1 23

Environmental Science and PollutionResearch ISSN 0944-1344 Environ Sci Pollut ResDOI 10.1007/s11356-015-4927-3

Cytotoxic and genotoxic effects ofabamectin, chlorfenapyr, and imidaclopridon CHOK1 cells

Ali S. Al-Sarar, Yasser Abobakr, AlaaE. Bayoumi & Hamdy I. Hussein

1 23

Your article is protected by copyright and

all rights are held exclusively by Springer-

Verlag Berlin Heidelberg. This e-offprint is

for personal use only and shall not be self-

archived in electronic repositories. If you wish

to self-archive your article, please use the

accepted manuscript version for posting on

your own website. You may further deposit

the accepted manuscript version in any

repository, provided it is only made publicly

available 12 months after official publication

or later and provided acknowledgement is

given to the original source of publication

and a link is inserted to the published article

on Springer's website. The link must be

accompanied by the following text: "The final

publication is available at link.springer.com”.

RESEARCH ARTICLE

Cytotoxic and genotoxic effects of abamectin, chlorfenapyr,and imidacloprid on CHOK1 cells

Ali S. Al-Sarar1 & Yasser Abobakr1 & Alaa E. Bayoumi1 & Hamdy I. Hussein1

Received: 22 January 2015 /Accepted: 17 June 2015# Springer-Verlag Berlin Heidelberg 2015

Abstract The cytotoxicity and genotoxicity of abamectin,chlorfenapyr, and imidacloprid have been evaluated on theChinese hamster ovary (CHOK1) cells. Neutral red incorpora-tion (NRI), total cellular protein content (TCP), and methyltetrazolium (MTT) assays were followed to estimate the mid-point cytotoxicity values, NRI50, TCP50, and MTT50, respec-tively. The effects of the sublethal concentration (NRI25) onglutathione S-transferase (GST), glutathione reductase(GRD), glutathione peroxidase (GPX), and total glutathionecontent have been evaluated in the presence and absence ofreduced glutathione (GSH), vitamin C, and vitamin E. Thegenotoxicity was evaluated using chromosomal aberrations(CA), micronucleus (MN) formation, and DNA fragmentationtechniques in the presence and absence of the metabolic acti-vation system, S9 mix. Abamectin was the most cytotoxicpesticide followed by chlorfenapyr, while imidacloprid wasthe least cytotoxic one. The glutathione redox cycle compo-nents were altered by the tested pesticides in the absence andpresence of the tested antioxidants. The results of genotoxicityindicate that abamectin, chlorfenapyr, and imidacloprid havepotential genotoxic effects on CHOK1 cells under the experi-mental conditions.

Keywords Cytotoxicity . Genotoxicity . Abamectin .

Chlorfenapyr . Imidacloprid . CHOK1 cells . Glutathioneredox cycle

Introduction

The extensive and/or improper uses of pesticides cause pollu-tion of water, air, soil, and food. The environmental pollutionwith pesticides results in detrimental effects on non-target or-ganisms (Gilden et al. 2010). Pesticide production workers,applicators, and farmers are the main risk groups of high-doseexposure; however, the common population is exposed tolower doses via the contaminated food and water or duringthe household use. It was documented that the exposure to lowdoses of pesticides produces various biochemical alterationsleading to adverse health effects in the exposed organisms(Banerjee et al. 1999; Al-Sarar et al. 2009, 2014).

The pesticides tested in the present study are systemic andbelong to three new classes with different modes of action.Abamectin, a macrocyclic lactone derived from the soil mi-croorganism Streptomyces avermitilis, is a mixture of B1a andB1b avermectins (Meister 1992). In agriculture, abamectin isused as a systemic acaricide and insecticide worldwide; it isalso used as a parasiticide for the lung worm and nasal botsand against gastrointestinal nematodes in cattle and sheep(Elbetieha and Da’as 2003; Novelli et al. 2012). Its mode ofaction is associated with its effect on the -aminobutyric acid(GABA) receptors and glutamate-gated chloride channels in-creasing the permeability of chloride ions, hyperpolarizing thenerve and muscle cells, and disturbing the neuromusculartransmission leading to death (Cully et al. 1994).

Chlorfenapyr, a novel N-substituted halogenated pyrrole, isa wide-spectrum insecticide with an acaricidal activity (Huntand Treacy 1998). Chlorfenapyr is a proinsecticide activatedby the in vivo oxidative elimination of its N-ethoxymethylgroup by mixed function oxidases (Black et al. 1994).Chlorfenapyr inhibits the production of mitochondrial ATPvia uncoupling the mitochondrial oxidative phosphorylations,interrupting the conversion of ADP to ATP, which might

Responsible editor: Markus Hecker

* Yasser Abobakrymohamed@ksu.edu.sa

1 Department of Plant Protection, College of Food and AgricultureSciences, King Saud University, P.O. Box 2460, Riyadh 11451,Saudi Arabia

Environ Sci Pollut ResDOI 10.1007/s11356-015-4927-3

Author's personal copy

generate reactive oxygen radicals. Although chlorfenapyr isclassified as slightly hazardous insecticide as per WHO(Tomlin 2000), it was banned in Europe because of its highpersistence in the environment (van Leeuwen et al. 2004).

Imidacloprid is a neonicotinoid insecticide. In 2010, theworld production of imidacloprid was estimated to be ca. 20,000 t; it was the world’s largest selling insecticide in 120countries with registered uses for more than 140 crops (Pol-lack 2011; Jeschke et al. 2011; Simon-Delso et al. 2015).Imidacloprid acts as a selective agonist of the nicotinic acetyl-choline receptor subtypes in insects; it is classified as a toxic-ity class II agent (EPA 1994; WHO 2002) with low mamma-lian toxicity (Tomizawa and Casida 2003). So, it is widelyused in animal health and crop protection with soil, seed,and foliar applications (Tomizawa and Casida 2005; Costaet al. 2009). However, imidacloprid was found to induceDNA damage in a dose-related manner in earthworms as wellas to increase the frequency of adducts in calf thymus DNA,indicating agent-induced genotoxicity (Shah et al. 1997; Zanget al. 2000).

In vitro toxicity tests, using animal cell cultures, are usefultools in the screening of environmental contaminants, riskassessment, and safety evaluation in a rapid and cost-effective way (Spielmann and Goldberg 1999; Bols et al.2005). Many cellular endpoints have been utilized as bio-markers such as cellular lysosomes via the neutral red incor-poration (NRI) assay, changes in total cellular protein (TCP)content, and mitochondrial function through methyl tetrazoli-um (MTT) assay (Saito et al. 1991; Wataha et al. 1991;Bertheussen et al. 1997).

The glutathione redox cycle components, glutathione S-transferase (GST), glutathione reductase (GRD), glutathioneperoxidase (GPX), and glutathione, are involved in the cellu-lar detoxification of xenobiotics; the oxidative stress of pesti-cides has been reported (Bayoumi et al. 2000, 2001; Garcia-Fernandez et al. 2002), and the role of GSH, vitamin C, andvitamin E in the protection of cells and tissues against theoxidative stress of xenobiotics was documented (Aly et al.2010; Elsharkawy et al. 2013).

The mutagenic and genotoxic behaviors of several pesti-cides have been studied both in vivo and in vitro using cyto-genetic endpoints such as chromosomal aberrations (CA),cytokinesis-block micronuclei (CBMN), sister chromatid ex-change (SCE), and DNA fragmentation assays (Soloneskiet al. 2008; Soloneski and Larramendy 2010; Calderón-Segura et al. 2012; Pandey and Guo 2014).

The increasing use of abamectin, chlorfenapyr, andimidacloprid and the presence of their residues in fruits, veg-etables, and milk (Dakova 2005; Arias et al. 2014) demanddetailed studies to evaluate their potential toxic risks to non-target organisms, particularly mammals. The present studywas conducted to evaluate the in vitro cytotoxicity andgenotoxicity of abamectin, chlorfenapyr, and imidacloprid

using the Chinese hamster ovary (CHOK1) cells. Weemployed three cytotoxicity assays: neutral red incorporation(NRI), total cellular protein (TCP) content, and methyl tetra-zolium (MTT) assays. Furthermore, the effects of the sublethalcytotoxicity value (NRI25) on GST, GRD, GPX, and totalcellular glutathione content were evaluated in the presenceand absence of GSH, vitamin C, and vitamin E. Thegenotoxicity assays CA, CBMN, and DNA fragmentationwere employed as different cytogenetic endpoints to evaluatethe genotoxic effects exerted by the tested pesticides.

Materials and methods

Chemicals

All reagent-grade chemicals and cell culture components werepurchased from Sigma-Aldrich (St. Louis, MO, USA). Fetalcalf serum (FCS) was obtained from Boehringer IngelheimGmbH (Germany). The pesticides abamectin, chlorfenapyr,and imidacloprid were purchased with purity of 98 % fromDr. Ehrenstorfer-Schafers (Augsburg, Germany).

Cell culture

CHOK1 cells were purchased from Cell Line Service Compa-ny (Eppelheim, Germany) and cultured in humidified atmo-sphere of 5 % CO2 at 37 °C. Culture medium (Ham F-12) wassupplemented with HEPES buffer (25 mM, pH 7.4), 10 %heat-inactivated FCS, 100 μg/ml streptomycin, and 100 U/ml penicillin. The cells were counted by an improvedNeubauer hemocytometer, and cell viability was checked bytrypan blue dye exclusion (Freshney 1987).

Pesticide treatments

The cytotoxicity of the tested compounds was determinedafter 24- and 48-h exposure times in a serum-free medium orin a medium supplemented with 10% FCS. Stock solutions ofpesticides (10 mM) were prepared in sterilized dimethyl sulf-oxide (DMSO), and a series of ten concentrations (0.97–500 μM) were freshly prepared. Cells were allowed to reach65 % confluence before being pulsed with indicatedconcentrations.

Cytotoxicity assays

Neutral red incorporation assay

According to Borenfreund and Puerner (1985), 200-μl medi-um containing 65 % confluence of CHOK1 cells was added to96-well tissue culture plates and incubated for 24 h. The me-dium was replaced with pesticide containing medium, serum-

Environ Sci Pollut Res

Author's personal copy

free, or supplemented with 10 % FCS. After 24 and 48 h, themedium was substituted with a medium containing 40 μg/mlNR dye, which had been prepared and preincubated overnightat 37 °C. Three hours after incubation, the cells were washedtwice with 0.5 % formaldehyde in 1 % CaCl2. To extract thedye from the cells, 200 μl acetic acid (1 %) in 50 % ethanolwas added. After 20 min of agitation, the absorbance wasmeasured at 570 nm using microplate reader (Dialab modelELx 800, Wiener Neudorf, Austria).

Total cellular protein content assay

TCP content was measured according to the method ofPelletier et al. (1988). After 24- and 48-h exposure times, thewells were washed with 200 μl of phosphate-buffered saline(PBS) and fixed with 200 μl of 10 % v/v formaldehyde inPBS. After 10 min, the cells were washed with 200 μl ofborate buffer (0.01 M, pH 8.4) and stained with 100 μl ofmethylene blue (1 % w/v in borate buffer). After 10 min, thedye was removed and the cells were washed five times withborate buffer. The stain was solubilized with 200 μl of 0.1 NHCl, and plates were shaken for 15 min. The optical densitywas measured at 660 nm.

Methyl tetrazolium assay

This assay was performed according to the method of Plumbet al. (1989) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye. After 24- and 48-h exposuretimes, the medium was removed and 50 μl of 5 mg/ml MTTdye in PBS was added. Plates were incubated at 37 °C for 4 h;the dye solution was removed, and 200 μl of DMSO wasadded followed by 25 μl Sorensen’s glycine buffer (0.1 Mglycine and 0.1 M NaCl, pH 10.5). After 15 min of agitation,the optical density was measured at 570 nm.

Determination of glutathione redox cycle parameters

Treatment and preparation of cellular extracts

Cells were plated (104 cells/cm2) in tissue culture Petri dishes.On the second day, the medium was replaced with serum-freemedium containing the antioxidants, GSH (1 mM), vitamin C(70 μM), and vitamin E (30 μM). After 24 h, an aliquot ofserum-free medium containing the tested concentration(NRI25) was added. After 24 h, the cells were trypsinizedusing 500 μl of a 0.25 % trypsin/EDTA solution in sterilizedPBS. Cells were washed twice via centrifugation at 990g for5 min at 4 °C using sterilized PBS (Bayoumi 1998). To deter-mine the enzymatic activities, the cells were resuspended in500 μl of a sucrose solution (0.25 M) containing 1 mMEDTA. Finally, the cell suspension was sonicated for threeintervals of 10 s separated by 10 s in the ice bath. The

suspension was recentrifuged for 15 min at 13,680g, and thesupernatant was used as the enzyme extract. For determinationof the total glutathione content, the same procedure wasfollowed after suspending the cells in 500 μl perchloric acid(1 M) containing EDTA (2 mM). The cell suspension wassonicated and recentrifuged as mentioned above; the superna-tant (100 μl) was used in the assay. Before determining theglutathione levels, all samples were neutralized to pH 7 usingKOH (2M) and MOPS buffer (0.3 M), according to Bayoumiet al. (2001).

Assessment of the enzymatic activity and glutathionecontent

Glutathione S-transferases

This assay was performed according to Habig et al. (1974).The reaction mixture consisted of 800 μl phosphate buffer(0.2 M, pH 6.5), 50 μl of GSH solution (20 mM), 50 μl of1-chloro-2,4-dinitrobenzene solution (20 mM), and 100 μl ofthe enzyme extract. Absorbance was recorded for 3 min at340 nm.

Glutathione reductase

The activity of GRD was determined according Carlbergand Mannervik (1985). The reaction mixture contained500 μl phosphate buffer (0.2 M, pH 7.0), 50 μl β-NADPH, (2 mM) in Tris-HCl (10 mM, pH 7.0), 50 μloxidized glutathione (20 mM), 300 μl redistilled water,and 100 μl of the enzyme extract. The reaction mixturewas incubated for 3 min at 30 °C; the absorbance wasmeasured for 3 min at 340 nm.

Glutathione peroxidase

GPX activity was determined according to Wendel (1981).The reaction mixture composed of 500 μl phosphate buffer,(0.25 M, pH 7.0), 100 μl GRD, 100 μl GSH (10 mM), 100 μlβ-NADPH (2.5 mM in 0.1 % NaHCO3), and 100 μl of theenzyme extract. After incubation for 10 min at 37 °C, 100 μlof tert-butyl hydroperoxide (12 mM) was added. The varia-tion in the absorbance was recorded at 366 nm for 3 min.

Total glutathione content

The method of Akerboom and Sies (1981) was followed. Thefollowing reagents were added to a quartz cuvette: 900 μlphosphate buffer (0.1 M) with EDTA (1 mM), 200 μl of thecellular extract, 50 μl NADPH (4 mg/ml in 0.5 % NaHCO3),20 μl of DTNB (1.5 mg/ml in 0.5 % NaHCO3), and 20 μl ofGRD. The variation of absorbance was measured at 412 nmafter 3 min.

Environ Sci Pollut Res

Author's personal copy

Total protein determination

The microassay procedure was performed according to themethod described by Bradford (1976) using bovine serumalbumin as standard.

Chromosome aberration assay

CHOK1 cells were seeded in 60-mm plates at 4×104 cells/dish. The cultures were treated at the NRI25 values for 24 hin the absence and presence of the S9 fraction. Methylmethanesulfonate (MMS, 40 μg/ml) and benzo[a]pyrene(BP, 2 μg/ml) were the positive controls in the absence andpresence of S9 fraction, respectively. The chromosomes wereprepared according to the conventional method (IAE 1986).Three hours before harvesting the culture, cells were treatedwith colchicine (0.2 μg/ml). The cells were trypsinized andcollected by centrifugation; then, the hypotonic solution(0.075 M KCl at 37 °C for 17 min) was added, followed byfixation in methanol/acetic acid (3:1). The suspension of cellswas dropped onto cold glass microscope slides and air-dried.The slides were stained with 5 % Giemsa in distilled water for10min and scored for structural chromosome aberrations. Onehundred metaphases were examined for each treatment.

Cytokinesis-block micronucleus assay

The CBMN test (Fenech 1993) was used to detect thegenotoxic effects of abamectin, chlorfenapyr, andimidacloprid. CHOK1 cells were seeded in cell culture dishes(60 mm) at 4×104 cells/dish and allowed 24 h to establishnormal growth. The cells were treated for 24 h with theNRI25 concentrations and were cultured with cytochalasin-B(4.5 μg/ml) for 24 h before trypsinization, centrifugation, andresuspension in KCl (0.075 M) at 37 °C for 5 min. The cellswere then fixed three times in methanol/glacial acetic acid(3:1). Subsequently, the cell solution was dropped ontoprecleaned slides; after air-drying, the slides were stained by5 % Giemsa for 10 min. The slides were analyzed under lightmicroscope at×600magnification. The number of binucleatedcells containing one, two, or more micronucleus (MN) wasscored in cytokinesis-blocked cells based on the observationof 500 cells per slide.

Evaluation of DNA fragmentation

The cell cultures were exposed to the NRI25 values for 24 h,followed by trypsinization and centrifugation at 1200×g for10 min to harvest the cells. The DNAwas extracted and puri-fied using DNA extraction kit (Wizard® Genomic DNA Puri-fication Kit, Promega Corporation, Madison, USA). The qual-ity and quantity of DNA were assessed by Nanodrop 2000UV-vis spectrophotometer (Thermo Scientific, Wilmington,

DE, USA). The DNA samples were analyzed on 1.1 % aga-rose gels containing ethidium bromide. The gels were visual-ized with a long wave transilluminator and photographed.

Statistical analysis

For estimation of cytotoxicity values, NRI25, NRI50, TCP50,and MTT50, the absorbance values were correlated to the per-centages of cell mortality by plotting the toxicity regressionlines in the form of a log/probit relationship according to themethod of Bayoumi (1998) using Sigma Plot® Version 2.0software (Systat Software Inc., CA, USA). The statistical anal-ysis of cytotoxicity, chromosome aberrations and MN resultswas performed by the Student’s t test. For effects of treatmentson glutathione redox cycle components, analysis of variance(ANOVA) and the Newman-Keuls test were used to determinesignificant differences between the treatment groups using thestatistical program (CoStat 2®, CoHort Software, Monterey,USA).

Results

Cytotoxicity

The median lethal cytotoxicity values, NRI50, TCP50, andMTT50, for abamectin, chlorfenapyr, and imidacloprid areshown in Table 1. The NRI assay showed that abamectin after24 and 48 h was the most cytotoxic pesticide followed bychlorfenapyr and imidacloprid; the cytotoxicity was time-de-pendent. The FCS caused a significant increase in the NRI50values of abamectin and chlorfenapyr calculated by 5.2- and2.4-fold after 24 h and 5.5- and 1.9-fold after 48 h, respective-ly. The TCP50 values showed that abamectin was the mostcytotoxic pesticide followed by chlorfenapyr andimidacloprid. The presence of FCS reduced the cytotoxicityof abamectin by 12.2- and 14-fold after 24 and 48 h, respec-tively; the TCP50 values of chlorfenapyr increased by 2.9- and2.1-fold after 24 and 48 h, respectively. The MTT50 valuesshowed that abamectin was significantly the most cytotoxicpesticide followed by chlorfenapyr and imidacloprid.Abamectin was more toxic than chlorfenapyr by 35.8- and661-fold and more toxic than imidacloprid by 297.1- and5513-fold after 24 and 48 h, respectively.

Effects on the glutathione redox cycle components

The effects of the sublethal concentration (NRI25) ofabamectin, chlorfenapyr, and imidacloprid on the glutathioneredox cycle components, after 24 h, in the absence and pres-ence of GSH, vitamin C, and vitamin E are illustrated inTable 2 and Fig. 1. For the effect of antioxidants on the un-treated cells, the extracellular GSH caused significant

Environ Sci Pollut Res

Author's personal copy

elevations in the activities of GST (26.8 %), GRD (93.4 %),and intracellular total glutathione content (34.7 %) comparedwith the antioxidant-free control. In addition, vitamins C and

E caused significant inductions by 175.4 and 94.96 % in GRDactivity and 26.1 and 29.2 % in GPX activity, respectively,compared with the antioxidant-free control.

Table 1 The midpointcytotoxicity values of abamectin,chlorfenapyr, and imidaclopriddetermined by neutral redincorporated (NRI), total cellularprotein (TCP) content, andmethyl tetrazolium (MTT) assaysafter 24- and 48-h exposureperiods of CHOK1 cells in serum-free medium and mediumsupplemented by 10 % fetal calfserum (FCS)

Pesticide Exposure period (h) Serum content Cytotoxicity values (μM)

NRI50 TCP50 MTT50

Abamectin 24 Free 4.3±2.1 2.4±0.94 0.16±0.02

10 % 22.4$$±4.7 28.6$$±1.7 ND

48 Free 4.5±0.7 2.1±0.6 0.02***±0.01

10 % 24.8$$±1.3 28.7$$±5.4 ND

Chlorfenapyr 24 Free 62.1±5.0 56.6±7.5 5.9±2.4

10 % 146.3$$±6.3 166.1$$±12.9 ND

48 Free 48.3*±2.7 93.9**±9.7 13.2*±3.6

10 % 89.9$±6.6 199.5$$±14.1 ND

Imidacloprid 24 Free 353.9±15.8 504.8±22.7 47.5±6.9

10 % 214.7$$±20.0 168.7$$±13.0 ND

48 Free 99.5**±4.7 111.2***±10.5 110.3**±10.5

10 % 102.1±4.4 328.5$$±33.4 ND

Each value of the NRI represents an average of four experiments ± SE

ND not determined

Comparison between values of serum-free medium after 24 and 48 h, ***highly significant p≤0.001, **moder-ately significant p≤0.01, and *significant p≤0.05. Comparison between values of serum-free medium andmedium containing FCS, $$$highly significant p≤0.001, $$moderately significant p≤0.01, and $significantp≤0.05

Table 2 Effect of abamectin, chlorfenapyr, and imidacloprid on thespecific activity of glutathione S-transferase, glutathione reductase,glutathione peroxidase, and glutathione content in the absence and

presence of extracellular GSH, vitamin C, and vitamin E in CHOK1

cells after 24-h exposure period

Pesticide (NRI25) Antioxidant Specific activity (nmol/min/mg protein) Glutathione content (μM)

GST GRD GPX

Abamectin (2.85 μM) Free 29.0 d±0.7 17.0 fg±1.6 55.5 f±4.9 29.3 cde±1.2

GSH (1 mM) 35.9 cd±0.4 33.2 c±1.5 69.9 def±8.9 36.3 a±1.2

Vit. C (70 μM) 26.2 d±0.1 29.2 cd±2.7 56.3 f±5.2 25.3 efg±1.5

Vit. E (30 μM) 49.9 bc±3.1 25.7 de±1.0 82.3 cd±5.6 30.7 bc±0.6

Chlorfenapyr (4.08 μM) Free 35.9 cd±5.5 21.2 ef±1.8 72.9 de±2.1 32.3 abc±5.0

GSH (1 mM) 32.1 cd±5.4 39.7 b±2.1 83.9 cd±4.7 35.7 a±2.1

Vit. C (70 μM) 44.7 bc±5.9 16.9 fg±1.4 85.1 cd±1.5 25.3 efg±1.2

Vit. E (30 μM) 43.6 bc±5.9 28.3 cd±3.4 62.9 ef±4.1 29.7 bcd±2.1

Imidacloprid (114.63 μM) Free 34.5 cd±0.3 14.1 gh±1.3 34.3 g±1.2 26.0 defg±1.0

GSH (1 mM) 43.3 bc±5.3 11.4 h±1.1 79.3 cd±5.5 33.7 ab±0.6

Vit. C (70 μM) 35.9 cd±4.5 15.9 gh±1.3 79.1 cd±1.9 23.7 g±0.6

Vit. E (30 μM) 46.2 bc±0.2 30.8 cd±1.8 92.3 c±6.5 28.3 cdef±3.2

Control Free 56.7 b±1.7 22.6 e±1.3 139.7 b±3.1 24.0 g±1.0

GSH (1 mM) 71.9 a±5.1 42.7 b±1.7 140.9 b±8.3 32.3 abc±2.5

Vit. C (70 μM) 56.2 b±3.6 62.2 a±2.1 176.1 a±3.9 22.0 g±2.0

Vit. E (30 μM) 55.0 b±3.9 44.1 b±0.8 180.5 a±7.0 25.0 fg±2.0

Each value represents the average of three replicates ± SD. Values with common letters are not significantly different at the 0.01 level

GST glutathione S-transferase, GRD glutathione reductase, GPX glutathione peroxidase, GSH reduced glutathione, Vit. C vitamin C, Vit. E vitamin E

Environ Sci Pollut Res

Author's personal copy

The three pesticides caused a significant inhibition in theactivity of GST in the antioxidant-free and GSH-treated cellscompared with the antioxidant-free or GSH-treated controlcells. Abamectin and imidacloprid significantly inhibited theactivity of GST in vitamin C-treated cells compared with thecorresponding control. In addition, the specific activity ofGST significantly increased in the presence of vitamin E inthe treatment of abamectin compared with antioxidant-free/abamectin-treated cells.

Abamectin and imidacloprid significantly inhibited the ac-tivity of GRD in the antioxidant-free and antioxidant-treatedcells compared with their corresponding controls;chlorfenapyr significantly inhibited the GRD activity in thepresence of vitamins C and E compared with their correspond-ing controls. A significant induction in the GRD activity wasobserved in abamectin-treated cells in the presence of GSH(95.9 %), vitamin C (72.4 %), and vitamin E (51.4 %) com-pared with the antioxidant-free/abamectin-treated cells. GSHand vitamin E significantly increased the activity of GRD inthe chlorfenapyr-treated cells by 87.1 and 33.3 %, respective-ly, compared with the antioxidant-free/chlorfenapyr-treatedcells. In the presence of vitamin E, imidacloprid induced theGRD activity by 118.6 % compared with the antioxidant-free/imidacloprid-treated cells.

All tested pesticides caused significant decreases inthe activity of GPX in the absence and presence of alltested antioxidants compared with their parallel controls.In the imidacloprid treatments, presence of GSH, vita-min C, and vitamin E caused inductions in the GPXactivity by 131.1, 130.3, and 168.7 % compared withthe antioxidant-free treatment, respectively. In addition,presence of vitamin E in the abamectin treatment in-creased the GPX activity by 48.3 % over the antioxi-dant-free/abamectin-treated cells.

Abamectin and chlorfenapyr significantly increased theglutathione content by 22.2 and 34.7 %, respectively,

compared with the antioxidant-free control. Moreover,abamectin and chlorfenapyr caused a significant elevation inthe glutathione content in the presence of vitamin E comparedwith the corresponding control. The presence of vitamin C inabamectin and chlorfenapyr treatments significantly reducedthe glutathione content compared with antioxidant-free/abamectin- and chlorfenapyr-treated cells, respectively, reduc-ing its level to that of the control. No significant differenceswere observed between all imidacloprid treatments and theircontrols.

Chromosome aberrations

The effects of abamectin, chlorfenapyr, and imidacloprid atthe sublethal concentrations (NRI25) on the chromosome ab-errations were evaluated in the CHOK1 cells in the presenceand absence of S9 mix (Table 3). Abamectin and chlorfenapyrsignificantly (p<0.05) induced the total CA compared withthe control in the absence and presence of S9 mix. Chromo-some and chromatid gaps were the most obvious aberrationsin all affected cells. Imidacloprid did not exert any significant(p>0.05) CA in the CHOK1 cells in the absence and presenceof S9 mix.

Induction of micronucleus

The results of pesticide-induced micronuclei in binucle-ated cytokinesis-blocked CHOK1 cells are shown inTable 4. There were significant increases (p<0.01) inthe frequencies of micronuclei in the positive controls,MMC-, or BP-treated cells, compared with the controlcells. Abamectin, chlorfenapyr, and imidacloprid signif-icantly induced the frequencies of micronuclei in theabsence of S9 mix (p<0.05). In the presence of S9mix, a highly significant induction (p<0.01) in themicronuclei frequencies was observed after exposure to

0.00

10.0

20.0

30.0

40.0

-10.0

-20.0

-30.0

-40.0

-50.0

-60.0

-70.0

-80.0

GST GRD GPX GSH

Tested antioxidants

Free GSH(1 mM)

Vit. C(70 M)

Vit. E(30 M)

Free GSH(1 mM)

Vit. C(70 M)

Vit. E(30 M)

Free GSH(1 mM)

Vit. C(70 M)

Vit. E(30 M)

A B C

Incr

ease

or

decr

ease

of g

luta

thio

nere

dox

cycl

e co

mpo

nent

(%)

*

* * *

* ** *

**

*

*

* *

*

*

*

*

* *

*

*

*

*

*

**

*

*

***

*

*

Control

Fig. 1 Effect of imidacloprid (A), chlorfenapyr (B), and abamectin (C) onthe glutathione redox cycle components, GST, GRD, GPX, and GSHcontent, in CHOK1 cells in absence and presence of the testedantioxidants, GSH, vitamin C, and vitamin E. *Significantly different

relative to controls at the 0.01 level. GST glutathione S-transferase,GRD glutathione reductase, GPX glutathione peroxidase, GSH reducedglutathione, Vit. C vitamin C, Vit. E vitamin E

Environ Sci Pollut Res

Author's personal copy

abamectin and chlorfenapyr, while imidacloprid treat-ment resulted in a significant induction (p<0.05). Cellswith one MN were predominant in the binucleated cells.No cells with more than two micronuclei were observedin all treatments.

DNA strand breakage

Using the DNA laddering technique, a single band was ob-tained in the treated CHOK1 cells for the three pesticides(Fig. 2). This single band is a characteristic of intact genomicDNA. So, no apparent DNA breakage was observed inabamectin, chlorfenapyr, and imidacloprid-treated CHOK1

cells using this technique.

Discussion

The present study was planned to assess the cytotoxic andgenotoxic effects of three systemic insecticides, abamectin,chlorfenapyr, and imidacloprid, using in vitro-culturedCHOK1 cells via ten different endpoints. In addition, the ame-liorative effect of the antioxidants, GSH and vitamins C and E,on the glutathione redox cycle components was evaluated.The results indicate that abamectin was the most cytotoxicpesticide in all cytotoxicity assays, followed by chlorfenapyrand imidacloprid. Maioli et al. (2013) reported a cytotoxiceffect of abamectin on isolated rat hepatocytes. Abamectin isa mixture of B1a and B1b avermectins (Meister 1992).Avermectin exerted in vivo and in vitro cytotoxic actions in

Table 3 Induced structuralchromosome aberrations incultured CHOK1 cells treated withabamectin, chlorfenapyr, andimidacloprid

Pesticides S9 mix Aberrations

Crg Crb Ctg Ctb Frg Dic Total (%)

Control − 1.5 0.0 1.0 0.0 0.0 0.0 2.5

+ 2.0 0.0 1.0 0.0 0.0 0.0 3.0

Abamectin (2.85 μM) − 9.0 0.0 2.0 0.0 0.0 1.5 12.5*

+ 7.0 0.0 4.0 0.0 0.0 2.0 13.0*

Chlorfenapyr (4.08 μM) − 5.5 0.0 4.0 0.0 1.0 2.0 12.5*

+ 4.5 0.0 5.5 0.0 0.0 1.0 11.0*

Imidacloprid (114.63 μM) − 1.5 0.0 2.5 0.0 0.0 0.0 4.0

+ 1.0 0.0 2.0 0.0 0.0 0.0 3.0

MMS (40 μg/ml)

BP (2 μg/ml)

− 8.0 9.0 2.0 1.0 0.0 2.0 22.0**

+ 2.0 2.0 3.0 4.0 0.0 3.0 14.0*

Crg chromosome gap, Crb chromosome break, Ctg chromatid gap, Ctb chromatid break, Frg fragment, Dicdicentric chromosome, MMS methyl methanesulfonate, BP benzo[a]pyrene

∗Significantly different from control (p<0.05); ∗∗significantly different from control (p<0.01)

Table 4 Micronucleusinductions in CHOK1 cells treatedwith abamectin, chlorfenapyr, andimidacloprid

Pesticide S9 mix Distribution of micronucleated cells MN frequenciesa

1 MN 2 MN >2 MN

Control − 8.5 0.0 0.0 8.5±0.5

+ 6.0 0.0 0.0 6.0±1.0

Abamectin (2.85 μM) − 17.0 1.5 0.0 18.5±1.5*

+ 14.5 0.0 0.0 14.5±0.5**

Chlorfenapyr (4.08 μM) − 22.5 5.5 0.0 28.0±3.0*

+ 23.5 3.5 0.0 27.0±1.0**

Imidacloprid (114.63 μM) − 21.0 1.0 0.0 22.0±3.0*

+ 18.0 0.0 0.0 18.0±2.0*

MMC − 30.0 4.5 0.5 35.0±2.0**

BP + 21.5 4.0 5.5 31.0±1.0**

MN micronucleus, MMC mitomycin C (4.5 μg/ml, positive control in absence of S9 mix), BP benzo(a)pyrene(2 μg/ml, positive control in presence of S9 mix)

∗p<0.05, ∗∗p<0.01; significant difference compared with controla Data are presented as mean ± SE of micronuclei/500 binucleated cytokinesis-blocked cells

Environ Sci Pollut Res

Author's personal copy

hepatocytes and brain neurons of King pigeon (Chen et al.2013; Zhu et al. 2013). Ivermectin, a member of theavermectin family, and its commercial formulation Ivomec®originated cytotoxic effects in CHOK1 cells (Molinari et al.2009). The present results showed that the MTTwas the mostsensitive tested bioassay for assessing the cytotoxicity ofabamectin. The MTT assay is an indicator for the mitochon-drial function. In accordance with our results, Maioli et al.(2013) reported that abamectin inhibited the mitochondrialactivity in the rat hepatocytes, decreasing ATP synthesis, lead-ing to the cell death. Little metabolism of avermectin has beenobserved in animals, and up to 80–98 % of the parent com-pound was found in the feces (Sun et al. 2005). Maioli et al.(2013) proved that abamectin, not its metabolites, is responsi-ble for the cytotoxic effect on isolated hepatocytes. The ultra-structural examination showed swollen mitochondria and un-clear structure of the inner membranes in abamectin-treated cells from Spodoptera frugiperda (Sf9) (Huang et al.2011). The authors supposed that mitochondria may beinvolved in the apoptosis initiation effect of abamectin. Ourresults indicate that abamectin cytotoxicity was time-depen-dent, particularly in the MTT assay (p<0.001). In agreementwith our findings, Huang et al. (2011) reported that abamectindecreased the cell viability in a time-dependent manner in Sf9cell line. Chlorfenapyr exhibited cytotoxic effects in the threeassays. Chlorfenapyr is a pyrrole proinsecticide; it is activatedby oxidative elimination of N-ethoxymethyl group,which inhibits mitochondrial ATP production throughuncoupling of the mitochondrial oxidative phosphorylationthat might produce reactive oxygen radicals (Black et al.1994). Studies on the cytotoxicity of chlorfenapyr are verylimited. Chlorfenapyr caused a significant inhibition in the cellgrowth and a marked decrease in the ATP concentration in Sf9cells (Saito 2005; Saito et al. 2005). Aljabr et al. (2014) re-ported a cytotoxic action of chlorfenapyr against the midgutcel l cul ture (RPW-1) f rom the red palm weevi lRhynchophorus ferrugineus. Our results showed thatimidacloprid is less cytotoxic to the CHOK1 cells comparedwith abamectin and chlorfenapyr, but it still displays acytotoxic action to some extent, especially with the MTT

assay. Su et al. (2007) showed that imidacloprid is able toinhibit the growth of flounder gill (FG) cell culture causingsevere injury to the mitochondria. They suggested that themitochondria are probably the primary target of imidacloprid.

The presence of FCS in the culture medium significantlyreduced the cytotoxicity of abamectin and chlorfenapyr in theNRI and TCP assays, indicating the important effect of theprotein binding on the availability of these compounds toreach their targets. On contrary, FCS increased thecytotoxicity of imidacloprid, indicating the toxifyinginteraction between FCS and imidacloprid. Freshney (1987)showed that FCS is a complex mixture containing differentprotein molecules, which may bind with the tested substanceand reduce its bioavailability and penetration to the targetcells; however, both reduction and enhancement of pesticidecytotoxicity by binding proteins were reported (Sogorb et al.2002; Gülden et al. 2003).

The glutathione redox cycle is playing a vital role inprotecting cells from oxidative injury (Meister 1983). Alter-ation in the activity of glutathione redox cycle components isaffected by the chemical structure and concentrations of pes-ticides that cells are exposed to (EL-Shenawy 2010). Thepresent results indicate that abamectin and imidacloprid sig-nificantly reduced the GRD activity in the presence and ab-sence of antioxidants compared with the control. However, alltested antioxidants in abamectin treatment and vitamin E inimidacloprid treatment ameliorated the GRD activity to levelsover that of the antioxidant-free control. Abdollahi et al.(2004) observed a reduction of GRD activity in pesticideworkers and explained this reduction by the implication of thisenzyme in regeneration of GSH. GRD is an important enzymethat reduces GSSG to its sulfhydryl form, GSH, which iscrucial for cell proliferation, viability, and protection fromoxidative damage (Tandoğan and Ulusu 2006). Inhibition ofGRD disturbs cellular prooxidant/antioxidant steadiness andmay contribute to the genesis of several diseases (Tandoğanand Ulusu 2006). Significant inhibition of GPX activity wasobserved with all tested pesticides in the absence or presenceof tested antioxidants compared with their corresponding con-trols. However, the tested antioxidants significantly increasedthe activity of GPX in imidacloprid-treated cells comparedwith the antioxidant-free/imidacloprid-treated cells. Also, vi-tamin E ameliorated the abamectin-reducing effect on GPXactivity. GPX is one of the most important enzymes of the cellantioxidant defense system; it catalyzes the GSH-dependentreduction of H2O2 and other peroxides (Pigeolet et al. 1990;Lei 2002). Reduced levels of GPX activity following pes-ticide exposure have been reported in the neuronal (SH-SY5Y) cells (Jia and Misra 2007). All the tested pesticidescaused a significant inhibition of GST activity in the presenceand absence of GSH compared with their corresponding con-trols. Abamectin and imidacloprid significantly inhibited theGST activity in the presence of vitamin C compared with the

1 2 3 4 5 6 7 8M1032

1.51

0.5

kbp

Fig. 2 Agarose gel electrophoresis of CHOK1 cells genomic DNA after24-h exposure to abamectin, chlorfenapyr, and imidacloprid. Lanes M:marker; 1 and 2: control (−S9 and+S9); 3 and 4: abamectin (−S9 and+S9); 5 and 6: chlorfenapyr (−S9 and+S9); 7 and 8 imidacloprid (−S9and+S9)

Environ Sci Pollut Res

Author's personal copy

parallel controls. With all tested pesticides, the ameliorativeeffect of vitamin E was clear; it increased the GST activity tolevels close to that of antioxidant-free control. GST plays akey role in the cellular detoxification of endogenous and xe-nobiotic chemicals (Chronopouloua et al. 2012). Inhibitionand induction of human and animal GST by pesticides havebeen reported (Ezemonye and Tongo 2010; Chronopoulouaet al. 2012); the differential alteration in the GST activity ispesticide- and tissue-specific (Ezemonye and Tongo 2010).Abamectin and chlorfenapyr significantly increased theglutathione content; the presence of vitamin C amelioratedthis effect. The induction of the glutathione content was alsoobserved by Barlow et al. (2005) in the rat PC12 cells afterexposure to the fungicide, maneb; the authors attributed thiseffect to the increase of -glutamyl cysteine synthetase, theenzyme involved in the production of GSH precursor. Gluta-thione is a critical constituent of the cellular antioxidant de-fense system, and its reduced form is the predominant reduc-ing agent in animal cells (Sebastià et al. 2003; Wu et al. 2004).Alterations of the glutathione status by toxic agents causeoxidative stress leading to mammalian cytotoxicity (Vettoriet al. 2005). Therefore, the in vitro studies with mammaliancells often depend on the cellular glutathione status as an earlybiomarker of cytotoxicity (Schoonen et al. 2005). Antioxi-dants differ in their binding site inside the organism as wellas in their affinity to the free radicals; for example, GSH andvitamin C are powerful scavengers of reactive oxygen species(ROS), particularly in aqueous medium. In contrast, vitamin Efunctions principally in the membrane lipid bilayers (Inoue2001). Therefore, no common effective antioxidant is foundas a universal treatment (Matkovics 2001)

The present results revealed a significant induction of CAand MN frequencies in CHOK1 cells exposed 24 h toabamectin. On the other hand, no DNA damage was observedfor the same treatment. Abamectin induced single-strandDNA breaks in the rat hepatocytes (EPA 1989); there are rarestudies on the genotoxicity of abamectin. Molinari et al.(2009, 2013) found that ivermectin did not induce SCE inthe CHOK1 cells; however, it induced single-DNA-strandbreaks. Ivermectin showed genotoxic effects on the somaticcells of the mother Wister rats and higher MN frequencies inthe red blood cells from the embryos’ umbilical cord com-pared with the control (El-Ashmawy et al. 2011).Chlorfenapyr caused significant inductions of CA and MNin the presence and absence of S9 mix. Studies on thegenotoxicity of CPF are very limited. The induction of stressprotein genes in cultured cells of cabbage armywormMamestra brassicae treated with chlorfenapyr wasconcentration- and time-dependent (Sonoda and Tsumuki2007); the authors attributed this induction to defend the cellsfrom the reactive oxygen radicals. The present results showedthat imidacloprid did not induce CA or DNA damage; how-ever, a significant induction of MN was detected in the

absence and presence of S9 mix. On contrary, Stivaktakiset al. (2010) reported no significant changes in the MN fre-quencies in the imidacloprid-treated blood cells. Fourneonicotinoid pesticides, including imidacloprid, significantlyinduced DNA damage when measured by the comet frequen-cy and the tail length (Calderón-Segura et al. 2012). Admire,the commercial product of imidacloprid, produced calf thy-mus DNA adducts upon activation by S9 mix (Shah et al.1997). Exposure of the human peripheral blood lymphocytesto imidacloprid (0.05 to 0.5 mg/l) significantly increased thelevels of SCE and MN and enhanced DNA strand breaks(Feng et al. 2005); however, imidacloprid (0.1 to 100 μg/ml)did not affect the frequency of SCE and MN formation(Demsia et al. 2007). In the presence and absence of S9 mix,imidacloprid (20 μM) significantly increased the frequency ofMN in the peripheral blood lymphocytes and DNA strandbreaks in the leukocytes (Costa et al. 2009). Recently, DeArcaute et al. (2014) demonstrated that imidacloprid can beconsidered a harmful agent with genotoxic effects at bothDNA and chromosomal levels. The molecular mechanismsmotivating the genotoxicity of the neonicotinoid insecticidesare generally unknown. In vitro studies of Yao et al. (2006)have indicated that acetamiprid may induce ROS gener-a t ion in bacter ia . However, the incubat ion ofimidacloprid with Yurkat cells and lymphocytes did notincrease the production of ROS (Costa et al. 2009).Although these results are inconsistent, Valko et al.(2006) suggested that the neonicotinoid insecticides aredirect genotoxic compounds that could act as a sourceof ROS or free radicals in the treated human cells.

Generally, the induction of genotoxicity may lead tofurther problems of mutagenic and carcinogenic activi-ties (Valko et al. 2006). Previous epidemiological stud-ies demonstrated a relationship between pesticide expo-sure and the occurrence of cancer (Dich et al. 1997).Therefore, the current awareness of the real or potentialhazards of pesticides considering their cytotoxic/genotoxic actions cannot be neglected. Thus, further in-vestigations are needed to gain a comprehensive andcomplete knowledge of the possible mechanismsth rough wh ich abamec t i n , ch lo r f enapy r, andimidacloprid exert their cytotoxic/genotoxic effects.

Acknowledgments This project was funded by the National Plan forScience, Technology and Innovation (MAARIFAH), King AbdulazizCity for Science and Technology, Kingdom of Saudi Arabia, AwardNumber (09-ENV837-02).

References

Abdollahi M, Ranjbar A, Shadnia S, Nikfar S, Rezaiee A (2004)Pesticides and oxidative stress: a review. Med Sci Monit 10:RA14–RA147

Environ Sci Pollut Res

Author's personal copy

Akerboom TPM, Sies H (1981) Assay of glutathione, glutathione disul-fide and glutathione mixed disulfides in biological samples. MethodEnzymol 77:373–382

Aljabr AM, Rizwan-ul-Haq M, Hussain A, Al-Mubarak AI, AL-AyiedHY (2014) Establishing midgut cell culture from Rhynchophorusferrugineus (Olivier) and toxicity assessment against ten differentinsecticides. In Vitro Cell Dev Biol Anim 50(4):296–303

Al-Sarar AS, Abobakr Y, Al-Erimah GS, Hussein HI, Bayoumi AE(2009) Hematological and biochemical alterations in occupationallypesticides-exposed workers of Riyadh municipality, Kingdom ofSaudi Arabia. Res J Environ Toxicol 3(4):179–185

Al-Sarar AS, Abobakr Y, Bayoumi AE, Hussein HI, Al-Ghothemi M(2014) Reproductive toxicity and histopathological changes inducedby lambda-cyhalothrin in male mice. Environ Toxicol 29(7):750–762

Aly N, EL-Gendy K,Mahmoud F, El-Sebae A (2010) Protective effect ofvitamin C against chlorpyrifos oxidative stress in male mice. PesticBiochem Physiol 97(1):7–12

Arias LA, Bojacá CR, Ahumada DA, Schrevens E (2014) Monitoring ofpesticide residues in tomato marketed in Bogota, Colombia. FoodControl 35:213–217

Banerjee BD, Seth V, Bhattacharya A, Pasha ST, Chakraborty AK (1999)Biochemical effects of some pesticides on lipid peroxidation andfree-radical scavengers. Toxicol Lett 107(1-3):33–47

Barlow BK, Lee DW, Cory-Slechta DA, Opanashuk LA (2005)Modulation of antioxidant defense systemsby the environmentalpesticide maneb in dopaminergic cells. Neurotoxicology 26:63–75

Bayoumi AE (1998) Utilization of the alternative methods in the toxico-logical evaluation of environmental contaminants. Dissertation,University of Leon, Spain

Bayoumi AE, Perez-Pertejo Y, Ordóñez C, Reguera RM, Balaña-FouceR, Ordóñez D (2000) Changes in the glutathione-redox balanceinduced by the pesticides heptachlor, chlordane and toxaphene inCHO-K1 cells. Bull Environ Contam Toxicol 65:748–755

Bayoumi AE, Garcia-Fernandez AJ, Ordonez C, Perez-Pertejo Y, CubriaJC, Reguera RM, Balana-Fouce R, Ordonez D (2001) Cyclodieneorganochlorine insecticide-induced alterations in the sulfur-redoxcycle in CHO-K1 cells. Comp Biochem Physiol C ToxicolPharmacol 130:315–323

Bertheussen K, Yousef MI, Figenschau Y (1997) A new sensitive cellculture test for the assessment of pesticide toxicity. J Environ SciHealth 32:195–211

Black BC, Hollingworth RM, Ahammadsahib KI, Kubkel CB, DonovanS (1994) Insecticidal action and mitochondrial uncoupling activityof AC-303, 603 and related halogenated pyrroles. Pestic BiochemPhysiol 50:115–128

Bols N, Dayeh V, Lee L, Schirmer K (2005) Use of fish cell lines in thetoxicology. In: Moon TW, Mommsen TP (eds) Biochemistry andmolecular biology of fishes. Elsevier Science, Amsterdam, pp43–84

Borenfreund E, Puerner JA (1985) Toxicity determined in vitro by mor-phological alteration and neutral red absorption. Toxicol Lett 24:119–124

Bradford M (1976) A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dyebinding. Anal Biochem 72:248–254

Calderón-Segura ME, Gómez-Arroyo S, Villalobos-Pietrini R, Martínez-Valenzuela C, Carbajal-López Y, Calderón-Ezquerro MD, Cortés-Eslava J, García-Martínez R, Florñes-Ramírez D, Rodríguez-Romero MI, Méndez-Pérez P, Bañuelos-Ruíz E (2012) Evaluationof genotoxic and cytotoxic effects in human peripheral blood lym-phocytes exposed in vitro to neonicotinoid insecticides news. JToxicol. doi:10.1155/2012/612647

Carlberg Y, Mannervik B (1985) Glutathione reductase. MethodEnzymol 113:484–490

Chen LJ, Sun BH, Qu JP, Xu S, Li S (2013) Avermectin induced inflam-mation damage in king pigeon brain. Chemosphere 93(10):2528–34

Chronopouloua EG, Papageorgiou AC,Markoglou A, Labrou NE (2012)Inhibition of human glutathione transferases by pesticides: develop-ment of a simple analytical assay for the quantification of pesticidesin water. J Mol Catal B Enzym 81:43–51

Costa C, Silvari V, Melchini A, Catania S, Heffron JJ, Trovato A, DePasquale R (2009) Genotoxicity of imidacloprid in relation to met-abolic activation and composition of the commercial product. MutatRes 672:40–44

Cully DF, Vassilatis DK, Liu KK, Paress PS, van der Ploeg LHT,Schaeffer JM, Arena JP (1994) Cloning of an avermectin-sensitiveglutamate-gated chloride channel from Caenorhabditis elegans.Nature 371(6499):707–11

Dakova T (2005) Abamectin and closantel residues in milk from sheeptreated with abantel-b. Trakia J Sci 3(5):17–19

De Arcaute CR, Pérez-Iglesiasa JM, Nikoloff N, Natale GS, Soloneski S,Larramendy ML (2014) Genotoxicity evaluation of the insecticideimidacloprid on circulating blood cells of Montevideo tree frogHypsiboas pulchellus tadpoles (Anura, Hylidae) by comet and mi-cronucleus bioassays. Ecol Indic 45:632–639

Demsia G, Vlastos D, Goumenou M, Matthopoulos DP (2007)Assessment of the genotoxicity of imidacloprid and metalaxyl incultured human lymphocytes and rat bone marrow. Mutat ResGenet Toxicol Environ Mutagen 634(1-2):32–39

Dich J, Zahm SH, Hanberg A, Adami HO (1997) Pesticides and cancer.Cancer Causes Control 8(3):420–443

El-Ashmawy IM, El-Nahas AF, Bayad AE (2011) Teratogenic and cyto-genetic effects of ivermectin and its interaction with P-glycoproteininhibitor. Res Vet Sci 90:116–123

Elbetieha A, Da’as SI (2003) Assessment of antifertility activities ofabamectin pesticide in male rats. Ecotoxicol Environ Saf 55(3):307–313

Elsharkawy EE, Yahia D, El-Nisr NA (2013) Sub-chronic exposure tochlorpyrifos induces hematological, metabolic disorders and oxida-tive stress in rat: attenuation by glutathione. Environ ToxicolPharmacol 35(2):218–227

El-Shenawy NS (2010) Effects of insecticides fenitrothion, endosulfanand abamectin on antioxidant parameters of isolated rat hepatocytes.Toxicol In Vitro 24(4):1148–57

EPA (1989) Avermectin (Agri-Mek, Affirm) pesticide fact sheet 9/89.Cornell University, Ithaca

EPA (1994) Office of Pesticide Programs, pesticide fact sheet:imidacloprid, Washington, DC

Ezemonye L, Tongo I (2010) Sublethal effects of endosulfan and diazi-non pesticides on glutathione-S-transferase (GST) in various tissuesof adult amphibians (Bufo regularis). Chemosphere 8:214–217

Fenech M (1993) The cytokinesis-block micronucleus technique and itsapplication to genotoxicity studies in human populations. EnvironHealth Perspect 101(3):101–107

Feng S, Kong Z, Wang X, Peng P, Zeng EY (2005) Assessing thegenotoxicity of imidacloprid and RH-5849 in human peripheralblood lymphocytes in vitro with comet assay and cytogenetic tests.Ecotoxicol Environ Saf 61(2):239–246

Freshney RI (1987) Culture of animal cells. A manual of basic technique.Wielly Liss press, New York, pp 72–81

Garcia-Fernandez AJ, Bayoumi AE, Perez-Pertejo Y, Romeros D,Ordóñez C, Reguera RM, Balaña-Fouce R, Ordóñez D (2002)Changes in glutathione-redox balance induced by hexachloro-cyclohexane and lindane in CHO-K1 cells. Xenobiotica32(11):1007–1016

Gilden RC, Huffling K, Sattler B (2010) Pesticides and health risks. JObstet Gynecol Neonatal Nurs 39:103–110

Gülden M, Mörchel S, Seibert H (2003) Serum albumin binding at cyto-toxic concentrations of chemicals as determined with a cell prolifer-ation assay. Toxicol Lett 137:159–168

Environ Sci Pollut Res

Author's personal copy

Habig WH, Pabst MJ, Jakoby WB (1974) Glutathione S-transferase: thefirst enzymatic step in mercapturic acid formation. J Biol Chem 249:7130–7139

Huang JF, Tian M, Lv CJ, Li HY, Muhammad R, Zhong GH (2011)Preliminary studies on induction of apoptosis by abamectin inSpodoptera frugiperda (Sf9) cell line. Pestic Biochem Physiol100(3):256–263

Hunt DA, Treacy MF (1998) Pyrrole insecticides: a new class of agricul-turally important insecticides functioning as uncouplers of oxidativephosphorylation. In: Ishaaya I, Degheele D (eds) Insecticides withnovel modes of action, mechanism and application. Springer Verlag,Heidelberg

IAE (1986) Biological dosimetry: chromosome aberration analysis fordose assessment, In: Technical reports series no. 260, InternationalAtomic Energy Agency, Vienna, pp 59–63

Inoue M (2001) Protective mechanisms against reactive oxygen species.In: Arias IM, Boyer JL, Chisari FV, Fausto N, Schachter D, ShafritzDA (eds) The liver: biology and pathobiology, 4th edn. LippincottWilliams & Wilkins, Philadelphia

Jeschke P, Nauen R, Schindler M, Elbert A (2011) Overview of the statusand global strategy for neonicotinoids. J Agric Food Chem 59:2897–2908

Jia ZH, Misra P (2007) Reactive oxygen species in in vitro pesticide-induced neuronal cell (SH-SY5Y) cytotoxicity: role of NFκB andcaspase-3. Free Radic Biol Med 42:288–298

Lei XG (2002) In vivo antioxidant role of glutathione peroxidase: evi-dence from knockout mice. Methods Enzymol 347:213–225

Maioli MA, de Medeiros HCD, Guelfi M, Trinca V, Pereira FTV,Mingatto FE (2013) The role of mitochondria and biotransformationin abamectin-induced cytotoxicity in isolated rat hepatocytes.Toxicol in Vitro 27:570–579

Matkovics A (2001) Antioxidánsok 2000- elmélet és gyakorlat. MagyBelorv Arch (54):35–40

Meister A (1983) Selective modification of glutathione metabolism.Science 220:472–477

Meister RT (1992) Farm chemicals handbook 92. Meister PublishingCompany, Willoughby

Molinari G, Soloneski S, Reigosa MA, Larramendy ML (2009) In vitrogenotoxic and cytotoxic effects of ivermectin and its formulationivome on Chinese hamster Ovary (CHO-K1) cells. J Hazard Mater165:1074–1082

Molinari G, Kujawski M, Scuto A, Soloneski S, Larramendy ML (2013)DNA damage kinetics and apoptosis in ivermectin-treated Chinesehamster ovary cells. J Appl Toxicol 33:1260–1267

Novelli A, Vieira BH, Cordeiro D, Cappelini LTD, Vieira EM, EspíndolaELG (2012) Lethal effects of abamectin on the aquatic organismsDaphnia similis, Chironomus xanthus and Danio rerio.Chemosphere 86(1):36–40

Pandey MR, Guo H (2014) Evaluation of cytotoxicity, genotoxicity andembryotoxicity of insecticide propoxur using flounder gill (FG) cellsand zebrafish embryos. Toxicol In Vitro 28:340–353

Pelletier B, Dhainaut F, Pauly A, Zahnd JP (1988) Evaluation of growthrate in adhering cell cultures using a simple colorimetric method.Biochem Biophys Methods 16:63–73

Pigeolet E, Corbisier P, Houbion A, Lambert D, Michiels C, Raes M,Zachary M, Remacle J (1990) Glutathione peroxidase, superoxidedismutase, and catalase inactivation by peroxides and oxygen de-rived free radicals. Mech Ageing Dev 51:283–297

Plumb JA, Milroy R, Kaye SB (1989) Effects of pH dependence of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide-formazan absorption on chemosensitivity determined by a noveltetrazolium-based assay. Cancer Res 49:4435–4440

Pollack P (2011) Fine chemicals: the industry and the business, 2nd edn.Wiley, New Jersey

Saito S (2005) Effects of pyridalyl on ATP concentrations in cultured Sf9cells. J Pestic Sci 30(4):403–405

Saito H, Iwami S, Shigeoka T (1991) In vitro cytotoxicity of 45 pesticidesto goldfish GF-scale (GFS) cells. Chemosphere 23:525–537

Saito S, Sakamoto N, Umeda K (2005) Effect of pyridalyl, a novel insec-ticide agent on cultured Sf9 cells. J Pestic Sci 30(1):17–21

Schoonen WGEJ, Westerink WMA, de Roos JADM, Debiton E (2005)Cytotoxic effects of 100 reference compounds on Hep G2 and HeLacells and of 60 compounds on ECC-1 and CHO cells. I. Mechanisticassays on ROS, glutathione depletion and calcein uptake. ToxicolIn Vitro 19:505–516

Sebastià J, Cristòfol R, Martín M, Rodríguez-Farré E, Sanfeliu C (2003)Evaluation of fluorescent dyes for measuring intracellular glutathi-one content in primary cultures of human neurons and neuroblasto-ma SH-SY5Y. Cytometry A 51:16–25

Shah RG, Lagueux J, Kapur S, Levallois P, Ayotte P, Tremblay M, Zee J,Poirier GG (1997) Determination of genotoxicity of the metabolitesof the pesticides guthion, sencor, lorox, reglone, daconil and admireby 32P-post labeling. Mol Cell Biochem 169:177–184

Simon-Delso N, Amaral-Rogers V, Belzunces LP, Bonmatin JM,Chagnon M, Downs C, Furlan L et al (2015) Systemic insecticides(neonicotinoids and fipronil): trends, uses, mode of action and me-tabolites. Environ Sci Pollut Res 22:5–34

Sogorb MA, Carrera V, Benabent M, Vilanova E (2002) Rabbit serumalbumin hydrolyzes the carbamate carbaryl. Chem Res Toxicol 15:520–526

Soloneski S, Larramendy ML (2010) Sister chromatid exchanges andchromosomal aberrations in Chinese hamster ovary (CHO-K1) cellstreated with the insecticide pirimicarb. J Hazard Mater 174(1-3):410–415

Soloneski S, Reigosa MA, Molinari G, González NV, Larramendy ML(2008) Genotoxic and cytotoxic effects of carbofuran and furadan®on Chinese hámster ovary (CHO-K1) cells. Mutat Res 656:68–73

Sonoda S, Tsumuki H (2007) Induction of heat shock protein genes bychlorfenapyr in cultured cells of the cabbage armyworm,Mamestrabrassicae. Pestic Biochem Physiol 89:185–189

Spielmann H, Goldberg AM (1999) In vitro methods. In: Marquardt H,Schafer SG, McClellan R, Welsch F (eds) Toxicology. Academicpress, USA, pp 113–138

Stivaktakis P, Vlastos D, Giannakopoulos E, Matthopoulos DP (2010)Differential micronuclei induction in human lymphocyte cultures byimidacloprid in the presence of potassium nitrate. Sci World J 10:80–89

Su F, Zhang S, Li H, Guo H (2007) In vitro acute cytotoxicity ofneonicotinoid insecticide imidacloprid to gill cell line of flounderParalichthy olivaceus. Chin J Oceanol Limnol 25(2):209–214

Sun Y, Diao X, ZhangQ, Shen J (2005) Bioaccumulation and eliminationof avermectin B1a in the earthworms (Eisenia foetida).Chemosphere 60:699–704

Tandoğan B, Ulusu NN (2006) Kinetic mechanisms and molecular prop-erties of glutathione reductase. FABAD J Pharm Sci 31:230–237

Tomizawa M, Casida JE (2003) Selective toxicity of neonicotinoids at-tributable to specificity of insect andmammalian nicotinic receptors.Annu Rev Entomol 48:339–364

Tomizawa M, Casida JE (2005) Neonicotinoid insecticide toxicology:mechanisms of selective action. Annu Rev Pharmacol Toxicol 45:247–268

Tomlin CDS (2000) The pesticide manual, a world compendium, 12thedn. British Crop Protection Council, London

Valko M, Rhodes CJ, Moncol J, Izakovic M, Mazur M (2006) Freeradicals, metals and antioxidants in oxidative stress induced cancer.Chem Biol Interact 160(1):1–40

van Leeuwen T, Stillatus V, Tirry L (2004) Genetic analysis and cross-resistance spectrum of a laboratory-selected chlorfenapyr resistantstrain of two-spotted spider mite (Acari: Tetranychidae). Exp ApplAcarol 32:249–261

Vettori MV, Caglieri A, Goldoni M, Castoldi AF, Dare E, Alinovi R,Ceccatelli S, Mutti A (2005) Analysis of oxidative stress in SK-N-

Environ Sci Pollut Res

Author's personal copy

MC neurons exposed to styrene 7,8-oxide. Toxicol In Vitro19:11–20

Wataha JC, Hanks CT, Craig RG (1991) The in vitro effects metal cationson eukaryotic cell metabolism. J Biomed Mater Res 25:1133–1149

Wendel A (1981) Glutathione peroxidase. Method Enzymol 77:325–333WHO (2002) The WHO recommended classification of pesticides by

hazard and guidelines to the classification 2000–2002. WorldHealth Organization, Geneva, pp 1–58

Wu G, Fang Y, Yang S, Lupton JR, Turner ND (2004) Glutathione me-tabolism and its implications for health. J Nutr 134:489–492

Yao XH, Min H, Lv ZM (2006) Response of superoxide dismutase,catalase, and ATPase activity in bacteria exposed to acetamiprid.Biomed Environ Sci 19(4):309–314

Zang Y, Zhong Y, Luo Y, Kong ZM (2000) Genotoxicity of two novelpesticides for the earthworm, Eisenia fetida. Environ Pollut 108:271–278

Zhu WJ, Li M, Liu C, Qu JP, Min YH, Xu SW, Li S (2013) Avermectininduced liver injury in pigeon: mechanisms of apoptosis and oxida-tive stress. Ecotoxicol Environ Saf 98:74–81

Environ Sci Pollut Res

Author's personal copy