Metabolism of diflubenzuron in lizard (Eremias argus) and

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Bioactive Constituents, Metabolites, and Functions

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 J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03713 • Publication Date (Web): 22 Oct 2018 Downloaded from http://pubs.acs.org on October 24, 2018

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Journal of Agricultural and Food Chemistry

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Metabolism of diflubenzuron in lizard (Eremias argus) and comparative

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toxicity of diflubenzuron and its metabolite

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Huili Wang a, Yun Xie a,b, Meng Jiao a,b, Xiao Hu a,b, Jitong Li a, Peng Xu a,

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Yanfeng Zhang a, Jing Chang a*

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a

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Shuangqing RD 18, Beijing 100085, China

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b University

Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences,

*

of Chinese Academy of Sciences, Yuquan RD 19 a, Beijing 100049, China

Corresponding author, email address: [email protected] 1

ACS Paragon Plus Environment


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Abstract

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The metabolic process of diflubenzuron in rat or fish has been well studied, but little

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is known about its elimination pathway in lizard. The current study predicted the

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metabolic route of diflubenzuron in lizard feces and compared the toxicity of

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diflubenzuron and 4-chloroaniline on lizard thyroid system. The amido bond cleavage

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was the major route for diflubenzuron elimination in lizard feces. 4-chloroaniline as the

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most toxic diflubenzuron metabolite was also abundant in feces. According to liver slices,

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4-chloroaniline exposure induced significant changes of nuclear shape while

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diflubenzuron exposure caused significant hepatocytes clustering. Based on thyroid

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hormone and thyroid related gene levels, triiodothyronine (T3) level in lizard liver was

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regulated by thyroid hormone receptors while thyroxine (T4) concentration was

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modulated by dio2 and udp genes after diflubenzuron or 4-chloroaniline exposure. These

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results showed that both diflubenzuron and 4-chloroaniline could disrupt lizard thyroid

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system which could provide evidences for lizard population decline.

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Key words: diflubenzuron; 4-chloroaniline; elimination; histopathology; thyroid

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Introduction

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From

2008-2018,

the

found

lizard

species

are

increasing

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(http://www.reptile-database.org/db-info/SpeciesStat.html). However, its populations are

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declining with years. The Mongolian racerunner (Eremias argus) is a small lacertid lizard

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species which is distributed widely in China, Korea, Monglia, and Russia1, 2. However, its

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populations in Korea are small and decreasing, and Eremias argus has been listed under

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the Protection of Wild Fauna and Flora Act in South Korea3. Compared with a previous

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survey in 1991, six species of reptiles including Eremias argus were not found in

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Songshan Nature Reserve, Beijing from 2009-2010 4. The reasons for Eremias argus

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populations decline are still unclear and few researches have been focused on this issue.

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Lizards are commonly found in agriculture areas5. Pesticides application has been

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regarded as one of the possible reasons to induce lizard population decline6. More than

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100 pesticides have been listed as confirmed or possible endocrine disruption chemicals

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(ECDs) by official source7. Previous studies on endocrine disrupting effects of pesticides

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have focused on their reproductive toxicity to non-target organisms, such as estrogen,

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androgens, anti-estrogen or anti-androgen effects8. In recent years, studies have found

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that

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hypothalamus-pituitary-thyroid (HPT) axis plays an important role in regulation of

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growth, metabolic and reproductive system10. In that case, the disruption of lizard thyroid

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system may contribute to the populations decline. However, we still don't know much

thyroid

axis

is

also

one

of

the

target

3


locations

of

ECDs9.

The


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about the mechanisms of interaction between pesticides and lizard thyroid system.

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Diflubenzuron acts as a chitin synthesis inhibitor, and chitin-synthesizing organisms

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are sensitive to this chemical during molting11-13. Because of its properties, diflubenzuron

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has been widely used to control grasshoppers in grassland. The half-lives of

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diflubenzuron in soil were ranged from 80-119 days14,

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enormous strain on higher organisms after bioaccumulation through the trophic chain.

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Eremias argus, which inhabits in grassland and takes grasshoppers as their main food,

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may accumulate diflubenzuron inevitably. Little is known about the influences of

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diflubenzuron on lizard population decline.

15.

Thereby, it can place an

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Although diflubenzuron can be accumulated through food chain, it is considered to

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show very low toxicity on mammals. However, when metabolized or degraded,

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diflubenzuron can produce an extremely toxic compound 4-chloroaniline16. 25 mg/L

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4-chloroaniline alone or 15 mg/L of mixture (75% 4-chloroaniline and 25%

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diflubenzuron) induced 50% mortality of tilapia (Oreochromis niloticus)17. In rat and

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mice, the major route of unabsorbed diflubenzuron elimination is via feces and from the

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liver via bile to feces or urine after absorption18. 4-chloroaniline is not a major metabolite

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in both fish19 and rat18. However, the metabolic process of diflubenzuron in lizard has not

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been studied. Moreover, the toxic effects of 4-chloroaniline on lizard compared with

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diflubenzuron is still unclear.

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In this study, we predicted the elimination pathway of diflubenzuron in lizard by 4


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investigating the metabolites in feces. The disruption of diflubenzuron and

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4-chloroaniline on lizard thyroid system were also compared. This study may provide

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some evidences to assess the risk of diflubenzuron and its metabolites to lizards.

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Materials and methods

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Reagents

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Diflubenzuron and 4-chloroaniline (purity >98%) were purchased from J&K

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Chemical Technology (Beijing, China). The solvents including acetonitrile, methanol,

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and n-haxane (HPLC grade) were obtained from Dikma (Beijing, China). TRNzol A+,

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reverse transcription (RT) Kit and qPCR SYBR Green Kit were purchased from

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TIANGEN Biotech (Beijing, China). The diflubenzuron or 4-chloroaniline stock solution

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was prepared with carrier solvent (methanol) and corn oil.

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Exposure experiment

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The 2-3 years male lizards (3.5-4.0 g) were collected in our breeding colony in

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Changping District, Beijing, China. Lizards were allowed to acclimate in experiment

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cages (60 cm×60 cm×40 cm) for 7 days before experiment. The temperature and

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humidity were set at 25-30 °C and 30-50%, respectively. Lizards were fed with

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mealworms (Tenebrio molitor) twice a day. The animal care and use procedure were

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approved by Research Center for Eco-Environmental Sciences, Chinese Academy of

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Sciences. Animal welfare and experimental procedures were following the Guide for the

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Care and Use of Laboratory Animals (Ministry of Science and Technology of China, 5



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2006).

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7-day diflubenzuron exposure: male lizards were randomly separated into control

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and exposure groups (n=6 for each group), which were orally administrated with corn oil

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and 20 mg/kg diflubenzuron respectively. After 7 days, the feces were collected and

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stored at -20 °C before analysis.

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21-day diflubenzuron and 4-chloroaniline exposure: male lizards were randomly

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separated into 3 groups, including control, diflubenzuron and 4-chloroaniline exposure

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groups (n=6 for each group). In the exposure groups, lizards were orally administrated

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with 20 mg/kg diflubenzuron or 4-chloroaniline per week. In the control group, only

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carrier solvent-methanol and corn oil were exposed. Lizards at both control and exposure

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groups were fed normally. After 3 weeks of exposure, lizards were killed through

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decollation. Blood was collected and centrifuged (2500 × g, 10 min) to collect the serum.

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Lizard liver was also collected and stored in RNA store. For histopathology analysis, a

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part of liver was fixed with 4% paraformaldehyde. Samples including serum and liver

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were stored at -20 °C before analysis.

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Chemical analysis

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The metabolic process of diflubenzuron was mainly studied through lizard excreted

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feces. It is an effective method to investigate the metabolites of diflubenzuron. In our

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study, lizard feces (1.5-2.0 g) were collected from 0-7 days after diflubenzuron exposure.

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The metabolites were extracted with 15 mL acetonitrile in a 50 mL polypropylene 6


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centrifuge tube. The tube was mixed on a vortex mixer for 2 min, ultrasonic for 10 min

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and then centrifuged at 8000 rpm for 5 min. The upper phase was collected, and then the

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steps above were repeated. The upper phases were combined and dehydrated with 2 g

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anhydrous magnesium sulfate. The extracts were dried on a vacuum rotary at 35 °C and

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redissolved in 1 mL n-haxane.

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The analysis of diflubenzuron metabolites were performed on a Thermo-TSQ 8000

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GC/MS/MS equipped with an electron-impact ionization (EI) source and Thermo column

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TR-35MS (0.25 mmΦ×30 mID×0.25 mm). The ramped temperature program was held at

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50 °C for 1 min, ramped at 20 °C/min to 200 °C for 1min, ramped at 5 °C/min to 260 °C

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for 10 min, ramped at 20 °C /min to 280 °C for 2 min. The mass spectrum was operated

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at full scan mode. The scan range was from 50-550 m/z.

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Histopathology

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Lizard liver sections were trimmed, placed in cassettes, embedded in paraffin,

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sectioned at 6 μm, stained with hematoxylin and eosin, and observed under light

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microscope (ZEISS, AXIO) according to the method described by MacFarland et al20.

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The liver lesions were quantified using semi-quantitative scoring method21, 22. Twelve

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tissue sections were randomly chosen under the light microscope in each group. The

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scoring system was defined as follows: - (no lesions); mild: + (1-4 lesions); moderate: ++

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(5-8 lesions); sever +++ (≥ 9 lesions).

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Thyroid hormones analysis 7



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The thyroxine (T4) and triiodothyronine (T3) levels were measured by

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enzyme-linked immunosorbent assay (ELISA) kit specified for lizards (T3 kit was from

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Elabscience Biotechnology Co.,LTD; T4 kit was from Cloud-Clone Corp.) according to

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the manuals. The competing ELISA principle was applied in both kits. Samples diluted 5

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folds were used in the T3 and T4 assay in order to fit the calibration curves.

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Quantitative real-time PCR

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The isolation of RNA, cDNA synthesis method has been described in our previous

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paper23. Briefly, total RNA in lizard liver was extracted using TRNzol-A+ reagent. The

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concentration and purity of extracted RNA was evaluated using NanoDrop 2000

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microvolume spectrophotometers. The reverse transcription was achieved using RT kit

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with gDNase.

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The hypothalamus-pituitary-thyroid axis-related genes including thyroid hormone

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receptors (trα, trβ), deiodinases (dio1, dio2), uridinediphosphate glucuronosyl-transferase

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(udp), and sulfotransferase (sult) and their respective primers were listed in Table S1.

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Quantitative real-time PCR was performed in a MX3005P real-time quantitative

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polymerase chain reaction system (Stratagene, USA) using the SYBR Green PCR Kit.

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The PCR system was operated according to the following protocols: denaturation at 95

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°C for 5 min, followed by 40 cycles of amplification at 95 °C for 30 s, at 54 °C for 40 s

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and at 72 °C for 40 s. The dissociation curve was obtained at 95 °C for 40 s, 54 °C for 40

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s, and a gradual heating to 95 °C. Relative quantification of each gene expression level

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was normalized according to the β-actin gene expression.

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Data analysis 8


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The body weight to body length ratio (BWL) was calculated as follows:

146 147 148

BWL= Body weight/body length The mass spectrum of predicted metabolites was compared with compounds in the NIST library.

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The statistical analysis was performed using SPSS software (Version 13.0, USA).

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Differences of BWL, thyroid hormone levels and gene expressions between control and

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exposure group were analyzed according to analysis of variance (ANOVA). A probability

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of p

Metabolism of diflubenzuron in lizard (Eremias argus) and

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