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Effect of exposure to diazinon on adult rat's brain Rashedinia Marzieh, Hossein Hosseinzadeh, Imenshahidi Mohsen, Lari Parisa, Razavi Bibi Marjan and Abnous Khalil Toxicol Ind Health published online 11 November 2013 DOI: 10.1177/0748233713504806 The online version of this article can be found at: http://tih.sagepub.com/content/early/2013/11/08/0748233713504806
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Effect of exposure to diazinon on adult rat’s brain
Toxicology and Industrial Health 1–7 © The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233713504806 tih.sagepub.com
Rashedinia Marzieh1, Hossein Hosseinzadeh1, Imenshahidi Mohsen1, Lari Parisa1, Razavi Bibi Marjan1 and Abnous Khalil2 Abstract Diazinon (DZN), a commonly used agricultural organophosphate insecticide, is one of the major concerns for human health. This study was planned to investigate neurotoxic effects of subacute exposure to DZN in adult male Wistar rats. Animals received corn oil as control and 15 and 30 mg/kg DZN orally by gastric gavage for 4 weeks. The cerebrum malondialdehyde and glutathione (GSH) contents were assessed as biomarkers of lipid peroxidation and nonenzyme antioxidants, respectively. Moreover, activated forms of caspase 3, -9, and Bax/ Bcl-2 ratios were evaluated as key apoptotic proteins. Results of this study suggested that chronic administration of DZN did not change lipid peroxidation and GSH levels significantly in comparison with control. Also, the active forms of caspase 3 and caspase 9 were not significantly altered in DZN-treated rat groups. Moreover, no significant changes were observed in Bax and Bcl-2 ratios. This study indicated that generation of reactive oxygen species was probably modulated by intracellular antioxidant system. In conclusion, subacute oral administration of DZN did not alter lipid peroxidation. Moreover, apoptosis induction was not observed in rat brain. Keywords Diazinon, caspase 3, caspase 9, Bax, Bcl-2, neurotoxicity, subacute toxicity
Introduction Organophosphorus insecticides (OPs) are intensively used to eliminate variety of insects in agriculture. Because of the widespread use of pesticides, their toxicity remains a major concern for public health. Human long-term exposure to OPs occurs during the agricultural activities or through domestic use and consumption of contaminated food and water (Binukumar and Gill, 2011; Nougade`re et al., 2011). Diazinon (DZN) is the most commonly used OPs (Garfitt et al., 2002).Also, a large proportion of people are continuously exposed to DZN. Therefore, studying the chronic adverse effects of exposure to this pesticide on different organs is an important priority. It is believed that the main unfavorable chronic toxic effect is in central nervous system (CNS). Primary mechanism of OPs toxicity is inhibition of acetylcholine esterase (AchE) activity and accumulation of endogenous acetylcholine in CNS (Mileson et al., 1998). Irreversible inhibition of AchE suggested that excess of acetylcholine may cause hyperstimulation
of cholinergic receptors, particularly in CNS. Consequently, OPs damage the nervous system by inducing excitotoxic neuronal death (Rush et al., 2010). In the body, DZN is metabolized to diazoxon through oxidative desulfuration, and cytochrome P450 is the major catalyzing agent that mediated this reaction. Diazoxon is responsible for inhibition of AChE and neuroˇ olovic´ et al., 2010). toxic signs of DZN (C
1
Department of Pharmacodynamy and Toxicology, Pharmaceutical Research Center, Mashhad University of Medical Sciences, Mashhad, Islamic Republic of Iran 2 Department of Medicinal Chemistry and Department of Biotechnology, Pharmaceutical Research Center, Mashhad University of Medical Sciences, Mashhad, Islamic Republic of Iran Corresponding author: Abnous Khalil, Department of Medicinal Chemistry and Department of Biotechnology, Pharmaceutical Research Center, Mashhad University of Medical Sciences, Mashhad, 917751365, Islamic Republic of Iran. Email: [email protected]
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It has been proposed that additional mechanisms, other than AchE inhibition, may mediate adverse health effects of chronic exposure to OPs. Various studies have revealed that OPs induced oxidative stress in some in vivo and in vitro studies (Giordano et al., 2007). OPs increased lipid peroxidation through formation of reactive oxygen species (ROS) (ElMazoudy et al., 2011; Giordano et al., 2007). ROS may affect cell membrane functions and metabolism due to interaction with biological macromolecules including proteins, nucleic acids, lipids, and carbohydrates. The enzymatic and nonenzymatic antioxidant defense system protects cells against ROS damages. Hence, imbalance between the rate of ROS production and protective effects of antioxidant systems cause oxidative stress (Ojha et al., 2011). It has been shown that acute exposure to DZN induced generation of free radicals in a dose-dependent manner. Moreover, inhibition of antioxidant enzyme activities and glutathione (GSH) depletion were observed in the heart, brain, and spleen of rats (Jafari et al., 2012). OPs compounds have also been reported to affect brain development through different mechanisms such as impact on expression and function of nuclear transcription factors, cell signaling pathway, neuronal cell interactions, and oxidative stress induction (Slotkin and Seidler, 2007). Numerous factors including dose of OPs, route of exposure, total absorption, physicochemical property, metabolism, and rate of detoxification play a role in severity and duration of poisoning (Karalliedde et al., 2003). Majority of reports are about acute and developmental toxicity of DZN via intraperitoneal injection. This study was designed to investigate effects of subacute exposure to orally administered DZN. In this study, the levels of lipid peroxidation and induction of apoptosis were investigated in adult rat brain after subacute exposure to DZN via gavage for 4 weeks.
Materials and methods Animals Adult male Wistar rats (weight: 200–250 g) were provided by Animal Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. Animals were maintained on a 12-h light/dark cycle with free access to food and water. All experiments were performed in accordance with Ethical Committee Acts, Mashhad University of Medical Sciences, Mashhad, Iran. As oral median lethal dose
(LD50) of DZN was reported to be 300–400 mg/kg in rats (Gokcimen et al., 2007), 1/10th and 1/20th of LD50 was used in this study. Rats were randomly divided into 3 groups of 6 animals each. The groups include: (1) control (corn oil), (2) DZN-treated group (15 mg/kg), and (3) DZN-treated group (30 mg/kg). Rats were daily gavaged DZN (Bazodin1, Syngenta, Basel, Switzerland, purity 96%) in corn oil or received corn oil alone. Rats were killed after 4 weeks of treatment, and brains were removed and quickly freezed in liquid nitrogen and stored at 80 C until use.
MDA measurement To measure the levels of lipid peroxidation, brain tissues were homogenized using Polytron homogenizer (Kinematica, Lucerne, Switzerland) at 4 C in 1.15% potassium chloride to make a final concentration of 10% (w/w). To this, 1 ml 0.6% thiobarbituric acid, 3 ml of 1% phosphoric acid (Merck, Germany), and 0.5 ml of supernatant were mixed and heated in boiling water bath for 45 min. Concentration of thiobarbituric acid reactive substances (TBARS) was spectrophotometrically determined using microplate reader (Synergy H4 Hybrid Reader, BioTek, Winooski, Vermont, Sigma-Aldrich Corp. St Louis, USA) by measuring absorbance at 532 nm (Kei, 1978). Concentrations of MDA in samples are expressed in nanomole per gram wet tissue.
GSH content measurement For estimation of brain tissue GSH, equal amount of tissue homogenate was mixed with 10% trichloroacetic acid and vortexed. After centrifugation, 0.5 ml of supernatant was mixed with a reaction buffer containing 3.0 ml of 0.3 M phosphate buffer (pH 8.4) and 0.5 ml 0.04% 5,50 dithiobis-(2-nitrobenzoic acid). Absorbance was measured at 412 nm within 10 min (Moron et al., 1979). A standard curve of GSH was plotted using commercially available standard GSH (Sigma, Sigma-Aldrich Corp., St Louis, USA). Levels of GSH were expressed in nanomole per gram wet tissue.
Western blot analysis Cerebrum tissues (0.2 g) were homogenized in the lysis buffer containing 50 mM Tris-hydrochloric acid, pH 8.8, 2 mM ethylenediaminetetraacetic acid, 2 mM ethylene glycol tetraacetic acid, 2% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-
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100, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 10 mM b-glycerophosphate, 10% v/v 2mercaptoethanol, 2 ml complete protease inhibitor cocktail (Sigma P8340), and 1 mM phenylmethylsulfonyl fluoride (PMSF). Homogenates were sonicated for 30 s and centrifuged at 16,000g for 10 min at 4 C. Total protein concentration of supernatants was determined using Bradford protein assay kit (Bio-Rad Laboratories, Hercules, California, USA). Samples were mixed with equal volumes of 2 SDS sample buffer containing 100 mM Tris-base, 4% w/v SDS, 20% v/v glycerol, 0.2% w/v bromophenol blue, and 10% v/v 2mercaptoethanol and then incubated in boiling water for 5 min. The same amounts of total proteins from each sample were loaded to SDS-polyacrylamide gel electrophoresis wells. Proteins were transferred to polyvinylidene difluoride membranes. Membranes were incubated with 5% skimmed milk in Tris-buffered saline–Tween 20 (TBST) for 1 h at room temperature. Blots were incubated with following antibodies at appropriate dilution according to manufacturer’s instruction for 2 h at room temperature: mouse monoclonal anti-caspase 9, rabbit polyclonal anti-caspase 3, rabbit polyclonal anti-Bax, rabbit monoclonal antiBcl-2, mouse and rabbit anti-b actin (all antibodies were purchased from Cell Signaling, USA). After several washes with TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibody. Protein bands were visualized using enhanced chemiluminescence ( Pierce, Rockford, Illinois, USA) and Alliane 4.7 gel doc (UK). Intensities of immunoreactive bands were quantified using UVI band software (UVtec, UK). Protein levels were normalized against corresponding b actin intensities.
Statistical analysis Results are expressed as mean + SD. Differences between test groups were examined using one-way analysis of variance followed by Tukey–Kramer. The value of p < 0.05 was considered statistically significant.
Results Effects of DZN on lipid peroxidation levels in brain tissues The administration of DZN for 4 weeks had no effect on malondialdehyde (MDA) concentration as oxidative stress biomarkers as compared to the control group (Table 1).
Table 1. Effects of DZN (4 weeks) on MDA and GSH levels in the cerebrum.a Group
MDA (nmol/g tissue)
GSH (nmol/g tissue)
Control DZN 15 mg/kg DZN 30 mg/kg
175.72 + 12.23 178.67 + 17.34 177.5 + 14.02
381.3 + 28.96 375.7 + 11.08 370.7 + 16.52
DZN: diazinon; MDA: malondialdehyde; GSH: glutathione. a Data are shown as mean + SD, n ¼ 6 independent determinations on different preparations of samples.
Effects of DZN on GSH level in brain tissue GSH level did not significantly change in the brain of experimental animals compared with the control (Table 1).
Immunoblot analysis of Bax, Bcl-2, and caspase 3 To investigate the effects of subacute DZN treatment on apoptosis state in brain tissue, active forms of caspase 3 were measured using Western blot. Our data showed that there were no significant differences between active forms of caspase 3 in brain of control and DZN-treated rats (Figure 1). Moreover, no significant changes were observed among caspase 9 and its active form in all experimental groups (Figure 2). Our data showed that Bax/Bcl-2 ratios remain unchanged among control and DZN-treated rats (Figure 3).
Discussion The present study was designed to evaluate neurotoxic effects of subacute exposure to DZN in the brain of rat. Our data showed that subacute exposure to 15 and 30 mg/kg DZN does not change MDA and GSH levels, as well as active form of caspase 3, -9, and Bax/Bcl2. Numerous studies have explained that accumulation of acetylcholine, after inhibition of AChE, causes neurotoxicity (Kaur et al., 2007; Milatovic et al., 2006; Roszczenko et al., 2012). Previous studies in our laboratory showed significant decrease in plasma cholinesterase activity after subacute administration of DZN in rat. Moreover, these studies showed that DZN increases level of MDA in aortic and liver tissues and 8-iso-prostaglandin in serum (Hariri et al., 2010; Lari et al., 2013; Hosseinzadeh et al., 2011). Some studies showed that OPs cause marked perturbations in the antioxidant defense system, induce oxidative damages, and enhanced lipid peroxidation in the
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Figure 1. Western blot analysis of caspase 3 in the cerebrum of control and DZN-treated rat groups. No significant differences were found among groups in the total and activated form of caspase 3 levels. Data are expressed relative to the control and are mean + SD of four separate experiments. p < 0.05 were considered statistically significant. DZN: diazinon.
brain (Binukumar and Gill, 2011; Kaur et al., 2007; ¨ ner et al., 2006). Formation of ROS and induction of U oxidative stress by OPs have also been revealed to be related to apoptosis in different tissues (Yu et al., 2008). Kaur et al. (2007) showed that elevation in calcium and ROS production could induce apoptotic neuronal degeneration following chronic exposure to dichlorvos in rat brain. Caughlan et al. (2004) indicated that chlorphyrifos induced apoptosis and perturbed mitochondrial function in rat cortical neurons through activation of few mitogen-activated protein kinases including ERK1/2, JNK, and p38. Also, it has been reported that subchronic oral administration of dichlorvos in rat increased level of MDA in the endometrium leading to endometrial damage and apoptosis (Mahaboob Khan and Kour, 2007). Our data showed that subacute oral exposure to DZN did not significantly change levels of MDA and lipid peroxidation in brain. This finding is not in agreement with the results of previous studies. Jafari et al. (2012) demonstrated that the concentration of MDA was significantly increased, GSH level was
Figure 2. Western blot analysis of caspase 9 in the cerebrum of control and DZN-treated rat groups. No significant differences were found among groups in the total and activated form of caspase 9 levels. Data are expressed relative to the control and are mean + SD of four separate experiments. p < 0.05 were considered statistically significant. DZN: diazinon.
decreased, and antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), and glutathione S-transferase activity changed in brain, heart, and spleen after acute exposure to DZN through intraperitoneal injection at high doses (100 and 200 mg/kg) in both Wistar and Norway rat strains. It was reported that dipping the animals in DZN for protection from scab mite (Psoropets ovis) cause impairment of the biochemical, physiological, and histopathological parameters in rabbits. In a later study, red Baladi rabbit dipped in water containing DZN (6.0 or 30 mg) for 10 s. Histopathological changes of brain, liver, and kidney were observed after dipping in high DZN concentration. Histopathological modification in brain lesions were seen with polymorphic and hyperchromatic nuclei with perinuclear cytoplasmic vacuoles in neuron cells and mild degenerative changes of ¨ ner et al. found that nerve fibers (Yehia et al., 2007). U exposure to sublethal concentrations of DZN (1 and 2 mg/l) caused inhibition of AChE activity and significant increase in MDA levels in the brain of freshwater ¨ ner fish, Oreochromis niloticus, after 7 and 15 days (U et al., 2006). The results of acute treatment single dose of DZN (335 mg/kg) in rat showed that DZN
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Figure 3. Western blots and densitometric analysis of Bax and Bcl-2 protein levels in cerebrum obtained from control and DZN-treated rat groups. Densitometric quantification of the Bax/Bcl-2 ratio. p < 0.05 were considered statistically significant. DZN: diazinon.
induced significant increased in the level of MDA in rat brain with no significant changes in activities of free radical scavenging enzymes SOD, CAT, and GSH peroxidase(Yilmaz et al., 2012). Moreover, it has been shown that DZN and chlorpyrifos could induce widespread changes in the genes related to oxidative stress such as SOD and CAT in differentiating PC12 cells (Slotkin and Seidler, 2009). We believe that variations in the animal types, breed and species, tissue types, doses, chemical structures, administration route, and duration of exposure to OPs have effects on DZN toxicity (Karalliedde et al., 2003). Activation of antioxidant enzymes like SOD and CAT are the first line of cell defense against free radicals and ROS exposure. Mammalian cells have a comprehensive set of antioxidant defense mechanisms to prevent free radical formation and also to limit their damaging effects. So, depending on the magnitude of the oxidative stress and dose of stressor, generation of ROS may get modulated by intracellular antioxidant systems (Isik and Celik, 2008). Elevated activity and gene expression of enzymes involved in oxidative stress that were observed in previous studies
and also nonsignificant reduction of GSH content and unchanged MDA level in our study suggest that compensatory mechanism and adaptive response are activated during subacute low oral dose exposure to DZN. Perhaps, the GSH acts as a powerful antioxidant in the neutralized free radical cells and protected cells against oxidative damage and toxic agents. It has been proved that OPs toxicity in rats is age dependent; as young animals are more sensitive to OPs. AChE inhibition is much stronger in young animals because of lower detoxification capability (Kousba et al., 2007; Padilla et al., 2004; Slotkin et al., 2006). Some developmental studies in neonatal rats showed that organophosphates induce neurotoxicity and reduce numbers of neurons through induction of proapoptotic and antimitotic mechanisms (Dam et al., 1998; Slotkin, 2004; Slotkin and Seidler, 2011, 2012).Gene expression profiling in forebrain of neonatal rats that were exposed to daily doses of DZN or chlorpyrifos showed that multiple mechanisms contribute to developmental neurotoxicity of organophosphates. This study indicated that DZN elicited major transcriptional changes in genes involved in the apoptosis pathways and oxidative stress. Wide range effects were observed on bax, bmf, casp1 and casp4, sod, gpx, and gst gene families. Moreover, DZN was reported to damage spatial learning memory and change expression of neurotrophic factors in the brain of neonatal rat (Slotkin et al., 2007). In vitro mechanistical study of DZN in cortical culture indicated that DZN induced apoptotic neuronal death. This effect was inhibited by the caspase inhibitor. Interestingly, some of DZN effects were independent from its inhibitory effect on the activity of cholinesterase (Rush et al., 2010). Development of neural cell is a critical phase with more sensitivity and susceptibly to external stimuli. During the early stages of life, many pesticides impact on the brain function that result in damage to various regions of brain or declined the number of neurons. Exposure to variety of cytotoxic and stress-related stimuli, including ROS has major role in initiation of mitochondrial apoptosis. Mitochondrial damage releases cytochrome c that activates caspase 9 and its downstream effector caspases 3 and 7. These process leads to DNA fragmentation and cell death (Allan and Clarke, 2009). Permeabilization of the mitochondrial membrane is partially controlled through Bcl family of protein apoptosis inhibitors such as Bcl-2 and Bcl-xL and apoptosis inducers such as Bax and Bid
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(Allan and Clarke, 2009; Franco et al., 2009). We did not observe significant changes in Bax/Bcl-2 ratio in DZN-treated group as compared to the control group. Overall, the results of our study showed that subacute exposure to 15 and 30 mg/kg DZN via gavage did not activate apoptosis pathway with no effects on the level of cleaved caspase 3 and -9 in brain of rat. This finding indicates that DZN has no or low effect on adult neural cells. Pharmacokinetic and pharmacodynamic differences may contribute to age-related OPs toxicities (Binukumar and Gill, 2011; Timchalk et al., 2006). In conclusion, according to our observation, nervous system in adult rats is less sensitive and less susceptible to subacute exposure to DZN toxicity. Perhaps dissimilarity in animal type, age, exposure duration, and administration route of DZN cause various results in different studies. As a consequence, DZN toxicity may be dose dependent, and higher antioxidant capacity of brain tissue in adult rats may protect the cell against its toxicity at low dose. Funding The authors thank the Vice Chancellor of Research, Mashhad University of Medical Sciences, Mashhad, Iran, for financially supporting the present work.
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Effect of exposure to diazinon on adult rat's brain Rashedinia Marzieh, Hossein Hosseinzadeh, Imenshahidi Mohsen, Lari Parisa, Razavi Bibi Marjan and Abnous Khalil Toxicol Ind Health published online 11 November 2013 DOI: 10.1177/0748233713504806 The online version of this article can be found at: http://tih.sagepub.com/content/early/2013/11/08/0748233713504806
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Effect of exposure to diazinon on adult rat’s brain
Toxicology and Industrial Health 1–7 © The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233713504806 tih.sagepub.com
Rashedinia Marzieh1, Hossein Hosseinzadeh1, Imenshahidi Mohsen1, Lari Parisa1, Razavi Bibi Marjan1 and Abnous Khalil2 Abstract Diazinon (DZN), a commonly used agricultural organophosphate insecticide, is one of the major concerns for human health. This study was planned to investigate neurotoxic effects of subacute exposure to DZN in adult male Wistar rats. Animals received corn oil as control and 15 and 30 mg/kg DZN orally by gastric gavage for 4 weeks. The cerebrum malondialdehyde and glutathione (GSH) contents were assessed as biomarkers of lipid peroxidation and nonenzyme antioxidants, respectively. Moreover, activated forms of caspase 3, -9, and Bax/ Bcl-2 ratios were evaluated as key apoptotic proteins. Results of this study suggested that chronic administration of DZN did not change lipid peroxidation and GSH levels significantly in comparison with control. Also, the active forms of caspase 3 and caspase 9 were not significantly altered in DZN-treated rat groups. Moreover, no significant changes were observed in Bax and Bcl-2 ratios. This study indicated that generation of reactive oxygen species was probably modulated by intracellular antioxidant system. In conclusion, subacute oral administration of DZN did not alter lipid peroxidation. Moreover, apoptosis induction was not observed in rat brain. Keywords Diazinon, caspase 3, caspase 9, Bax, Bcl-2, neurotoxicity, subacute toxicity
Introduction Organophosphorus insecticides (OPs) are intensively used to eliminate variety of insects in agriculture. Because of the widespread use of pesticides, their toxicity remains a major concern for public health. Human long-term exposure to OPs occurs during the agricultural activities or through domestic use and consumption of contaminated food and water (Binukumar and Gill, 2011; Nougade`re et al., 2011). Diazinon (DZN) is the most commonly used OPs (Garfitt et al., 2002).Also, a large proportion of people are continuously exposed to DZN. Therefore, studying the chronic adverse effects of exposure to this pesticide on different organs is an important priority. It is believed that the main unfavorable chronic toxic effect is in central nervous system (CNS). Primary mechanism of OPs toxicity is inhibition of acetylcholine esterase (AchE) activity and accumulation of endogenous acetylcholine in CNS (Mileson et al., 1998). Irreversible inhibition of AchE suggested that excess of acetylcholine may cause hyperstimulation
of cholinergic receptors, particularly in CNS. Consequently, OPs damage the nervous system by inducing excitotoxic neuronal death (Rush et al., 2010). In the body, DZN is metabolized to diazoxon through oxidative desulfuration, and cytochrome P450 is the major catalyzing agent that mediated this reaction. Diazoxon is responsible for inhibition of AChE and neuroˇ olovic´ et al., 2010). toxic signs of DZN (C
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Department of Pharmacodynamy and Toxicology, Pharmaceutical Research Center, Mashhad University of Medical Sciences, Mashhad, Islamic Republic of Iran 2 Department of Medicinal Chemistry and Department of Biotechnology, Pharmaceutical Research Center, Mashhad University of Medical Sciences, Mashhad, Islamic Republic of Iran Corresponding author: Abnous Khalil, Department of Medicinal Chemistry and Department of Biotechnology, Pharmaceutical Research Center, Mashhad University of Medical Sciences, Mashhad, 917751365, Islamic Republic of Iran. Email: [email protected]
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It has been proposed that additional mechanisms, other than AchE inhibition, may mediate adverse health effects of chronic exposure to OPs. Various studies have revealed that OPs induced oxidative stress in some in vivo and in vitro studies (Giordano et al., 2007). OPs increased lipid peroxidation through formation of reactive oxygen species (ROS) (ElMazoudy et al., 2011; Giordano et al., 2007). ROS may affect cell membrane functions and metabolism due to interaction with biological macromolecules including proteins, nucleic acids, lipids, and carbohydrates. The enzymatic and nonenzymatic antioxidant defense system protects cells against ROS damages. Hence, imbalance between the rate of ROS production and protective effects of antioxidant systems cause oxidative stress (Ojha et al., 2011). It has been shown that acute exposure to DZN induced generation of free radicals in a dose-dependent manner. Moreover, inhibition of antioxidant enzyme activities and glutathione (GSH) depletion were observed in the heart, brain, and spleen of rats (Jafari et al., 2012). OPs compounds have also been reported to affect brain development through different mechanisms such as impact on expression and function of nuclear transcription factors, cell signaling pathway, neuronal cell interactions, and oxidative stress induction (Slotkin and Seidler, 2007). Numerous factors including dose of OPs, route of exposure, total absorption, physicochemical property, metabolism, and rate of detoxification play a role in severity and duration of poisoning (Karalliedde et al., 2003). Majority of reports are about acute and developmental toxicity of DZN via intraperitoneal injection. This study was designed to investigate effects of subacute exposure to orally administered DZN. In this study, the levels of lipid peroxidation and induction of apoptosis were investigated in adult rat brain after subacute exposure to DZN via gavage for 4 weeks.
Materials and methods Animals Adult male Wistar rats (weight: 200–250 g) were provided by Animal Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. Animals were maintained on a 12-h light/dark cycle with free access to food and water. All experiments were performed in accordance with Ethical Committee Acts, Mashhad University of Medical Sciences, Mashhad, Iran. As oral median lethal dose
(LD50) of DZN was reported to be 300–400 mg/kg in rats (Gokcimen et al., 2007), 1/10th and 1/20th of LD50 was used in this study. Rats were randomly divided into 3 groups of 6 animals each. The groups include: (1) control (corn oil), (2) DZN-treated group (15 mg/kg), and (3) DZN-treated group (30 mg/kg). Rats were daily gavaged DZN (Bazodin1, Syngenta, Basel, Switzerland, purity 96%) in corn oil or received corn oil alone. Rats were killed after 4 weeks of treatment, and brains were removed and quickly freezed in liquid nitrogen and stored at 80 C until use.
MDA measurement To measure the levels of lipid peroxidation, brain tissues were homogenized using Polytron homogenizer (Kinematica, Lucerne, Switzerland) at 4 C in 1.15% potassium chloride to make a final concentration of 10% (w/w). To this, 1 ml 0.6% thiobarbituric acid, 3 ml of 1% phosphoric acid (Merck, Germany), and 0.5 ml of supernatant were mixed and heated in boiling water bath for 45 min. Concentration of thiobarbituric acid reactive substances (TBARS) was spectrophotometrically determined using microplate reader (Synergy H4 Hybrid Reader, BioTek, Winooski, Vermont, Sigma-Aldrich Corp. St Louis, USA) by measuring absorbance at 532 nm (Kei, 1978). Concentrations of MDA in samples are expressed in nanomole per gram wet tissue.
GSH content measurement For estimation of brain tissue GSH, equal amount of tissue homogenate was mixed with 10% trichloroacetic acid and vortexed. After centrifugation, 0.5 ml of supernatant was mixed with a reaction buffer containing 3.0 ml of 0.3 M phosphate buffer (pH 8.4) and 0.5 ml 0.04% 5,50 dithiobis-(2-nitrobenzoic acid). Absorbance was measured at 412 nm within 10 min (Moron et al., 1979). A standard curve of GSH was plotted using commercially available standard GSH (Sigma, Sigma-Aldrich Corp., St Louis, USA). Levels of GSH were expressed in nanomole per gram wet tissue.
Western blot analysis Cerebrum tissues (0.2 g) were homogenized in the lysis buffer containing 50 mM Tris-hydrochloric acid, pH 8.8, 2 mM ethylenediaminetetraacetic acid, 2 mM ethylene glycol tetraacetic acid, 2% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-
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100, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 10 mM b-glycerophosphate, 10% v/v 2mercaptoethanol, 2 ml complete protease inhibitor cocktail (Sigma P8340), and 1 mM phenylmethylsulfonyl fluoride (PMSF). Homogenates were sonicated for 30 s and centrifuged at 16,000g for 10 min at 4 C. Total protein concentration of supernatants was determined using Bradford protein assay kit (Bio-Rad Laboratories, Hercules, California, USA). Samples were mixed with equal volumes of 2 SDS sample buffer containing 100 mM Tris-base, 4% w/v SDS, 20% v/v glycerol, 0.2% w/v bromophenol blue, and 10% v/v 2mercaptoethanol and then incubated in boiling water for 5 min. The same amounts of total proteins from each sample were loaded to SDS-polyacrylamide gel electrophoresis wells. Proteins were transferred to polyvinylidene difluoride membranes. Membranes were incubated with 5% skimmed milk in Tris-buffered saline–Tween 20 (TBST) for 1 h at room temperature. Blots were incubated with following antibodies at appropriate dilution according to manufacturer’s instruction for 2 h at room temperature: mouse monoclonal anti-caspase 9, rabbit polyclonal anti-caspase 3, rabbit polyclonal anti-Bax, rabbit monoclonal antiBcl-2, mouse and rabbit anti-b actin (all antibodies were purchased from Cell Signaling, USA). After several washes with TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibody. Protein bands were visualized using enhanced chemiluminescence ( Pierce, Rockford, Illinois, USA) and Alliane 4.7 gel doc (UK). Intensities of immunoreactive bands were quantified using UVI band software (UVtec, UK). Protein levels were normalized against corresponding b actin intensities.
Statistical analysis Results are expressed as mean + SD. Differences between test groups were examined using one-way analysis of variance followed by Tukey–Kramer. The value of p < 0.05 was considered statistically significant.
Results Effects of DZN on lipid peroxidation levels in brain tissues The administration of DZN for 4 weeks had no effect on malondialdehyde (MDA) concentration as oxidative stress biomarkers as compared to the control group (Table 1).
Table 1. Effects of DZN (4 weeks) on MDA and GSH levels in the cerebrum.a Group
MDA (nmol/g tissue)
GSH (nmol/g tissue)
Control DZN 15 mg/kg DZN 30 mg/kg
175.72 + 12.23 178.67 + 17.34 177.5 + 14.02
381.3 + 28.96 375.7 + 11.08 370.7 + 16.52
DZN: diazinon; MDA: malondialdehyde; GSH: glutathione. a Data are shown as mean + SD, n ¼ 6 independent determinations on different preparations of samples.
Effects of DZN on GSH level in brain tissue GSH level did not significantly change in the brain of experimental animals compared with the control (Table 1).
Immunoblot analysis of Bax, Bcl-2, and caspase 3 To investigate the effects of subacute DZN treatment on apoptosis state in brain tissue, active forms of caspase 3 were measured using Western blot. Our data showed that there were no significant differences between active forms of caspase 3 in brain of control and DZN-treated rats (Figure 1). Moreover, no significant changes were observed among caspase 9 and its active form in all experimental groups (Figure 2). Our data showed that Bax/Bcl-2 ratios remain unchanged among control and DZN-treated rats (Figure 3).
Discussion The present study was designed to evaluate neurotoxic effects of subacute exposure to DZN in the brain of rat. Our data showed that subacute exposure to 15 and 30 mg/kg DZN does not change MDA and GSH levels, as well as active form of caspase 3, -9, and Bax/Bcl2. Numerous studies have explained that accumulation of acetylcholine, after inhibition of AChE, causes neurotoxicity (Kaur et al., 2007; Milatovic et al., 2006; Roszczenko et al., 2012). Previous studies in our laboratory showed significant decrease in plasma cholinesterase activity after subacute administration of DZN in rat. Moreover, these studies showed that DZN increases level of MDA in aortic and liver tissues and 8-iso-prostaglandin in serum (Hariri et al., 2010; Lari et al., 2013; Hosseinzadeh et al., 2011). Some studies showed that OPs cause marked perturbations in the antioxidant defense system, induce oxidative damages, and enhanced lipid peroxidation in the
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Figure 1. Western blot analysis of caspase 3 in the cerebrum of control and DZN-treated rat groups. No significant differences were found among groups in the total and activated form of caspase 3 levels. Data are expressed relative to the control and are mean + SD of four separate experiments. p < 0.05 were considered statistically significant. DZN: diazinon.
brain (Binukumar and Gill, 2011; Kaur et al., 2007; ¨ ner et al., 2006). Formation of ROS and induction of U oxidative stress by OPs have also been revealed to be related to apoptosis in different tissues (Yu et al., 2008). Kaur et al. (2007) showed that elevation in calcium and ROS production could induce apoptotic neuronal degeneration following chronic exposure to dichlorvos in rat brain. Caughlan et al. (2004) indicated that chlorphyrifos induced apoptosis and perturbed mitochondrial function in rat cortical neurons through activation of few mitogen-activated protein kinases including ERK1/2, JNK, and p38. Also, it has been reported that subchronic oral administration of dichlorvos in rat increased level of MDA in the endometrium leading to endometrial damage and apoptosis (Mahaboob Khan and Kour, 2007). Our data showed that subacute oral exposure to DZN did not significantly change levels of MDA and lipid peroxidation in brain. This finding is not in agreement with the results of previous studies. Jafari et al. (2012) demonstrated that the concentration of MDA was significantly increased, GSH level was
Figure 2. Western blot analysis of caspase 9 in the cerebrum of control and DZN-treated rat groups. No significant differences were found among groups in the total and activated form of caspase 9 levels. Data are expressed relative to the control and are mean + SD of four separate experiments. p < 0.05 were considered statistically significant. DZN: diazinon.
decreased, and antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), and glutathione S-transferase activity changed in brain, heart, and spleen after acute exposure to DZN through intraperitoneal injection at high doses (100 and 200 mg/kg) in both Wistar and Norway rat strains. It was reported that dipping the animals in DZN for protection from scab mite (Psoropets ovis) cause impairment of the biochemical, physiological, and histopathological parameters in rabbits. In a later study, red Baladi rabbit dipped in water containing DZN (6.0 or 30 mg) for 10 s. Histopathological changes of brain, liver, and kidney were observed after dipping in high DZN concentration. Histopathological modification in brain lesions were seen with polymorphic and hyperchromatic nuclei with perinuclear cytoplasmic vacuoles in neuron cells and mild degenerative changes of ¨ ner et al. found that nerve fibers (Yehia et al., 2007). U exposure to sublethal concentrations of DZN (1 and 2 mg/l) caused inhibition of AChE activity and significant increase in MDA levels in the brain of freshwater ¨ ner fish, Oreochromis niloticus, after 7 and 15 days (U et al., 2006). The results of acute treatment single dose of DZN (335 mg/kg) in rat showed that DZN
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Figure 3. Western blots and densitometric analysis of Bax and Bcl-2 protein levels in cerebrum obtained from control and DZN-treated rat groups. Densitometric quantification of the Bax/Bcl-2 ratio. p < 0.05 were considered statistically significant. DZN: diazinon.
induced significant increased in the level of MDA in rat brain with no significant changes in activities of free radical scavenging enzymes SOD, CAT, and GSH peroxidase(Yilmaz et al., 2012). Moreover, it has been shown that DZN and chlorpyrifos could induce widespread changes in the genes related to oxidative stress such as SOD and CAT in differentiating PC12 cells (Slotkin and Seidler, 2009). We believe that variations in the animal types, breed and species, tissue types, doses, chemical structures, administration route, and duration of exposure to OPs have effects on DZN toxicity (Karalliedde et al., 2003). Activation of antioxidant enzymes like SOD and CAT are the first line of cell defense against free radicals and ROS exposure. Mammalian cells have a comprehensive set of antioxidant defense mechanisms to prevent free radical formation and also to limit their damaging effects. So, depending on the magnitude of the oxidative stress and dose of stressor, generation of ROS may get modulated by intracellular antioxidant systems (Isik and Celik, 2008). Elevated activity and gene expression of enzymes involved in oxidative stress that were observed in previous studies
and also nonsignificant reduction of GSH content and unchanged MDA level in our study suggest that compensatory mechanism and adaptive response are activated during subacute low oral dose exposure to DZN. Perhaps, the GSH acts as a powerful antioxidant in the neutralized free radical cells and protected cells against oxidative damage and toxic agents. It has been proved that OPs toxicity in rats is age dependent; as young animals are more sensitive to OPs. AChE inhibition is much stronger in young animals because of lower detoxification capability (Kousba et al., 2007; Padilla et al., 2004; Slotkin et al., 2006). Some developmental studies in neonatal rats showed that organophosphates induce neurotoxicity and reduce numbers of neurons through induction of proapoptotic and antimitotic mechanisms (Dam et al., 1998; Slotkin, 2004; Slotkin and Seidler, 2011, 2012).Gene expression profiling in forebrain of neonatal rats that were exposed to daily doses of DZN or chlorpyrifos showed that multiple mechanisms contribute to developmental neurotoxicity of organophosphates. This study indicated that DZN elicited major transcriptional changes in genes involved in the apoptosis pathways and oxidative stress. Wide range effects were observed on bax, bmf, casp1 and casp4, sod, gpx, and gst gene families. Moreover, DZN was reported to damage spatial learning memory and change expression of neurotrophic factors in the brain of neonatal rat (Slotkin et al., 2007). In vitro mechanistical study of DZN in cortical culture indicated that DZN induced apoptotic neuronal death. This effect was inhibited by the caspase inhibitor. Interestingly, some of DZN effects were independent from its inhibitory effect on the activity of cholinesterase (Rush et al., 2010). Development of neural cell is a critical phase with more sensitivity and susceptibly to external stimuli. During the early stages of life, many pesticides impact on the brain function that result in damage to various regions of brain or declined the number of neurons. Exposure to variety of cytotoxic and stress-related stimuli, including ROS has major role in initiation of mitochondrial apoptosis. Mitochondrial damage releases cytochrome c that activates caspase 9 and its downstream effector caspases 3 and 7. These process leads to DNA fragmentation and cell death (Allan and Clarke, 2009). Permeabilization of the mitochondrial membrane is partially controlled through Bcl family of protein apoptosis inhibitors such as Bcl-2 and Bcl-xL and apoptosis inducers such as Bax and Bid
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(Allan and Clarke, 2009; Franco et al., 2009). We did not observe significant changes in Bax/Bcl-2 ratio in DZN-treated group as compared to the control group. Overall, the results of our study showed that subacute exposure to 15 and 30 mg/kg DZN via gavage did not activate apoptosis pathway with no effects on the level of cleaved caspase 3 and -9 in brain of rat. This finding indicates that DZN has no or low effect on adult neural cells. Pharmacokinetic and pharmacodynamic differences may contribute to age-related OPs toxicities (Binukumar and Gill, 2011; Timchalk et al., 2006). In conclusion, according to our observation, nervous system in adult rats is less sensitive and less susceptible to subacute exposure to DZN toxicity. Perhaps dissimilarity in animal type, age, exposure duration, and administration route of DZN cause various results in different studies. As a consequence, DZN toxicity may be dose dependent, and higher antioxidant capacity of brain tissue in adult rats may protect the cell against its toxicity at low dose. Funding The authors thank the Vice Chancellor of Research, Mashhad University of Medical Sciences, Mashhad, Iran, for financially supporting the present work.
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