Related Genes. The the toxicity of 4-chloroaniline has not been investigated on expression of thyroid system-related genes was influenced after lizard. In that case, we studied the toxic effects of 4-chloroaniline and diflubenzuron exposure. The fold changes diflubenzuron and 4-chloroaniline on the lizard thyroid system. of trα, trβ, dio1, dio2, udp, and sult genes were shown in Figure The results are shown below. 4. The levels of thyroid hormone receptors were significantly Body Weight to Body Length (SVL) Ratio. No mortality decreased after diflubenzuron or its metabolite exposure. In the was found during the experiment. The body weight to body 4-chloroaniline exposure group, trα and trβ gene expressions

Figure 2. Chromatography and mass spectrum of predicted metabolites of diﬂubenzuron in lizard feces.

11642 DOI: 10.1021/acs.jafc.8b03713 J. Agric. Food Chem. 2018, 66, 11640−11646 Journal of Agricultural and Food Chemistry Article

Table 1. Body Weight/Body Length Ratio (BWL) and Thyroid Hormone Levels in Both Control and Exposure Groups

groups BWL (g/mm) T4 (μg/L) T3 (ng/L) control 8.78 ± 0.69 1378.80 ± 124.20 34.86 ± 3.50 4-chloroaniline exposure 7.95 ± 0.46 992.11 ± 250.04 106.02 ± 12.40a diflubenzuron exposure 8.69 ± 0.51 2386.38 ± 213.42a 155.89 ± 16.72a aIndicated significant difference as compared to control group (one-way ANOVA, p < 0.05).

Figure 3. Sections of male lizards stained with hematoxylin and eosin (HE). (A and a) Liver sections of control groups representing normal liver (10×,20×, respectively). (B and b) Liver sections of 4-chloroaniline exposure groups (10×,20×, respectively). (C and c) Liver sections of diﬂubenzuron exposure groups (10×,20×, respectively). Black arrows represented kupﬀer cells, black circles represented hepatocytes clustering, and black short arrows represented nuclear changes.

Figure 5. Clustered heat map of thyroid hormone and related mRNA log 2-fold changes between 4-chloroaniline or diﬂubenzuron treatment and control conditions. Target genes were thyroid hormone receptors (trα, trβ), deiodinases (dio1, dio2), uridinediphosphate glucuronosyl-transferase (udp), and sulfotransferase (sult).

According to correlation analysis, the serum T3 concentrations in exposure groups were negatively correlated with trα mRNA Figure 4. Log 2-fold changes of thyroid system-related gene expressions (Pearson, R2 = −0.98, p < 0.05), while the T4 fl expressions in lizard liver after 4-chloroaniline or di ubenzuron levels were positively correlated with dio2 mRNA expression exposure. (Pearson, R2 = 0.99, p < 0.05). were −1.68-, −3.96-fold down-regulated, and diflubenzuron ■ DISCUSSION exposure made them decrease −2.17- and −5.11-fold, It is the first time to report the elimination process of respectively. In contrast, the expression of dio1, dio2, udp, diflubenzuron in lizards. Similar to the metabolic pathway of and sult genes was opposite between the 4-chloroaniline and diflubenzuron in rats, hydroxylation and cleavage of amido diflubenzuron exposure group. The dio1, dio2, and udp gene bond were the two primary methods. However, the cleavage of expressions were 3.77-, −1.99-, −3.76-fold changed after 4- amido bond was the major route (approximately 90%) in chloroaniline exposure. Diflubenzuron exposure induced lizards, which only occupied 20% in rats.18 The result indicates −2.93-, 1.90-, and 3.81-fold changes of the dio1, dio2, and that the metabolic enzymes between lizards and rats are sult gene level. different. Although as one of the most toxic metabolites, 4- Relationship between Thyroid Hormone and Tested chloroaniline was not at a high level in lizard tissues,24 it is one Genes. Overall thyroid hormone and mRNA response profiles of the most abundant metabolites in lizard feces. Excreting to related to 4-chloroaniline or diflubenzuron were presented as a the environment may also be a danger to lower trophic level heat map (Figure 5). The clustering analysis was conducted organisms. In that case, the toxicity of 4-chloroaniline and between thyroid hormones and tested genes for better diflubenzuron on the lizard thyroid system was further studied. visualization. Two major clusters were observed. In cluster 1, The body size of reptiles, including body weight and length, T3 and dio1 mRNA were pooled together. In cluster 2, T4 and is primarily affected by food availability.25 In this study, the trα, trβ, dio2, udp, sult mRNA were grouped together. BWL was not significantly changed after 4-chloroaniline or

11643 DOI: 10.1021/acs.jafc.8b03713 J. Agric. Food Chem. 2018, 66, 11640−11646 Journal of Agricultural and Food Chemistry Article

Figure 6. Proposed thyroid hormone regulation pathways altered by 4-chloroaniline (4CA) and diflubenzuron (DFB) treatment. Note: The fold changes are shown in different colors. The color from light to dark indicates the increased fold changes. Thyroid hormone changes are depicted by square, and thyroid system-related genes are depicted by rhombus. diflubenzuron exposure. The food ingestion of lizards during and decreased without significance in the 4-chloroaniline exposure showed no significant changes. These results exposure group. As 100% T4 in lizard serum comes from indicated that 4-chloroaniline or diflubenzuron exposure did thyroid, the thyroid gland activities could be predicted from not influence the appetite of lizards. As food ingestion is also a the T4 levels in lizard serum. According to the results, 4- good indicator for hyperthyroidism or hypothyroidism, the chloroaniline exposure may cause less toxicity in lizard thyroid influences of 4-chloroaniline and diflubenzuron on the lizard gland. Consistent with the changes of T4 levels, dio2 and udp thyroid system may not show on individual levels. mRNA gene expressions were decreased in the 4-chloroaniline In this study, 4-chloroaniline or diflubenzuron exposure exposure group and increased in the diflubenzuron exposure showed different morphological changes in lizard liver. 4- group. The T3 synthesis is mainly from the conversion from Chloroaniline exposure mainly induced narrowed sinusoid, T4 to T3, which are modulated by dio2 enzyme. After 4- decreased kupffer cell, and changed nuclear shape. Previous chloroaniline or diflubenzuron exposure, the dio2 gene studies have demonstrated that kupffer cells, which are located expression was significantly altered, which indicated that the in sinusoid, are involved in modulating the growth of nearby dio2 enzyme activity may be also affected by 4-chloroaniline hepatocytes.26,27 In that case, the nuclear changes may be and diflubenzuron. Moreover, the fold changes of trα and trβ caused by decreased kupffer cells in 4-chloroaniline exposure mRNA in lizard liver remained uniform with the changes of T3 group. Moreover, as kupffer cells are considered traditional levels in serum, although the T3 levels did not show significant members of the innate immune system,28 the decrease of correlation with trβ mRNA levels. This result showed that both kupffer cells after 4-chloroaniline exposure may increase the 4-chloroaniline and diflubenzuron disturbed the thyroid risk of hepatic inflammation. Similar to our results, the hormone receptor genes. irregular outline of nucleus was also observed in the liver of The regulation mechanism of HPT axis after 4-chloroaniline zebrafish larvae after chronic 4-chloroaniline exposure.29 In or diflubenzuron exposure can be predicted as follows. In lizard contrast, hepatocytes clustering was obviously seen by liver, 100% of T4 is from serum, and T3 is converted from T4 diflubenzuron exposure. Consistent with our previous study, or transported from serum by thyroid hormone receptors lizards exposed to diflubenzuron contaminated soil also show (Figure 6). In addition, T3 is the only biological active hepatocytes clustering phenomenon.14 Hepatocytes clustering hormone to regulate the lizard growth and metabolism.31 The may be a good marker for increased metabolic rate in lizard HPT axis-related genes in lizard liver focus on the regulation of liver after xenobiotic invasion, considering the relative low T4 and T3 levels. In this study, the T4 levels in lizard serum concentration of diflubenzuron in lizard liver.24 A previous could represent the T4 concentrations in lizard liver at some study suggested that diflubenzuron acted as a peroxisome extent. The dio2 and udp genes in lizard liver mainly control proliferator-actived receptor γ (PPARγ) agonist and showed the conversion and degradation of T4.23 Thus, the tran- diverse hepatotoxic effects by disrupting hepatic energy scription changes of dio2 and udp genes after 4-chloroaniline or metabolism.30 Thus, these histological changes in lizard may diflubenzuron exposure were to modulate the normal T4 levels indicate that the energy metabolism in liver may be influenced in lizard liver as feedback effect. The transportation of T3 from by diflubenzuron exposure. Although diflubenzuron and 4- lizard serum to liver must be carried out by the thyroid chloroaniline exposure induced different lesions to liver, the hormone receptors. The T3 levels in lizard serum were hepatocytes proliferation and growth were influenced in both significantly up-regulated after 4-chloroaniline or diflubenzur- exposure groups. on exposure. To control the T3 concentration in lizard liver to The HPT axis of lizards can regulate the growth, metabolic, normal levels, the expression of trα and trβ genes was and reproductive systems of lizards.31 The influences of significantly down-regulated. It indicated that the normal T3 pesticides on the lizard thyroid system may cause multiple levels in lizard liver were mainly regulated by thyroid hormone functional damages. In that case, the toxic effects of 4- receptor genes and T4 was primarily controlled by dio2 and chloroaniline and diflubenzuron on lizard thyroid system were udp genes after 21 days of exposure by 4-chloroaniline or compared. Although both 4-chloroaniline and diflubenzuron diflubenzuron. exposure up-regulated the T3 levels in lizard serum, the T4 Although 4-chloroaniline exposure did not show significant levels were significantly increased by diflubenzuron exposure changes of T4 levels, it is difficult to make a conclusion of

11644 DOI: 10.1021/acs.jafc.8b03713 J. Agric. Food Chem. 2018, 66, 11640−11646 Journal of Agricultural and Food Chemistry Article whether 4-chloraoniline was less toxic than diflubenzuron to (11) Merzendorfer, H.; Kim, H. S.; Chaudhari, S. S.; Kumari, M.; lizards because of the complicated changes of related gene Specht, C. A.; Butcher, S.; Brown, S. J.; Manak, J. R. Genomic and expression. These results gave us a hint that both proteomic studies on the effects of the insect growth regulator diflubenzuron and its metabolites 4-chloroaniline could disrupt diflubenzuron in the model beetle species Tribolium castaneum. Insect − the thyroid system of lizards. Moreover, this study is helpful to Biochem. Mol. Biol. 2012, 42, 264 276. assess the risk of diflubenzuron and 4-chloroaniline to lizards. (12) Merzendorfer, H. Chitin synthesis inhibitors: old molecules and new developments. Insect Sci. 2013, 20, 121−138. ■ ASSOCIATED CONTENT (13) Merzendorfer, H. Insect chitin synthases: a review. J. Comp. Physiol., B 2006, 176,1−15. *S Supporting Information (14) Chang, J.; Li, J.; Hao, W.; Wang, H.; Li, W.; Guo, B.; Li, J.; The Supporting Information is available free of charge on the Wang, Y. The body burden and thyroid disruption in lizards (Eremias ACS Publications website at DOI: 10.1021/acs.jafc.8b03713. argus) living in benzoylurea pesticides-contaminated soil. J. Hazard. Mater. 2018, 347, 218−226. Two tables (PDF) (15) Levot, G. W. Degradation of diflubenzuron, ivermectin, cyromazine and temephos in soil following surface disposal of sheep ■ AUTHOR INFORMATION dipping and jetting solutions. Anim. Prod. Sci. 2011, 51, 996−1001. (16) Olsvik, P. A.; Samuelsen, O. B.; Erdal, A.; Holmelid, B.; Corresponding Author * Lunestad, B. T. Toxicological assessment of the anti-salmon lice drug E-mail: [email protected]. diflubenzuron on Atlantic cod Gadus morhua. Dis. Aquat. Org. 2013, ORCID 105,27−43. Yanfeng Zhang: 0000-0002-8916-0996 (17) Dantzger, D. D.; Jonsson, C. 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11646 DOI: 10.1021/acs.jafc.8b03713 J. Agric. Food Chem. 2018, 66, 11640−11646