Assessment of neurohepatic DNA damage in male Sprague–Dawley rats exposed to organophosphates and pyrethroid insecticides

The current work was undertaken to test the genotoxic potential of chlorpyrifos (CPF), dimethoate, and lambda cyhalothrin (LCT) insecticides in rat brain and liver using the single cell gel electrophoresis (comet assay). Three groups of adult male Sprague-Dawley rats were exposed orally to one third LD₅₀of CPF, dimethoate, or LCT for 24 and 48 h while the control group received corn oil. Serum samples were collected for estimation of malondialdehyde (MDA) and glutathione peroxidase (GPx); the brain and liver samples were used for comet assay and for histopathological examination. Results showed that signs of neurotoxicity appeared clinically as backward stretching of hind limb and splayed gait in dimethoate and LCT groups, respectively. CPF, LCT, and dimethoate induced oxidative stress indicated by increased MDA and decreased GPx levels. CPF and LCT caused severe DNA damage in the brain and liver at 24 and 48 h indicated by increased percentage of DNA in tail, tail length, tail moment, and olive tail moment. Dimethoate induced mild DNA damage in the brain and liver at 48 h. Histopathological changes were observed in the cerebrum, cerebellum, and liver of exposed rats. The results concluded that CPF, LCT, and dimethoate insecticides induced oxidative stress and DNA damage associated with histological changes in the brain and liver of exposed rats.

CPF; Dimethoate; LCT; DNA damage; Comet; Oxidative stress; Brain; Liver

Insecticides are widely produced all over the world and used for control of agricultural and household pests, which put human and animals under the risk of exposure. Manufacturing workers, field applicators, and the public are exposed to insecticides by the use of synthetic insecticides such as organophosphates (OP) and pyrethroids (Wang et al. [85] ). Commonly used OP insecticides in Egypt include chlorpyrifos (CPF) and dimethoate. Organophosphate pesticides produce their effect on the nervous system by inhibition of acetylcholinesterase (AChE) in cholinergic synapses and in neuromuscular junctions (Wang [84] ; Ballesteros et al. [11] ). Accumulation of AChE in the synapses results in death due to asphyxia and loss of respiratory control, which attributed to hyperactivity in cholinergic pathways (Sparling and Fellers [78] ).

Dimethoate is a widely used insecticide and acaricide; it is frequently used as systemic and contact pesticide. It is used on agricultural crops and ornamental plants to control insects and mites. Previous reports indicated that dimethoate caused cellular injury, lipid peroxidation, free radicals release, and oxidative stress in rats (Sharma et al. [72] ; Singh et al. [74] ).

Toxic effects of OP include genotoxicity, hepatic dysfunction, embryotoxicity, teratogenicity, neurochemical, and neurobehavioral changes (Goel et al. [34] ). It was reported that both acute and chronic exposures to CPF resulted in liver damage in rats (Mansour and Mossa [52] ; Elsharkawy et al. [26] ; Ezzi et al. [27] ). Furthermore, acute and subchronic exposures to dimethoate caused pathological changes in the brain and liver of rats (Astiz et al. [8] ; Saafi et al. [69] ).

Pyrethroid insecticides have a limited persistence in soil and low toxicity to mammals and birds, which encouraged their widespread application all over the world in agriculture as a potent against pests (Glickman and Lech [33] ; Kidd and James [44] ). Moreover, pyrethroid is used for the control of a broad range of ectoparasites on cattle and sheep (Abd Elkawy et al. [1] ).

Lambda-cyhalothrin (LCT) is a type II synthetic pyrethroid insecticide. It is neurotoxic, and it produces its effect by altering sodium channels (Soderlund et al. [75] ; Naravaneni and Jamil [59] ; Ray and Fry [68] ) and also affects chloride and calcium channels, which are essential for appropriate nerve function (He et al. [37] ). Reportedly, LCT induced genotoxicity, chromosomal aberrations, and micronucleus formation in the bone marrow cells of rat (Fahmy and Abdallah [28] ; Celik et al. [17] , [18] ) and in human lymphocytes cultured in vitro (Naravaneni and Jamil [59] ).

Many research studies suggested that pesticides produce oxidative stress through the formation of oxygen free radical (Bagchi et al. [10] ; Monteiro et al. [56] ; Modesto and Martinez [55] ; Lee et al. [46] ). The generation of reactive oxygen species (ROS) including hydroxyl, superoxide radicals, and hydrogen peroxide cause damage to different membranous components of the cells (Bebe and Panemangalore [13] ) and inactivate biological macromolecules (Meister [53] ). Normally, antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) provide the first-line defense against oxidative stress (Livingstone [48] ). The GPx enzyme is the mayor antioxidant molecule that involved in detoxification of hydrogen peroxide (H2O2), with the help of reduced glutathione and glutathione reductase (Sharma et al. [73] ).

Damage to membrane lipids results in lipid peroxidation, which is considered as one of the mechanisms involved in pesticide toxicity and plays an important role as a biomarker for oxidative stress (Kavitha and Rao [43] ; Ballesteros et al. [11] ). Lipid peroxidation product in the form of malondialdehyde (MDA) is used as a biomarker for lipid (Ayala and Munoz [9] ; Arce et al. [7] ). There is a relationship between SOD, catalase, and GSH-Px activities as the superoxide radical (O.) dismuted to hydrogen peroxide (H2O2) by SOD; H2O2 is then removed by GSH-Px and catalase (Felton and Summers [29] ). Catalase is present in the peroxisome. However, glutathione peroxidase performs functions in the cytosol. This makes that catalase has a limited role in comparison to the antioxidant function of GSH-Px (DeLeve and Kaplowitz [21] ).

Pesticides have been considered potential mutagens and have genotoxic properties leading to mutations and DNA damage (Bolognesi [15] ). OP compounds are known also to cause DNA damage by their alkylating properties and alkylating agents (Braun et al. [16] ).

Comet assay known as the alkaline single cell gel electrophoresis (SCGE) is a rapid and sensitive technique that is used for quantitating DNA lesions in mammalian cells (Tsuda et al. [80] ). This assay allows the assessment of genetic damage in a great variety of cells (Kassie et al. [42] ). It is used to detect DNA strand breakage in any nucleated cell and has the advantages of being a straight forward technique, sensitive, and requires small numbers of cells (Tice et al. [79] ).

The brain is the main target organ for organophosphates and pyrethroid insecticides, and the liver is the organ responsible for detoxification of xenobiotics. The brain and liver are considered the most sensitive organs for OP and pyrethroid insecticides toxicity; so, the goal of the current study is to evaluate the genotoxic effect of CPF, dimethoate, and LCT in the brain and liver by comet assay and the relation between DNA damage and oxidative stress induced by acute exposure to these insecticides in addition to histopathological changes in the brain and liver of rats.

The commercial formulations of insecticides obtained from a local market were used in this study, because they are often more toxic than the pure grades of pesticide compound; they contain organic solvents, surface active ingredients, activity enhancers, dyes, and stabilizers, with poorly characterized toxicity (Ducolomb et al. [23] ; Dogan et al. [22] ; Ezzi et al. [27] ).

Lambda cyhalothrin (2.5 g/100ml) a synthetic pyrethroid insecticide with the trade name Dolf 2.5 EC (Star Chem. Co, Egypt). Chlorpyrifos (Clorzan 48% EC) and dimethoate (Saydon/Cheminova 40% EC) organophosphate insecticides (Kafre Elzayat KZ Co, Egypt).

A total number of 40 healthy adult Sprague-Dawley male rats aged 8-10 weeks with an average body weight of 100-120 g were used in this study. Rats were purchased from the Experimental Animal Center located at Faculty of Medicine, Assiut University, Egypt.

The animals were kept in plastic cages and allowed to adapt for a week before treatment. Animal facilities were operated under controlled temperature (24-26 °C) and a 12-h light-dark cycle. Rats were fed on standard food pellets, and tap water was supplied ad libitum. The design of the experiment was in agreement with the ethical rules prescribed by Assiut University.

Animals were divided randomly into four groups of ten animals each. The three pesticides (Clorzan, Saydon and Dolf) were dissolved in corn oil. The first group was exposed to chlorpyrifos with oral LD50 155 mg/kg (Worthing and Walker [88] ). The second group was exposed to dimethoate with oral LD50 310 mg/kg (US EPA [82] ). The third group was exposed to lambda cyhalothrin with oral LD50 79 mg/kg (Southwood [77] ), and the fourth group was kept as control and treated with corn oil. All treated animals were exposed to one third of the LD50 by oral gavage for 24 and 48 h. Animals were observed after administration for any clinical signs or mortality. Five animals from each group were euthanized under diethyl ether anesthesia 24 h after the first exposure, whereas another five animals were euthanized 24 h after the second exposure (48 h). Blood samples were collected from the descending aorta, and serum samples were harvested and kept at − 20 °C until analysis. The brain and liver specimens were collected, and each organ was divided into two parts; the first part was used for comet assay, and the second part was preserved in 10% neutral buffered formalin for histopathological examination.

Determination of serum MDA was done using colorimetric kit supplied by Bio-diagnostics (Dokki, Giza, Egypt). This assay is based on the reaction of MDA with chromogenic reagent at 45 °C to yield a stable chromophore with absorbance at 534 nm, which is measured using UV spectrophotometer (Optizen 3220 UV, Mecasys Co. Ltd., Korea). The rate of lipid peroxidation was expressed as nanomoles of reactive substance formed per min per milligram of protein (Grotto et al. [36] ).

Measurement of glutathione peroxidase activity in serum samples was done by using test kits supplied by Bio-diagnostic (Dokki, Giza, Egypt). The test is an indirect measure of the rate of reduction of organic peroxide by GPx, which results in the formation of oxidized glutathione (GSSG); the latter is recycled to its reduced state by the enzyme glutathione reductase. The oxidation of NADPH to NADP by glutathione reductase is accompanied by a decrease in absorbance at 340 nm, providing a spectrophotometric means (UV spectrophotometer (Optizen 3220 UV, Mecasys Co., Ltd., Korea) for monitoring GPx enzyme activity (Abd Ellah et al. [2] ).

DNA damage in the brain and liver was detected using comet assay according to the methods of Sasaki et al. ([71] ) as follows:

Homogenization

The brain and liver specimens (0.5 g) were minced separately, suspended in chilled homogenizing buffer (0.075 M NaCl, 0.024 M Na2EDTA, pH 7.5), and then homogenized gently using bench top homogenizer (PRO Scientific, USA) surrounded by ice at 800 rpm. To obtain the nuclei, the homogenate was then centrifuged at 1500 rpm for 10 min at 0 °C, and the precipitate was re-suspended in chilled homogenizing buffer and allowed to settle for 1-2 min.

Slide preparation

Fully frosted slides (Matsunami Glass Ind, Japan) were layered twice with 100 μL of 1% GP-42 normal agarose (NacalaiTesqe, Inc., Kyoto, Japan). An amount of 75 μL of nuclear suspension (supernatant) was mixed with 75 μL of 2% low melting (LGT) agarose (NacalaiTesqe, Inc., Kyoto, Japan) at 40 °C, and the mixture was layered on the slide using a cover slide. Finally, 100 μL of agarose GP-42 was quickly layered on the surface and covered with another slide and left to gel.

Lysing

The slides were placed immediately into a chilled lysing solution (2.5 M NaCl, 100 mM Na4EDTA, 10 mM Trizma, 0.1% sodium lauryl sulfate (SDS), 10% dimethyl sulfoxide, and Triton X-100) and kept at 4 °C in the dark for 60 min.

Unwinding and electrophoresis

The slides were placed on a horizontal gel electrophoresis platform (Cleaver Scientific Ltd., UK) and covered with chilled alkaline solution (300 mM NaOH and 1 mM Na2 EDTA, pH 13) in the dark at 0 °C for 10 min, electrophoresis was conducted at 0 °C in the dark for 15 min at 25 V and approximately 300 mA, and then the slides were rinsed with 400 mM Tris buffer (Wako Pure Chemical Industries, Ltd., Japan) with pH 7.5 for 7 min to neutralize the excess alkali. The neutralized slides were kept in ethanol for 5 min and then allowed to dry at room temperature and then stained with 50 μL (20 μg/mL) ethidium bromide (Wako Pure Chemical Industries, Ltd.) just before microscopical examination.

The nuclei on the slides were examined at a 200 fold magnification using a fluorescence microscope (Olympus BX-43, Japan) equipped with a green filter. The image of the cells was captured using digital camera. At least 50 nuclei per slide were analyzed using Comet Assay Software Project (CASP) to measure the diameter of the head and the length of tail of the comet to obtain DNA migration.

Fresh specimens from the brain and liver of rats from all experimental groups were collected and fixed in 10% neutral buffered formalin. The tissues were dehydrated in a graded alcohol series, cleared with methyl benzoate, embedded in paraffin wax, sectioned at 4 μ thickness, and stained with hematoxylin and eosin. Toluidine blue stain was applied to brain tissue sections as specific stain for nerve cells (Sakonlaya et al. [70] ). Histopathological examination was performed by light microscopy (Olympus CX31, Japan) and photographed using digital camera (Olympus, Camedia C-5060, Japan) (Bancroftet al. [12] ). In addition, all the microscopic findings for each group were presented in tables to demonstrate the type of lesion, severity, and percentages according to Radadet al. ([67] ) as follows: severe: +++, moderate: ++, slight: +, and 0: no change.

Statistical analyses were conducted using SPSS software package version 16.0. Data were analyzed by using one-way analysis of variance (ANOVA) followed by post-hoc lowest significant difference (LSD) multiple range test for comparison between control and exposed groups. All data were expressed as mean ± SE for all experimental and control animals. P < 0.05 was considered significant compared to control.

Sprague-Dawley rats exposed to CPF for 24 and 48 h did not show any clinical signs while rats exposed to dimethoate for 48 h showed hind limb disorder appeared as backward stretching of the hind limb (Fig. 1a). On the other hand, rats exposed to LCT pronounced hind limb disorder appeared as tiptoe and splayed gait for half the number of rats after 24 h exposure which died after the second dose, while the other half are still alive and showed the same signs (Fig. 1b).Hind limb disorder in rats exposed to dimethoate for 48 h (a) and LCT for 24 h (b)

Table 1 shows that MDA level significantly increased in dimethoate (P < 0.01), CPF, and LCT (P < 0.05) treated rats at 24 and 48 h. On the other hand, Table 2 shows the activities of glutathione peroxidase (GPx); it decreased (P < 0.01) in CPF and LCT groups at 24 and 48 h while in dimethoate group, GPx decreased at 48 h (P < 0.05).

Results of this study clearly showed that oral exposure of Sprague-Dawley rats to one third of LD50 of CPF or LCT for 24 h caused significantly marked DNA damage in the brain tissue when compared with control group. DNA damage was indicated by decreased head DNA% and increased tail DNA%, tail length, tail moment, and olive tail moment (Table 3). Different degrees of DNA damage are shown in Fig. 2, and the method of measuring DNA damage by CASP software is presented in Fig. 3.Different degrees of DNA damage from 0 to 4 according to the tail length in comet appearance. a Zero degree indicates undamaged DNA. b Degree I mild DNA damage. c Degree II moderate. d Degree III severe. e Degree IV is extensive DNA damageMeasuring DNA damage in cells using CASP lab software showing different degrees of DNA migration indicated by comet parameters as comet head and tail. a No DNA migration in undamaged cell. b Mild increase in DNA migration to the tail. c Moderate increase in DNA migration. d Severe DNA damage with increased DNA migration and tail length. e Extensive DNA damage appeared as small head and very long tail due to extensive DNA migration from the head to the tail

Single cell gel electrophoresis assay (comet assay) showed that exposure of Sprague-Dawley rats to one third LD50 from each dimethoate, CPF, and LCT for 48 h caused significant DNA damage in the brain tissue of all exposed groups with highest increase in CPF group followed by LCT then dimethoate group in comparison with control one (Table 4). Figure 4a shows increased tail length in exposed groups at 24 and 48 h.DNA migration (tail length, μm) in control, CPF, dimethoate, and LCT-exposed groups in the brain (a) and liver tissue (b) after 24 and 48 h. *P < 0.05 and **P < 0.01 as compared with control group

Comet assay parameters indicated that DNA damage was significantly increased in hepatic tissue of rats exposed to dimethoate, CPF, or LCT for 24 and 48 h with highest increase in LCT group followed by CPF then dimethoate group (P < 0.01) in comparison with control group (Tables 5 and 6). Figure 4b demonstrated that hepatic DNA damage gradually increased at 24 and 48 h according to LD50 of each compound.

The histopathological examination of cerebellum of rats exposed to dimethoate for 24 h showed degeneration with pyknosis of nucleus of moderate number of purkinje cells surrounded with perineuronal space associated with angiopathic changes demonstrated as congestion of blood vessels in medulla while the stellate and basket cells in the molecular layer and the Golgi cells in the granular layer were normal (Fig. 5a). Cerebellum of rats exposed to dimethoate for 48 h showed degeneration with pyknotic nucleus of large number of purkinje cells while others showed chromatolysis where Nissl granules were dissolved with swollen eccentric nucleus, this was confirmed by toluidine blue stain (Fig. 5b). These changes were associated with angiopathic changes in all cases. In LCT-exposed group for 24 h, cerebellum showed severe degeneration of purkinje cells with pyknotic nucleus associated with vacuolation along its monolayer accompanied with hemorrhage in the cerebellar medulla (Fig. 5c). These changes accompanied with slight increase in number of astrocyte cells, which increased in its severity after 48 h. The purkinje cells dissociated and showed slight disorganization with karyolysis of its nucleus (Fig. 5d). The cerebellar cortex of CPF intoxicated group after 24 h showed severe degeneration of purkinje cells with pyknotic nucleus surrounded by perineuronal spaces and others necrosed and denuded leaving empty spaces (Fig. 5e). The same changes were observed after 48 h which are characterized by disorganization and pyknosis of nucleus of purkinje cells with proliferation of astrocyte cells in all cases (Fig. 5f).Cerebellar cortex. a Dimethoate-exposed rats for 24 h showing degeneration of purkinje cells with pyknotic nucleus surrounded by perineuronal spaces (notched arrow), congestion of blood vessels in medulla (arrow) (H&E, bar = 50 μm). b Dimethoate group after 48 h showing degeneration of purkinje cells with pyknotic nucleus (arrow), chromatolysis of other purkinje cells (notched arrow). c LCT-exposed rats for 24 h showing degeneration of purkinje cells with pyknosis of nucleus (arrow), hemorrhage in medulla (star). d LCT group after 48 h showing dissociation of purkinje cells with karyolysis of its nucleus (notched arrow) (toluidine blue). e CPF group after 24 h showing degeneration of purkinje cells with pyknotic nucleus surrounded by perineuronal spaces (notched arrow), and others completely dead leaving empty spaces (arrow). f CPF group after 48 h showing degeneration of purkinje cells with pyknotic nucleus (arrow), proliferation of astrocytes (notched arrow)

Cerebral cortex of dimethoate-exposed group for 24 h showed degeneration with pyknotic, shrunken, and hyperchromatic nuclei of a large number of neurons in all cases (Fig. 6a). The angiopathic changes were observed after 48 h as submeningeal perivascular hemorrhage in some areas accompanied with thrombosis of other blood vessels (Fig. 6b). These changes demonstrated in the cerebrum of LCT-exposed group after 24 h associated with decrease in the thickness of the pyramidal layer of hippocampus which showed severely damaged neurocytes in the form of pyknotic, shrunken, and hyperchromatic nuclei of hippocampal neurons (Fig. 6c). After 48 h, cerebral cortex of LCT-intoxicated group showed demyelination in the form of small clear vacuoles (Fig. 6d). The cerebral cortex of CPF-exposed group for 24 h showed proliferation of glial cells with condensed nucleus forming glial nodules (Fig. 6e). The changes increased in its severity after 48 h as degeneration of cerebral neurons with pyknotic nucleus; while others showed complete karyolysis of neuronal nucleus, some neurons are swollen with eccentric nuclei (Fig. 6f). Scoring of the histological findings in the brain sections are presented in Table 7.Cerebral cortex. a Dimethoate-exposed group for 24 h showing degeneration of large number of neurons with pyknosis of nucleus (arrow) (H&E, bar = 50 μm). b Dimethoate group after 48 h showing perivascular meningeal hemorrhage (star), thrombosis of others (arrow) (H&E, bar = 50 μm). c LCT group after 24 h showing degeneration with pyknotic nucleus of hippocampal neurons (arrow), (H&E, bar = 50 μm). d LCT group after 48 h showing demyelination (arrow). e CPF group after 24 h showing proliferation of glial cells (notched arrow). f Cerebral cortex, CPF intoxicated group after 48 h showing degeneration of neurons with pyknosis of nucleus (notched arrow), complete karyolysis of neuronal nucleus (star), and periphery positioned nucleus in others (arrow) (toluidine blue, bar = 50 μm)

The effect of dimethoate on liver after 24 h showed angiopathic changes which are demonstrated by dilatation of large blood vessels with dilatation of hepatic sinusoids (Fig. 7a). The same lesions were detected after 48 h accompanied by thrombosis of some blood vessels with vacuolar degeneration of endothelial cells of the wall of blood vessels (Fig. 7b). In LCT-exposed group after 24 h, the liver showed congestion of blood vessels associated with focal areas of proliferative active Kupffer cells (Fig. 7c). The damaged hepatocytes were observed after 48 h, which expressed by slight vacuolar degeneration of hepatocytes around congested blood vessels (Fig. 7d). CPF-exposed rats for 24 h showed vascular changes characterized by mixed thrombosis of blood vessel consisted of RBCs, WBCs, and fibrin in the blood vessels with damaged endothelial cells causing slight hemorrhage (Fig. 7e). After 48 h, focal areas of necrosis in hepatocytes with complete absence of nucleus were observed in addition to coagulative necrosis with pyknosis of nucleus (Fig. 7f).a Liver, Dimethoate group after 24 h showing congestion of large blood vessels (star) and dilatation of hepatic sinusoids (arrow). b Dimethoate group after 48 h showing thrombosis (star), with vacuolar degeneration of endothelial cells of the wall of blood vessel (arrow). c LCT group after 24 h showing congestion of blood vessels (star), proliferation of Kupffer cells (arrow). d LCT group after 48 h showing congestion of blood vessels (star) and slight vacuolar degeneration of hepatocytes (arrow). e CPF group after 24 h showing thrombosis of blood vessels (star), damaged endothelial cells (notched arrow) causing hemorrhage (arrow) (notched arrow). f CPF group after 48 h showing focal areas of necrosis (star) and coagulative necrosis of hepatocytes with pyknosis of nucleus (arrow) (H&E, bar = 50 μm)

Organophosphorus and pyrethroid insecticides are neurotoxic in nature; OP act as inhibitor of neuronal cholinesterase activity (Oral et al. [63] ), while pyrethroids exert their neurotoxic effects through voltage-dependent sodium channels (Soderlund et al. [75] ; Ray and Fry [68] ).

In the current study, a change in animal gate with backward stretching of the hind limb was observed in dimethoate group after 48 h and splayed gait with tiptoe in LCT group after 24 h indicating that both insecticides could affect the nervous system in a certain degree. The effect of LCT on the hind limbs of rats may be related to the neurotoxic effect of LCT, which delay the closure of sodium channels and thus increase cell membrane excitability; the extended opening of sodium channels lowers the threshold of sensory nerve fibers for the activation of further action potentials, resulting in repetitive firing of sensory nerve endings (Vijverberg and van den Bercken [83] ) that may progress to hyperexcitability of the entire nervous system (Narahashi et al. [58] ). Histological changes were found in the brain of rats exposed to LCT; cerebellum showed degeneration with pyknosis of nucleus of purkinje cells and medullary hemorrhage accompanied by degeneration of hippocampal neurons in the cerebral cortex at 24 h. These changes in association with the effect on sodium channels may be the major causes of the clinical signs of splayed gait and tiptoe. Our results agreed with a study on animals exposed to lambda cyhalothrin insecticide, which induced noticeable significant changes in the liver and brain (Abdel-Mobdy and Abdel-Rahim [4] ; Mani et al. [51] ).

The clinical signs observed in dimethoate group may be attributed to the action of dimethoate as an OP insecticide, which acts through irreversible inhibition to acetylcholinesterase enzyme leading to accumulation of acetylcholine at neuromuscular junction and in the autonomic and central nervous system (Wiener and Hoffman [87] ; Oral et al. [63] ). Dogan et al. ([22] ) explained the neurotoxicity of dimethoate and inhibition of acetylcholinesterase enzyme by the formation of oxon metabolite (dimethoxon). In addition, dimethoate induced histological changes in the brain of rats at 24 and 48 h indicated by degenerative changes in purkenji cells, as degeneration with nuclear pyknosis and vascular changes which appeared after 24 h and continue until 48 h. Cerebral cortex showed degeneration with pyknotic nucleus of neurons and severe vascular changes as perivascular meningeal hemorrhage and thrombosis. These changes were in agreement with other studied by Oppenheimer and Esiri ([62] ) and Lowe et al. ([50] ). Inhibition of Ach E and histological changes in the brain may explain the neurotoxicity of dimethoate insecticide. Meanwhile, CPF-exposed groups showed complete absence of purkinje cells leaving empty spaces in the cerebellar cortex with proliferation of astrocyte cells in all cases. On other hand, glial nodules were formed of in the cerebral cortex. Soderlundet al. ([75] ) reported that pesticides are hydrophobic in nature so they accumulate in the biological membranes, especially in the phospholipid bilayers and in lipid rich internal tissues including the body fat, liver, kidneys, ovaries, and elements of the central and peripheral nervous systems.

Oxidative stress, as a probable mechanism of pesticides toxicity, has become a focus of toxicological research, as it plays critical pathophysiological mechanism in different human pathologies, including cancer, immunosuppression, and neurodegenerative diseases (Piperakis et al. [64] ; Mostafalou and Abdollahi [57] ; Deeba et al. [20] ). OP insecticides can develop oxidative stress by inhibiting antioxidant defenses (Soltaninejad and Abdollahi [76] ; Hussain [39] ; Lee et al. [46] ). The present results are in harmony with all the previous reports; our results revealed that exposure to CPF and dimethoate insecticides was associated with significant increase in serum MDA levels at 24 and 48 h and a concomitant decrease in GPx activity at 24 and 48 h for CPF but dimethoate reduced GPx activity at 48 h only. Some authors suggested that the basis of OP toxicity in the production of oxidative stress may be due either to their redox-cycling activity, where they readily accept an electron to form free radicals and then transfer them to oxygen to generate superoxide anions and hence hydrogen peroxide through disputation reactions or may be due to ROS generation via changes in normal antioxidant homeostasis resulting in antioxidant depletion (Abd Ellah et al. [2] ). Another source for the oxidative stress is the excess levels of oxidants that are not efficiently removed by antioxidant defense systems, which react with biological macromolecules causing enzyme inactivation, DNA damage, and lipid peroxidation in tissues (Abdollahi et al. [5] ; Ogut et al. [61] ).

In this study, LCT pyrethroid induced oxidative stress at 24 and 48 h, which is indicated by the significant increase in MDA level accompanied by reduction in GPx activity; these results are in agreement with other reports of LCT toxicity in mammals and the ability of this pesticide to induce oxidative stress in vivo and in vitro (El-Demerdash [25] ; Fetoui et al. [30] , [31] , [32] ; Abdallah et al. [3] ). The increase of MDA levels and the impairment of antioxidant enzyme activities clearly indicated that LTC had the potency to cause oxidative damage. Nasuti et al. ([60] ) and Prasanthiet and Rajini ([66] ) reported that oxidative damage, induced by LCT pyrethroid, might be due to their lipophilicity, whereby they could penetrate easily to the cell membrane and caused lipid peroxidation. Kale et al. ([40] ) suggested that pyrethroid metabolism might produce reactive oxygen species (ROS), which in turn could lead to enhanced peroxidation of lipids. Furthermore, WHO ([86] ) reported that toxicity of LTC might be attributed to the release of cyanohydrins, which are unsteady under physiological circumstances and further decay to cyanides and aldehydes, which consequently could act as a source of free radicals. Thus, our findings are consistent with those of other studies in which the oxidative stress was increased by the toxicity of synthetic organophosphates and Pyrethroid insecticides (Prakasam and Sethupathy [65] ; Lee et al. [46] ).

Oxidative stress and free radicals formed by insecticides could produce DNA damage (Bertram and Hass [14] ; Tuzmen et al. [81] ; Heikal et al. [38] ; Lee et al. [46] ). Comet assay is used commonly as an indicator for the occurrence of DNA damage and used widely in the field of genetic toxicology and environmental biomonitoring. The DNA damage detected by the comet assay could be due to DNA single strand breaks, double strand breaks, adduct formations, DNA-DNA, and DNA-protein cross-links (Mitchelmore and Chipman [54] ). DNA damage using comet assay is generally quantified by comet tail length and tail moment. Tail length is the distance of DNA migration from the body of the nuclear core while tail moment is calculated as tail length multiplied by the percentage of DNA in the tail (Collins et al. [19] ; Tice et al. [79] ). Tail length can be used to indicate initial DNA damage and confirm exposure to a genotoxin, while tail moment and the percentage of DNA in the tail can be used to indicate the intensity of damage (Knopper et al. [45] ).

The current study used alkaline comet assay for detection of DNA damage in the brain and liver tissues. DNA% in head was decreased, and tail DNA% increased; this indicates DNA migration from head to the tail of the comet and indicates some degree of DNA damage; also, tail length is the most famous parameter that gives an indication about the length of migrated DNA, but tail moment is the most accurate because it is a product of multiplication of tail length and tail DNA%. We found that DNA damage in the brain tissue was severe in CPF and LCT-exposed groups at 24 and 48 h, while dimethoate group showed the lowest degree of DNA damage only at 48 h indicated by increased tail length; this damage has a direct relation with oxidative stress parameters as MDA increased and GPx decreased in CPF and LCT groups; on the other hand, dimethoate caused DNA damage in the brain at 48 h only associated with similar change in GPx value, which was decreased at 48 h, and this result may confirm the rule of oxidative stress in induction of DNA damage. All these results confirm that CPF in the brain showed the highest degree of DNA damage at 24 and 48 h; this indicated that neurotoxicity of CPF is greater than LCT regardless of LD50, which is supported by the explanation of Linn ([47] ), who stated that CPF is bioactivated to a more potent cholinesterase inhibitor, chlorpyrifos oxon (CPO), by a cytochrome P450-mediated desulfuration reaction; this induces DNA damage alone or in combination with CPF, which might be responsible for producing DNA damage. Additional and more reactive oxygen atom of CPO may also be a source of ROS, which can damage DNA. Also, Braun et al. ([16] ) reported that OP compounds show alkylating properties which can induce DNA damage.

Hepatic DNA damage was detected by single cell gel electrophoresis. LCT and CPF groups showed the highest degree of DNA damage at 24 and 48 h, while dimethoate group showed mild DNA damage at 24 h indicated by increased tail length only without significant changes in other parameters, but at 48 h, dimethoate induced severe DNA damage in the liver tissue. This result is in agreement with oxidative stress data that showed increased MDA level and reduction in GPx activity in serum in the same manner with DNA damage. Finally, it was observed that the degree of DNA damage in the liver tissue was in direct relation with LD50 of each compound: LCT, CPF, and then dimethoate.

Pesticides are known to induce various histopathological changes in the liver tissues (Al-Awthanet al. [6] ; Elsharkawy et al. [26] ; Ezzi et al. [27] ). In the present study, the effect of dimethoate on the liver was characterized by angiopathic changes, which include congestion of blood vessels as well as thrombosis of some blood vessels. These results are in agreement with Al-Awthan et al. ([6] ) and Lone et al. ([49] ), who found that exposure to dimethoate in pigs and rats caused histological changes in hepatic tissue accompanied by increased lipid peroxidation and decreased antioxidant levels; these changes may be due to the direct toxic effect of dimethoate such as lipid peroxidation in the cell membrane with stimulation of lipids to accumulate in hepatocytes (Lone et al. [49] ). Dimethoate usually accumulated in the liver after absorption causing damage to hepatic tissue (El-Damaty et al. [24] ). In LCT group, the observed vascular changes were accompanied with proliferation of active Kupffer cells and vacuolar degeneration (Mani et al. [51] ). The changes were more pronounced in the liver of rats exposed to CPF that includes mixed thrombosis with focal areas of necrosis in hepatocytes.

This result agreed with Elsharkawy et al. ([26] ), who reported that subchronic exposure of rats to CPF caused hemorrhage, vacuolar degeneration, dilation of sinusoids, vascular congestion, and necrosis of hepatic cells. Necrosis of hepatic cells was reported by Kammon et al. ([52] ) in chickens exposed to chlorpyrifos. It is possible that dimethoate, LCT, and CPF behave like several other insecticides and affects adversely on the cytochrome P450 system or the mitochondrial membrane transport system of hepatocytes. Exposure to CPF may lead to membrane damage in hepatic cells and eventual loss of membrane integrity (Gokcimen et al. [35] ).

CPF, dimethoate, and LCT insecticides have the potential to cause oxidative stress and mutagenic effects by causing DNA damage in addition to histopathological changes in the brain and liver.

The authors declare that they have no conflict of interest.

All procedures involving animals were done in accordance with the ethical standards of Assiut University. All rats were handled according to the standard guidelines for care and use of experimental animals.

1 Abd Elkawy M, Oliman G, Abd-El Rehim E, Effect of Solanum nigrum Linn against lambda cyhalothrin-induced toxicity in rats, IOSR J Pharm Bio Sci, 2013, 5, 55, 62

• 2 Abd Ellah MR, Nishimori K, Goryo M, Okada K, Yasuda J, Glutathione peroxidase and glucose 6 phosphate dehydrogenase activities in bovine blood and liver, J Vet Med Sci, 2004, 66, 10, 1219, 1221, 10.1292/jvms.66.1219
• 3 Abdallah F, Fetoui H, Fakhfakh F, Keskes L, Caffeic acid and quercetin protect erythrocytes against the oxidative stress and the genotoxic effects of lambda-cyhalothrin in vitro, Hum Exp Toxicol, 2012, 31, 1, 92, 100, 10.1177/0960327111424303
• 4 Abdel-Mobdy YE, Abdel-Rahim EA, Toxicological influences of lambda cyhalothrin and evaluation of the toxicity ameliorative effect of pomegranate in albino rats, global, Veterinaria, 2015, 14, 6, 913, 921
• 5 Abdollahi M, Ranjbar A, Shadnia S, Nokfar S, Rezaie A (2004) Pesticides and oxidative stress: a review,. Med SciMonit.10: 141, -147
• 6 Al-Awthan YS, Mohamed A, Gamal HE, Esam MA, Dimethoate-induced oxidative stress and morphological changes in the liver of Guinea pig and the protective effect of vitamin C and E, Asian J Biol Sci, 2012, 5, 9, 19, 10.3923/ajbs.2012.9.19
• 7 Arce ZR, Rojas-García AE, Benitez-Trinidad A, Herrera-Moreno JF, Medina-Díaz IM, Barrón-Vivanco BS, Villegas GP, Hernández-Ochoa I, Sólis Heredia MJ, Bernal-Hernández YY, Oxidative stress and genetic damage among workers exposed primarily to organophosphate and pyrethroid pesticides, Environ Toxicol, 2017, 32, 1754, 1764, 10.1002/tox.22398
• 8 Astiz M, Alaniz MJ, Marra CA, Effect of pesticides on cell survival in liver and brain rat tissues, Ecotoxicol Environ Saf, 2009, 72, 2025, 2032, 10.1016/j.ecoenv.2009.05.001
• 9 Ayala A, Munoz MF (2014) Arguelles S. Lipid peroxidation production, metabolism and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxidative Med Cell Longevity 1-31
• 10 Bagchi D, Balmoori J, Bagchi M, Ye X, Williams CB, Stohs SJ, Comparative effects of TCDD, endrin, naphthalene and chromium (IV) on oxidative stress and tissue damage in the liver and brain tissues of mice, Toxicology, 2002, 175, 73, 82, 10.1016/S0300-483X(02)00062-8
• 11 Ballesteros ML, Durando PE, Nores ML, Diaz MP, Bistoni MA, Wunderlin DA, Endosulfan induces changes in spontaneous swimming activity and acetylcholinesterase activity of Jenynsia multidentata (Anablepidae, Cyprinodontiformes), Environ Pollut, 2009, 157, 1573, 1580, 10.1016/j.envpol.2009.01.001
• 12 Bancroft D, Stevens A, Turmer R (1996) Theory and practice of histological technique, 4, thed, Churchill Living Stone, Edinburgh, London, Melbourne. pp. 47-67
• 13 Bebe FN, Panemangalore M, Exposure to low doses of endosulfan and chlorpyrifos modifies endogenous antioxidants in tissue of rats, J Environ Sci Heal B, 2003, 38, 349, 363, 10.1081/PFC-120019901
• 14 Bertram C, Hass R, Cellular responses to reactive oxygen species induced DNA damage and aging, BiolChem, 2008, 389, 211, 220
• 15 Bolognesi C, Genotoxicity of pesticides: a review of human biomonitoring studies, Mutat Res/Rev Mutat Res, 2003, 543, 251, 272, 10.1016/S1383-5742(03)00015-2
• 16 Braun R, Schoneich J, Weissflog L, Debek W, Activity of organophosphorus insecticides in bacterial tests for mutagenicity and DNA repair-direct alkylation vs metabolic activation and breakdown. I: butanoate, vinylbutonate, dichlorvos, demethyldichlorovos and demethyl vinylbutonate, Chem Biol Interact, 1982, 39, 339, 350, 10.1016/0009-2797(82)90050-3
• 17 Celik A, Mazmanci BC, Amlica Y, Askin AC, Omelekoglu U, Cytogenitic effects of lambda-cyhalothrin on wistar rat bone marrow, Mutat Res, 2003, 539, 91, 97, 10.1016/S1383-5718(03)00159-1
• 18 Celik A, Mazmanci BC, Amlica YC, Omelekoglu U, Askin A, Evaluation of cytogenetic effects of lambda-cyhalothrin on Wistar rat bone marrow by gavage administration, Ecotoxicol Environ Saf, 2005, 61, 128, 133, 10.1016/j.ecoenv.2004.07.009
• 19 Collins AR, Dobson VL, Dusinská M, Kennedy G, Stĕtina R, The comet assay: what can it really tell us?, Mutat Res, 1997, 375, 2, 183, 193, 10.1016/S0027-5107(97)00013-4
• 20 Deeba F, Raza I, Muhammad N (2016) Chlorpyrifos and lambda cyhalothrin-induced oxidative stress in human erythrocytes: in vitro studies. Toxicol Ind Health:1-11
• 21 DeLeve LD, Kaplowitz N, Glutathione metabolism and its role in hepatotoxicity, Pharmacol Ther, 1991, 52, 287, 305, 10.1016/0163-7258(91)90029-L
• 22 Dogan D, Can C, Kocyigit A, Dikilitas M, Taskin A, Bilinc H, Dimethoate-induced oxidative stress and DNA damage in Oncorhynchus mykiss, Chemosphere, 2011, 84, 39, 46, 10.1016/j.chemosphere.2011.02.087
• 23 Ducolomb Y, Casas E, Valdez A, Gonzalez G, Altamirano- Lozano M, Betancourt M, In vitro effect of malathion and diazinon on oocytes fertilization and embryo development in porcine, Cell Biol Toxicol, 2009, 25, 623, 633, 10.1007/s10565-008-9117-3
• 24 El-Damaty EM, Farrag AH, Rowayshed G, Fahmy HM, Biochemical and Histopathological effects of systemic pesticides on some functional organs of male albino rats, J Appl Sci Res, 2012, 8, 11, 5459, 5469
• 25 El-Demerdash F, Lambda-cyhalothrin-induced changes in oxidative stress biomarkers in rabbit erythrocytes and alleviation effect of some antioxidants, Toxicol in Vitro, 2007, 21, 3, 392, 397, 10.1016/j.tiv.2006.09.019
• 26 Elsharkawy EE, Yahia D, El-Nisr N, Sub-chronic exposure to chlorpyrifos induces hematological, metabolic disorders and oxidative stress in rat: attenuation by glutathione, Environ Toxicol Pharmacol, 2013, 35, 218, 227, 10.1016/j.etap.2012.12.009
• 27 Ezzi L, Salah IB, Haouas Z, Sakly A, Grissa I, Histopathological and genotoxic effects of chlorpyrifos in rats, Environ SciPollut Res, 2016, 23, 4859, 4867, 10.1007/s11356-015-5722-x
• 28 Fahmy AM, Abdalla EF, Cytogenetic effects by the natural pyrethrin and the synthetic lambda-cyhalothrin in mice in vivo, Cytologia, 2001, 66, 139, 149, 10.1508/cytologia.66.139
• 29 Felton GW, Summers CB, Antioxidant systems in insects, Arch Insect Biochem Physiol, 1995, 29, 187, 197, 10.1002/arch.940290208
• 30 Fetoui H, Garoui M, Makni-Ayadi F, Zeghal N, Oxidative stress induced by lambda-cyhalothrin (LTC) in rat erythrocytes and brain: attenuation by vitamin C, Environ Toxicol Pharmacol, 2008, 26, 2, 225, 231, 10.1016/j.etap.2008.04.002
• 31 Fetoui H, Garoui MZ, eghal N, Lambda-cyhalothrin-induced biochemical and histopathological changes in the liver of rats: ameliorative effect of ascorbic acid, Exp Toxicol Pathol, 2009, 61, 189, 196, 10.1016/j.etp.2008.08.002
• 32 Fetoui H, Makni M, Garoui M, Zeghal N, Toxic effects of lambda-cyhalothrin, a synthetic pyrethroid pesticide, on the rat kidney: involvement of oxidative stress and protective role of ascorbic acid, Exp Toxicol Pathol, 2010, 62, 6, 593, 599, 10.1016/j.etp.2009.08.004
• 33 Glickman AH, Lech JJ, Differential toxicity of trans-permethrin in rainbow trout and mice. II. Role of target organ sensitivity, Toxicol Appl Pharmacol, 1982, 66, 162, 171, 10.1016/0041-008X(82)90281-2
• 34 Goel A, Chanuhan DP, Dhawan DK, Protective effects of zinc in chlorpyrifos induced hepatotoxicity; a biochemical and trace element study, Bio Trace Elemen Res, 2000, 74, 171, 183, 10.1385/BTER:74:2:171
• 35 Gokcimen A, Gulle K, Demirin H, Bayram D, Kocak A, Altuntas I, Effects of diazinon at different doses on rat liver and pancreas tissues, Pestic Biochem Physiol, 2007, 87, 103, 108, 10.1016/j.pestbp.2006.06.011
• 36 Grotto D, Maria LS, Valentini J, Paniz C, Schmitt G, Garcia SC, Pomblum VJ, Rocha JB, Farina M, Importance of the lipid peroxidation biomarkers and methodological aspects FOR malondialdehyde quantification, Química Nova, 2009, 32, 1, 169, 174, 10.1590/S0100-40422009000100032
• 37 He L, Troiano J, Wang A, Goh K, Environmental chemistry, ecotoxicity, and fate of lambda cyhalothrin, Rev Environ Contam Toxicol, 2008, 195, 71, 91
• 38 Heikal TM, Mossa AH, Nawwar GA, MEl-sherbiny M, Ghanem HZ, Protective effect of a synthetic antioxidant “acetyl gallate derivative” against dimethoate induced DNA damage and oxidant antioxidant status in male rats, Environ Anal Toxicol, 2012, 2, 155
• 39 Hussain LA, Role of oxidative stress in organophosphate insecticide toxicity—short review, Pest Biochem Physiol, 2010, 98, 145, 150, 10.1016/j.pestbp.2010.07.006
• 40 Kale M, Rathore N, John S, Bhatnagar D, Lipid peroxidative damage on pyrethroid exposure and alterations in antioxidant status in rat erythrocytes: a possible involvement of reactive oxygen species, Toxicol Lett, 1999, 105, 197, 205, 10.1016/S0378-4274(98)00399-3
• 41 Kammon AM, Brar RS, Banga HS, Sodhi S, Patho-biochemical studies on hepatotoxicity and nephrotoxicity on exposure to chlorpyrifos and imidacloprid in layer chickens, Vet Arhi v, 2010, 80, 663, 672
• 42 Kassie F, Parzefall W, Knasmuller S, Single cell gel electrophoresis assay: a new technique for human biomonitoring studies, Mutat Res, 2000, 463, 13, 31, 10.1016/S1383-5742(00)00041-7
• 43 Kavitha P, Rao JV, Toxic effects of chlorpyrifos on antioxidant enzymes and target enzyme acetylcholinesterase interaction in mosquito fish, Gambusia affinis, Environ Toxicol Pharm, 2008, 26, 192, 198, 10.1016/j.etap.2008.03.010
• 44 Kidd H, James DR, The agrochemicals handbook, 1991, 3, Cambridge, Royal Society of Chemistry Information Services, 27, 33
• 45 Knopper LD, Mineau P, McNamee JP, Lean DR, Use of comet and micronucleus assays to measure genotoxicity in meadow voles (Microtus pennsylvanicus) living in golf course ecosystems exposed to pesticides, Ecotoxicology, 2005, 14, 3, 323, 335, 10.1007/s10646-004-6369-4
• 46 Lee KM, Park SY, Lee K, Oh S, Ko S, Pesticide metabolite and oxidative stress in male farmers exposed to pesticide, Ann Occup Environ Med, 2017, 29, 5, 10.1186/s40557-017-0162-3
• 47 Linn S, DNA damage by iron and hydrogen peroxide in vitro and in vivo, Drug Metab Rev, 1998, 30, 313, 326, 10.3109/03602539808996315
• 48 Livingstone DR, Contaminant-stimulated reactive oxygen species production and oxidative damage in aquatic organisms, Mar Pollut Bull, 2001, 42, 656, 666, 10.1016/S0025-326X(01)00060-1
• 49 Lone MY, Baba BA, Raj P, Shrivastava VK, Bhide M, Haematological and hepatopathological changes induced by dimethoate in Rattus rattus, Indo Am J Pharm Res, 2013, 3, 4360, 4365
• 50 Lowe J, Lennox G, Leigh PN, Graham DI, Lantos PL, Disorders of movement and system degenerations, Greenfield’s neuropathology, 1997, 6, London, Arnold, 312, 314
• 51 Mani VM, Ahmed AN, Ali AL, Gokulakrishnan A, Vinoth K, Hussain AA, Hepatoprotective effect of quercetin on lambda-cyhalothrin induced hepatotoxicity in male Wistar rats, Int J Sci Humanit, 2016, 2, 401, 416
• 52 Mansour SA, Mossa AT, Oxidative damage, biochemical and histopathological alterations in rats exposed to chlorpyrifos and the antioxidant role of zinc, Pestic Biochem Physiol, 2010, 96, 14, 23, 10.1016/j.pestbp.2009.08.008
• 53 Meister A (1988) Glutathione metabolism and its selective modification. J Biol Chem 263(33):17205-17208
• 54 Mitchelmore CL, Chipman JK, DNA strand breakage in aquatic organisms and the potential value of the comet assay in environmental monitoring, Mutat Res, 1998, 399, 135, 147, 10.1016/S0027-5107(97)00252-2
• 55 Modesto KA, Martinez CB, Effects of roundup transorb on fish: hematology, antioxidant defenses and acetylcholinesterase activity, Chemosphere, 2010, 81, 781, 787, 10.1016/j.chemosphere.2010.07.005
• 56 Monteiro DA, Almeida JA, Rantin FT, Kalinin AL, Oxidative stress biomarkers in the freshwater characid fish, Brycon cephalus, exposed to organophosphorus insecticide Folisuper 600 (methyl parathion), Comp Biochem Phys, 2006, 143, 141, 149, 10.1016/j.cbpa.2006.01.053
• 57 Mostafalou S, Abdollahi M, Pesticides and human chronic diseases: evidences, mechanisms and perspectives, Toxicol Appl Pharmacol, 2013, 268, 157, 177, 10.1016/j.taap.2013.01.025
• 58 Narahashi T, Carter DB, Frey J, Ginsburg K, Hamilton BJ, Nagata K, Roy ML, Song JH, Tatebayashi H, Sodium channels and GABAA receptor-channel complex as targets of environmental toxicants, Toxicol Lett, 1995, 82-83, 239, 245, 10.1016/0378-4274(95)03482-X
• 59 Naravaneni R, Jamil K, Evaluation of cytogenetic effects of lambda-cyhalothrin on human lymphocytes, J Biochem Mol Toxicol, 2005, 19, 5, 304, 310, 10.1002/jbt.20095
• 60 Nasuti C, Cantalamessa F, Falcioni G, Gabbianelli R, Different effects of type I and type II pyrethroids on erythrocyte plasma membrane properties and enzymatic activity in rats, Toxicology, 2003, 191, 233, 244, 10.1016/S0300-483X(03)00207-5
• 61 Ogut S, Gultekin F, Kisioglu AN, Kucukoner E, Oxidative stress in the blood of farm workers following intensive pesticide exposure, Toxicol Ind Health, 2011, 27, 820, 825, 10.1177/0748233711399311
• 62 Oppenheimer DR, Esiri MM, Adams JH, Duchen LW, Diseases of the basal ganglia, cerebellum and motor neurons, Greenfield’s neuropathology, 1992, 5, London, Arnold, 988, 1045
• 63 Oral B, Guney M, Demirin H, Endometrial damage and apoptosis in rats induced by dichlorvos and ameliorating effect of antioxidant vitamin E and C, Reprod Toxicol, 2006, 22, 783, 790, 10.1016/j.reprotox.2006.08.003
• 64 Piperakis SM, Kontogianni K, Siffel C, Piperakis MM, Measuring of effects of pesticides on occupationally exposed humans with the comet assay, Environ Toxicol, 2005, 21, 355, 359, 10.1002/tox.20191
• 65 Prakasam AS, Sethupathy SL, Plasma and RBCs antioxidant status in occupational male pesticide sprayers, Clin Chim Acta, 2001, 310, 107, 112, 10.1016/S0009-8981(01)00487-9
• 66 Prasanthi K, MuralidharaRajini PS, Morphological and biochemical perturbations in rat erythrocytes following in vitro exposure to Fenvalerate and its metabolite, Toxicol in Vitro, 2005, 19, 449, 456, 10.1016/j.tiv.2004.12.003
• 67 Radad K, Hassanein K, Moldzio R, Rausch WD, Vascular damage mediates neuronal and non-neuronal pathology following short and long-term rotenone administration in Sprague-Dawley rats, Exp Toxicol Pathol, 2013, 65, 41, 47, 10.1016/j.etp.2011.05.008
• 68 Ray DE, Fry JR, A reassessment of the neurotoxicity of pyrethroid insecticides, Pharmacol Ther, 2006, 111, 174, 193, 10.1016/j.pharmthera.2005.10.003
• 69 Saafi EB, Louedi M, Elfeki A, Zakhama A, Najjar MF, Hammami M, Achour L, Protective effect of date palm fruit extract (Phoenix dactylifera L.) on dimethoate induced-oxidative stress in rat liver, Exp Toxicol Pathol, 2011, 63, 433, 441, 10.1016/j.etp.2010.03.002
• 70 Sakonlaya D, Apisarnthanarak A, Yamada N, Tomtitchong P, Modified toluidine blue: an alternative stain for Helicobacter pylori detection in routine diagnostic use and post-eradication confirmation for gastric cancer prevention, Asian Pac J Cancer Prev, 2014, 15, 6983, 6987, 10.7314/APJCP.2014.15.16.6983
• 71 Sasaki YF, Izumiyama F, Nishidate E, Matsusaka N, Tsuda S, Detection of rodent liver carcinogen genotoxicity by the alkaline single cell gel electrophoresis (Comet) assay in multiple mouse organs (liver, lung, spleen, kidney and bone marrow), Mutat Res, 1997, 391, 201, 214, 10.1016/S1383-5718(97)00072-7
• 72 Sharma Y, Bashir S, Irshad M, Gupta SD, Dogra TD, Effects of acute dimethoate administration on antioxidant status of liver and brain of experimental rats, Toxicology, 2005, 206, 49, 57, 10.1016/j.tox.2004.06.062
• 73 Sharma RK, Upadhyay G, Siddiqi NJ, Sharma B, Pesticides-induced biochemical alterations in occupational North Indian suburban population, Hum Exp Toxicol, 2013, 32, 1213, 1227, 10.1177/0960327112474835
• 74 Singh M, Sandhir R, Kiran R, Erythrocyte antioxidant enzymes in toxicological evaluation of commonly used organophosphate pesticides, Indian J Exp Biol, 2006, 44, 580, 583
• 75 Soderlund DM, Clarc GM, Sheets LP, Mullin LS, Piccirillo VJ, Mechanisms of pyrethroid neurotoxicity: implications for cumulative risk assessment, Toxicology, 2002, 171, 3, 59, 10.1016/S0300-483X(01)00569-8
• 76 Soltaninejad K, Abdollahi M, Current opinion of the science of organophosphate pesticides and toxic stress: a systematic review, Med Sci Monit, 2009, 15, 75, 90
• 77 Southwood J (1985) Acute oral toxicity studies. Unpublished report no. CTL/P/1102 from Central Toxicology Laboratory, Macclesfield, England. Submitted to WHO by Syngenta Crop Protection AG. PP321
• 78 Sparling DW, Fellers G, Comparative toxicity of chlorpyrifos, diazinon, malathion and their oxon derivatives to larval Rana boylii, Environ Pollut, 2007, 147, 535, 539, 10.1016/j.envpol.2006.10.036
• 79 Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, Sasaki YF, Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing, Environ Mol Mutagen, 2000, 35, 3, 206, 221, 10.1002/(SICI)1098-2280(2000)35:3<206::AID-EM8>3.0.CO;2-J
• 80 Tsuda S, Kosaka Y, Matsusaka N, Sasaki YF, Detection of pyrimethamine-induced DNA damage in mouse embryo and maternal organs by the modified alkaline single cell gel electrophoresis assay, Mutat Res, 1998, 415, 69, 77, 10.1016/S1383-5718(98)00057-6
• 81 Tuzmen N, Candan N, Kaya E, Demiryas N, Biochemical effects of chlorpyrifos and deltamethrin on altered antioxidative defence mechanisms and lipid peroxidation in rat liver, Cell Biochem Funct, 2008, 26, 119, 124, 10.1002/cbf.1411
• 82 US EPA (1988) Fact sheet number 171: karate (PP321). Washington, DC
• 83 Vijverberg HP, Bercken J, Neurotoxicological effect and the mode of action of pyrethroid insecticides, Crit Rev Toxicol, 1990, 21, 105, 126, 10.3109/10408449009089875
• 84 Wang WX, Interaction of trace metals and different marine food chains, Mar Ecol Prog Ser, 2002, 243, 295, 309, 10.3354/meps243295
• 85 Wang X, Xing H, Li X, Xu S, Wang X, Effects of atrazine and chlorpyrifos on the mRNA levels of IL-1 and IFN-γ 2b in immune organs of common carp, Fish Shellfish Immunol, 2011, 31, 126, 133, 10.1016/j.fsi.2011.04.015
• 86 WHO (1990) Cyhalothrin, environmental health criteria, 99; Geneva, Switzerland
• 87 Wiener SW, Hoffman RS, Nerve agents: a comprehensive review, J Intensive Care Med, 2004, 19, 1, 22, 37, 10.1177/0885066603258659
• 88 Worthing CR, Walker SB (1987) The Pesticide manual: a world compendium. 8th ed, Thornton Heath, [Surrey, England]: British Crop Protection Council

PHOTO (COLOR)

PHOTO (COLOR)

PHOTO (COLOR)

PHOTO (COLOR)

PHOTO (COLOR)

PHOTO (COLOR)

PHOTO (COLOR)