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Endosulfan-induced neurotoxicity and serum acetylcholinesterase inhibition in rabbits: The protective effect of Vit C
Pesticide Biochemistry and Physiology 96 (2010) 108–112
Contents lists available at ScienceDirect
Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest
Endosulfan-induced neurotoxicity and serum acetylcholinesterase inhibition in rabbits: The protective effect of Vit C Firdevs Mor a, Ozlem Ozmen b,* a b
Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Mehmet Akif Ersoy, 15100 Burdur, Turkey Department of Pathology, Faculty of Veterinary Medicine, University of Mehmet Akif Ersoy, 15100 Burdur, Turkey
a r t i c l e
i n f o
Article history: Received 10 January 2009 Accepted 16 October 2009 Available online 24 October 2009 Keywords: Endosulfan toxicity Serum acetylcholinesterase activity Pathology Rabbits Brain Immunohistochemistry
a b s t r a c t The neurotoxic effects and acetylcholinesterase inhibition induced by endosulfan, and the amelioration of these effects by Vitamin C (Vit C), were studied in the brains of New Zealand white rabbits. The cerebrum and cerebellum of each rabbit was examined grossly and histopathologically, and caspase-3 activity was determined by immunohistochemical methods. Twenty-four rabbits were divided into four groups (n = 6). Rabbits in Group I (END) were given a sublethal dose of endosulfan (1 mg/kg bw) in corn oil daily by oral gavage for 6 weeks. Group II (END + C) received the same dose of endosulfan and also Vit C (20 mg/kg bw) every second day during the 6 week period. Group III (OIL + C) received oral corn oil daily and Vit C every second day for 6 weeks. Group IV (OIL) received corn oil daily by oral gavage throughout the experiment. A significant reduction in acetylcholinesterase activity was observed in the END group, which was ameliorated in the END + C group. Hyperemia and slight hemorrhages in brains and cerebellums were seen in some rabbits in the END group. There were no gross cerebral or cerebellar lesions in the other groups. Hemorrhages, degenerations and slight gliosis were the marked histopathological findings of some rabbits belonging to the END group. A positive caspase-3 reaction was more severe in the END group than in the others. An ameliorating effect of Vit C on gross, histopathological, and immunohistochemical findings was observed in the END + C group. Thus, although endosulfan could cause neurotoxic effects in rabbits, this toxicity was decreased by Vit C treatment, which increased serum acetylcholinesterase activity. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Endosulfan, a chlorinated organic pesticide, is currently classified by the WHO as Class II (moderately hazardous to human health) and as category Ib (highly hazardous) by the United States Environmental Protection Agency [1]. Despite having caused many environmental tragedies and although it is now either banned or severely restricted in use in some countries, endosulfan is still widely used in many areas of the world including Turkey [2,3]. Commercial preparations of endosulfan consist of a- and b-isomers, constituting 70% and 30%, respectively. In the natural environment, mixed pesticide use and toxicity is common. However, cases do exist where only one particular pesticide, such as endosulfan, has been aerially spread over crops two to three times a year for more than 20 years. In these types of instances, toxicity in humans and livestock specifically due to endosulfan has been confirmed [1,4].
* Corresponding author. Fax: +90 248 2344505. E-mail address: [email protected] (O. Ozmen). 0048-3575/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2009.10.004
Acetylcholinesterase (AChE; EC 3.1.1.7), which is well known for its function at cholinergic synapses, is a multimeric protein with many facets. AChE terminates neurotransmission through the neurosynaptic cleft in both the central and the peripheral nervous system. AChE prevents the accumulation of acetylcholine that may overstimulate nicotinic and/or muscarinic receptors [5,6]. During neuronal development, AChE most likely is involved in regulating neural growth in various cellular systems. From in vitro studies using cells and explants, it has been concluded that AChE can regulate neural growth by a nonenzymatic mechanism [7]. Cholinesterases have also been identified in the serum (SAChE) [8]. Vitamin C (Vit C) is a water-soluble antioxidant that effectively scavenges reactive oxygen radicals (ROS), thus preventing tissue damage [9]. It can act to overcome oxidative stress, forming part of the antioxidant system. Many studies have reported that Vit C can reduce the lipid peroxidation (LPO) induced by toxic substances [10]. ROS readily damage biological molecules, ultimately leading to apoptotic and necrotic cell death [11]. The role of the Vit C in ameliorating oxidative stress reactions, such as induction of apoptosis and necrosis, has been indicated by numerous studies [12,13]. One mode of action may involve caspases, a family of
F. Mor, O. Ozmen / Pesticide Biochemistry and Physiology 96 (2010) 108–112
cysteine proteases that are activated when apoptosis is initiated and that play essential roles at various stages of apoptosis [14]. The aim of the present study was to examine the neurotoxic effect of endosulfan on acetylcholinesterase activity following experimental toxication of rabbits and to determine if Vit C might exert a protective effect against endosulfan toxicity. 2. Materials and methods The experiment was approved by the Institutional Animal Use and Care Committee of the Akdeniz University, and was performed in accordance with the National Institutes of Health Guidelines for the Care and Handling of Animals. 2.1. Animals Male New Zealand white rabbits, 6–8 months old, were used for this study. Rabbits were fed on standard rabbit chow and tap water ad libitum and were housed in cages at a controlled temperature (22 °C) and 12-h light/dark cycles throughout the study. The physical condition of each rabbit was assessed daily for any obvious signs of illness. 2.2. Experimental design Twenty-four rabbits were randomly allocated to four groups (n = 6). Group I (END) animals were exposed daily to sublethal concentrations of endosulfan (1 mg/kg bw) in corn oil for 6 weeks. Group II (END + C) received 1 mg/kg bw endosulfan daily and 20 mg/kg bw Vit C (ascorbic acid, Vit C) every second day during this period. Group III (OIL + C) received oral corn oil daily and 20 mg/kg bw Vit C every second day for 6 weeks. Group IV (OIL) was the control group and received daily corn oil only. All treatments were administered by oral gavage. 2.3. Histopathological evaluation Rabbits were sacrificed at 1 week after the last treatment. Before euthanasia, intracardiac blood samples were taken, and then necropsy was performed all the animals. Samples of cerebrum and cerebellum were fixed in 10% formalin, routinely processed, paraffin embedded, and stained with hematoxylin and eosin for by light microscope examination. Histopathological changes were graded in a blinded manner.
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temperature. After three washes with PBS, streptavidin peroxidase was added and the samples were incubated for 10 min at room temperature, and then rinsed four times with PBS. Tissues were further incubated for 20 min at room temperature in a solution of DAB (3,30 -diaminobenzidine) chromogen. After washing with PBS, tissues were counterstained with Mayer’s hematoxylin, washed with water, and coverslips were applied with mounting media. 2.5. Serum acetylcholinesterase (AChE) activity assay Acetylcholinesterase activity was determined according to Ellman et al. [15], with some modifications [16]. Hydrolysis rates were measured at 25 °C in 1 mL assay solutions containing 0.8 mM acetylthiocholine (S), 30 mM phosphate buffer, pH 7.5, and 1.0 mM DTNB. Fifty microliters of serum was added to the reaction mixture and preincubated for 3 min. The hydrolysis reaction was monitored by the formation of the thiolate dianion of DTNB at 412 nm for 2–3 min (recorded at 30 s intervals). All samples were run in duplicate. 2.6. Statistical analysis The Kruskal–Wallis Test was used for statistical treatment of data: within-and between-run coefficients of variation (cvs) were calculated for characterization assay. Mann–Whitney U-test analysis was performed to establish significant changes in cholinesterase activity after endosulfan incubation. All statistical analyses were run using SPSS 10.0 software program pack. 3. Results Clinical depression and slight nervous symptoms such as teeth grinding and hyperexcitability were observed in some rabbits in the END group after 4 weeks of endosulfan administration. There were no clinical symptoms in the other groups. Gross examination of the brains revealed marked hyperemia at the meningeal vessels and slight hemorrhages in brains and cerebellums in some rabbits from the END group. These findings were more prominent in those rabbits that had shown clinical nervous symptoms. There were no gross cerebral or cerebellar lesions in the other groups.
2.4. Immunohistochemical evaluation For evaluation of apoptosis, slides were stained for the presence of caspase-3 activity by immunohistochemical methods [Neomarker–California (caspase-3 (CPP32) Ab-4, 1:100 dilution] using a routine streptavidin–biotin peroxidase technique. For immunohistochemical examination, paraffin blocks were sectioned at 5 lm and sections were attached to glass slides coated with poly-L-lysine. The slides were dried overnight at 37 °C for optimization of adhesion. Sections were deparaffinized in multiple xylene baths, and tissues were rehydrated in a graded ethanol series. To reduce nonspecific background staining due to endogenous peroxidase, slides were incubated in hydrogen peroxide in methanol for 10 min. The sections were washed twice in phosphate buffer solution (PBS), then boiled in 1:100 citrate buffer solution for 10 min and cooled for 20 min. The tissues were washed in PBS for four times, then blocked with serum for 5 min. The primary antibody was then applied and the tissues were incubated for 30 min at room temperature. After four rinses with PBS, biotinylated antipolyvalent antibody was added and incubated for 10 min at room
Fig. 1. Marked edema, with enlargement of Virchow Robin spaces, in a rabbit from the END group, HE, bar = 200 lm.
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F. Mor, O. Ozmen / Pesticide Biochemistry and Physiology 96 (2010) 108–112
Fig. 2. Degeneration and slight gliosis in the brain of a rabbit belonging to the END group, HE, bar = 50 lm.
Histopathologically, marked lesions were seen in the END group. These included hemorrhages, marked edema with enlargement of Virchow Robin spaces, degenerations, and slight gliosis (Figs. 1 and 2). Hemorrhages were more severe and numerous in the END group, with slight perivascular cuffing observed in two animals in this group. A marked amelioration of these lesions was seen in brains in the END + C group (Fig. 3). There were no cerebral or cerebellar lesions in the OIL or the OIL + C group. Immunohistochemistry revealed a strong caspase-3 positive reaction in neurons and microglial cells in rabbits in the END group (Figs. 4 and 5). There was a slight caspase-3 expression in microglial cells in rabbits in the END + C group but no reaction in neurons was observed, indicating decreased apoptotic activity in this group (Fig. 6). Only a few microglial cells expressed caspase-3 in the OIL and OIL + C groups; there were no immunopositive neurons observed in either of these two groups. Decreases in SAChE activity in the endosulfan treated rabbits were observed at the end of the study in both the END and the END + C groups, but the strongest declines occurred in the END group. Vit C prevented the loss of SAChE activity in the END + C group and the differences between the END and the END + C groups were statistically significant. SAChE activity did not change
Fig. 3. Relatively normal brain histology in a rabbit from the END + C group, HE, bar = 200 lm.
Fig. 4. Caspase-3 positive reaction in neurons (arrows) in a rabbit from the END group. ABP method, with DAB, Harris hematoxylin counterstain, bar = 200 lm.
Fig. 5. Caspase-3 positive reaction in microglial cells in a rabbit belonging to the END group. ABP method, with DAB, Harris hematoxylin counterstain, bar = 100 lm.
Fig. 6. A few caspase-3 positive reactions in microglial cells (arrows) in a rabbit from the END + C group. ABP method, with DAB, Harris hematoxylin counterstain, bar = 50 lm.
F. Mor, O. Ozmen / Pesticide Biochemistry and Physiology 96 (2010) 108–112 Table 1 Serum acetylcholinesterase (SAChE) activities (U/L) in rabbits intoxicated with endosulfan (1 mg/kg body weight) after ascorbic acid treatment. Experimental groups
END END + C OIL + C OIL
Serum acetylcholinesterase activity Minimal
Maximal
Mean
126 248 19 234
211 1157 4576 5811
182 ± 13.55a 562 ± 68.70b 931 ± 70.60b 765 ± 34.37b
All values were expressed as mean ± SE. There is no statistical differences between the means having same superscripts. Highly significant differences between a and b, P < 0.0001.
in the OIL group but a slight increase was observed in the OIL + C group above the normal values (Table 1). In this study, increases in SAChE activities were statistically significant, p < 0.001, between the END and the END + C, OIL + C, and OIL groups. 4. Discussion Organochlorine pesticides, such as endosulfan, are of environmental significance due to their high toxicity to animals [17]. Endosulfan is known to affect many organs and tissues [17–20]. It can penetrate the brain and its neurotoxic effects are manifested by clonic convulsions in experimental animals. Convulsant and proconvulsant effects of endosulfan can become apparent after its chronic exposure at low dose levels [18]. The present results support and extend previous findings demonstrating neurotoxicity due to endosulfan [18–20]. In this study, clinical neural symptoms were not severe, appearing only as teeth grinding and hyperexcitability in some rabbits in the END group. The main reason for the minor degree of nervous symptoms was attributed to the low dose and the subacute toxicity of the endosulfan administered. Neural symptoms were not observed in the END + C group, which was attributed to the ameliorating affect of Vit C on the neuronal cells. The gross and histopathological lesions indicated toxicity in the nervous system, which was supported by the high apoptotic activity observed in the END group. To the best of our knowledge, the present study is the first to show caspase-3 activity by immunohistochemical methods in nervous systems of rabbits following endosulfan exposure. In the present study, apoptotic activity of endosulfan in neurons was observed only in the END group, while glial cell positivity was seen in the END + C group. These findings showed that Vit C had an ameliorative effect on neuronal cell damage following endosulfan exposure. Acetylcholine is the neurotransmitter hydrolyzed by AChE and this hydrolysis reaction is significantly reduced by endosulfan exposure [21]. The present results were in agreement with previous studies that showed a clear association between endosulfan exposure and significant decreases in AChE activity. The inhibition of AChE in the brain is important for increasing cholinergic neurotransmission [22]. Vit C (ascorbic acid) is a low molecular weight antioxidant that defends the cellular compartment against water-soluble oxygen and nitrogen radicals. It is an effective antioxidant of the hydrophilic phase [18]. Since many insecticides are hydrophobic molecules that bind extensively to biological membranes, especially to phospholipid bilayers [23], it was considered likely that Vit C treatment would exert an effect on cellular responses to pesticide exposure. Oxidative stress is known to be an important etiopathological factor in a variety of pathological lesions. The imbalance between antioxidant capacity and ROS production is known as oxidative stress and it has been shown to precede pathological lesions in
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neurons [23,24]. ROS destroy cells by inducing apoptosis that may perturb the natural cellular antioxidant defense system [25]. This can be a result of one of three factors; namely, an increase in ROS, an impairment of antioxidant defense systems, or an insufficient capacity to repair oxidative damage [26]. The brain is especially vulnerable to oxidative damage because of its high content of easily peroxidizable unsaturated fatty acids, its high oxygen consumption rate, and its relative paucity of antioxidant enzymes [24]. Endosulfan was identified as a chemical that could induce alterations in the activity of enzymes involved in oxidative stress and lipid peroxidation [27,28]. Our results suggest that endosulfan toxication can cause inhibition of serum AChE activity, due to ROS and cellular damage. Because Vit C is an antioxidant, it was able to reduce the inhibition of AChE in serum. We evaluated apoptotic activity of the neuronal cells by caspase-3 activity revealed by an immunohistochemical method. Endosulfan had an apoptotic effect on neuronal cells. The mechanisms underlying the Vit C neuroprotective effects are not fully understood; however, our results are in agreement with the general neuroprotective actions of Vit C. In this study, an ameliorating effect of Vit C on gross, histopathological, and immunohistochemical neurotoxic results of endosulfan exposure. Endosulfan-induced inhibition of SAChE was prevented by Vit C treatment in our study. Ballesteros et al. reported that endosulfan toxicity could cause oxidative stress in different organs of fish, with the brain being the most severely affected [29]. Numerous studies have shown the induction of oxidative stress and its markers in response to endosulfan toxicity. All of these studies have reported the same results and the same pattern of increase in oxidative stress markers such as malondialdehyde (MDA) and myelopeoxidase (MPO) [29– 32]. For this reason, these markers were not examined in the present study. The formation of ROS can be prevented by the operation of an antioxidant system, which can include low molecular mass antioxidants (e.g., ascorbic acid, glutathione, and tocopherols), enzymes that regenerate the reduced forms of antioxidants, and ROS-interacting enzymes such as superoxide dismutases (SOD), peroxidases, and catalases [33]. In the current study, we examined the caspase reaction and an ameliorative effect of Vit C on endosulfan-induced neurotoxicity was supported by the immunohistochemical data obtained for caspase activity. Several studies have previously reported that Vit C has ameliorative effects on the sperm toxicity and genotoxicity of endosulfan [13,34,35]. The present study demonstrated a protective and ameliorative effect of Vit C on neurotoxicity caused by endosulfan exposure. Although several types of pesticides have potential to contribute to neurodegenerative diseases, our results revealed that endosulfan can cause notable neurotoxic effects in rabbits, even at subacute exposures. This included the induction of apoptosis in neurons, as well as in microglial cells. However, toxicity was decreased by Vit C treatment, which increased serum acetylcholinesterase activity.
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Contents lists available at ScienceDirect
Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest
Endosulfan-induced neurotoxicity and serum acetylcholinesterase inhibition in rabbits: The protective effect of Vit C Firdevs Mor a, Ozlem Ozmen b,* a b
Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Mehmet Akif Ersoy, 15100 Burdur, Turkey Department of Pathology, Faculty of Veterinary Medicine, University of Mehmet Akif Ersoy, 15100 Burdur, Turkey
a r t i c l e
i n f o
Article history: Received 10 January 2009 Accepted 16 October 2009 Available online 24 October 2009 Keywords: Endosulfan toxicity Serum acetylcholinesterase activity Pathology Rabbits Brain Immunohistochemistry
a b s t r a c t The neurotoxic effects and acetylcholinesterase inhibition induced by endosulfan, and the amelioration of these effects by Vitamin C (Vit C), were studied in the brains of New Zealand white rabbits. The cerebrum and cerebellum of each rabbit was examined grossly and histopathologically, and caspase-3 activity was determined by immunohistochemical methods. Twenty-four rabbits were divided into four groups (n = 6). Rabbits in Group I (END) were given a sublethal dose of endosulfan (1 mg/kg bw) in corn oil daily by oral gavage for 6 weeks. Group II (END + C) received the same dose of endosulfan and also Vit C (20 mg/kg bw) every second day during the 6 week period. Group III (OIL + C) received oral corn oil daily and Vit C every second day for 6 weeks. Group IV (OIL) received corn oil daily by oral gavage throughout the experiment. A significant reduction in acetylcholinesterase activity was observed in the END group, which was ameliorated in the END + C group. Hyperemia and slight hemorrhages in brains and cerebellums were seen in some rabbits in the END group. There were no gross cerebral or cerebellar lesions in the other groups. Hemorrhages, degenerations and slight gliosis were the marked histopathological findings of some rabbits belonging to the END group. A positive caspase-3 reaction was more severe in the END group than in the others. An ameliorating effect of Vit C on gross, histopathological, and immunohistochemical findings was observed in the END + C group. Thus, although endosulfan could cause neurotoxic effects in rabbits, this toxicity was decreased by Vit C treatment, which increased serum acetylcholinesterase activity. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Endosulfan, a chlorinated organic pesticide, is currently classified by the WHO as Class II (moderately hazardous to human health) and as category Ib (highly hazardous) by the United States Environmental Protection Agency [1]. Despite having caused many environmental tragedies and although it is now either banned or severely restricted in use in some countries, endosulfan is still widely used in many areas of the world including Turkey [2,3]. Commercial preparations of endosulfan consist of a- and b-isomers, constituting 70% and 30%, respectively. In the natural environment, mixed pesticide use and toxicity is common. However, cases do exist where only one particular pesticide, such as endosulfan, has been aerially spread over crops two to three times a year for more than 20 years. In these types of instances, toxicity in humans and livestock specifically due to endosulfan has been confirmed [1,4].
* Corresponding author. Fax: +90 248 2344505. E-mail address: [email protected] (O. Ozmen). 0048-3575/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2009.10.004
Acetylcholinesterase (AChE; EC 3.1.1.7), which is well known for its function at cholinergic synapses, is a multimeric protein with many facets. AChE terminates neurotransmission through the neurosynaptic cleft in both the central and the peripheral nervous system. AChE prevents the accumulation of acetylcholine that may overstimulate nicotinic and/or muscarinic receptors [5,6]. During neuronal development, AChE most likely is involved in regulating neural growth in various cellular systems. From in vitro studies using cells and explants, it has been concluded that AChE can regulate neural growth by a nonenzymatic mechanism [7]. Cholinesterases have also been identified in the serum (SAChE) [8]. Vitamin C (Vit C) is a water-soluble antioxidant that effectively scavenges reactive oxygen radicals (ROS), thus preventing tissue damage [9]. It can act to overcome oxidative stress, forming part of the antioxidant system. Many studies have reported that Vit C can reduce the lipid peroxidation (LPO) induced by toxic substances [10]. ROS readily damage biological molecules, ultimately leading to apoptotic and necrotic cell death [11]. The role of the Vit C in ameliorating oxidative stress reactions, such as induction of apoptosis and necrosis, has been indicated by numerous studies [12,13]. One mode of action may involve caspases, a family of
F. Mor, O. Ozmen / Pesticide Biochemistry and Physiology 96 (2010) 108–112
cysteine proteases that are activated when apoptosis is initiated and that play essential roles at various stages of apoptosis [14]. The aim of the present study was to examine the neurotoxic effect of endosulfan on acetylcholinesterase activity following experimental toxication of rabbits and to determine if Vit C might exert a protective effect against endosulfan toxicity. 2. Materials and methods The experiment was approved by the Institutional Animal Use and Care Committee of the Akdeniz University, and was performed in accordance with the National Institutes of Health Guidelines for the Care and Handling of Animals. 2.1. Animals Male New Zealand white rabbits, 6–8 months old, were used for this study. Rabbits were fed on standard rabbit chow and tap water ad libitum and were housed in cages at a controlled temperature (22 °C) and 12-h light/dark cycles throughout the study. The physical condition of each rabbit was assessed daily for any obvious signs of illness. 2.2. Experimental design Twenty-four rabbits were randomly allocated to four groups (n = 6). Group I (END) animals were exposed daily to sublethal concentrations of endosulfan (1 mg/kg bw) in corn oil for 6 weeks. Group II (END + C) received 1 mg/kg bw endosulfan daily and 20 mg/kg bw Vit C (ascorbic acid, Vit C) every second day during this period. Group III (OIL + C) received oral corn oil daily and 20 mg/kg bw Vit C every second day for 6 weeks. Group IV (OIL) was the control group and received daily corn oil only. All treatments were administered by oral gavage. 2.3. Histopathological evaluation Rabbits were sacrificed at 1 week after the last treatment. Before euthanasia, intracardiac blood samples were taken, and then necropsy was performed all the animals. Samples of cerebrum and cerebellum were fixed in 10% formalin, routinely processed, paraffin embedded, and stained with hematoxylin and eosin for by light microscope examination. Histopathological changes were graded in a blinded manner.
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temperature. After three washes with PBS, streptavidin peroxidase was added and the samples were incubated for 10 min at room temperature, and then rinsed four times with PBS. Tissues were further incubated for 20 min at room temperature in a solution of DAB (3,30 -diaminobenzidine) chromogen. After washing with PBS, tissues were counterstained with Mayer’s hematoxylin, washed with water, and coverslips were applied with mounting media. 2.5. Serum acetylcholinesterase (AChE) activity assay Acetylcholinesterase activity was determined according to Ellman et al. [15], with some modifications [16]. Hydrolysis rates were measured at 25 °C in 1 mL assay solutions containing 0.8 mM acetylthiocholine (S), 30 mM phosphate buffer, pH 7.5, and 1.0 mM DTNB. Fifty microliters of serum was added to the reaction mixture and preincubated for 3 min. The hydrolysis reaction was monitored by the formation of the thiolate dianion of DTNB at 412 nm for 2–3 min (recorded at 30 s intervals). All samples were run in duplicate. 2.6. Statistical analysis The Kruskal–Wallis Test was used for statistical treatment of data: within-and between-run coefficients of variation (cvs) were calculated for characterization assay. Mann–Whitney U-test analysis was performed to establish significant changes in cholinesterase activity after endosulfan incubation. All statistical analyses were run using SPSS 10.0 software program pack. 3. Results Clinical depression and slight nervous symptoms such as teeth grinding and hyperexcitability were observed in some rabbits in the END group after 4 weeks of endosulfan administration. There were no clinical symptoms in the other groups. Gross examination of the brains revealed marked hyperemia at the meningeal vessels and slight hemorrhages in brains and cerebellums in some rabbits from the END group. These findings were more prominent in those rabbits that had shown clinical nervous symptoms. There were no gross cerebral or cerebellar lesions in the other groups.
2.4. Immunohistochemical evaluation For evaluation of apoptosis, slides were stained for the presence of caspase-3 activity by immunohistochemical methods [Neomarker–California (caspase-3 (CPP32) Ab-4, 1:100 dilution] using a routine streptavidin–biotin peroxidase technique. For immunohistochemical examination, paraffin blocks were sectioned at 5 lm and sections were attached to glass slides coated with poly-L-lysine. The slides were dried overnight at 37 °C for optimization of adhesion. Sections were deparaffinized in multiple xylene baths, and tissues were rehydrated in a graded ethanol series. To reduce nonspecific background staining due to endogenous peroxidase, slides were incubated in hydrogen peroxide in methanol for 10 min. The sections were washed twice in phosphate buffer solution (PBS), then boiled in 1:100 citrate buffer solution for 10 min and cooled for 20 min. The tissues were washed in PBS for four times, then blocked with serum for 5 min. The primary antibody was then applied and the tissues were incubated for 30 min at room temperature. After four rinses with PBS, biotinylated antipolyvalent antibody was added and incubated for 10 min at room
Fig. 1. Marked edema, with enlargement of Virchow Robin spaces, in a rabbit from the END group, HE, bar = 200 lm.
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Fig. 2. Degeneration and slight gliosis in the brain of a rabbit belonging to the END group, HE, bar = 50 lm.
Histopathologically, marked lesions were seen in the END group. These included hemorrhages, marked edema with enlargement of Virchow Robin spaces, degenerations, and slight gliosis (Figs. 1 and 2). Hemorrhages were more severe and numerous in the END group, with slight perivascular cuffing observed in two animals in this group. A marked amelioration of these lesions was seen in brains in the END + C group (Fig. 3). There were no cerebral or cerebellar lesions in the OIL or the OIL + C group. Immunohistochemistry revealed a strong caspase-3 positive reaction in neurons and microglial cells in rabbits in the END group (Figs. 4 and 5). There was a slight caspase-3 expression in microglial cells in rabbits in the END + C group but no reaction in neurons was observed, indicating decreased apoptotic activity in this group (Fig. 6). Only a few microglial cells expressed caspase-3 in the OIL and OIL + C groups; there were no immunopositive neurons observed in either of these two groups. Decreases in SAChE activity in the endosulfan treated rabbits were observed at the end of the study in both the END and the END + C groups, but the strongest declines occurred in the END group. Vit C prevented the loss of SAChE activity in the END + C group and the differences between the END and the END + C groups were statistically significant. SAChE activity did not change
Fig. 3. Relatively normal brain histology in a rabbit from the END + C group, HE, bar = 200 lm.
Fig. 4. Caspase-3 positive reaction in neurons (arrows) in a rabbit from the END group. ABP method, with DAB, Harris hematoxylin counterstain, bar = 200 lm.
Fig. 5. Caspase-3 positive reaction in microglial cells in a rabbit belonging to the END group. ABP method, with DAB, Harris hematoxylin counterstain, bar = 100 lm.
Fig. 6. A few caspase-3 positive reactions in microglial cells (arrows) in a rabbit from the END + C group. ABP method, with DAB, Harris hematoxylin counterstain, bar = 50 lm.
F. Mor, O. Ozmen / Pesticide Biochemistry and Physiology 96 (2010) 108–112 Table 1 Serum acetylcholinesterase (SAChE) activities (U/L) in rabbits intoxicated with endosulfan (1 mg/kg body weight) after ascorbic acid treatment. Experimental groups
END END + C OIL + C OIL
Serum acetylcholinesterase activity Minimal
Maximal
Mean
126 248 19 234
211 1157 4576 5811
182 ± 13.55a 562 ± 68.70b 931 ± 70.60b 765 ± 34.37b
All values were expressed as mean ± SE. There is no statistical differences between the means having same superscripts. Highly significant differences between a and b, P < 0.0001.
in the OIL group but a slight increase was observed in the OIL + C group above the normal values (Table 1). In this study, increases in SAChE activities were statistically significant, p < 0.001, between the END and the END + C, OIL + C, and OIL groups. 4. Discussion Organochlorine pesticides, such as endosulfan, are of environmental significance due to their high toxicity to animals [17]. Endosulfan is known to affect many organs and tissues [17–20]. It can penetrate the brain and its neurotoxic effects are manifested by clonic convulsions in experimental animals. Convulsant and proconvulsant effects of endosulfan can become apparent after its chronic exposure at low dose levels [18]. The present results support and extend previous findings demonstrating neurotoxicity due to endosulfan [18–20]. In this study, clinical neural symptoms were not severe, appearing only as teeth grinding and hyperexcitability in some rabbits in the END group. The main reason for the minor degree of nervous symptoms was attributed to the low dose and the subacute toxicity of the endosulfan administered. Neural symptoms were not observed in the END + C group, which was attributed to the ameliorating affect of Vit C on the neuronal cells. The gross and histopathological lesions indicated toxicity in the nervous system, which was supported by the high apoptotic activity observed in the END group. To the best of our knowledge, the present study is the first to show caspase-3 activity by immunohistochemical methods in nervous systems of rabbits following endosulfan exposure. In the present study, apoptotic activity of endosulfan in neurons was observed only in the END group, while glial cell positivity was seen in the END + C group. These findings showed that Vit C had an ameliorative effect on neuronal cell damage following endosulfan exposure. Acetylcholine is the neurotransmitter hydrolyzed by AChE and this hydrolysis reaction is significantly reduced by endosulfan exposure [21]. The present results were in agreement with previous studies that showed a clear association between endosulfan exposure and significant decreases in AChE activity. The inhibition of AChE in the brain is important for increasing cholinergic neurotransmission [22]. Vit C (ascorbic acid) is a low molecular weight antioxidant that defends the cellular compartment against water-soluble oxygen and nitrogen radicals. It is an effective antioxidant of the hydrophilic phase [18]. Since many insecticides are hydrophobic molecules that bind extensively to biological membranes, especially to phospholipid bilayers [23], it was considered likely that Vit C treatment would exert an effect on cellular responses to pesticide exposure. Oxidative stress is known to be an important etiopathological factor in a variety of pathological lesions. The imbalance between antioxidant capacity and ROS production is known as oxidative stress and it has been shown to precede pathological lesions in
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neurons [23,24]. ROS destroy cells by inducing apoptosis that may perturb the natural cellular antioxidant defense system [25]. This can be a result of one of three factors; namely, an increase in ROS, an impairment of antioxidant defense systems, or an insufficient capacity to repair oxidative damage [26]. The brain is especially vulnerable to oxidative damage because of its high content of easily peroxidizable unsaturated fatty acids, its high oxygen consumption rate, and its relative paucity of antioxidant enzymes [24]. Endosulfan was identified as a chemical that could induce alterations in the activity of enzymes involved in oxidative stress and lipid peroxidation [27,28]. Our results suggest that endosulfan toxication can cause inhibition of serum AChE activity, due to ROS and cellular damage. Because Vit C is an antioxidant, it was able to reduce the inhibition of AChE in serum. We evaluated apoptotic activity of the neuronal cells by caspase-3 activity revealed by an immunohistochemical method. Endosulfan had an apoptotic effect on neuronal cells. The mechanisms underlying the Vit C neuroprotective effects are not fully understood; however, our results are in agreement with the general neuroprotective actions of Vit C. In this study, an ameliorating effect of Vit C on gross, histopathological, and immunohistochemical neurotoxic results of endosulfan exposure. Endosulfan-induced inhibition of SAChE was prevented by Vit C treatment in our study. Ballesteros et al. reported that endosulfan toxicity could cause oxidative stress in different organs of fish, with the brain being the most severely affected [29]. Numerous studies have shown the induction of oxidative stress and its markers in response to endosulfan toxicity. All of these studies have reported the same results and the same pattern of increase in oxidative stress markers such as malondialdehyde (MDA) and myelopeoxidase (MPO) [29– 32]. For this reason, these markers were not examined in the present study. The formation of ROS can be prevented by the operation of an antioxidant system, which can include low molecular mass antioxidants (e.g., ascorbic acid, glutathione, and tocopherols), enzymes that regenerate the reduced forms of antioxidants, and ROS-interacting enzymes such as superoxide dismutases (SOD), peroxidases, and catalases [33]. In the current study, we examined the caspase reaction and an ameliorative effect of Vit C on endosulfan-induced neurotoxicity was supported by the immunohistochemical data obtained for caspase activity. Several studies have previously reported that Vit C has ameliorative effects on the sperm toxicity and genotoxicity of endosulfan [13,34,35]. The present study demonstrated a protective and ameliorative effect of Vit C on neurotoxicity caused by endosulfan exposure. Although several types of pesticides have potential to contribute to neurodegenerative diseases, our results revealed that endosulfan can cause notable neurotoxic effects in rabbits, even at subacute exposures. This included the induction of apoptosis in neurons, as well as in microglial cells. However, toxicity was decreased by Vit C treatment, which increased serum acetylcholinesterase activity.
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