Chronic exposure to imidacloprid induces inflammation and oxidative stress in the liver & central nervous system of rats

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Chronic exposure to imidacloprid induces inﬂammation and oxidative stress

in the liver & central nervous system of rats

Vesile Duzguner

b,⇑

_{, Suat Erdogan}

a a

Mustafa Kemal University, Veterinary Faculty, Biochemistry Department, 31040 Antakya, Hatay, Turkey b

Ardahan University, Health Services Vocational School, 75000 Ardahan, Turkey

a r t i c l e

i n f o

Article history:

Received 13 October 2011 Accepted 27 June 2012 Available online 27 July 2012 Keywords: Imidacloprid Inﬂammation Insecticide Nitric oxide Oxidative stress

a b s t r a c t

Imidacloprid is the most important example of the neonicotinoid insecticides known to target the nico-tinic acetylcholine receptor (nAChR) in insects, and potentially in mammals. In the present study, oxidant and inflammatory responses to chronic exposure of imidacloprid was studied in rats. Wistar rats were randomly allocated into two groups as control and imidacloprid-exposed group (n = 10 rat/each group). 1 mg/kg/BW/day imidacloprid was administrated orally by gavage for 30 days. After exposure, rats were euthanized and liver and brain samples were surgically removed for analyses. Imidacloprid application caused a significant increase in nitric oxide production in brain (p < 0.05) and liver (p < 0.001). The quan-titative analyses of mRNA confirmed the finding that imidacloprid induced the mRNA transcriptions of the three isoforms of nitric oxide synthases (iNOS, eNOS, nNOS) in brain and two isoforms (iNOS, eNOS) in the liver. Exposure to imidacloprid caused significant lipid peroxidation in plasma, brain (p < 0.001) and liver (p < 0.003). While the superoxide-generating enzyme xanthine oxidase activity was elevated in both tissues (p < 0.001), myeloperoxidase activity was increased only in the liver (p < 0.001). Antioxi-dant enzyme activities showed various alterations following exposure, but a significantly depleted anti-oxidant glutathione level was detected in brain (p < 0.008). Evidence of chronic inflammation by imidacloprid was observed as induction of pro-inflammatory cytokines such as TNF-a, 1b, 6, IL-12 and IFN-cin the liver and brain. In conclusion, chronic imidacloprid exposure causes oxidative stress and inflammation by altering antioxidant systems and inducing pro-inflammatory cytokine production in the liver and central nervous system of non-target organisms.

1. Introduction

The neonicotinoids are a new major class of highly potent insec-ticides that are used for crop protection against piercing–sucking insects of cereals, vegetables, tea and cotton, and for ﬂea control in cats and dogs. Currently the best known neonicotinoid is imida-cloprid [IMI, 1-(6-chloro-3-pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine] which is an active substance in such commercial insecticide preparations as Conﬁdor, Gaucho, Prestige, Admire, and Premier which are increasingly used in agriculture[1,2]. Imi-dacloprid and its analogs are remarkably potent neurotoxic insec-ticides, which act as nicotinic acetylcholine receptor agonists (nAChRs)[3]. nAChRs play a central role in rapid cholinergic synap-tic transmission and are important targets of insecsynap-ticides [3,4]. Most neonicotinoids are partial agonists of native and recombinant nAChRs in both mammals and insects with differential selectivity conferred by only minor structural changes [5,6]. Tomizawa [7]

suggests that nAChRs may be up-regulated in mammals by chronic exposure to imidacloprid or its metabolites.

It is also known that free radicals play an important role in the toxicity of pesticides and environmental chemicals[8]. Pesticide chemicals such as insecticides may induce oxidative stress leading to generation of free radicals and alterations in antioxidants or free radical scavenging enzyme systems[9,10]. The data on experimen-tal animals either in vivo or in vitro[11–13]indicate that the en-zymes associated with antioxidant defense mechanisms are altered under the inﬂuence of pesticides. Moreover, oxidative stress and DNA damage have been proposed as mechanisms link-ing pesticide exposure to health effects such as cancer and neuro-logical diseases. During metabolism of the insecticides reactive oxygen species (ROS) and reactive nitrogen species (RNS) such as nitric oxide (NO) can be generated. Nitric oxide is a diatomic free radical which plays critical roles in the homeostatic regulation of cardiovascular, neuronal and immune systems. Despite its impor-tant physiological functions, NO is also a well-known toxic agent [14]. Subsequently there is onset of an oxidative stress in central nervous system (CNS) structures. These structures include the hip-pocampus, cortex, striatum and cerebellum where mitochondrial

⇑Corresponding author. Tel.: +90 478 211 37 50.

E-mail addresses: v.duzguner@hotmail.com (V. Duzguner), serdogan1967@

hotmail.com(S. Erdogan).

Contents lists available atSciVerse ScienceDirect

Pesticide Biochemistry and Physiology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / p e s t

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respiratory chain dysfunctions have been noted. Oxidative mecha-nisms play an important role in insecticide-induced tissue damage not only by balancing oxidant-antioxidant status but also by inhib-iting neutrophil inﬁltration and regulating inﬂammatory media-tors[15,16].

Insecticides are associated with direct or indirect modulation of major and vital immune mechanisms and inﬂammatory activation might be an important mechanism underlying neurotoxicity of pesticides[17]. Its known that exposure to different insecticides could accelerate the synthesis of cytokines such as interferon gam-ma-

c

(IFN) and tumor necrosis factor-

a

(TNF), reduce anti-inﬂam-matory cytokine interleukin-10 (IL) and also inhibit signal transduction correlated with their toxicity[10,18,19].

Few studies have been performed in mammals with neonicoti-noid insecticides and their relationship with oxidative and inflam-matory events. Tomizawa [7] suggested that stimulation of the nervous system by acute or sustained exposure to these chemicals may lead to synaptic plasticity or attenuated neuronal functions. We have previously demonstrated that acute exposure to imida-cloprid leads to oxidative and inflammatory effects in rats[10]. Although imidacloprid is the most frequently used insecticide amongst the pesticides, it is possible that its chronic effects in non-target organisms such as mammals remain to be elucidated. In this study, we investigated the potential oxidative and chronic inflammatory effects of imidacloprid on the central nervous sys-tem and liver in rats.

2. Materials and methods 2.1. Animals and study design

Ten-week-old female Wistar rats (150 ± 25 g) were acclima-tized for 10 days before starting the experimental procedure. The rats were assigned randomly to either control (n = 10) or imidaclo-prid (n = 10) group and housed in a temperature controlled room (21–22 °C) with a 12 h dark–light cycle during the study.

Concentration of imidacloprid was calculated based on 1/15 of its LD50values and on body weight data[20]. Imidacloprid (Riedel,

Sigma–Aldrich) was suspended in corn oil and administered to rats at the dose of 1 mg/kg/bw-day by gavage during a 30 day period, whilst controls were treated with corn oil as vehicle. At the end of the exposure, the rats were anesthetized by intramuscular injec-tion of 50 mg/kg ketamine hydrochloride and blood was taken by puncturing the heart ventricle. The brain and liver were taken from each rat, washed with ice-cold physiological saline and used for biochemical studies. The protocols were approved by the Animals Experiments Ethics Committee of Firat University, Elazig, Turkey. 2.2. Biochemical assays

Tissue samples were homogenized in ice-cold homogenization buffer (10 mM Tris, 1 mM EDTA, 25 mM MgCl2, 0.1 mM

dithiothre-itol, 0.25 M sucrose, pH 7.4) containing complete protease inhibitor mixture (aprotinin, phenylmethylsulphonyl ﬂuoride, leupeptin, so-dium ﬂoride) (Sigma, Germany). Homogenates were centrifuged at 4 °C, 15 000 rpm for 10 min and the soluble fraction was retained. Protein concentrations of supernatants were measured by the method of Bradford[21]using bovine serum albumin as a stan-dard. The degree of lipid peroxidation was assessed by measuring malondialdehyde (MDA) levels in plasma and tissue samples[22]. Total superoxide dismutase (SOD) activity in the homogenates and plasma samples was determined according to the method of Sun et al.[23]. Nitric oxide concentration in plasma and tissue samples was analyzed indirectly by measuring the nitrite levels based on Griess reaction[24].

Xanthine oxidase (XO) activity was analyzed spectrophotomet-rically by the formation of uric acid from xanthine through in-creased absorbency at 293 nm, according to the method of Prajda and Weber [25]. Myeloperoxidase (MPO) activity was measured by the method of Andrews and Krinski[26]. Catalase activity was measured according to the method of Luck[27]. Glutathione per-oxidase (GSH-Px) activity was determined by a kinetic method using a commercial kit (RANSEL, Randox Laboratorius). The meth-od is based on Paglia and Valentine’s study[28].

Reduced glutathione (GSH) concentrations found in liver and brain homogenates were determined according to Sedlak and Lind-say[29]. GSH was reacted with 5,5-dithiobis-2-nitrobenzoic acid resulting in the formation of a product with a maximal absorbance at 410 nm. The results were expressed as

l

mol/mg protein.

To reveal the relationship between activation of nicotinic recep-tors on neurons and intracellular calcium levels, plasma calcium concentrations were determined using a commercial kit (Teco Diagnostics, USA). Finally, the activities of liver enzymes such as alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were measured using commer-cial kits (Thermo Inﬁnity, USA).

2.3. RNA preparation and reverse transcriptase polymerase chain reaction (RT–PCR)

For the evaluation of mRNA expression, real-time PCR was per-formed in a Strategene Mx 3005P QPCR system. Total RNA was ex-tracted from brain and liver samples using TRIZOL reagent (Sigma) according to the manufacturer’s instructions. Two micrograms of total RNA was reverse transcribed in a reaction volume of 20

l

l using a reverse transcriptase kit (Fermentase). One microgram of each cDNA was used as templates for amplification using SYBER Green PCR amplification reagent and gene-specific primers. The primer sets used were from Thermo Electron Corporation (Ger-many). The specific primers for TNF-

a

, IFN-

c

, IL-1b, IL-6, IL-12, neuronal- (nNOS), inducible- (iNOS), and endothelial nitric oxide synthase (eNOS) and the PCR reaction conditions are given in Ta-ble 1. The threshold cycle (Ct) number for the transcripts is nor-malized to b-actin by subtracting the average Ct number for each treatment. Expression of this housekeeping gene should not alter in response to imidacloprid exposure. Each PCR reaction was per-formed in triplicate. The mean relative expressions of the targeting genes were calculated and the differences were determined using the 2DDCt

method[30]. 3. Results

3.1. The effect of imidacloprid on oxidant and antioxidant systems Nitric oxide is produced from the amino acidL-arginine by the

enzymatic action of NOS. Whilst it has physiological actions, abnormal production of NO can damage numerous molecules (including lipids, proteins and DNA) causing alterations in the functioning of target cells potentially leading to cell death. In the present study, we found that long term exposure to imidacloprid signiﬁcantly increased NO production by approximately 23% in brain (p < 0.05) and 50% in liver (p < 0.001) (Table 2). The level of lipid peroxidation, as indicated by malondialdehyde (MDA) forma-tion, was markedly increased by 1.24-, 1.45-, 2.35-fold respectively in liver (p < 0.003), brain and plasma (p < 0.001) when imidacloprid exposed animals were compared to control (Table 2). Similar to NO production and MDA formation, imidacloprid signiﬁcantly stimu-lated the activity of oxidant generating enzymes such as xanthine oxidase in the brain and liver (p < 0.001), and myeloperoxidase in the liver (p < 0.001) (Table 3). Catalase activity was also elevated

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almost 2-fold in brain (p < 0.05), though unchanged in liver ( Ta-ble 3). However, neither superoxide dismutase (SOD) nor glutathi-one peroxidase (GSH-Px) activities were affected by imidacloprid exposure (Table 3). A reduced antioxidant capacity in the brain was also evidenced by the 8% decrease in intracellular GSH concen-tration (p < 0.008) (Table 2).

To investigate the possible involvement of calcium mobiliza-tion, we measured Ca2+_{levels and found that imidacloprid}

expo-sure increased plasma Ca2+_{concentration from 8.19 to 8.62 mg/}

dl (p < 0.05;Table 4). The exposure to imidacloprid also decreased the activities of liver enzymes such as aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) by 1.5-, and 2.6-fold, respectively (p < 0.001), but did not change alanine aminotransfer-ase (ALT) activity (Table 4).

3.2. The effects of imidacloprid on iNOS, nNOS, eNOS and cytokine transcription

The mRNA transcription of three isoforms of nitric oxide syn-thase enzymes (iNOS, nNOS, eNOS) was up-regulated by 4.76-,

1.13- and 3.97-fold, respectively in the brain. Inducible NOS (1.10-fold) and eNOS (1.16-fold) expressions were also enhanced in the liver by imidacloprid treatment (Fig. 1A and B). Quantitative mRNA analyses have demonstrated that imidacloprid administra-tion caused up-regulaadministra-tion of pro-inﬂammatory cytokine mRNAs

Table 1

Primer sequences and quantitative RT-PCR reaction conditions.

Transcript Primer sequences Reaction conditions

TNF-a F

R

50_{-TAC TGA ACT TCG GGG TGA TTG GTC C- 3}0

50_{-CAG CCT TGT CCC TTG AAG AGA ACC-3}0

94 °C-1 min/60 °C-45 s/72 °C-2 min (33 cycle)

IFN-c F

R

50_{-ATC TGG AGG AAC TGG CAA AAG GAC G-3}0

50_{-CCT TAG GCT AGA TTC TGG TGA CAG C-3}0

94 °C-15 s/60 °C-75 s/72 °C-30 s (35 cycle)

iNOS F

R

50_{-GGC AGA CTG GAT TTG GCT GGT C-3}0

50_{-AGG TGT TCC CCA GGT AGG TAG C-3}0

94 °C-20 s/59 °C-10 s/72 °C-1 min (35 cycle)

nNOS F

R

50_{-ACC CCG TCC TTT GAA TAC CAG-3}0

50_{-GAC GCT GTT GAA TCG GAC CTT-3}0

92 °C-1 min/50 °C-1 min/72 °C-1 min (30 cycle)

eNOS F

R

50_{-AAG ACA AGG CAG CGG TGG AA-3}0

50_{-GCA GGG GAC AGG AAA TAG TT-3}0

94 °C-30 s/60 °C-30 s/72 °C-90 s (35 cycle)

IL-12 F

R

50_{-AGA TGA CAT CAC CTG GAC CT-3}0

50_{-CTT TGG TTC AGT GTG ACC TTC-3}0

94 °C-30 s/60 °C-30 s/72 °C-1 min (35 cycle)

IL-1b F

R

50_{-ATA GCA GCT TTC GAC AGT GAG-3}0

50_{-GTC AAC TAT GTC CCG ACC ATT-3}0

94 °C-30 s/50 °C-45 s/72 °C-90 s (30 cycle)

IL-6 F

R

50_{-TTG CCG AGT AGA CCT CAT AGT GAC C-3}0

50_{-CAA GAG ACT TCC AGC CAG TTG C-3}0

94 °C-30 s/55 °C-30 s/72 °C-1 min (35 cycle)

b-actin F

R

50_{-CAT CGT CAC CAA CTG GGA CGA C-3}0

50_{-CGT GGC CAT CTC TTG CTC GAA G-3}0

95 °C-60 s/55 °C-70 s/72 °C-100 s 35 (35 cycle)

Table 2

Nitric oxide (NO), malondialdehyde (MDA) and glutathione (GSH) levels in control (unexposed) and imidacloprid-treated rats.

NO MDA GSH Plasma (lmol/L) Brain (lmol/mg protein) Liver (lmol/mg protein) Plasma (lmol/L) Brain (lmol/mg protein) Liver (lmol/mg protein) Brain (lmol/mg protein) Liver (lmol/mg protein) Control 38.57 ± 3.20 3.35 ± 0.26 1.32 ± 0.20 2.42 ± 0.15 7.53 ± 0.46 16.43 ± 0.21 0.37 ± 0.01 3.90 ± 0.04 Imidacloprid 37.53 ± 2.24 4.35 ± 0.25*** 2.80 ± 0.007* 5.71 ± 0.60* 10.95 ± 0.39* 20.40 ± 1.00** 0.34 ± 0.004*** 4.01 ± 0.06 Values are mean ± S.E. from seven rats in each group. Control vs. imidacloprid-treated group within the same tissue.

*_{p < 0.001.}

** _{p < 0.003.}

***_{p < 0.05.}

Table 3

Antioxidant and oxidant enzyme activities in brain and liver homogenates of control (unexposed) and imidacloprid (IMI)-treated rats.

XO (U/g protein) MPO (U/g protein) CAT (k/g protein) SOD (U/mg protein) GSH-Px (U/mg protein)

Brain-control 9.4 ± 0.5 12.57 ± 0.43 0.261 ± 0.002 1.801 ± 0.009 173.28 ± 8.98

Brain-IMI 13.5 ± 0.2* _{12.95 ± 0.35} _{0.451 ± 0.005}** _{1.665 ± 0.002} _{200.39 ± 25.88}

Liver-control 58 ± 1.2 9.13 ± 0.50 6.82 ± 0.21 2.38 ± 0.07 173.99 ± 4.25

Liver-IMI 78 ± 3.4* _{12.18 ± 0.33}* _{6.56 ± 0.28} _{2.26 ± 0.03} _{181.07 ± 4.95}

Values are mean ± S.E. from seven rats in each group. Control vs. imidacloprid (IMI)-treated group within the same tissue, XO: xanthine oxidase, MPO: myeloperoxidase, CAT: catalase, SOD: superoxide dismutase, GSH-Px: glutathione peroxidase.

*_{p < 0.001.}

** _{p < 0.05.}

Table 4

The effect of imidacloprid exposure to liver enzyme activities and plasma calcium

(Ca2+_{) levels.}

ALT AST LDH Ca2+

mg/dl

Control 37.11 ± 3.60 147.06 ± 6.48 256.79 ± 2.11 8.19

Imidacloprid 46.01 ± 5.74 97.84 ± 2.73* _{98.24 ± 5.21}* _8.62**

Values are mean ± S.E. from seven rats in each group. Control vs. imidacloprid treated, alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH).

*_{p < 0.001.}

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such as TNF-

a

, IL-6, IL-1b and IFN-

c

transcripts (1.65-, 14.32-, 7.51-, 4.19-folds, respectively) and down regulated IL-12 in brain (Fig. 2A). On the other hand imidacloprid exposure stimulated

TNF-

a

, IL-12, IL-1b and IFN-

c

expressions (2.05-, 5.54-, 1.51-, 1.63-fold, respectively) while suppressing IL-6 in the liver (Fig. 2B).

4. Discussion

Pesticide usage constitutes the principal method of insect or weed control, however environmental contamination by these chemicals can lead to damage in non-target organisms [31]. In-deed, several mechanisms have been proposed for the primitive ef-fects of these pesticides on vertebrates, including oxidative stress, interference with dopamine transporters, mitochondrial dysfunc-tion and inﬂammadysfunc-tion [10,32]. However, despite the increased attention to the ecotoxicological effects of neonicotinoids, there re-mains a lack of sufﬁcient information on their toxicodynamics and, in particular, their negative effects in mammals. Neonicotinoids such as imidacloprid are characterized by their high potency against sucking insects and many other pests, combined with their relatively low mammalian toxicity. However, as these insecticides affect insects by interfering with nAChRs, this suggests that these receptors may also be a target in mammals[33].

In the present study NO levels were found to be increased in the brain and liver of imidacloprid-exposed rats. Transcriptional anal-yses demonstrated that mRNA expressions of NO producing en-zymes were stimulated in these two tissue types: iNOS, nNOS and eNOS in brain, and iNOS and eNOS in liver. A previous study with thiacloprid (a neonicotinoid) and deltamethrin (a pyretiroid) had demonstrated that NO levels were increased in polymorpho-nuclear leukocytes and plasma of rats[34]. Deltamethrin-induced testicular apoptosis was also shown to be reversed by a NOS inhib-itor (NG_{-nitro monomethyl}

L-arginine hydrochloride; L-NMMA),

demonstrating that apoptosis was in fact caused by NO production [35]. Han et al.[36]showed that endosulfan (a cyclodiene insecti-cide) stimulates the production of NO, elevates the expression of iNOS and enhances proinﬂammatory cytokine release.

Nicotine exerts its central actions by regulating cationic fluxes through nAChRs, just like imidacloprid. It also modifies events occurring beyond the nAChR, including the regulation of NO syn-thesis[37]. Research by Pogun et al.[38]indicates that acute and chronic administration of nicotine increased the levels of NO in the brain of male and female rats with different degrees of stimu-lation based on gender. In our previous study, acute imidacloprid treatment significantly increased NO levels in liver, brain and plas-ma[10]. The effects of imidacloprid on production of NO, as is the case with nicotine, may depend on nicotinamide dihydrogen phos-phate (NADPH) or oxygen radical interactions with NOS. Increased plasma calcium might stimulate calcium/calmodulin dependent NOS (nNOS) activity which produces NO.

Calcium responses generated following activation of nAChRs facilitate the interface with many intracellular processes [39]. Rathouz et al.[40]demonstrated that agonist stimulated activation of neuronal nicotinic receptors can produce substantial increases in intracellular calcium levels by direct passage of calcium via voltage gated Ca2+_{channels. When multiple classes of nicotinic receptors}

are expressed by the same neuron, each appears capable of increas-ing calcium in the cell. In their studies on rat brains Tsuneki et al. [41]reported that the Ca2+_{inﬂux due to nAChR activation is}

subse-quently ampliﬁed by the recruitment of intracellular Ca2+_stores.

This Ca2+_{mobilization may possibly contribute to the long-term}

ef-fects of nicotine. In this study calcium levels were increased by long term exposure to imidacloprid in plasma samples and the results therein are supported by studies with other pesticides [42,43]. Tomizawa and Casida[44]utilized desnitro-imidacoprid, a metab-olite of imidacloprid, to ascertain that neonicotinoids activate an extracellular signal-regulated kinase (ERK) cascade. This cascade was triggered by primary action at the nAChR with subsequent

0 1 2 3 4 5 6

iNOS nNOS eNOS

re la ti v e m R N A i n br a in ( fol d c h a nge ) 0 0,2 0,4 0,6 0,8 1 1,2 1,4 eNOS iNOS re la ti v e mR N A in liv e r ( fo ld c h a n g e )

Fig. 1. RT–PCR analyses of NOS in imidacloprid treated groups compared with control in brain (4.76 ± 0.07; 1.13 ± 0.01; 3.97 ± 0.07, respectively in (A) and iNOS 1.1 ± 0.06 and eNOS 1.16 ± 0.03 in liver in (B)).

Fig. 2. Quantitative RT–PCR analyses of inﬂammatory cytokine mRNA transcripts in

imidacloprid treated groups compared with control (TNF-a, IL-6, IL-1b and IFN-c

transcripts: 1.65, 14.32, 7.51, 4.19-folds, respectively also 21.55-fold down

regu-lation of IL-12 in brain in (A) and stimuregu-lation of TNF-a, IL-12, IL-1b and IFN-c

expressions: 2.05, 5.54, 1.51, 1.63-fold, respectively while suppressing IL-6 by

4.95-fold in the liver in (B). TNF-a: tumor necrosis factor-alpha, 6: interleukin-6,

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involvement of intracellular Ca2+_{mobilization, possibly mediated}

by inositol-3-phosphate. The same group has also suggested that intracellular Ca2+activates a sequential pathway from protein ki-nase C (PKC) to ERK. Studies indicate that stimulation of this path-way through nAChR may activate Ca2+_{-dependent eNOS}_[43]_and

consequently increase production of NO[45].

Exposure to pesticides has been associated with many harmful effects, including: acute and chronic toxicities in human and ani-mals, liver and heart diseases, hormonal disorders, mutagenic and carcinogenic events and effects on lipid peroxidation [46]. Oxygen free radicals generated due to exposure to pesticides can cause tissue damage by triggering several oxidative mechanisms and lipid peroxidation [9]. Indeed, Atessahin et al.[47] reported that cypermetrin leads to lipid peroxidation in brain, kidney and blood, whilst Fortunato et al.[48]showed that lipid peroxidation is increased in cerebrospinal ﬂuid and brain samples in Malathion exposed rats. The signiﬁcant increase of MDA, the index of lipid peroxidation, in brain, liver and plasma following imidacloprid treatment is important evidence of oxidative stress in the present study.

Xanthine oxidase catalyses the last steps in purine catabolism, namely the conversion of hypoxanthine to xanthine and that of xanthine to uric acid; the byproduct of these processes is a toxic superoxide radical[49]. Xanthine oxidase activity and superoxide anion generation by XO have been shown to increase in the pres-ence of nicotine, an agonist of nAChR like imidacloprid[50,51]. In addition, various insecticides such as organophosphate poisons are known to stimulate the generation of superoxide anions by increasing XO activity[49,52,53].

Imidacloprid treatment increased the activity of catalase but no statistical changes were observed in SOD and GSH-Px activity. Even though some studies show an increase in intracellular antioxidants to compensate for the generation of free radicals induced by pesti-cide exposure[54], others have reported that the elevation in oxi-dant molecule production causes an inhibition of antioxioxi-dant enzyme activity[9,55]. Different studies have demonstrated that long term pesticide exposure leads to a significant decrease in GSH levels[56,57]. The decreased brain GSH content observed in our current study may reflect, at least partially, GSH conjugation or oxidation of GSH to glutathione disulfide (GSSG) due to the pes-ticide-induced generation of oxygen free radicals and their by-products.

Myeloperoxidase, present in the granules of neutrophils, catal-yses the production of hypochlorous acid which has bacteriocidal properties[58]. It is reported that chronic exposure to endosul-phane causes an increase in MPO activity as a result of neutrophil activation[59]. Gabbianelli et al.[60]have suggested that superox-ide anion levels and activation of the hydrogen peroxsuperox-ide-myelo- peroxide-myelo-peroxidase system were increased 33- and 67-fold, respectively in long term permethrin-exposed rats. Based on these observations it is suggested that the increased activity of MPO following chronic imidacloprid exposure may evidence the intensity of the oxidative process and the presence of inﬂammation.

In the present study, the mRNA transcriptions of TNF-

a

, 6, IL-1b and IFN-

c

were up regulated and that of IL-12 was down regu-lated in brain. Additionally, although TNF-

a

, IL-1b, IL-12 and IFN-

c

mRNA transcriptions were enhanced in liver, IL-6 transcription was inhibited. A raft of evidence exist linking exposure to pesti-cides to changes in cytokine activity. For example, Omurtag et al. [61]reported that endosulfan administration resulted in hepatox-icity related to proinﬂammatory cytokine expression (TNF-

a

and IFN-

c

) which in turn was increased by oxidative stress. It has also been demonstrated that subcutaneous injection of dieldrin (10 mg/ kg/day for 7 days), an organochloride, inhibited IL-6 and IL-12 pro-duction and c-JUN activation in a dose dependent manner [62]. Diazinon treatment is reported to deplete IL-2, IL -4, IL-12 and

IFN-

c

synthesis in mice[63], whilst DDT metabolites trigger ROS production, leading to oxidative stress and enhanced activation of the NF-

j

B pathway[64]. Additionally, DDT induces signiﬁcant production of TNF-

a

and NO in macrophages and thus contributes to inﬂammatory reactions, cytokine imbalance and immune-dysregulation[65]. Videla et al.[66]have proposed that lindane-induced oxidative stress in liver triggers DNA binding activity of NF-

j

B, with a consequent increase in the expression of

NF-j

B-dependent genes for TNF-

a

and IL-1

a

, therein identifying factors that may mediate the hepatotoxic effect of insecticides. In our study the observed increase of NO levels in liver and brain, and the stimulation of proinﬂammmatory cytokine expression suggests that, as with other insecticides, imidacloprid may mediate its effect through the NF-

j

B pathway in the chronic phase of inﬂammation.

The liver plays a major role in metabolism and carries out vital functions such as detoxiﬁcation, so in order to evaluate hepatic damage serum enzymes such as ALT, AST and LDH were moni-tored. It was noted that chronic imidacloprid exposure markedly inhibited AST and LDH activities whilst ALT activity was un-changed. Supporting our ﬁndings, chlorpyrifos treatment has been shown to decrease the activity of ALT and AST[67,68], whilst Ribe-iro et al. [69] have shown parathion treatment to decrease the activities of LDH and acetylcholinesterase: parameters that may be used as criteria for toxicity tests. Our results may therefore be taken as evidence of altered liver function due to imidacloprid exposure.

In conclusion, the ﬁndings obtained in the present study show that chronic exposure to imidacloprid, which is generally accepted as being a less toxic compound in comparison with other insecti-cides, may indeed induce oxidative stress and trigger chronic inﬂammation in non-target organisms.

Acknowledgments

This work was ﬁnancially supported by Mustafa Kemal Univer-sity, Scientiﬁc Research Projects Committee (Project No. 08L0201). The authors thank Altug Kucukgul (Antakya, Turkey) for his techni-cal assistance. Authors wish to thank Dr. Sandra Spence for her helpful reading of the manuscript.

References

[1] A. Elbert, R. Nauen, W. Leicht, Imidacloprid a Novel Chloronicotinyl Insecticide: Biological Activity and Agricultural Importance, in: I. Ishaaya, D. Degheele (Eds.), Insecticides with Novel Modes of Action, Mechanism, and Application, Springer, Verlag, 1998, pp. 50–73.

[2] J.N. Tasei, J. Lerin, G. Ripault, Sub-lethal effects of imidacloprid on bumblebees, Bombus terrestris (Hymenoptera: Apidae), during a laboratory feeding test, Pest Manage. Sci. 56 (2000) 784–788.

[3] K. Matsuda, M. Shimomura, M. Ihara, M. Akamatsu, D.B. Sattelle,

Neonicotinoids show selective and diverse actions on their nicotinic receptor targets: electrophysiology, molecular biology, and receptor modeling studies, Biosci. Biotechnol. Biochem. 69 (2005) 1442–1452.

[4] D.B. Sattelle, H. Breer, Cholinergic nerve terminals in the central nervous system of insects: molecular aspects of structure, function and regulation, J. Neuroendocrinol. 2 (1990). 241–156.

[5] M. Shimomura, M. Yokota, M. Ihara, M. Akamatsu, D.B. Sattelle, K. Matsuda, Role in the selectivity of neonicotinoids of insect-speciﬁc basic residues in loop D of the nicotinic acetylcholine receptor agonist binding site, Mol. Pharmacol. 70 (2006) 1255–1263.

[6] M. Ihara, L.A. Brown, C. Ishida, H. Okuda, D.B. Sattelle, K. Matsuda, Actions of

imidacloprid, clothianidin and related neonicotinoids on nicotinic

acetylcholine receptors of American cockroach neurons and their

relationships with insecticidal potency, J. Pestic. Sci. 31 (1) (2006) 35–40. [7] M. Tomizawa, Neonicotinoids and derivatives: effects in mammalian cells and

mice, J. Pestic. Sci. 29 (3) (2004) 177–183.

[8] V. D’Almeida, D.C. Hipólide, L.A. Azzalis, L.L. Lobo, V.B.C. Junqueira, S. Tuﬁk, Absence of oxidative stress following paradoxical sleep deprivation in rats, Neurosci. Lett. 235 (1997) 25–28.

[9] M. Kanbur, B.C. Liman, G. Eraslan, S. Altinordulu, Effects of cypermethrin, propetamphos, and combination involving cypermethrin and propetamphos on lipid peroxidation in mice, Environ. Toxicol. 23 (4) (2008) 473–479.

(6)

[10] V. Duzguner, S. Erdogan, Acute oxidant and inﬂammatory effects of imidacloprid on the mammalian central nervous system and liver in rats, Pest. Biochem. Physiol. 97 (2010) 13–18.

[11] M. Singh, R. Sandhir, R. Kiran, Erythrocyte antioxidant enzymes in toxilogical evaluation of commonly used organophasphate pesticides, Indian J. Exp. Biol. 44 (2006) 580–583.

[12] S. John, M. Kale, N. Rathore, D. Bhatnagar, Protective effect of vitamin E in dimethoate and malathion induced oxidative stress in rat erythrocytes, J. Nutr. Biochem. 12 (2001) 500–504.

[13] A. Thapar, R. Sandhir, R. Kiran, Acephate induced oxidative stress in erythrocytes, Indian J. Exp. Biol. 40 (2002) 963–996.

[14] M.B. Grisham, D. Jourd’Heuil, D.A. Wink, Nitric Oxide 431 I. Physiological chemistry of nitric oxide and its metabolites: implications in inﬂammation, Am. J. Physiol. 433 Gastrointest. Liver Physiol. 276 (1999) G315–G321. [15] J.F. Muniz, L. McCauley, J. Scherer, M. Lasarev, M. Koshy, Y.W. Kow, V.

Nazar-Stewart, G.E. Kisby, Biomarkers of oxidative stress and DNA damage in agricultural workers: a pilot study, Toxicol. Appl. Pharmacol. 227 (2008) 97–107. [16] E.H.B. Delgado, E.L. Streck, J.L. Quevedo, F. Dal-Pizzol, Mitochondrial respiratory dysfunction and oxidative stress after chronic malathion exposure, Neurochem. Res. 31 (2006) 1021–1025.

[17] R.L. Miller, M. James-Kracke, G.Y. Sun, A.Y. Sun, Oxidative and inﬂammatory pathways in Parkinson’s disease, Neurochemistry 34 (2009) 55–65. [18] A.M. Alluwaimi, Y. Hussein, Diazinon immunotoxicity in mice. Modulation of

cytokines level and their gene expression, Toxicology 236 (2007) 123–131. [19] I. Ikizceli, Y. Yurumez, L. Avsarog˘ullari, C. Kucuk, E.M. Sozuer, I. Soyuer, Y.

Yavuz, S. Muhtaroglu, Effect of interleukin-10 on pancreatic damage caused by organophosphate poisoning, Regul. Toxicol. Pharmacol. 42 (2005) 260–264. [20] OECD Guidelines for the Testing of Chemicals/Section 4: Health Effects, Test

No. 408: Repeated Dose 90-Day Oral Toxicity Study in Rodents (1998), OECD Publishing.

[21] M. Bradford, A rapid and sensitive method for the quantiﬁcation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem. 72 (1976) 248–254.

[22] T. Yoshioka, K. Kawada, T. Shimada, M. Mori, Lipid peroxidation in maternal and cord blood and protective mechanism against activated-oxygens toxicity in the blood, Am. J. Obstet. Gynecol. 135 (1979) 372–376.

[23] Y. Sun, L.W. Oberley, L.A. Ying, Simple method for clinical assay of superoxide dismutase, Clin. Chem. 34 (1988) 497–500.

[24] N.K. Cortas, N.W. Wakid, Determination of inorganic nitrate in serum and urine by a kinetic cadmium-reduction method, Clin. Chem. 36 (1990) 1440– 1443.

[25] N. Prajda, G. Weber, Malignant transformation-linked imbalance: decreased XO activity in hepatomas, FEBS Lett. 59 (1975) 245–249.

[26] P.C. Andrews, N.I. Krinski, Quantitative determination of myeloperoxidase using tetramethylbenzidine as substrate, Anal. Biochem. 127 (1982) 346–350. [27] H. Luck, Catalase, in: H.U. Bergmeyer (Ed.), Methods in Enzyme Analysis,

Verlag-Chemic, Weinheim/Bergstrasse, Germany, 1965.

[28] D.E. Paglia, W.N. Valentine, Studies on the quantitative and qualitative characterization of erythrocyte GSH-Px, J. Lab. Clin. Med. 70 (1) (1967) 158– 169.

[29] J. Sedlak, R.H. Lindsay, Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman’s reagent, Anal. Biochem. 25 (1) (1968) 192–205.

[30] M.W. Pfafﬂ, A new mathematical model for relative quantiﬁcation in real-time RT–PCR. Nucleic Acids Res. 1, 29(9) (2001), e45.

[31] H. Qian, W. Chen, L. Sun, Y. Jin, W. Liu, Z. Fu, Inhibitory effects of paraquat on photosynthesis and the responseto oxidative stress in Chlorella vulgaris, Ecotoxicology 18 (2009) 537–543.

[32] A. Ascherio, H. Chen, M.G. Weisskopf, E. O’Reilly, M.L. McCullough, E.E. Calle, M.A. Schwarzschild, M.J. Thun, Pesticide exposure and risk for Parkinson’s disease, Ann. Neurol. 60 (2006) 197–203.

[33] S.L. Chao, J.E. Casida, Interaction of imidacloprid metabolites and analogs with the nicotinic acetylcholine receptor of mouse brain in relation to toxicity, Pest. Biochem. Physiol. 58 (1997) 77–88.

[34] B. Aydin, Effects of thiacloprid, deltamethrin and their combination on oxidative stress in lymphoid organs, polymorphonuclear leukocytes and plasma of rats, Pest. Biochem. Physiol. 100 (2011) 165–171.

[35] M. El-Gohary, W.M. Awara, S. Nassar, S. Hawas, Deltamethrin-induced testicular apoptosis in rats: the protective effect of nitric oxide synthase inhibitor, Toxicology 132 (1999) 1–8.

[36] E.H. Han, Y.P. Hwang, H.G. Kim, H.G. Jeong, Inﬂammatory effect of endosulfan via NF-jB activation in macrophages, Biochem. Biophys. Res. Commun. 355 (2007) 860–886.

[37] B.H. Tonnessen, S.R. Severson, R.D. Hurt, V.M. Miller, Modulation of nitric-oxide synthase by nicotine, JPET 295 (2000) 601–606.

[38] S. Pogun, S. Demirgoren, D. Taskiran, L. Kanit, O. Yilmaz, E.O. Koylu, B. Balkan,

E.D. London, Nicotine modulates nitric oxide in rat brain, Eur.

Neuropsychopharmacol. 10 (2000) 463–472.

[39] F. Dajas-Bailador, S. Wonnacott, Nicotinic acetylcholine receptors and the regulation of neuronal signalling, Trends in Pharmacol. Sci. 25 (2004) 317–324. [40] M.M. Rathouz, S. Vijayaraghavan, D.K. Berg, Elevation of intracellular calcium levels in neurons by nicotinic acetylcholine receptors, Mol. Neurobiol. 12 (1996) 117–131.

[41] H. Tsuneki, R. Klink, C. Léna, H. Korn, J.P. Changeux, Calcium mobilization elicited by two types of nicotinic acetylcholine receptors in mouse substantia nigra pars compacta, Eur. J. Neurosci. 12 (2000) 2475–2485.

[42] L. Imamura, M. Yasuda, K. Kuramitsu, D. Hara, A. Tabuchi, M. Tsuda, Deltamethrin, a pyrethroid insecticide, is a potent inducer for the activity-dependent gene expression of brain-derived neurotrophic factor in neurons, JPET 316 (2006) 136–143.

[43] N. Pourkhalilii, S. Pournourmohammadi, F. Rahimi, S. Vosough-Ghanbari, M.B. Seyed, Comparatıve effects of calcium channel blockers, autonomic nervous

system blockers, and free radical scavengers on diazinon-induced

hyposecretion of insulin from isolated islets of langerhans in rats, Arh. Hig. Rada. Toksikol. 60 (2009) 157–164.

[44] M. Tomizawa, J.E. Casida, Desnitro-imidacloprid activates the extracellular signal-regulated kinase cascade via the nicotinic receptor and intracellular calcium mobilization in N1E–115 cells, Toxicol. Appl. Pharmacol. 184 (3) (2002) 180–186.

[45] C. Heeschen, M. Weis, A. Aicher, S. Dimmeler, J.P. Cooke, A novel angiogenic pathway mediated by non-neuronal nicotinic acetylcholine receptors, J. Clin. Invest. 110 (2002) 527–536.

[46] T.S.S. Dikshith, Toxicology of pesticides in animals, CRC Press (1991) Boca Raton, Boston.

[47] A. Atessahin, S. Yilmaz, I. Karahan, I. Pirinci, The effects of vitamin E and selenium on cypermethrin induced oxidative stress in rats, Turk. J. Vet. Anim. Sci. 29 (2005) 385–391.

[48] J.J. Fortunato, F.R. Agostinho, G.Z. Réus, F.C. Petronilho, F.D. Pizzol, J. Quevedo, Lipid peroxidative damage on malathion exposure in rats, Neurotox. Res. 9 (2006) 23–28.

[49] C.E. Berry, J.M. Hare, Xanthine oxidoreductase and cardiovascular disease: molecular mechanisms and pathophysiological implications, J. Physiol. 555 (3) (2004) 589–606.

[50] A. Cormier, C. Morin, R. Zini, J.P. Tillement, G. Lagrue, Nicotine protects rat brain mitochondria against experimental injuries, Neuropharmacology 44 (2003) 642–652.

[51] U.S. Kayyali, R. Budhiraja, C.M. Pennella, S. Cooray, J.J. Lanzillo, R. Chalkley, P.M. Hassoun, Upregulation of xanthine oxidase by tobacco smoke condensate in pulmonary endothelial cells, Toxicol. Appl. Pharmacol. 188 (2003) 59–68.

[52] A. Terzi, M. Iraz, S. Sahin, A. Ilhan, N Idiz, E. Fadilliog˘lu, Protective effects of erdosteine on rotenone-induced oxidant injury in liver tissue, Toxicol. Indust. Health. 20 (6–10) (2004) 141–147.

[53] L.M. Domico, K.R. Cooper, L.P. Bernard, G.D. Zeevalk, Reactive oxygen species generation by the ethylene-bis-dithiocarbamate (EBDC) fungicide mancozeb and its contribution to neuronal toxicity in mesencephalic cells, Neuro. Toxicol. 28 (2007) 1079–1091.

[54] A.E. Bayoumi, A.J. Garcia-Fernandez, C. Ordonez, Y. Perez-Pertejo, J.C. Cubria, R.M. Reguera, R. Balana-Fouce, D. Ordonez, Cyclodiene organochlorine insecticide induced alterations in the sulfur-redox cycle in CHO-K1 cells, Comp. Biochem. Physiol. Part C. 130 (2001) 315–323.

[55] M. Panemangalore, F.N. Bebe, Dermal exposure to pesticides modiﬁes antioxidant enzymes in tissues of rats, J. Environ. Sci. Health B 35 (4) (2000) 399–416.

[56] B.D. Banerjee, V. Seth, A. Bhattacharya, S.T. Pasha, A.K. Chakraborty, Biochemical effects of some pesticides on lipid peroxidation and free-radical scavengers, Toxicol. Lett. 107 (1999) 33–47.

[57] R.S. Ahmed, V. Seth, S.T. Pasha, B.D. Banerjee, Inﬂuence of dietary ginger (Zingiber ofﬁcinales Rosc) on oxidative stress induced by malathion in rats, Food Chem. Toxicol. 38 (2000) 443–450.

[58] M. Tokarska-Rodak, S. Tos-Luty, A. Haratym-Maj, Selected parameters of immunological response in hop growers during the period of intensive application of pesticides, Ann. Agric. Environ. Med. 11 (2004) 227–231.

[59] F. Mor, O. Ozmen, Endosulfan-induced neurotoxicity and serum

acetylcholinesterase inhibition in rabbits: The protective effect of Vit C, Pest. Biochem. Physiol. 96 (2010) 108–112.

[60] R. Gabbianelli, M.L. Falcioni, C. Nasuti, F. Cantalamessa, I. Imada, M. Inoue. Effect of permethrin insecticide on rat polymorphonuclear neutrophils, Chem. Biologic. Int. 182 (2009) 245–252.

[61] G.Z. Omurtag, A. Tozan, A.O. Sehirli, G. Sener, Melatonin protects againsts endosulfan induced oxidative tissues damage, J. Pineal Res. 44 (2008) 432– 438.

[62] S.B. Pruett, R. Fan, S. Oppenheimer, Greater than additive suppression of TLR3-induced IL-6 responses by administration of dieldrin and atrazine, J. Immunotoxicol. 3 (4) (2006) 253–262.

[63] F. Diel, B. Horr, H. Borck, T. Irman-Florjanc, Pyrethroid insecticides inﬂuence the signal transduction in T helper lymphocytes from atopic and nonatopic subjects, Inﬂamm. Res. 52 (2003) 154–163.

[64] Y. Shi, Y. Song, Y. Wang, X. Liang, Y. Hu, X. Guan, J. Cheng, K. Yang, p, p-DDE Induces Apoptosis of Rat Sertoli Cells via a FasL-Dependent Pathway, J. Biomed. Biotechn. 2009 (2009) 1–11.

[65] R. Dutta, A.M. Mondal, V. Arora, T.C. Nag, N. Das, Immunomodulatory effect of DDT (bis[4-chlorophenyl]-1,1,1-trichloroethane) on complement system and macrophages, Toxicology 252 (2008) 78–85.

[66] L.A. Videla, G. Tapia, P. Varela, P. Cornejo, J. Guerrero, Y. Israel, V. Fernández,

Effects of acutec-hexachlorocyclohexane intoxication in relation to the redox

regulation of nuclear factor-jB, cytokine gene expression, and liver injury in

the rat, Antiox. Redox Signal. 6 (2) (2004) 471–480.

[67] T. Barna-Lloyd, J.R. Szabo, N.L. Davis, Chlorpyrifos-methyl rat subchronic dietary toxicity and recovery study Unpublished report TXT: K-046193-0,31 from Dow chemical, Texas, USA. Submitted to WHO by Dow Elenco, (1990), Indianapolis, USA.

(7)

[68] S. Ambali, D. Akanbi, N. Igbokwe, M. Shittu, M. Kawu, J. Ayo, Evaluation of subchronic chlorpyrifos poisoning on hematological and serum biochemical changes in mice and protective effect of vitamin C, J. Toxicol. Sci. 32 (2) (2007) 111–120.

[69] S. Ribeiro, L. Guilhermino, J.P. Sousa, A.M.V.M. Soares, Novel bioassay based on acetylcholinesterase and lactate dehydrogenase activities to evaluate the toxicity of chemicals to soil isopods, Ecotoxicol. Environ. Saf. 44 (3) (1999) 287–293.