The Body Burden and Thyroid Disruption in Lizards (Eremias Argus)

Home , Eremias, Eremias argus

Journal of Hazardous Materials 347 (2018) 218–226

Contents lists available at ScienceDirect

Journal of Hazardous Materials

jo urnal homepage: www.elsevier.com/locate/jhazmat

The body burden and thyroid disruption in lizards (Eremias argus)

living in benzoylurea pesticides-contaminated soil

a,b a,b a,b a a a

Jing Chang , Jitong Li , Weiyu Hao , Huili Wang , Wei Li , Baoyuan Guo ,

a a a,∗

Jianzhong Li , Yinghuan Wang , Peng Xu

Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Shuangqing RD 18, Beijing, 100085, China

University of Chinese Academy of Sciences, Yuquan RD 19A, Beijing, 100049, China

h i g h l i g h t s g r a p h i c a l a b s t r a c t

•

Diﬂubenzuron degraded faster than

ﬂufenoxuron in the soil.

•

The SPFs of ﬂufenoxuron were

greater than diﬂubenzuron.

•

The body burden of BPUs was related

with LogKow and molecular weight.

•

BPUs exposure disturbed both thy-

roid and metabolism system of

lizards.

a r t i c l e i n f o a b s t r a c t

Article history: Dermal exposure is regarded as a potentially signiﬁcant but understudied route for pesticides uptake

Received 1 November 2017

in terrestrial reptiles. In this study, a native Chinese lizard was exposed to control, diﬂubenzuron or

Received in revised form −1

ﬂufenoxuron contaminated soil (1.5 mg kg ) for 35 days. Tissue distribution, liver lesions, thyroid hor-

19 December 2017

mone levels and transcription of most target genes were examined. The half-lives of diﬂubenzuron and

Accepted 3 January 2018

ﬂufenoxuron in the soil were 118.9 and 231.8 days, respectively. The accumulation of ﬂufenoxuron in

Available online 4 January 2018

−1

the liver, brain, kidney, heart, plasma and skin (1.4–35.4 mg kg ) were higher than that of diﬂubenzuron

−1

(0–1.7 mg kg ) at all time points. The skin permeability factor of ﬂufenoxuron was more than 20-fold

Keywords:

Diﬂubenzuron greater than that of diﬂubenzuron at the end of exposure. However, the liver was more vulnerable in

Flufenoxuron the diﬂubenzuron exposure group. The alterations of triiodothyronine (T3) and thyroxine (T4) level after

Reptile diﬂubenzuron or ﬂufenoxuron exposure were accompanied with the changes in the transcription of tar-

Endocrine disruption get genes involved not only in hypothalamus-pituitary-thyroid (HPT) axis (sult, dio2, tr and udp) but

Tissue distribution also in metabolism system (cyp1a and ahr). These results indicated that ﬂufenoxuron produced greater

body burdens to lizards through dermal exposure, whereas both diﬂubenzuron and ﬂufenoxuron have

the potential to disturb metabolism and thyroid endocrine system.

Abbreviations: BPUs, benzoylurea pesticides; HPT, hypothalamus-pituitary-thyroid; tr˛, thyroid hormone receptor ˛; trˇ, thyroid hormone receptor ˇ; dio1, deiodinase

type 1; dio2, deiodinase type 2; udp, uridine diphosphate glucuronosyl transferase; sult, sulfotransferase; (cyp1a, cyp2a and cyp3a), cytochrome P450; (ahr), aryl hydrocarbon

receptor; T3, triiodothyronine; T4, thyroxine. ∗

Corresponding author.

E-mail address: [email protected] (P. Xu).

https://doi.org/10.1016/j.jhazmat.2018.01.005

J. Chang et al. / Journal of Hazardous Materials 347 (2018) 218–226 219

1. Introduction 2. Materials and methods

Benzoylurea pesticides (BPUs) act as a chitin synthesis inhibitor 2.1. Chemicals

to inﬂuence the molting of insects [1]. BPUs are mainly used

to control locusts and grasshoppers on rangeland and crop- Diﬂubenzuron and ﬂufenoxuron (Purity: 98%, Fig. S1) were

lands worldwide [2]. Due to their effectiveness as insecticides purchased from J&K Scientiﬁc Ltd. (Beijing, China). The solvents

and low mammalian toxicity, the commercial development and including methanol, acetonitrile, acetone and n-hexane (HPLC

use in agriculture practice has increased [3]. Diﬂubenzuron was grade) were obtained from Dikma (Beijing, China).

shown to be persistent on conifer foliage after aerial application

[4]. In rat, diﬂubenzuron was eliminated rapidly from the tis- 2.2. Test lizards and husbandry

sues [5] and was not cytotoxic to the cells [6]. In contrast, ﬁsh

can accumulate diﬂubenzuron to concentrations more than 70 Although E. argus has been listed as endangered by the Korean

times greater than that of the water content when exposed at Ministry of Environment in 2005 due to its limited distribution

−1

environmental concentration (2.5 mg L ) [7]. Moreover, diﬂuben- and decreased population sizes [22], it is still regard as the most

zuron impairs reproduction in crustaceans, as shown in mysid abundant lizard species in China. The species is easily maintained

shrimp [8]. Flufenoxuron is one of the third generation BPUs in a laboratory and will readily breed in captivity. We collected

that is metabolized more slowly than ﬁrst generation compounds, juvenile E. argus from the natural landscape in the Inner Mongolia

such as diﬂubenzuron by insects [9]. The bioconcentration factor Province and fed them in our breeding base from July 2009 till the

(BCF) of ﬂufenoxuron is more than 100-fold higher than that of start of our experiments. We tried to mimic a micro ecosystem

diﬂubenzuron in aquatic organisms [3]. Flufenoxuron displayed lit- for lizards and kept them in enclosures with pesticide-free sand

−1

tle or no acute toxicity up to 1000 ␮g L whereas developmental and soil to let them reproduce naturally. The experimental lizards

effects were seen under environmentally relevant concentrations were captured from the enclosures. Females were excluded from

−1

(3.2–320 ng L ) in exposed copepods [10]. Flufenoxuron has also the exposure experiment, as they were in the timing of reproduc-

shown very high toxicity against Daphnia magna, and the EC50 value tion. As body weight and length are good indicators of the age of

−1

is 0.000908 mg L [3]. Although the effects of BPUs on mammals lizards [22], the 2–3 years old mature male E. argus (progeny of the

or aquatic organisms are well-studied, reptiles remain neglected in previous wild caught females, body weight: 3.2–3.8 g, snout-vent

ecotoxicology studies. In fact, reptiles are not routinely included in length: 3.5–4.5 cm) were picked out according to body weight and

toxicity tests and there is little information about their sensitivity length [23]. Prior to the experiment, lizards lived in the uncontam-

to pesticides. inated soil for two weeks to acclimate in experimental glass tanks

Lizards account for 59.93% of reptile species [11]. A previous (40 × 25 × 15 cm). Ultraviolet lamps (Canada, Exo Terra, UVB 150,

−2

study conducted in European Union (EU) demonstrated that lizards 13 W, the intensity is 150 ␮w cm measured 30 cm beneath the

seem to display higher exposure sensitivity than other reptile sub- lamp) were set on 12 h light/dark cycles to provide enough light,

orders, such as snakes and tortoises [12]. Lizards primarily inhabit promote vitamin D protection, and maintain the needed tempera-

◦

grasslands in Asian countries [13], where BPUs could be applied ture. The heat lamp created a gradient of approximately 26–34 C

in order to control massive locusts [14]. However, there is no data in the container when the lamps were on. When the lamps were off,

◦

about the degradation kinetic of BPUs in lizard tissues and the toxic temperatures were maintained at 25 ± 4 C. Live mealworms (Tene-

effects of pesticides on lizards are usually predicted from the toxi- brio molitor) were put in food containers every day to feed lizards.

city threshold established for birds [15]. Using birds as a surrogate We sprayed water containing mixed vitamin D3 and calcium car-

might not be effective because of the lizard’s unique combination of bonate (Repti calcium, made in USA; 0.5 g powder dissolved in 1 kg

physical and life history characteristics [16,17]. For example, lizards water) four times a day on the tank wall in order to provide enough

were suspected to receive higher relative exposure than birds due water for lizards (lizards prefer to lick the water on the wall rather

to the greater percentage body contact with soil and vegetation if than get water from the provided water containers).

dermal route is explicitly considered [18]. In that case, it would be

meaningful to study the body burden and toxicity of BPUs in the 2.3. Exposure experiment and sample collection

lizards under dermal exposure.

Thyroid hormones play an important role in metabolic regu- Aerial application resulted in deposition level of diﬂubenzuron

−1

lation, growth, protein synthesis, activity of others hormones in on soil surface ranging from 1.07–3.84 mg kg [24]. The nominal

mammals [19]. The standard metabolic rate and cardiac muscle exposure concentration in both diﬂubenzuron and ﬂufenoxuron

−1

mass in lizards also depend on the thyroid function [20]. A ﬁeld exposure group is 1.50 mg kg in sandy soil, which is similar with

study already showed that lizards exposed to agricultural areas the environmental concentration. In order to make sure that the

have thyroid disrupting effects that ultimately inﬂuenced the male 5.5 kg of sandy soil was spiked homogeneously with the chem-

reproduction system [21]. Therefore, the disturbance of thyroid ical, we did the procedure in steps. Firstly, the acetone solution

hormone levels by pesticides may affect the normal reproduction (only the carrier solvents - acetone was used in the control group)

and metabolism of lizards. BPUs are known to inhibit the growth was added slowly to the soil (0.5 kg, organic content, 1.0 ± 0.6%;

of insects through disruption of molting [1] while their effects on sand (2000–20 ␮m), 71.2 ± 5.0%; slit (<20–2 ␮m), 5 ± 2.5%; clay

lizard development are little known. (<2 ␮m), 23.8 ± 2.5%; pH, 7.5 ± 0.2), and then mixed for 15 min

In this study, we used a Chinese native lacertid species-Eremias using a mechanical shaker. The contaminated soil was left in a fume

argus (E. argus) to investigate the effects of diﬂubenzuron and cupboard overnight to evaporate acetone. Secondly, 5 kg of uncon-

ﬂufenoxuron on lizard thyroid endocrine system. The bioaccumu- taminated medium was mixed thoroughly with the contaminated

lation of the two BPUs in lizard plasma and tissues were measured 0.5 kg soil using the mechanical shaker. The depth of soil in the

after dermal exposure with environmentally relevant concentra- tanks was 3 cm.

tion. Furthermore, liver histopathology was conducted, thyroid After acclimating, the male lizards were impartially separated

hormone levels and transcriptional expressions of the target genes into control, diﬂubenzuron, and ﬂufenoxuron exposure tanks (total

were also determined. Our results facilitate understanding of the 3 groups, a total of 144 lizards were evenly separated into 9 tanks, 3

potential effects of BPUs on lizard thyroid endocrine system. tanks for each group with 3 replicates). Lizards in both control and

exposure groups were fed as usual. Six individuals (two lizards from

220 J. Chang et al. / Journal of Hazardous Materials 347 (2018) 218–226

each tank) from each treatment were weighed and sampled at 0, 2, 2.7. Isolation of mRNA and quantitative real-time polymerase

4, 7, 14, 21, 28 and 35 days. The decollation and blood collection of chain reaction

lizards were based on the method mentioned by Amaral et al. [25].

Blood was immediately collected and plasma was obtained by cen- As the tra, trb, dio1, dio2, udp and sult genes showed relatively

◦

trifugation and stored at −20 C before analysis. Lizard brain, heart, high expression in lizard liver [29], liver samples were homoge-

kidney, liver, skin, and gonad were collected, then weighed and nized and prepared for total RNA isolation using TRIzol reagent (Life

◦

frozen at −20 C before tissue distribution analysis of BPUs. Given technology, Beijing, China). The cDNA was synthesized by reverse

that tissue from one lizard was not enough to analyze BPUs resid- transcription reactions according to the manufacturer’s instruc-

ual concentration, the tissues of two lizards sampled from one tanks tions.

were combined as one replicate. When sampled at 35 days, a por- The selected target genes in the HPT axis and their respective

tion of the liver was stored in the RNA store for gene transcriptional primers are listed in Table S2. All the primers were designed by the

analysis, another portion of liver was stored in 4% paraformalde- authors using online National Center for Biotechnology Informa-

hyde (4 g paraformaldehyde dissolved in 100 mL phosphate buffer tion (NCBI). Real-time PCR was performed in a MX3005P real-time

saline) for hepatic pathological analysis. The soil (5 g, 3 replicates) quantitative polymerase chain reaction system (Stratagene, USA) in

was also sampled at 0, 2, 4, 7, 14, 21, 28 and 35 days in both the a total volume of 20 ␮L, including the SYBR Green RealMasterMix,

◦

control and exposure groups and stored at −20 C. 1 ␮M forward primer and 1 ␮M reverse primer. The thermal cycle

◦ ◦ ◦

parameters were: 95 C for 5 min, 40 cycles of 95 C for 30 s, 54 C

◦

for 40 s and 72 C for 40 s. The thyroid hormone receptors (tr˛, trˇ),

deiodinases (dio1, dio2), uridinediphosphate glucuronosyltrans-

2.4. Chemical analysis

ferase (udp), sulfotransferase (sult), transthyretin (ttr), cytochrome

450 (cyp1a, cyp2a and cyp3a) and aryl hydrocarbon receptor (ahr)

The soil, blood plasma (50–100 ␮L) or homogenized tissue

mRNA expression were normalized by ˇ-actin mRNA expression. A

matrix (0.05–1.5 g) was put into a 50 mL polypropylene centrifuge

dissociation curve analysis was performed for each gene to check

tube with 15 mL acetonitrile as extracting solution. The extraction

the ampliﬁcation of the untargeted fragments. Only one peak was

was conducted following the protocol described in Chang et al. [23].

observed for the amplification, indicating the specific amplifica-

The extract was diluted with 1 mL methanol and passed through a

tion of the target gene. The gene expression data showed changes

0.22 ␮m ﬁlter membrane.

relative to the control animals for the same treatment period.

Diﬂubenzuron and ﬂufenoxuron were detected by

HPLC/MS/MS. HPLC-MS/MS analyses were performed on a

TSQ QUANTUM ACCESS MAX triple quadrupole MS and an Accela

600 pump/auto sampler HPLC (Thermo Electron, Hopkinson, MA).

2.8. Data analysis

A C18 column was used and the mobile phase was combined

−1

with methanol and 5 mmol L ammonium acetate water (90:10,

Statistical analysis of the data including body weight, soil con-

−1

v/v) at a ﬂow rate of 0.2 mL min . Chromatographic analysis

centration, thyroid hormone levels and gene expressions was

◦

was conducted at 20 C and the injection volume was 10 ␮L. The

performed using analysis of variance (ANOVA), followed by the

analytes were detected by multiple reaction monitoring (MRM)

Least Signiﬁcant Difference (LSD) post-hoc test by SPSS (version

mode using negative electro spray ionization mass spectrometry

13.0; USA). A probability of p < 0.05 was considered statistically

(ESI-MS). The relative standard deviation (RSD) for the quality signiﬁcant.

control (QC) samples was <12.91%. The extraction blanks were also

The proportion of individuals exhibiting the different liver

performed and no target compound was detectable.

lesions among exposed and control groups were compared using

For determination of the precision and accuracy of the method,

Fisher’s Exact Tests.

the blank soil and tissue samples (3 replicates) were spiked with

The relationship between thyroid hormone level and the tran-

three levels of diﬂubenzuron or ﬂufenoxuron respectively. The

scription of genes was analyzed by principle component analysis

mean recoveries of samples were listed in Table S1.

(PCA). Furthermore, correlation analysis was conducted with Pear-

son’s test to conﬁrm the relationship between thyroid hormone

level and the gene expression level.

2.5. Liver histopathology The elimination equations of diﬂubenzuron and ﬂufenoxuron

in soil were performed by SASPAL 3.0 (Hu Liangping, China). The

The liver was processed for parafﬁn wax embedding. Sections equations matched with one compartment model.

were cut, stained with haematoxylin and eosin (HE), and then The calculation of skin permeability factor (SPF) was calculated

measured by light microscopy [26]. Generally, assessment of each as follows [30]:

sample involved an examination of 12 randomly chosen ﬁelds

of view. The histopathology lesions were classiﬁed with a semi-

SPF = (CL × BWL/SAL)/ACS (1)

quantitative scoring system [27,28]: - (no lesion), mild: + (1–4

lesions), moderate: ++ (5–8 lesions) and severe: +++ (≥9 lesions).

ACS = CS × SW/H/(W × L) × 1 cm (2)

2.6. Thyroid hormone analysis

where CL is the lizard whole body tissue concentration of BPUs

−1 −1

(mg g ), CS is the soil concentration of BPUs (mg g ), BWL is the

The plasma samples were collected after 35 days from both

body weight (g) of the lizard, SW is the soil weight in the tank (g),

control and exposure groups. The triiodothyronine (T3) and thy-

and ACS is the applied concentration in the soil referring to the soil

roxine (T4) levels in the plasma were analyzed by enzyme-linked −2

surface (W: width (cm); L: length (cm)) with 1 cm depth (mg cm ).

immunosorbent assay (ELISA) kit (purchased from Elabscience 2

SAL is the surface area of the lizard (cm ) calculated as follows [18]:

Biotechnology Co.,LTD, Wuhan, China) following the manufacture’s

instructions. No signiﬁcant cross reactivity or interference was

0.694

= . × . ×

observed for the kits. SAL 0 3 8 42 BW (3)

J. Chang et al. / Journal of Hazardous Materials 347 (2018) 218–226 221

control the inﬂuences of pollutants on the body weight of lizards depend

diflubenzuron on different exposure patterns and different kinds of chemicals. flufenoxuron

3.2. Degradation and bioaccumulation studies

3.2.1. The degradation of diﬂubenzuron and ﬂufenoxuron in the 3 soil

* *

The nominal exposure concentration of diﬂubenzuron and

−1

ﬂufenoxuron in the sandy soil is 1.50 mg kg while the quan-

2 −1

tiﬁed concentrations were 1.40 and 1.61 mg kg , respectively

(Fig. 2a–b). Generally, the concentration of the two BPUs decreased

with time. The concentration of diﬂubenzuron was relatively sta-

ble from 0 to 14 days but there was a signiﬁcant decrease especially

at 28 and 35 days compared with the concentration at 0 day (one-

way ANOVA, p = 0.013 < 0.05 at 28 days, p = 0.029 < 0.05 at 35 days).

In contrast, the concentration of ﬂufenoxuron decreased gradually

0 5 10 15 20 25 30 35

from the beginning while no signiﬁcant difference was observed

between different time points. The half-lives of diﬂubenzuron and

Fig. 1. The changes of body weight during 35 days in the control and exposure ﬂufenoxuron in the sandy soil which were calculated through one

± ≥ ∗

groups. The data are expressed as the mean S.D. of each treatment (n 6). rep- compartment model were 118.9 and 231.8 days, respectively. This

resents statistically signiﬁcant difference from the control (Multivariate ANOVA,

result indicated that the ﬂufenoxuron was more persistent than

p < 0.05).

diﬂubenzuron in the sandy soil. The high residual level of ﬂufenox-

uron could be explained by the low capacity of microorganisms to

degrade ﬂufenoxuron [32] or its relatively high octanol-water parti-

tion coefﬁcient (Log Kow) value [33]. Due to their different residual

3. Results and discussion

levels in soil, it can be predicted that the toxic effects of the two

BPUs in lizards would be different.

3.1. Mortality and body weight

No mortality was observed in the control or exposure groups 3.2.2. The bioaccumulation of diﬂubenzuron and ﬂufenoxuron in

during the experiment. Lizards of the control group gradually lizard plasma and tissues

gained weight throughout the study (Fig. 1). This phenomenon Lizards exposed to the diﬂubenzuron or ﬂufenoxuron contami-

indicated the growth of lizards under unexposed condition. No nated soil showed accumulation in tissues. Although lizards could

signiﬁcant difference was observed between the body weight take up contaminants through incidental or intentional soil inges-

of control and ﬂufenoxuron exposed group (Repeated Measures tion, we predicted that lizard skin penetration was the main route

ANOVA, p > 0.05). Only diﬂubenzuron exposure showed signiﬁcant to uptake BPUs as adequate food was provided for tested lizards.

difference on body weight when comparing with the control group The concentration-time proﬁles estimated the adsorption, elimi-

(Repeated Measures ANOVA, p = 0.019 < 0.05). The body weight nation and distribution of diﬂubenzuron (Fig. 3a) and ﬂufenoxuron

showed signiﬁcant differences between control and diﬂuben- (Fig. 3b) in different tissues.

zuron exposure group especially at 28 (Multivariate ANOVA, During diﬂubenzuron exposure, the highest concentration was

p = 0.009 < 0.05) and 35 days (Multivariate ANOVA, p = 0.025 < 0.05). observed in the brain followed by the liver and skin. The maxi-

−1

Lizards were fed as usual during exposure and no decrease in mum concentration of diﬂubenzuron in the liver (1.1 mg kg ) or

−1

appetite was observed. These results showed that the growth of brain (1.4 mg kg ) occurred at approximately 5 days while the con-

lizards was inﬂuenced when exposed to diﬂubenzuron contam- centration in the skin quickly reached a peak within 2 days. After

inated soil for 35 days. Similar to this result, oral exposure to that, the concentration of diﬂubenzuron in the three tissues experi-

diﬂubenzuron signiﬁcantly reduced the body weight of lizards at 21 enced a progressive decrease and another peak occurred at 28 days

days [29]. However, the glyphosate-contaminated water spraying for the skin and liver and at 23 days for the brain. The appear-

didn’t signiﬁcantly affect lizard body mass [31]. It suggested that ance of the second peak may be correlated with the changes of

Fig. 2. The concentration-time profiles of diflubenzuron (a) and flufenoxuron (b) in the soil during 35 days. The data was expressed as mean ± S.D. (n = 3). * p < 0.05 indicates

signiﬁcant difference compared to the soil concentration at the begining (one-way ANOVA, p < 0.05).

222 J. Chang et al. / Journal of Hazardous Materials 347 (2018) 218–226

Fig. 3. The tissue concentration-time profiles of diflubenzuron (a) and flufenoxuron (b) in the lizards tissues during 35 days. The data was expressed as mean ± S.D. (n = 3).

metabolic enzyme activities in the lizard tissues or the consecu- skin shedding during the exposure period. In this case, the ﬂuctua-

tive penetration of diﬂubenzuron from the contaminated soil to tion of BPUs concentrations in lizard skin may due to the depuration

the tissues. After 35 days of exposure, diﬂubenzuron in the liver of BPUs through skin shedding. Another hypothesis is that the abil-

−1

was comparatively low, whereas the brain (0.94 mg kg ) and skin ity of skin to absorb contaminants was varied between different

−1

(0.62 mg kg ) still had accumulations of diﬂubenzuron at high lev- stages (before or after skin shedding) [29]. The SPFs were used to

els. This phenomenon could be explained by the large amounts assess lizard dermal adsorption ability. It increased with increasing

of metabolizing enzymes in the liver or ongoing dermal uptake molecular mass and Log Kow and decreasing water solubility of the

effect. Another interesting ﬁnding is that diﬂubenzuron was only two BPUs (Table S3), which is opposite to that observed in a study

detectable in the kidney after 21 days of exposure and it was not on the human skin [39]. In addition, the SPFs of ﬂufenoxuron were

detectable in the plasma and heart at any time-point. It is possible greater than 1.0 after 2 days of exposure, which indicated the skin’s

that diﬂubenzuron was rapidly removed from the blood by the liver high permeability with respect to ﬂufenoxuron. Lizard tissues also

(brain) prior to overwhelming the liver (brain) detoxification mech- showed higher concentration of flufenoxuron than diflubenzuron

anisms and then only transferring to the kidney. A previous study after oral exposure [40]. Considering that lizards in natural envi-

on swine showed that more than 80% of the administrated diﬂuben- ronments will be exposed to insecticides not only from soil but also

zuron were recovered from the faeces and urine [34]. The result from treated insects, it is predicted that lizards could accumulate

from rats suggested that diﬂubenzuron was eliminated rapidly and even more ﬂufenoxuron in the natural environment.

not accumulated in the tissues at a high level after oral exposure [5]. The tendency of ﬂufenoxuron to accumulate in the heart,

In this study, the accumulation of diﬂubenzuron in the brain, skin liver and kidney was similar but the highest concentration

−1

and kidney of lizards may be related with 1) the exposure route, (38.4 mg kg ) was in the heart. Both diﬂubenzuron and ﬂufenox-

diﬂubenzuron slowly enter systemic circulation mainly through uron concentrations in lizard kidney were very high, especially

skin permeation; 2) as birds and reptiles show lower monooxyge- after 14 days of exposure. Overall, considering all the accumula-

nase activities than mammals [35], they may be more susceptible tion results, we presume that the kidney is an active site of BPUs

to diﬂubenzuron (lipophilic environmental chemicals) which are accumulation during long-term exposure.

detoxiﬁed by this system. The concentration of ﬂufenoxuron in the brain reached to a

In contrast to the diﬂubenzuron exposures, lizards exposed with steady state after 21-day exposure. It was presumed that ﬂufenox-

ﬂufenoxuron showed very high residual levels in almost all tis- uron can cross the blood-brain barrier and deposit in the brain.

sues. The maximum concentration in the tissues was ranked from A previous study regarded blood as a non-destructive tissue for

high to low in the following orders: heart, kidney, brain, liver, skin the analysis of contaminants in snakes because of the strong

and plasma. The bimodal phenomena appeared in the liver, brain, positive relationship between blood and target tissues [41]. The

heart and kidney. The ﬁrst peak was around 7 days and the second ﬂufenoxuron concentration in lizard brain was also well correlated

peak was at 28 days. However, the highest level of ﬂufenoxuron (R = 0.94, p = 0.006 < 0.05) with plasma concentration. Thus, blood

was at the second peak which was not consistent with the ten- might also be a suitable non-lethal tissue for the pollutants analysis

dency of diﬂubenzuron in lizard tissues. This difference may be in lizards.

associated with three equally important factors: 1) The residual

concentration of ﬂufenoxuron in soil was relatively high especially

3.3. The toxic effects of diﬂubenzuron and ﬂufenoxuron on HPT

after 14 days; 2) ﬂufenoxuron had a higher propensity to pene-

axis

trate through lizard skin, as its skin permeability factors (SPFs)

were greater than diﬂubenzuron at all time points (Fig. S2); 3)

3.3.1. Liver histopathology

the metabolic rate of diﬂubenzuron was higher in lizard tissues. It

In the control group, the liver showed obvious sinusoids of hep-

can be concluded that the ﬂufenoxuron-contaminated soil induced

atocytes. The hepatocytes were distributed evenly with clear cell

greater body burden in lizard tissues than diﬂubenzuron.

boundary and distinct outline of nucleolus (Fig. 4a).

Consistent with the trend of diﬂubenzuron in the skin, the peak

The histopathology changes such as ﬁbrosis (Fisher’s Exact Test,

value of ﬂufenoxuron was reached within four days which was

for diﬂubenzuron exposure, p < 0.001; for ﬂufenoxuron exposure,

faster than any other tissues. Similar with this result, the snake

p = 0.003), narrow sinusoid (Fisher’s Exact Test, for diﬂubenzuron

skin also permitted fairly rapid permeation of pyrethroid and some

exposure, p < 0.001; for ﬂufenoxuron exposure, p = 0.014), hepato-

signs of intoxication were seen within 1 h [36]. Previous studies

cytes clustering (Fisher’s Exact Test, for diﬂubenzuron exposure,

showed that organochlorines [37] and heavy metals [38] in the

p = 0.001; for ﬂufenoxuron exposure, p = 0.064) were presented in

snakes were excreted into the shed skin. The lizards also showed

both diﬂubenzuron and ﬂufenoxuron exposure group (Fig. 4b–c,

J. Chang et al. / Journal of Hazardous Materials 347 (2018) 218–226 223

Fig. 4. Lizard liver sections after 35 days of dermal exposure to benzoylurea pesticides (BPUs). (a) liver section from the control group representing a normal liver. (b) example

of diflubenzuron treated group showing narrow sinusoid, fibrosis (white arrows) and hepatocyte clustering (white circle). (c) example of flufenoxuron treated group showing

narrow sinusoid and ﬁbrosis (white arrows).

Table 1

that diﬂubenzuron exposure suppressed the thyroid gland activity

The concentration of triiodothyronine (T3) and thyroxine (T4) in the lizard plasma

while ﬂufenoxuron exposure stimulated the thyroid gland activity.

of both control and exposure groups at 35 days.

The alterations of thyroid gland activities may be attributed to the

−1 −1 −5

Exposure groups T3/ng L T4/␮g L T3/T4/10

different concentrations of the two BPUs in the plasma. The two

control 16.42 ± 1.54 804.42 ± 85.32 2.04 ± 0.03 BPUs exposure might inﬂuence the synthesis and metabolism of

* * *

± ± ±

diﬂubenzuron 8.38 1.29 64.40 10.56 13.02 0.27 T4 in different mechanisms. Only in the diﬂubenzuron exposure

* *

ﬂufenoxuron 58.72 ± 6.38 1764.39 ± 204.21 3.33 ± 0. 02

group showed a signiﬁcant increase in the ratio of T3 to T4. The

The data were expressed as mean S.D. (n = 3). deiodinase enzymes catalyse the conversion from T4 to T3. Thus,

p < 0.05 indicates signiﬁcant difference compared to the control (one-way the alteration of T3/T4 in diﬂubenzuron exposure group is supposed

ANOVA).

to be an indicator for the changes of deiodinase enzymes activities

[43]. Thyroid hormones could regulate the cardiac muscle mass in

lizards [20]. The human ingested a ﬂufenoxuron-containing insec-

Table S4). According to the series of Fisher’s Exact Test, it revealed

ticide presented severe elevation of cardiac enzymes and global left

signiﬁcant differences regarding the occurrence of ﬁbrosis and nar-

ventricular hypokinesia [44]. Therefore, the high accumulation of

row sinusoid between the control and exposure group. Only in

ﬂufenoxuron in lizard heart and the up-regulation of thyroid hor-

the diﬂubenzuron exposure group did the incidence of hepatocytes

mone levels after ﬂufenoxuron exposure may also cause cardiac

clustering approach signiﬁcance when comparing with the control.

lesions, which needs further research.

As the concentration of diﬂubenzuron was lower than ﬂufenox-

uron in the liver, the cell clustering may be a sign of the increased

metabolic rate for diﬂubenzuron detoxiﬁcation. Lizards sampled

3.3.3. Gene transcriptional proﬁles in lizard liver

from agriculture land where pesticides had been applied also pre-

The transcriptional proﬁles of HPT axis-related genes (tr˛, trˇ,

sented with liver lesions as well as some indicators of changed

dio1, dio2, udp, sult and ttr) and metabolism-related genes (cyp1a,

detoxiﬁcation rate [42]. Our previous study stated that only the oral

cyp2a, cyp3a and ahr) in lizard liver exposed to diﬂubenzuron or

ﬂufenoxuron treatment showed serious injuries in lizard liver [40].

ﬂufenoxuron at 35 days are shown in Fig. 5. The thyroid hormones

According to the results, the different exposure patterns may cause

(T3 and T4) interact with thyroid hormone receptors (tr˛, trˇ) to

different liver lesions and dermal exposure should be considered

elicit their speciﬁc biological response in lizard tissues [45]. No

as an important exposure route.

signiﬁcant difference was found in the tr˛ gene expression after

diﬂubenzuron or ﬂufenoxuron exposure (p > 0.05) while both expo-

3.3.2. T3 and T4 content sure groups caused signiﬁcantly decrease of trˇ gene expression

The plasma T3 and T4 levels are shown in Table 1. In the diﬂuben- (p < 0.05, for the df, F and p values, please see Table S5). Diﬂuben-

zuron exposure group, the T3 and T4 levels were signiﬁcantly zuron exposure signiﬁcantly increased the expression of dio2 gene

decreased (one-way ANOVA, T3: p = 0.002; T4: p < 0.001), whereas and ﬂufenoxuron exposure signiﬁcantly decreased the dio2 level

in the ﬂufenoxuron exposure group, both the T3 and T4 contents (p < 0.05). The dio2 mainly catalyzes the transfer from T4 to T3.

were signiﬁcantly up-regulated (one-way ANOVA, T3: p < 0.001; The changes of dio2 gene expression may explain the changes

T4: p = 0.002). The thyroid hormone level is an important indica- of T3 level. The dio1 and ttr genes in the liver were more sensi-

tor for the thyroid gland activity of lizards. This result indicated tive (increased gene expression) to diﬂubenzuron compared with

224 J. Chang et al. / Journal of Hazardous Materials 347 (2018) 218–226

The metabolic rate in lizards has a close connection with thy-

c ontrol

diflubenzuron * roid function [20]. The cytochrome P450 genes (CYP450), such as

flufe noxuron

10 cyp1a, cyp2a, cyp3a, play an important role in the liver metabolism.

The CYP450 expression is controlled by several nuclear receptors,

8 such as ahr [47]. As shown in Fig. 5, all the metabolism-related

gene expressions (cyp1a, cyp2a, cyp3a and ahr) were signiﬁcantly

increased after ﬂufenoxuron exposure compared with control

group. The relatively high residual level of ﬂufenoxuron in the *

* liver or the increased thyroid hormone levels may be related with *

4 * * the changes in metabolism system [27]. For diﬂubenzuron expo-

sure, only cyp2a and cyp3a gene levels were signiﬁcantly increased

* and the ahr gene level was signiﬁcantly decreased (p < 0.05). The

2 * *

metabolism of diﬂubenzuron in the liver may be mainly modulated * *

* *

* * by the cyp2a and cyp3a genes. The cyp1a and ahr gene expression

was positively correlated with the thyroid hormone levels. The

detoxiﬁcation rate in liver may be regulated by the thyroid hor-

mones. Yukie et al. [48]. have indicated that the ahr gene regulates

Fig. 5. The relative gene expression of HPT axis related genes in the liver and brain

the T4 level via adjusting its target gene-udp in mice. The alter-

after 35 days of diﬂubenzuron or ﬂufenoxuron exposure. The data was expressed as

ations of ahr and udp mRNA expression in lizard liver highlight the

mean ± S.D. (n = 3). * p < 0.05 indicates signiﬁcant difference compared to the control

importance of ahr and udp on the metabolism of T4.

(one-way ANOVA, p < 0.05).

Abbreviations: housekeeping gene ( -actin); thyroid hormone receptors (tr˛, trˇ);

deiodinases (dio1, dio2); uridinediphosphate glucuronosyl transferase (udp); sulfo-

transferase (sult) transthyretin (ttr), cytochrome 450 (cyp1a, cyp2a and cyp3a) and 3.3.4. The relationship between thyroid hormone levels and

aryl hydrocarbon receptor (ahr). tested genes

In order to understand the relationship between thyroid hor-

mone levels and the tested genes, the data mentioned above were

ﬂufenoxuron exposure. The two genes (dio1 and ttr) may not be examined by PCA (Fig. 6). All parameters were distinguished from

controlled by the thyroid hormone levels but may still be affected the score plot corresponding to the ﬁrst and second principal com-

by the concentration of BPUs in the liver. The expression of udp ponents (71.9% and 28.1%, respectively). According to the results,

and sult gene was signiﬁcantly increased in diﬂubenzuron expo- the T3 and T4 levels were positively correlated with the mRNA

sure group while signiﬁcantly decreased in ﬂufenoxuron exposure levels of cyp1a and ahr but were negatively related with the gene

group (p < 0.05). Generally, the udp and sult genes regulate the elim- expression of sult, dio2, tr˛ and udp. The correlation analysis fur-

ination of thyroid hormones [46]. The changes of their expressions ther conﬁrmed the observed relationships (Table S6). These results

in lizard liver exposed to BPUs may explain the alterations of thy- further supported our discussion above that the thyroid hormone

roid hormones. levels were mainly regulated by the sult, dio2, tr˛ and udp genes and

Fig. 6. Scatter plots of individual scores on the principle component analysis (PCA) of the transcription of genes involved in the HPT axis in lizard liver after diﬂubenzuron

or ﬂufenoxuron exposure for 35 days.

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