Original Article

Acetamiprid-induced Cyto- and Genotoxicity in the AR42J Pancreatic Cell Line


  • Mehtap KARA
  • Ezgi ÖZTAŞ
  • Gül ÖZHAN

Received Date: 04.03.2019 Accepted Date: 04.04.2019 Turk J Pharm Sci 2020;17(5):474-479 PMID: 33177926


Neonicotinoid insecticides, 30% of insecticides marketed worldwide, have selective toxicity on insects through α4p2 nicotinic acetylcholine receptors. Although it is known that acetamiprid exerts toxicity on several organ systems, its toxic effects on the pancreas and its mechanism of action have not been clarified yet. Therefore, in the present study, the cytotoxic and genotoxic potentials of acetamiprid on the AR42J pancreatic cell line were evaluated.

Materials and Methods:

The (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) (MTT) assay and comet assay were conducted for the cyto- and genotoxicity evaluations, respectively. Reactive oxygen species (ROS) production was assessed by flow cytometry and glutathione (GSH) levels were determined by ELISA for oxidative damage potential, which is thought to be an underlying mechanism of cyto-/genotoxic effects.


To reveal the dose-response relationship the concentration range of 1-6 mM was selected for the assays. Cell viability decreased in a dose-dependent manner and the inhibitory concentration 50 value was calculated as 12.61 mM by the MTT assay. Acetamiprid induced DNA damage in all concentrations tested in a dose-depending manner. The mean tail intensity values were 3.84 and ≤32.96 for the control and exposure groups, respectively. There was no significant difference for ROS production; however, the GSH level was reduced at the highest concentration.


It is thought that the present study will contribute to the literature due to the lack of data on the potential toxic effects of acetamiprid on the pancreas. To better understand acetamiprid toxicity, further studies including a wide range of mechanistic parameters are needed.

Keywords: Acetamiprid, AR42J pancreatic cell line, cytotoxicity, genotoxicity, oxidative damage


Many xenobiotics pose important threats for both human and environmental health. Pesticides, the most common pollutants, are harmful for biological structures via several mechanisms in acute and long-term exposure.1,2,3 Neonicotinoid pesticides as a new class of insecticides that are commonly used instead of organophosphate, and carbamate pesticides have selectively neurotoxic effects on nicotinic acetylcholine receptor. Neonicotinoid pesticides are highly effective insecticides that can disperse in all parts of plants, plant fluids, and fruits that grow on plants. Recent studies revealed that neonicotinoid pesticides can be associated with several adverse effects including decreased sperm production and function, decreased pregnancy rates, increased embryo death, stillbirth, and premature birth in vertebrate and invertebrate species.4,5,6

Acetamiprid [(E)-N-[(6-chloro-3- pyridyl) methyl]-N-cyano-N-methylacetamidine] is one of the most commonly used neonicotinoid class insecticides in many countries for crop pests on agricultural products. In general, acetamiprid has been considered a safe insecticide; however, several different adverse health effects may occur after exposure to acetamiprid as well as other neonicotinoids.3 In previous studies, it has been reported that acetamiprid showed teratogenic, mutagenic, and genotoxic effects via induction of oxidative stress. However, the data about its cyto- and genotoxic potentials are contradictory.7,8,9,10,11,12 As is well known, the worldwide rate of diabetes continues to rise, and the major molecular mechanisms underlying diabetes are increased oxidative stress and altered enzyme functions in pancreatic tissue.13 Indeed, no association between diabetes risk and neonicotinoid pesticides has been reported. Furthermore, there has been no study on the toxic effects of acetamiprid on the pancreas. Therefore, for the first time, we aimed to investigate the cytotoxic and genotoxic effects of acetamiprid on the AR42J pancreatic cell line and evaluated its oxidative damage potential as an underlying molecular mechanism.



Acetamiprid, dimethyl sulfoxide (DMSO), sodium dodecyl sulfate (SDS), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), and 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) were purchased from Sigma Chemical Co. Ltd. (St. Louis, Missouri, USA). The cell culture medium [Roswell Park Memorial Institute (RPMI 1640)] and other chemicals were purchased from Multicell Wisent (Quebec, Canada), while the disposable materials were purchased from Corning (Amsterdam, the Netherlands). All other chemicals at required biological grade were purchased from Merck (New Jersey, USA).

Cell culture and treatments

The AR42J (CRL1492) cell line was obtained from the American Type Culture Collection (ATCC, Virginia, USA), and for all cell applications incubation was carried out according to the manufacturer’s instructions. The cells were grown with 1640 cell culture medium including 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin in a humidified incubator in 5% CO2 at 37 °C. Subculturing was performed every 2-3 days when the cells reached confluence. Prior to exposure cells were seeded into appropriate plasticware and incubated overnight to ensure cell attachment.

Acetamiprid stock solution was prepared by dissolving in 100% DMSO, and stored at -20 °C until the day the assays were conducted. Before the cell treatments acetamiprid was diluted with culture medium to the desired concentrations, and DMSO concentration finalized as 1%. Treatments were performed at a concentration range for 24 h to evaluate dose-dependent effects. All experiments were performed in triplicate on three separate days.

MTT cytotoxicity test

The AR42J cells were placed into 96-well plates (1x104 cells/100 µL cell culture medium/well). The cells were treated with acetamiprid following overnight incubation at the concentration range of 1-50 mM for 24 h. After 5 mg/mL MTT was added to each well, the cells were again kept for 3 h at 37 °C in the dark. Cell culture medium was used as a growth control, 1% DMSO was used as a solvent control, and 10% SDS was used as a positive control. The wells were washed with poly (butylene succinate) (PBS) twice after 3 h. Following the washing step, 100 µL of DMSO was added, followed by incubation for 5 min on an orbital shaker (150 rpm) for evenly dissolved formazan crystals and optical densities were measured using a microplate reader (Biotek, Epoch, Vermont, USA) at 570 nm. The percentage of inhibition of cell viability for each concentration and the inhibitory concentration 50 (IC50) value were determined.

Comet genotoxicity assay

AR42J cells were placed into 6-well plate as 5x105 cells/2 mL cell culture medium/well and kept overnight for incubation. The cells were exposed to acetamiprid at 1, 2, 4, and 6 mM concentrations and 1% DMSO as a negative control for 24 h. After the cells were detached with trypsin-ethylenediaminetetraacetic acid (EDTA) and washed with PBS twice, the viability of cells was evaluated with the trypan blue test, and cell viability was determined as ≥80% for all concentrations. Next 100 µL of single cell suspension was mixed with 100 µL of prewarmed 0.65% low-melting agarose and then layered on microscope slides coated with 1.5% normal-melting point agarose. After lysing for 1 h at 4 °C, the slides were incubated in cold fresh electrophoresis buffer (0.3 M NaOH, 1 mM EDTA, pH 13) at 4 °C for 20 min for DNA unwinding. The electrophoresis conditions were 4 °C for 20 min (20 V/300 mA). The slides were neutralized in 0.4 M tris-HCl buffer (pH 7.5). DNA staining was performed with 20 mg/mL ethidium bromide dye and evaluated under a fluorescent microscope (Olympus BX53, Olympus, Tokyo, Japan) at 40x10 magnification by Comet Assay IV, Perceptive software (Suffolk, UK). One hundred cells were counted and scored for each concentration and %TDNA and tail intensity were evaluated.14

Oxidative stress parameters

A total reactive oxygen species (ROS) assay was performed via (2’-7’-dichlorodihydrofluorescein diacetate) analysis by flow cytometry. Next 5x105 cells/2 mL cell culture medium/well were placed into a 6-well plate. After one day of incubation, the cells were exposed to acetamiprid at 1, 2, 4, and 6 mM concentrations and 1% DMSO as a negative control for 24 h. After 24 h the plates were washed with PBS twice. The cells were incubated with 20 µM H2DCF-DA at 37 °C for 30 min on a shaker in the dark. They were detached from the plates via trypsinization and resuspended in 150 µL of PBS with 1% bovine serum albumin. Fluorescence intensity was measured by FITC channel with excitation 488 nm and emission 530 nm via an ACEA NovoCyte flow cytometer (San Diego, California, USA). The results were shown as median fluorescence intensity.

Glutathione (GSH) levels were determined by [5,50-dithiobis-2-nitrobenzoic acid) (DTNB)] reagent method described by Beutler.15 This method is based on DTNB reduction by free SH groups of GSH to 5-mercapto-2-nitrobenzoate. After treatment with acetamiprid, 1 mL of cell lysates was deproteinated with 1.67 g of metaphosphoric acid, 0.2 g of Na2 EDTA, and 30 g of NaCl solved in distilled water. After that, 2.4 mL of Na2HPO4 and 0.3 mL of DTNB were added, followed by centrifugation for 10 min at 3000x g. 5-Thio-2-nitrobenzoic acid formation was measured at 412 nm by spectrophotometer. GSH results were expressed as µmol/g protein.15

Statistical analysis

The experimental results were analyzed by One-Way ANOVA post hoc Dunnett’s t-test and given as mean ± standard deviation. The level of statistical significance was set as p≤0.05. All analyses were performed using SPSS version 20.0 (SPSS Inc., Chicago, Illinois, USA).


Cell viability

The MTT assay is one of the most frequently used, simple, and rapid colorimetric cell viability/cytotoxicity assays and yields quantitative data. This assay is based on reduction of water-soluble yellow tetrazolium salt by the mitochondrial succinate dehydrogenase enzyme in metabolically active/live cells dehydrogenase and quantified color intensity of dissolved formazan crystals by spectrophotometer.16

The cytotoxicity of acetamiprid on the AR42J cell line was evaluated with the MTT assay in the dose range of 1-50 mM after 24 h exposure and the IC50 value was determined as 12.61 mM (Figure 1).

Comet assay

The alkaline comet assay is a very common method for measuring DNA damage in a single cell suspension via migration of DNA under electrophoresis conditions. It has been reported that the tail intensity value is the most recommended end point for an alkaline comet assay in a dose-dependent manner.17

According to the results of the comet assay in the concentration range of 1-6 mM, acetamiprid significantly increased DNA damage in a dose-dependent manner. The mean tail intensity values were significantly increased in all exposure groups compared to the control group (Figure 2).

Oxidative stress parameters

Oxidative damage via ROS plays a key role in different human diseases such as cancer, cardiovascular diseases, diabetes, and neurodegeneration. Dichlorodihydrofluorescein diacetate (DCFH-DA) is a widely used assay that enables direct measurement of the redox state in the cells. This method is very sensitive, easy to use, and cheap and can be used to follow changes in ROS over time.18

There were no significant differences between the control and exposure groups according to total ROS levels, which were evaluated by H2DCF-DA with a flow cytometer. However, the GSH level was significantly reduced in the 6 mM group compared to the control group. It was observed that 6 mM of acetamiprid dramatically reduced GSH level by 98.07% (Figure 3).


Widespread use of acetamiprid in agriculture alone or in combination with other insecticides may cause pesticide spread into the environment and the food chain, resulting in toxicity in humans and animals. An increased risk of pancreatic cancer is found in those in agricultural occupations; however, pesticides’ effects on oncogenesis mechanisms have not been extensively evaluated yet.19,20 There are limited data about the effects of neonicotinoids on pancreatic tissue; moreover, there are no data about acetamiprid’s effects on the pancreas. Khalil et al.13 reported that 0.5 and 1.0 mg/kg bw imidacloprid over 60 days disrupted glucose homeostasis in male rats. In treated groups, the GLUT4 mRNA expression level was decreased and pancreatic islets shrinkage and infiltration of round cells, interlobular congestion, and hemorrhage were observed with histopathologic examination, and also decreased insulin expression in the pancreatic β cells was detected in the imidacloprid-treated groups.

To further examine the potential associations between their usage and toxic profiles for neonicotinoid pesticides, we conducted a study on in vitro acetamiprid exposure in a pancreatic cell line. Although there are a few studies on acetamiprid’s cyto- and genotoxicity potentials in the literature, there is no in vitro/in vivo study focusing on the pancreas. It has been reported that the LD50 value is the range of 140-417 mg/kg b.w. in rodents and the NOAEL level is 400 ppm in 13 week old mice for acetamiprid.21 It has been reported in another study that IC50 values of acetamiprid on SH-SY5Y and HepG2 cell lines were 2.16 and 3.61 mM, respectively.22 In our study, the cytotoxicity was evaluated by MTT assay in acetamiprid (1-50 mM) treated AR42J cells after 24 h exposure. The IC50 value was calculated as 12.61 mM.

In the same study, significantly increased DNA damage was shown at 500 µM in SH-SY5Y cells.22 In another study, >50 µM acetamiprid significantly increased micronuclei formation and DNA breaks in IMR-90 human lung cells.8 Genotoxic effects of acetamiprid on sister chromatid exchange, micronucleus, and chromosomal aberration analysis with 25, 30, 35, and 40 µg/mL doses for 24 and 48 h were shown in human peripheral blood lymphocytes. Micronucleus formation was significantly induced compared to the control group while the proliferation index was decreased.12 It has been reported that acetamiprid increased micronuclei per cell and chromosomal aberrations in Swiss albino male mice bone marrow depending on concentration with acetamiprid treatment over 60 and 90 days at 4.6 and 2.3 mg/kg/day i.p.23 According to the results of the comet assay performed with the concentration range of 1-6 mM, acetamiprid significantly induced DNA damage depending on concentration. The different results obtained from several studies can be related to cell types, study duration, and/or method selection.

Oxidative stress mechanisms could underlie the cyto- and genotoxic potentials of neonicotinoid pesticides.7,8,12 It has been reported that pesticides may impair the redox balance effects in different cells. However, the mechanisms underlying oxidative stress are still not fully understood.16 There are several studies about the effects of acetamiprid on oxidative stress parameters in different species such as rodents, bacteria, plants, and fish.24,25,26,27,28,29,30 In Wistar rat brain tissue 3.14 mg/kg acetamiprid exposure resulted in increased mitochondrial oxidative stress status that was significant. Decreased oxidative stress parameters were GSH level, GSH peroxidase, and catalase activities. Increased parameters determined were malondialdehyde level, GSH s-transferase, and superoxide dismutase activities.31 In our study, no significant difference was found between the control and exposure groups according to total ROS levels. However, the GSH levels were significantly different compared to the control at the highest concentration. It was observed that 6 mM acetamiprid dose dramatically reduced GSH level by 98.07%. In earthworms, it has been demonstrated that different concentrations of acetamiprid (0, 0.05, 0.10, 0.25, and 0.50 mg/kg of soil) with different exposure periods (7, 14, 21, and 28 days) increased the ROS levels to varying degrees. Olive tail moment, which indicates DNA damage in the comet assay, increased in a dose-dependent manner, indicating that subchronic acetamiprid exposure might cause oxidative stress and induce DNA damage in earthworms.24

Acetamiprid is classified as an “unlikely” human carcinogen according to EPA guidelines and its target organ toxicity data are not clear yet. Acetamiprid’s acute oral toxicity category is “II” for rats and its acute inhalation toxicity category is “III” for rabbits. Its NOAEL value for rats is 12.4/14.6 mg/kg/day (M/F) and its LOAEL value for rat is 50.8/56.0 mg/kg/day (M/F). The chronic carcinogenicity NOAEL value for rats is 7.1/8.8 mg/kg/day (M/F). According to the EPA acetamiprid is not yet classified as genotoxic.32 A target organ toxicity assessment for further subchronic and chronic in vivo studies may clarify the risk caused by acetamiprid for pancreas tissue-based diseases.


Under different durations in the pancreatic cell line acetamiprid may affect these oxidative stress parameters significantly. To clarify the oncogenic potential of acetamiprid on pancreatic tissue it is necessary to perform further in vivo studies with subchronic or chronic studies with molecular mechanistic observations.

Conflicts of interest: No conflict of interest was declared by the authors. The authors alone are responsible for the content and writing of the paper.


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