Environmental Science and Pollution Research https://doi.org/10.1007/s11356-018-1776-x RESEARCH ARTICLE Assessment of neurohepatic DNA damage in male Sprague–Dawley rats exposed to organophosphates and pyrethroid insecticides Doha Yahia 1 & Marwa F. Ali 2 Received: 28 January 2018 / Accepted: 13 March 2018 # Springer-Verlag GmbH Germany, part of Springer Nature 2018 Abstract 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. Keywords CPF . Dimethoate . LCT . DNA damage . Comet . Oxidative stress . Brain . Liver Introduction Insecticides are widely produced all over the world and used for control of agricultural and household pests, which put h um a n an d an i m a l s u n de r t he ri s k of ex po s u r e . 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. 2011). 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 2002; Ballesteros et al. 2009). Responsible editor: Philippe Garrigues * Doha Yahia dohayahia@yahoo.com 1 Department of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt 2 Department of Veterinary Pathology and Clinical Pathology, Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt 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 2007). 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. 2005; Singh et al. 2006). Toxic effects of OP include genotoxicity, hepatic dysfunction, embryotoxicity, teratogenicity, neurochemical, and neurobehavioral changes (Goel et al. 2000). It was reported that both acute and chronic exposures to CPF resulted in liver damage in rats (Mansour and Mossa 2010; Elsharkawy et al. 2013; Ezzi et al. 2016). Furthermore, acute and subchronic exposures to dimethoate caused pathological changes in the brain and liver of rats (Astiz et al. 2009; Saafi et al. 2011). 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 1982; Kidd and James 1991). Moreover, pyrethroid is used for the control of a Environ Sci Pollut Res broad range of ectoparasites on cattle and sheep (Abd Elkawy et al. 2013). 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. 2002; Naravaneni and Jamil 2005; Ray and Fry 2006) and also affects chloride and calcium channels, which are essential for appropriate nerve function (He et al. 2008). Reportedly, LCT induced genotoxicity, chromosomal aberrations, and micronucleus formation in the bone marrow cells of rat (Fahmy and Abdallah 2001; Celik et al. 2003, 2005) and in human lymphocytes cultured in vitro (Naravaneni and Jamil 2005). Many research studies suggested that pesticides produce oxidative stress through the formation of oxygen free radical (Bagchi et al. 2002; Monteiro et al. 2006; Modesto and Martinez 2010; Lee et al. 2017). 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 2003) and inactivate biological macromolecules (Meister 1988). Normally, antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) provide the first-line defense against oxidative stress (Livingstone 2001). 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. 2013). 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 2008; Ballesteros et al. 2009). Lipid peroxidation product in the form of malondialdehyde (MDA) is used as a biomarker for lipid (Ayala and Munoz 2014; Arce et al. 2017). 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 1995). 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 1991). Pesticides have been considered potential mutagens and have genotoxic properties leading to mutations and DNA damage (Bolognesi 2003). OP compounds are known also to cause DNA damage by their alkylating properties and alkylating agents (Braun et al. 1982). 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. 1998). This assay allows the assessment of genetic damage in a great variety of cells (Kassie et al. 2000). 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. 2000). 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. Materials and methods Chemicals 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. 2009; Dogan et al. 2011; Ezzi et al. 2016). 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). Animals and treatment 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 1987). The second group was exposed to dimethoate with oral LD50 310 mg/kg (US EPA 1988). The third group was exposed to lambda cyhalothrin with oral LD50 79 mg/kg (Southwood 1985), and the fourth group Environ Sci Pollut Res Fig. 1 Hind limb disorder in rats exposed to dimethoate for 48 h (a) and LCT for 24 h (b) 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 malondialdehyde (MDA) level 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. 2009). Table 1 MDA (nmol/mL) levels in serum of rats exposed to CPF, dimethoate, or LCT in comparison with control 24 h 48 h Control CPF Dimethoate LCT 5.29 ± 1.37 6.02 ± 1.59 8.56 ± 1.83* 10.33 ± 2.50* 10.59 ± 0.23** 17.73 ± 0.80** 8.93 ± 2.16* 10.25 ± 0.01* Data are presented as means ± SD *P < 0.05 and **P < 0.01 as compared with control group Determination of serum glutathione peroxidase (GPx) activity 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. 2004). Comet assay (single cell gel electrophoresis) DNA damage in the brain and liver was detected using comet assay according to the methods of Sasaki et al. (1997) as follows: A. 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 resuspended in chilled homogenizing buffer and allowed to settle for 1–2 min. B. 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 Environ Sci Pollut Res Table 2 GPx (U/L) levels in serum of rats exposed to CPF, dimethoate, or LCT in comparison with control Control CPF Dimethoate LCT 24 h 85.38 ± 7.56 41.34 ± 1.99 ** 84.13 ± 1.96 54.04 ± 4.32 ** 48 h 84.30 ± 7.48 59.77 ± 1.26** 62.27 ± 5.79 * 38.90 ± 1.18 ** Data are expressed as means ± SD *P < 0.05 and **P < 0.01 as compared with control group 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. 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. Histopathological examination C. 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. D. 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. Examination of the slides The nuclei on the slides were examined at a 200 fold magnification using a fluorescence microscope (Olympus BX-43, Table 3 Comet assay parameters in the brain of rats exposed to CPF, dimethoate, or LCT for 24 h 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. 2014). Histopathological examination was performed by light microscopy (Olympus CX31, Japan) and photographed using digital camera (Olympus, Camedia C-5060, Japan) (Bancroftet al. 1996). 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. (2013) as follows: severe: +++, moderate: ++, slight: +, and 0: no change. Statistical analysis 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. Control CPF Dimethoate LCT Head DNA% 95.85 ± 0.42a 77.03 ± 3.56b 94.96 ± 0.72 ac 93.06 ± 0.97 ac Tail DNA% Tail length (μm) Tail moment Olive tail moment 4.15 ± 0.42a 8.05 ± 0.69a 0.38 ± 0.17a 1.46 ± 0.46a 22.97 ± 3.56b 31.04 ± 1.33b 11.34 ± 7.22b 15.22 ± 7.55b 5.04 ± 0.72a 13.77 ± 4.81a 0.99 ± 0.57 ac 2.20 ± 1.03 ac 6.94 ± 0.97a 17.93 ± 1.40c 1.41 ± 0.71 ac 3.38 ± 1.77a Data are presented as mean ± SE. Values followed by different letters mean significant; a, b, and c indicate differences among exposed groups (P < 0.05) Environ Sci Pollut Res Fig. 2 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 damage Results Clinical signs of acute exposure to CPF, dimethoate, and LCT 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). 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). DNA damage in the brain tissue after 24 h 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. Results of lipid peroxidation and antioxidant status in serum DNA damage in the brain tissue after 48 h 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 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 Fig. 3 Measuring 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 Environ Sci Pollut Res Table 4 Comet assay parameters in the brain of rats exposed to CPF, dimethoate, or LCT for 48 h Control CPF Dimethoate LCT Head DNA% Tail DNA% Tail length (μm) 96.51 ± 0.44a 3.49 ± 0.44a 7.69 ± 0.85a 58.77 ± 4.67b 41.23 ± 4.67b 65.85 ± 2.38 b 93.58 ± 1.03a 6.42 ± 1.03a 21.61 ± 6.34 c 80.01 ± 2.42c 19.99 ± 2.42c 43.16 ± 3.85d Tail moment Olive tail moment 0.29 ± 0.13a 1.27 ± 0.53a 31.31 ± 4.10b 26.29 ± 3.49b 2.14 ± 1.22a 2.83 ± 1.49a 12.99 ± 2.34c 13.25 ± 5.32c Data are presented as mean ± SE. Values followed by different letters mean significant; a, b, c and d indicate differences among exposed groups (P < 0.05) 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. Hepatic DNA damage after 24 and 48 h exposure 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. Histopathological findings in the brain Cerebellum 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. 4 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 (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 LCTexposed 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). Cerebrum 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 Environ Sci Pollut Res Table 5 Comet assay parameters in the liver of rats exposed to CPF, dimethoate or LCT for 24 h Control CPF Dimethoate LCT Head DNA% Tail DNA% Tail length (μm) 97.86 ± 0.60a 2.14 ± 0.60a 5.42 ± 0.72a 93.21 ± 1.28b 6.79 ± 1.28b 15.95 ± 1.73bc 97.67 ± 0.55a 2.33 ± 0.55a 11.73 ± 1.71b 92.33 ± 1.38b 7.67 ± 1.38b 18.52 ± 2.26c Tail moment Olive tail moment 0.27 ± 0.08a 0.70 ± 0.55a 1.69 ± 1.09b 3.10 ± 1.82b 0.57 ± 0.5a 1.12 ± 0.81a 2.08 ± 2.05b 3.67 ± 2.06b Data are presented as mean ± SE. Values followed by different letters mean significant; a, b and c indicate differences among exposed groups (P < 0.05) 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 LCTexposed 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. Histopathological findings in the liver 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 Table 6 Comet assay parameters in the liver of rats exposed to CPF, dimethoate, or LCT for 48 h 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). Discussion Organophosphorus and pyrethroid insecticides are neurotoxic in nature; OP act as inhibitor of neuronal cholinesterase activity (Oral et al. 2006), while pyrethroids exert their neurotoxic effects through voltage-dependent sodium channels (Soderlund et al. 2002; Ray and Fry 2006). 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 Control CPF Dimethoate LCT Head DNA% 98.36 ± 0.59a 95.08 ± 0.67b 94.61 ± 0.91b 86.46 ± 1.24c Tail DNA% Tail length (μm) Tail moment Olive tail moment 1.64 ± 0.59a 5.02 ± 0.80a 0.20 ± 0.06a 0.56 ± 0.18a 4.92 ± 0.67b 16.23 ± 1.58b 1.59 ± 1.43a 2.08 ± 1.11b 5.39 ± 0.91b 13.61 ± 1.52b 1.12 ± 0.96a 2.5 ± 1.25b 13.54 ± 1.24c 37.09 ± 2.29c 5.59 ± 1.93b 7.61 ± 2.24c Data are presented as mean ± SE. Values followed by different letters mean significant; a, b and c indicate differences among exposed groups (P < 0.05) Environ Sci Pollut Res Table 7 Scoring of the histological findings in the brain sections Lesions Cerebellum Cerebrum Dimethoate LCT CPF 24 h 48 h 24 h 48 h 24 h 48 h Pyknosis of nucleus of purkinje cells All cases ++ All cases +++ All cases ++ All cases ++ All cases ++ All cases +++ Angiopathic changes All cases +++ All cases +++ All cases +++ All case +++ One case +++ Two cases ++ Proliferation of astrocytes 0 _ One case ++ Two cases ++ One case ++ One case +++ Two cases ++ Pyknosis of nucleus of motor neurons All cases ++ One case + One case + One case ++ All cases ++ All cases + One case + All cases +++ All cases ++ All cases ++ One case + One case ++ All cases +++ All cases ++ All cases ++ All cases ++ One case +++ One case ++ All cases ++ One case ++ All cases ++ Two cases ++ All cases +++ Two cases ++ All cases +++ All cases +++ All cases +++ All cases +++ All cases +++ All cases +++ Demyelination Pyknosis of nucleus of hippocampal neurons Glial cell reaction Angiopathic changes The number of rats in each group (N = 4) 0 no change, + slight, ++ moderate, +++ severe 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 1990) that may progress to hyperexcitability of the entire nervous system (Narahashi et al. 1995). 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 2015; Mani et al. 2016). 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 2004; Oral et al. 2006). Dogan et al. (2011) 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 (1992) and Lowe et al. (1997). 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. (2002) 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. 2005; Mostafalou and Abdollahi 2013; Deeba et al. 2016). OP insecticides can develop oxidative stress by inhibiting antioxidant defenses (Soltaninejad and Abdollahi 2009; Hussain 2010; Lee et al. 2017). 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 Environ Sci Pollut Res Fig. 5 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) 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. 2004). 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. 2004; Ogut et al. 2011). 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 2007; Fetoui et al. 2008, 2009, 2010; Abdallah et al. 2012). 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. (2003) and Prasanthiet and Rajini (2005) 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. (1999) suggested that pyrethroid metabolism might produce reactive oxygen species (ROS), which in turn could lead to enhanced peroxidation of lipids. Furthermore, WHO (1990) 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 Environ Sci Pollut Res Fig. 6 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) increased by the toxicity of synthetic organophosphates and Pyrethroid insecticides (Prakasam and Sethupathy 2001; Lee et al. 2017). Oxidative stress and free radicals formed by insecticides could produce DNA damage (Bertram and Hass 2008; Tuzmen et al. 2008; Heikal et al. 2012; Lee et al. 2017). 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 1998). 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. 1997; Tice et al. 2000). 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. 2005). 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 (1998), 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 Environ Sci Pollut Res Fig. 7 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) 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. (1982) 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. 2012; Elsharkawy et al. 2013; Ezzi et al. 2016). 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. (2012) and Lone et al. (2013), 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. 2013). Dimethoate usually accumulated in the liver after absorption causing damage to hepatic tissue (El-Damaty et al. 2012). In LCT group, the observed vascular changes were accompanied with proliferation of active Kupffer cells and vacuolar degeneration (Mani et al. 2016). 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. (2013), 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. (2010) in chickens exposed to chlorpyrifos. It is possible that dimethoate, LCT, and CPF behave like several other insecticides and affects Environ Sci Pollut Res 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. 2007). 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