Mechanisms of the Toxicity of Chiral Pesticides Dinotefuran

Enantiomers on Esiena fetida Earthworm

Yaofang Zhang

1 a

, Youpu Cheng

1 b

, Yu Cheng

1,2, c

, Lijuan Lv

1 d

, Yanling Xu

1 e

and Chengling Gao

1 f

Tianjin Agricultural University, No. 22 Jinjing Road, Tianjin, China

Tianjin Institute of Agricultural Quality Standard and Testing Technology, Jinjing Road, Tianjin, China

Keywords:

Dinotefuran, Enantiomers, Eisenia foetida, Acute Toxicity, DNA Damage.

Abstract: As a promising insecticide, dinotefuran has been commercialized and widely used around the world. In this

study, the acute toxicity of Rac-dinotefuran and its two enantiomers on earthworm were estimated by

artificial soil method according to the OECD criteria at the individual-tissue-cell-molecule level. The

14d-LC50 values were 2.372 mg/kg for Rac-(±)-dinotefuran, 1.158 mg/kg for S-(+)-Dinotefuran and 6.002

mg/kg for R-(-)-Dinotefuran respectively. E. foetida was exposed to different concentrations of

Rac-dinotefuran and its two enantiomers and the enzyme activities, DNA damage and gene expression were

measured on 3, 7, 14, 21, and 28 days of post treatments, respectively. The results showed that

Rac-dinotefuran and its two enantiomers caused obvious modulations on DNA damage, enzyme activities

and gene expression. Additionally, the toxicity of Rac-dinotefuran and its two enantiomers behaved in time-

dose-dependent manner.

1 INTRODUCTION

Neonicotinoid pesticides (NNs) are nitroguanidine

systemic insecticides that are commonly used to

protect seedling from leaf feeding by early season

pests (

Jeschke, 2008)

. Because of the high

selectivity, high efficiency and low toxicity to

mammals, neonicotinoid insecticides are now the

most widespread used pesticides around the world.

(Morrissey, 2015, Sparks, 2015). Neonicotinoid

insecticides including imidacloprid, clothianidin,

thiamethoxam, acetamiprid, and dinotefuran are

among the most effective insecticide recently

introduced to control pest with novel modes of

action (Lina, 2012). As the third generation

neonicotinoid insecticide, dinotefuran is deemed to

be a promising insecticide with emproved chemical

and biological properties, such as wide spectra of

targets, high insecticidal efficacies, and environment

https://orcid.org/0000-0001-7677-9207

https://orcid.org/0000-0002-0171-0165

https://orcid.org/0000-0002-8607-9981

https://orcid.org/0000-0002-3217-7535

https://orcid.org/0000-0001-6190-9566

https://orcid.org/0000-0001-5057-780X

safety (

Hem, 2012)

. Given the differences of chiral

pesticides enantiomers in the bio-activities,

toxicities, and environmental behavior, many

researchers have been vigilant to the security of

chiral pesticides (

Qi, 2015)

. However, dinotefuran

inevitably permeates into the natural environment

and sap the quality of soil. Although, the toxicity of

some pesticides can be partially and slowly

mitigated by some abiotic factors, such as

degradation, migration and transformation, the

contamination remains a long-lasting problem since

the pesticides including dinotefuran are notoriously

clumsy to be completely purged (

Morrissey, 2015)

Therefore, the acute toxicity of Rac-dinotefuran

and its two enantiomers on earthworems were

studied. Meanwhile, we compared the changes of

enzyme activities, DNA damage levels and the

modifications of gene expression of E.foetida with

and without the pesticides. The results provided

scientific basis for an evaluation of the

environmental safety on soil ecosystems.

Zhang, Y., Cheng, Y., Cheng, Y., Lv, L., Xu, Y. and Gao, C.

Mechanisms of the Toxicity of Chiral Pesticides Dinotefuran Enantiomers on Esiena fetida Earthworm.

DOI: 10.5220/0011232400003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 547-552

ISBN: 978-989-758-595-1

547

2 ATERIALS AND METHODS

2.1 Materials and Reagents

Earthworms (E. fetida) were purchased from an

earthworm cultivating farm (Tianjin, China).

Dinotefuran (98.0%) were provided by the ministry

of agriculture pesticide identification.

R-(-)-dinotefuran and S-(+)-dinotefuran were

prepared in our laboratory. The experimental soil

was artificial soil and prepared according to the

method described in the OECD guideline (

OECD,

2014)

2.2 Acute Toxicity Testing using

Earthworm E.fetida

The acute toxicity was conducted according to the

OECD standard method (

OECD, 2014)

. For the

media lethal concentrations (LC

) of

Rac-(±)-Dinotefuran and its enantiomers caculation,

seven test concentrations were used,

Rac-(±)-Dinotefuran (0.829, 1.077, 1.401, 1.821,

2.367, 3.077 and 4.000 mg/kg), S-(+)-Dinotefuran

(0.491, 0.638, 0.829, 1.077, 1.401 1.821 and 2.367

mg/kg) and R-(-)-Dinotefuran (2.072, 2.693, 3.501,

4.552, 5.917, 7.692 and 10.000 mg/kg). Each

concentration contained ten healthy earthworm and

with three replicates.

2.3 Experimental Design

Based on the acute toxicity experiment results, three

different concentrations (0.1, 0.5 and 1.0 mg/kg) and

three replicates for each concentration with artificial

soil were used in the present study. The control

groups were prepared similarly but without

insecticide. There are 0.5 g of dry cow dung was

added onto the artificial soil surface weekly from

days 1 to 28 and the same dose of artificial soils

were replaced on the days of 14. All the treatments

were cultured at 20±1 ℃ in 80 %-85 % relative

humidity for 16 h in light and 8 h in the dark, and

five exposure periods (3, 7, 14, 21 and 28 d) were

tested. Each earthworm was washed with distilled

water, gently dried with absorbent paper, and stored

at -80 ℃ before analysis.

2.4 Determination of Enzyme Activities

One Gut-cleaned earthworm was randomly selected

and homogenized in 100 mM phosphate buffer (pH

7.2). The supernatant was collected after

centrifuging at 10000 rpm for 30 min (at 4 ℃). SOD

activity was tested according with the method of

Song et al. (

Song, 2009)

. CAT activity was

determined by measuring the consumption of H

(

Xu, 1997)

. POD activity was determined using the

method of Kochba et al. (

Kochba, 1977)

2.5 Comet Assay

Earthworm coelomocytes were performed as

Eyambe et al. Described (

Eyambe, 1991)

. The

comet assay was described by the method of Mahsa

et al. (

Mahsa, 2014)

, which was used to determine

the degree of DNA damage. After electrophoresis,

each slide was neutralized with neutralizing buffer

every 5 min for 3 times, dehydrated with 95 %

ethanol and stained with SYBR green. At last, the

slides were observed under fluorescence microscope

(Olympus, BX51, Japan).

2.6 Real-time PCR Analysis

Total RNA was obtained using the Total RNA

extraction kit and reverse-transcribed to first-strand

cDNA was performed using the PrimeScript™ RT

reagent Kitr. The synthesized cDNA was stored at

-80 ℃ prior to use for real-time PCR. TransStart

Top Green qPCR SuperMix was used in real-time

PCR experiments, which was performed on a

real-time PCR system. The expression of five target

genes (SOD, MT, HSP70, TCTP) were compared to

the expression of the housekeeping gene (β-actin)

and presented as relative gene expression compared

to the control. The relative gene expression level

was calculated using the 2

△△

method (

Lukkari,

2004)

2.7 Statistical Analysis

Each treatment was analyzed with three replicates.

The data were analyzed with SPSS 17.0 statistical

software and the results were presented as

means

± standard deviation (SD). The comet images

were analyzed using CASP software. Olive tail

moment (OTM) value was used to determine the

degree of DAN damage.

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

548

3 RESULTS AND DISCUSSION

3.1 Acute Toxicity

After 14 days exposure, no mortality was observed

in control group. Dinotefuran and its enantiomers

showed different degree of toxicity on earthworms.

The values of 14d-LC

were Rac- (±)-Dinotefuran

2.372 mg/kg, S-(+)-Dinotefuran 1.158 mg/kg and

R-(-)-Dinotefuran 6.002 mg/kg. The results showed

that Rac-dinotefuran and its enantiomers are

moderately toxic to Eisenia foetida.

The data

showed that the mortality is both

concentration-dependent and application

time-dependent for all insecticides test. The acute

toxicity is

S-(+)-Dinotefuran>Rac-(±)-Dinotefuran>R-(-)-Dino

tefuran.

3.2 Enzyme Activities Assay

As antioxidant enzymes, SOD, CAT and POD, play

an important role in scavenging excess reactive

oxygen species (ROS) and promoting the growth of

healthy cells (

Ye, 2016)

. SOD plays a key role in

decomposing O

·-

to H

and O

, which is thought

to be the first line to prevent the harm of ROS

(

Liu,

2016)

. CAT can decompose H

to H

O and O

which is considered to play an important role in

detoxification of free radicals derived from oxygen.

As one type of redox enzyme, POD can eliminate

and other organic hydroperoxides to protect

the body from the damage posed by ROS (

Niu,

2013)

. The possible biochemical effects of

dinotefuran and its two enantiomers on the activities

of SOD, CAT and POD in E.foetida were measured

on the 3

, 7

, 14

, 21

, and 28

day respectively.

As is shown in Figure 1(A, B and C), After 7

days exposure, the SOD and CAT activities showed

the same trend of variation under high concentration

of S-(+)-Dinotefuran treatment groups which

showed significant pre-inhibiting and post-activating

effects. Their results suggested that a high

concentration of S-(+)-Dinotefuran may induce

excess toxicity which inhibited activities of SOD,

CAT and POD. At the same time, a high

concentration of S-(+)-Dinotefuran can induce

oxidative stress and induce the expression of

antioxidant enzymes to overcome the stress caused

by pollution. Finally, the SOD and CAT exhibited

higher activities, resulting in eliminating the

excessive ROS production. However, the POD

activities in the high concentration of

S-(+)-Dinotefuran treatment groups were inhibited

in the whole exposure time (P<0.001). Maybe the

toxicity is stronger to POD than to other antioxidant

enzymes. After 28 days of exposure, the SOD, CAT

and POD activities in 1 mg/kg and 0.5 mg/kg

S-(+)-Dinotefuran treatment groups changed more

significantly than the other groups and exhibited

dose-dependent elevate effect. The results showed

that the S-(+)-Dinotefuran is more toxic than

Rac-(±)-Dinotefuran and R-(-)-Dinotefuran.

Figure 1: The effect of dinotefuran and its enantiomers on

SOD (A), CAT (B) and POD (C) activity of Eisenia

foetida. Data are described as mean±SD (n=3). Statistical

significance compared with controls:

P<0.05,

P<0.01,

***

P<0.001.

3.3 DNA Damage Induced by

Dinotefuran and Its Enantiomers

The genotoxicity of dinotefuran and its enantiomers

to Eisenia foetida were evaluated by comet assay. As

is shown in Figure 2, after 3 d treatments, there were

no significant difference in OTM values, indicating

that after transit exposure did not cause DNA

damage. On the 7

day, the OTM values of the

S-(+)-Dinotefuran and 1 mg/kg Rac-(±)-Dinotefuran

treatment groups were significantly higher than

other treatment groups and control group (P<0.001,

P<0.05). After 14 d treatments, a significant

increase in the OTM values was observed for 1

mg/kg treatment groups and the OTM values

increased with the increase of dosage and exposure

time. The OTM values are

Mechanisms of the Toxicity of Chiral Pesticides Dinotefuran Enantiomers on Esiena fetida Earthworm

549

S-(+)-Dinotefuran>Rac-(±)-Dinotefuran>R-(-)-Dino

tefuran. However, the OTM did not change

significantly with the treatments of the low

concentration of dinotefuran and its enantiomers

compare the 21

day. The study indicated that the

DNA was damaged significantly by high

concentrations (1 mg/kg and 0.5 mg/kg) of exposure

to dinotefuran and its enantiomers and the toxicity

was S-(+)-Dinotefuran > Rac-(±)-Dinotefuran >

R-(-)-Dinotefuran.

Figure 2: The effect of dinotefuran and its enantiomers on DNA damage degree in earthworms. Data are described as

mean±SD (n=3). Statistical significance compared with controls:

P<0.05,

P<0.01,

***

P<0.001.

3.4 Real-time PCR Analysis of

Dinotefuran and Its Enantiomers

As is shown in Figure 3A, the relative expression of

SOD gene was significantly up-regulated on the 7

and 14

day (P<0.001). After 28 days treatments,

the expression levels of the SOD gene was more

significantly decreased by the treatment of the high

concentration of Rac-(±)-Dinotefuran and

S-(+)-Dinotefuran groups (1.0 mg/kg and 0.5 mg/kg)

than other treatment groups. The significant changes

of the SOD gene showed that dinotefuran and its

enantiomers caused toxic effects on earthworms and

had a positive correlation with the exposure time

and the dose.

The expression of MT gene is modulated by

environmental stress, so it has been used to assess

the eco-toxicity of contaminants (

Fisker, 2016)

. As

is shown in Figure 3B, after exposure for 7 days, a

significant up-regulation trend was observed and the

expression levels increased with an increase in the

dose and exposure time (P<0.001). However, on the

day, the up-regulation trend in high

concentration of S-(+)-Dinotefuran (1 mg/kg and 0.5

mg/kg) and 1mg/kg Rac-(±)-Dinotefuran, treatment

groups disappeared. Until being exposured for 28

days, the expression levels of the MT gene

down-regulated significantly on 1 mg/kg and 0.5

mg/kg treatments. Especially on the 28

day,

S-(+)-Dinotefuran (1 mg/kg and 0.1 mg/kg),

R-(-)-Dinotefuran (1 mg/kg) and

Rac-(±)-Dinotefuran (1 mg/kg and 0.5 mg/kg)

treatment groups were markedly lower than other

treatment groups.

Heat shock protein 70 (HSP70) play an essential

role in protecting cells from damages induced by

environment pressure

(

Wang, 2015)

. As is shown in

Figure 3C, after exposure for 3 days, a significant

increase of expression of HSP70 gene was observed

in the dinotefuran and its enantiomers treatment

groups (P<0.001). After 14 days treatments, the

relative expression levels of the HSP70 gene began

to wane. On the 28

day, the relative expression

levels of the HSP70 gene in all dinotefuran and its

enantiomers treatment groups were starkly lower

than the control group (P<0.001). The significant

changes in HSP70 expression indicated that

dinotefuran and its enantiomers caused stress in

earthworms.

TCTP plays an important role in preventing cell

apoptosis and causing tumor reversion (

Wang,

2015)

. As is shown in Figure 3D, a significant

up-regulation of the TCTP gene was observed in the

presence of S-(+)-Dinotefur, R-(-)-Dinotefuran (1

mg/kg) and Rac-(±)-Dinotefuran (1 mg/kg and 0.5

mg/kg) on the days of 7 and 14 (P<0.001). On the

days of 21 and 28, the relative expression levels of

the TCTP gene were markedly lower than the

control group except 0.1 mg/kg Rac-(±)-Dinotefuran

treatments (P<0.001). The up-regulation at first and

the following down-expression indicated that

dinotefuran and its enantiomers may influence cell

growth and lead to cell apoptosis at the final stage of

the exposure.

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

550

Figure 3: The effect of dinotefuran and its

enantiomers(S-(+)-Dinotefuran and R-(-)-Dinotefuran) on

the relative expression quantity of SOD (A), MT (B),

HSP70 (C) and TCTP (D). Data are described as

mean±SD (n=3). Statistical significance compared with

controls:

P<0.05,

P<0.01,

***

P<0.001.

4 CONCLUSIONS

In this study, the biochemical and genetic toxicity of

dinotefuran and its enantiomers on Eisenia foetida

were evaluated from the individual, tissue, cell and

molecule levels. The results showed that,

dinotefuran and its enantiomers are moderately toxic

to Eisenia foetida and have negative impacts on the

earthworm at different levels. Both the exposure

dose and time had obvious impacts on the toxicity.

Moreover, increasing dose and time of dinotefuran

and its enantiomers could induce redundant

production of ROS, resulting in significant changes

in antioxidant enzyme activities, DNA damage and

the relative expression of functional genes.

This study explored the toxicity mechanisms

underlying the toxicity of dinotefuran and its

enantiomers on earthworms, and provided scientific

basis references for devising and developing new

environment-friendly pesticides.

ACKNOWLEDGEMENTS

The authors acknowledge the financial support by

the National Natural Science of Foundation of China

(31572034) and (M2042003).

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