Sage Journals: Discover world-class research

Abstract

JS-118 is an extensively used insecticide in China. The present study investigated the genotoxic effect of JS-118 on whole blood at 24, 48, 72 and 96 h by using alkaline comet assay. Male Kunming mice were given 6.25, 12.5, 25, 50 and 100 mg/kg BW of JS-118 intraperitoneally. A statistically significant increase in all comet parameters indicating DNA damage was observed at 24 h post-treatment (p < 0.05). A clear concentration-dependent increase of DNA damage was revealed as evident by the OTM (arbitrary units), tail length (µm) and tail DNA (%). From 48 h post-treatment, a gradual decrease in mean comet parameters was noted. By 96 h of post-treatment, the mean comet tail length reached control levels indicating repair of damaged DNA. This study on mice showed different DNA damage depending on the concentration of JS-118 and the period of treatment. The present study provided further information of the potential risk of the genetic damage caused by JS-118.

Keywords

JS-118 SCGE genotoxicity male Kunming mice leucocytes

Introduction

Pesticides are the main means of protecting seeds and crops from insects and diseases, but they have been considered potential chemical mutagens: experimental data have revealed that various agrochemical ingredients possess properties inducing mutations, chromosomal alterations or DNA damages,¹ which may lead to adverse effects on animals and human populations. Different studies have shown that many pesticides have the capacity to cause DNA damage.^{1
–3}

The single-cell gel electrophoresis(SCGE) assay, which is also named the comet assay, based on the fact that broken DNA migrates more easily in an electric field than intact molecules, is a rapid, simple and sensitive technique for visualizing and measuring DNA damage leading to strand breakage in individual cells. It is becoming an important tool for evaluating the genotoxic potential of compounds in vitro and is also in vivo.^{4

–9} Under alkaline condition (pH > 13), the assay is able to evaluate DNA damage, i.e. single and double-stranded breaks or other lesions, such as alkali-labile sites, DNA-protein and DNA-DNA cross-links and incomplete excision repair events.¹⁰ The genotoxic effect of pesticides can be evaluated by using a relatively small sample of cells.¹¹

The insecticide JS-118 [CAS NO. 467427-81-1, N-(2,3-dihydro-2,7-dimethyl-6-benzo-furancarboxyl)-N′-(1,1-dimethylethyl)-N′-(3,5-dimethyl-benzoyl)-hydrazine], a recently introduced diacylhydrazines-type insect growth regulator to control Lepidoptera, developed by Jiangsu Pesticide Research Institute, China, in 1990s, has been granted patents in China (ZL01181611.9). Its chemical structure is shown in Figure 1 . JS-118 belongs to high-efficiency, low-toxin and pollution-free agricultural chemicals. It has almost no toxicity to mammal, bird and bee, i.e. the acute oral LD₅₀ >5000 mg/kg to rats, LD₅₀ (7d) >5000 mg/kg avoirdupois to partridge, LC₅₀ (48 h) >500 mg/L to bee. As JS-118 is now extensively used in China, studies are needed to evaluate the potential genotoxic risk of JS-118 to the health of animals and human.

Figure 1.

The chemical structure of JS-118.

In our study, we examined the genotoxic effect of JS-118 in leucocytes of mice by using comet assay and provided further knowledge of the potential risk of the genetic damage caused by this insecticide.

Materials and methods

Chemical

JS-118 standard (purity 97%) was obtained from Jiangsu Pesticide Research Institute, China, and used without further purification.

Normal melting agarose (NMA), low-melting agarose (LMA), dimethyl sulfoxide (DMSO), Triton X-100, N-Lauroyl sarcosine sodium, ethidium bromide (EtBr), tris, ethylmethane sulfonate and trypan blue were purchased from Amresco Inc.; ethylenediaminetetracetic acid disodium salt (Na₂EDTA) and ethanol were purchased from Chemical factory, Beijing; camellia oil was purchased from Longyou County Tianyushan tea oil development corporation Ltd; cyclophosphamide (CP) was obtained from The Academy of Military Medical Sciences. All other chemicals were obtained locally and were of analytical reagent grade.

Animals and treatment

Male Kunming mice (~4-week-old, 25.0 ± 2.0 g) were obtained from the Peking University Health Science Center (Beijing, China). They were housed in group of 10 in polypropylene cages in an air-conditioned room. The animal room was maintained 25 ± 2°C and 50%−70% relative humidity with a 12 h light-dark cycle. They were bred on a laboratory diet, with drinking distilled water ad libitum.

JS-118 was dissolved in camellia oil and was intraperitoneally injected to mice. The concentration selection was based on LD₅₀ value which was 5000 mg/kg for JS-118. The mice used in the comet assay were divided into seven groups (three mice in each group) as follows:

Group 1: negative control which received damallia oil alone (20 mL/kg BW).

Group 2: positive control which received cyclphosphamide (100 mg/kg BW).

Group 3−7 were treated by injected intraperitoneally with the JS-118 concentration of 6.25, 12.5, 25, 50 and 100 mg/kg BW, respectively. Details were as follows:

Group 3: 6.25 mg/kg BW;

Group 4: 12.5 mg/kg BW;

Group 5: 25 mg/kg BW;

Group 6: 50 mg/kg BW;

Group 7: 100 mg/kg BW.

No mortality was observed throughout the experiments.

Blood sample preparation and viability assay

Forty to sixty microlitres of blood were collected from the orbital vessels of each mouse at 24, 48, 72 and 96 h. Cell viability for the JS-118 was determined by the trypan blue.

Comet assay

The alkaline single-cell gel electrophoresis was performed as a three-layer procedure¹² with slight modifications.¹³ The frosted ends of conventional slides were used and were covered with 100 µL of 0.7% NMA. After application of a cover slip, the slides were allowed to gel at 4°C for 10 min. Meanwhile, 20 µL of whole blood was added to 100 µL of 0.65% LMA. After carefully removing the cover slips, the second layer of 100 µL of sample mixture was spread onto the precoated slides and allowed to solidify at 4°C for 10 min. The cover slips were removed and the third layer of 0.6% of 100 µL of LMA was pipetted out on the slides and allowed to gel at 4°C for 10 min. Finally, the cover slips were removed and the slides immersed in freshly prepared, cold, chilled lysing solution (2.5 M NaCl, 100 mM Na₂EDTA, 10 mM Tris, 1% sodium N-lauroyl sarcosinate, pH 10, with 1% Triton X-100 and 10% DMSO added just before use). The slides remained in the lysing solution overnight at 4°C. Slides were then placed in a horizontal gel electrophoresis tank with fresh and chilled alkaline buffer (1 mM Na₂EDTA and 300 mM NaOH, pH 13). Then, the slides were immersed into the buffer solution for 30 min to allow DNA unwinding and expressing of alkali-labile sites as DNA strand breaks. Electrophoresis was conducted at 25V (0.66V/cm) adjusted to 300 mA by raising or lowering the buffer level in the tank. After electrophoresis, the slides were drained and placed horizontally in a tray and washed slowly with three changes of 5 min each of neutralization buffer (0.4M Tris-HCl, pH 7.5). DNA was precipitated and slides were dehydrated in absolute methanol for 10 min and dried at room temperature. The whole procedure was carried out in dim-light to avoid additional DNA damage.

Preparation of stained slides

Slides were stained with 50 µL EtBr (20 mg/L) and examined by using an Olympus BX51 fluorescence microscope equipped with a wide band excitation filter of 515−560 nm and a barrier filter of 590 nm. Twenty-five cells from each of three replicated slides were scored at ×400 magnification.

Statistical analysis

Descriptive statistical analysis was carried out to study the significance of various concentrations of JS-118 compared to controls. The data were analyzed by using one-way analysis of variance (ANOVA). Lee et al. have reported various parameters for describing the comet assay.⁸ In our study, Olive Tail Moment, arbitrary units (OTM), tail length (µm) and tail DNA (% of DNA in tail) were selected to estimate DNA damage. In all cases p < 0.05 was considered significant compared with the controls, respectively.

Results

In all experiments, the cell viability by the trypan blue exclusion technique was relatively stable and ranged from 92% to 95%. It was the first time that the potential genotoxicity caused by JS-118 was evaluated by using the comet assay. Data of three parameters, OTM (Table 1), tail-DNA (Table 2) and tail length (Table 3) were obtained.

Table 1.

Mean comet Olive tail moment (arbitrary units) of leucocytes exposed to different concentrations of JS-118 at various time intervals^a

Groups	24 hours	48 hours	72 hours	96 hours
Camellia oil^b	2.21 ± 0.11	2.16 ± 0.10	2.07 ± 0.14	2.19 ± 0.14
6.25 mg/kg BW	2.74 ± 0.20^c	2.75 ± 0.12^c	2.83 ± 0.19^c	2.81 ± 0.11^c
12.5 mg/kg BW	4.20 ± 0.20^d	3.55 ± 0.21^d	3.13 ± 0.13^d	2.65 ± 0.16^c
25 mg/kg BW	6.20 ± 0.08^d	5.81 ± 0.15^d	4.05 ± 0.13^d	3.27 ± 0.16^d
50 mg/kg BW	7.07 ± 0.22^d	6.37 ± 0.17^d	4.41 ± 0.22^d	3.09 ± 0.15^d
100 mg/kg BW	9.20 ± 0.22^d	7.40 ± 0.45^d	5.31 ± 0.29^d	3.66 ± 0.13^d
CP^e	10.55 ± 0.94

^a Values represent mean ± S.E. of three animals in each group

^b Camellia oil: negative control (20 mg/kg BW).

^c p < 0.05, when compared with control.

^d p < 0.01, when compared with control.

^e CP: positive control (100 mg/kg BW).

Table 2.

Mean comet tail DNA (%) leucocytes exposed to different concentrations of JS-118 at various time intervals^a

Groups	24 hours	48 hours	72 hours	96 hours
Camellia oil^b	6.90 ± 0.82	6.74 ± 0.24	4.39 ± 0.22	7.01 ± 0.42
6.25 mg/kg BW	6.47 ± 0.25^c	7.54 ± 0.29^c	7.18 ± 0.91^c	7.32 ± 0.31^c
12.5 mg/kg BW	11.55 ± 0.32^d	9.19 ± 0.45^d	9.23 ± 0.28^d	8.95 ± 0.30^c
25 mg/kg BW	17.41 ± 0.27^d	14.57 ± 0.52^d	10.84 ± 0.41^d	8.11 ± 0.49^d
50 mg/kg BW	19.07 ± 0.22^d	11.91 ± 1.13^d	11.48 ± 0.63^d	8.12 ± 0.39^d
100 mg/kg BW	22.50 ± 0.71^d	16.47 ± 0.93^d	11.67 ± 0.73^d	9.08 ± 0.40^d
CP^e	25.70 ± 1.21

^a Values represent mean ± S.E. of three animals in each group.

^b Camellia oil: negative control (20 mg/kg BW).

^c p < 0.05, when compared with control.

^d p < 0.01, when compared with control.

^e CP: positive control (100 mg/kg BW).

Table 3.

Mean comet tail length (µm) of leucocytes exposed to different concentrations of JS-118 at various time intervals^a

Groups	24 Hours	48 Hours	72 Hours	96 Hours
Camellia oil^b	18.33 ± 1.23	18.60 ± 0.78	17.67 ± 1.03	18.13 ± 1.46
6.25 mg/kg BW	19.20 ± 0.41^c	21.40 ± 0.77^c	20.80 ± 0.85^c	23.80 ± 1.18^c
12.5 mg/kg BW	33.13 ± 0.62^d	29.27 ± 0.86^d	22.27 ± 1.12^c	18.33 ± 0.84^c
25 mg/kg BW	35.07 ± 1.23^d	29.84 ± 1.14^d	33.33 ± 1.55^d	22.00 ± 0.79^c
50 mg/kg BW	37.33 ± 1.06^d	31.07 ± 0.88^d	30.87 ± 0.84^d	25.47 ± 1.31^d
100 mg/kg BW	49.08 ± 1.93^d	40.60 ± 1.52^d	33.07 ± 1.28^d	26.67 ± 0.87^d
CP^e	67.62 ± 2.75

^a Values represent mean ± SE of three animals in each group.

^b Camellia oil: negative control (20 mg/kg BW).

^c p < 0.05, when compared with control.

^d p < 0.01, when compared with control.

^e CP: positive control (100 mg/kg BW).

From Table 1, the mice exposed to various concentrations of JS-118, at 24 and 48 h post-treatment, exhibited significantly different OTM (p < 0.01) when compared to respective negative control group. CP as the positive control induced a significantly higher DNA damage than the negative control (p < 0.01).

The study also showed statistically significant (p < 0.05) concentration-dependent increase in DNA damage in leucocytes of mice exposed to JS-118. At the same duration post-treatment, the OTM values increased for studied cells from 6.25 mg/kg BW to the maximum concentration group (100 mg/kg BW). The OTM values increased as exposed concentrations of JS-118 (Table 1, Figure 1). The highest DNA damage (9.20 ± 0.22) was recorded at 24 h at the maximum concentration.

From 48 h post-treatment, a gradual decrease in the mean OTM values was observed compared to the 24 h post-treatment with all concentrations of JS-118. A further decrease in the mean OTM values was observed at 72 h post-treatment. By 96 h of post-treatment, the mean OTM values had reached control levels for all the concentrations of JS-118.

A similar result was observed for tail length and tail DNA (Tables 2 and 3).

Discussion

The alkaline SCGE assay (the comet assay) could detect low levels of DNA damage in individual cells and required relatively shorter period to complete an experiment, so the comet assay was popularly used in the assessment of genotoxicity.¹⁰ Numerous studies have reported that the comet assay was successfully employed for estimating in vivo DNA damage by using mice as a model for environmental biomonitoring.^{11,14
–16} In our studies, the comet assay was employed to evaluate the DNA damage induced by JS-118 in leucocytes of mice, the results have shown that the assay was highly sensitive for the detection of DNA damage caused by JS-118 in leucocytes.

Based on the results, a significant DNA damage was observed with JS-118 in the form of comet induction at 24 h and 48 h post-treatment in comparison to controls. A statistically significant (p < 0.05) concentration-dependent increase was also shown in DNA damage in leucocytes of mice exposed to JS-118 at the same post-treatment time. A time-dependent decrease in DNA damage after treatment with JS-118 was observed, and it showed DNA repair of the leucocytes of mice. Both concentration-dependence and time-dependence were reported by many researchers. Rahman et al. reported the DNA damaging effect of two organophosphorus pesticides in leucocytes of mice in vivo. A clear concentration-dependence was observed at 24 h post-treatment as evident from mean tail length, and a gradual decrease was noted from 48 h post-treatment.¹⁴ Both metabolites malaoxon and isomalathion of malathion were reported to induce DNA breakage in a concentration-dependent manner in human lymphocytes in vitro.¹⁹ Researchers reported that cypermethrin induced systemic genotoxicity in mammal as it caused DNA damage in vital organs like brain, bone marrow, spleen and so forth using comet assay, and a statistically significant (p < 0.05) concentration-dependent increase was observed as evident from comet assay parameters.¹⁶ Gabbianelli reported the cypermethrin and permethrin showed different lymphocyte DNA damage depending on the type of pesticide, the concentration and the period of treatment.¹¹ Their results of concentration-dependence and time-dependence were consistent with ours.

Our results demonstrated the potential of JS-118 to induce DNA damage in leucocytes in vivo. The DNA damage could originate from DNA single-strand breaks, DNA double-strand breaks, DNA adducts formation, DNA-DNA and DNA-protein cross-links,¹⁷ resulting from the interaction of pesticides or their metabolites with DNA.¹⁸ The exact mechanism of JS-118-induced DNA damage was not known and required further studies. According to the findings reported by Sarabia et al., the hydrogen bonds of the DNA double chain were the most sensitive structures to chromatin-damaging agents.¹⁵ One of the mechanisms that may be involved in the generation of these lesions is oxidative damage.^20–21 It has been demonstrated that ROS may cause DNA damage, which could lead to single-strand breaks and mutation.²²

The comet assay is not the only way to measure DNA damage, but it is one of the most sensitive and accurate methods. Since the literature has inadequate and limited human data, the present investigation could be useful for increasing information on the potential toxicity of JS-118.

Footnotes

This work was supported by the Major State Basic Research Development Program of China (No. 2006CB101907 and No. 2003CB114400), National Natural Science Foundation of China (No. 20777078) and the 863 high-tech key project of China (2006AA10A203).

References

Bolognesi

. Genotoxicity of pesticides: a review of human biomonitoring studies. Mutat Res 2003; 543: 251−272.

Kornuta

Bagley

Nedopitanskaya

. Genotoxic effects of pesticides. J Environ Pathol Toxicol Oncol 1996; 15: 75−78.

Shuka

Taneja

. Mutagenic potential of cypermethrin in mouse dominant lethal assay. J Environ Pathol Toxicol Oncol 2002; 21: 259−265.

Collins

Dustinska

Franklin

. Comet assay in human biomonitoring studies: reliability, validation, and applications. Environ Mol Mutagen 1997; 30: 139−146.

Collins

Dustinska

Horska

. Detection of alkylation damage in human lymphocyte DNA with the comet assay. Acta Biochim Pol 2001; 48: 611−614.

Moretti

Marcarelli

Villarini

. In vitro testing for genotoxicity of the herbicide terbutryn: cytogenetic and primary DNA damage. Toxicol In Vitro 2002: 16: 81–88.

Moretti

Villarini

Scasselati-Sfrozolini

. Extent of DNA damage in density-separated trout erythrocytes assessed by “Comet” assay. Mutat Res 1998; 397: 353−360.

Lee

Steinert

Najkayama

. Use of the single cell gel electrophoresis/comet assay for detecting DNA damage in aquatic (marine and freshwater) animals. Mutat Res 2003; 544: 43−64.

Tice

Agurell

Anderson

. Single-cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 2000; 35: 206−221.

10.

Gecid

Ewen

SWB

Collins

. Single cell gel electrophoresis applied to analysis of UV-C damage and its repair in human cells. Int J Radiat Biol 1992; 62: 310−320.

11.

Gabbianelli

Nasuti

Falcioni

. Lymphocyte DNA damage in rats exposed to pyrethroids: effect of supplementation with Vitamins E and C. Toxicology 2004; 203: 17−26.

12.

Singh

McCoy

Tice

. A single technique for the quantation of low level of DNA damage in individual cells. Radiat Res 1988; 177: 79−92.

13.

Klaude

Eriksson

Nygren

. The comet assay: mechanisms and technical considerations. Mutat Res 1996; 363: 89−96.

14.

Rahman

Mahboob

Danadevi

. Assessment of genotoxic effects of chloropyriphos and acephate by the comet assay in mice leucocytes. Mutat Res 2002; 516: 139−147.

15.

Sarabia

Maurer

Bustos-Obregon

. Melatonin prevents damage elicited by the organophosphorous pesticide diazinon on mouse sperm DNA. Ecotoxicol Environ Saf 2009; 72: 663−338.

16.

Patel

Pandey

Bajpayee

. Cypermethrin-induced DNA damage in organs and tissues of the mouse: evidence from the comet assay. Mutat Res 2006; 607: 176−183.

17.

Blasiak

Fessard

Trzeciak

. In vitro studies on the genotoxicity of the organophosphorus insecticide malathion and its two analogues. Mutat Res 1999; 445: 275−283.

18.

Mitchelmore

Chipman

. DNA strand breakage in aquatic organisms and the potential value of the comet assay in environmental monitoring. Mutat Res 1998; 399: 135−147.

19.

Fairbairn

Olive

O’neill

. The comet assay: a comprehensive review. Mutat Res 1995; 339: 37-59.

20.

Delaney

Green

Lowe

. Use of the comet assay to investigate possible interactions of nitric oxide and reactive oxygen species in the induction of DNA damage and inhibition of function in an insulin-secreting cell line. Mutat Res 1997; 375: 137−146.

21.

Rojas

Lopez

Valverde

. Single cell gel electrophoresis assay: methodology and applications. J Chrom B 1999; 722: 225−254.

22.

Zegura

Lah

Filipic

. The role of reactive oxygen species in microcystin-LR-induced DNA damage. Toxicology 2004; 200: 59−68.

Leucocytes DNA damage in mice exposed to JS-118 by the comet assay

Abstract

Keywords

Introduction

Materials and methods

Chemical

Animals and treatment

Blood sample preparation and viability assay

Comet assay

Preparation of stained slides

Statistical analysis

Results

Discussion

Footnotes

References