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CNS & Neurological Disorders - Drug Targets, 2014, 13, 1397-1405 1397
Sub-Chronic Exposure of Non-Observable Adverse Effect Dose of Terbufos Sulfone: Neuroinflammation in Diabetic and Non-Diabetic Rats
Syed M. Nurulain
1, Ernest Adeghate
2, Azimullah Sheikh
1, Javed Yasin
3, Mohammad A. Kamal
4, Charu Sharma
3, Abdu Adem
1and Shreesh Ojha
*,1Departments of Pharmacology and Therapeutics1, Department of Anatomy2 and Department of Internal Medicine3, United Arab Emirates University, P.O. Box # 17666, Al Ain, Abu Dhabi, United Arab Emirates
4King Fahd Medical Research Center, King Abdulaziz University, P.O. Box # 80216, Jeddah 21589, Saudi Arabia
Abstract: Neuroinflammation (NI) contributes to the pathogenesis of several neurodegenerative disorders.
Epidemiological and a few animal studies have shown that chronic exposure of organophosphorus compounds (OPC) may cause neuronal injury and predispose to neuro- as well as psychotic disorders in conjunction with NI. However, in vivo studies are meager and do not represent the entire toxicologically diversified OPC. The present study aimed to investigate the result of non-observable adverse effect level dose of a highly toxic OPC, terbufos sulfone (TBS), on sub-chronic exposure on the status of proinflammatory cytokines; interleukin-1β, interleukin-6 and tumor necrosis factor-α in rats brain. In addition, lactate dehydrogenase, nitric oxide and reduced glutathione were also determined in brain. Red blood cell acetylcholinesterase was measured weekly. Total of four groups’ saline control, diabetes control, non-diabetes TBS and diabetes treated with TBS were employed in the study. Control groups received saline and the experimental groups were injected with TBS intraperitonealy for fifteen days daily. Twenty four hours after the last injection, the animals were euthanized for collection of brain and serum samples. The study showed significant elevation of interleukin-6, tumor necrosis factor-α and lactate dehydrogenase in brain of TBS treated groups, while the presence of interleukin-1β was significantly greater in the non-diabetes TBS treated group when compared with saline control. The increase was observed to be independent of acetylcholinesterase level and diabetes condition. The change in reduced glutathione was modest as compared with control. Based on the findings, the study concludes that the non-observable adverse effect level dose of TBS has potential to cause NI and subsequent neurodegeneration, a remarkable sign of many chronic neuronal and psychotic disorders. Further studies with prolonged exposure and other neurodegenerative parameters are warranted.
Keywords: Neuroinflammation, neurodegeneration, organophosphorus compounds, terbufos sulfone.
INTRODUCTION
The organophosphorus compounds (OPCs) are used for public health purposes to control disease vectors. Over the years, its application has surged enormously. They are used as insecticides, pesticides, helminthicides, acaricides, nematocides and herbicides. Up to this time, there are over 100 different OPCs available, all with a similar generalized chemical structure. The acute toxicity of different OPC ranges from that of extremely toxic nerve gas to less than that of Table salt [1]. The toxicity of all the OPCs is due to the irreversible inhibition of the neurotransmitter enzyme, acetylcholinesterase (AChE). Experimental evidence indicates that acute toxic manifestation of OPC is associated with activation of microglia and astrocytes [2]. The triggering of microglia produces proinflammatory cytokines and chemokines [3], which may have a detrimental effect on the CNS in the context of an important role of AChE in neurodevelopment [4]. However, chronic OPC exposure has been shown to induce inflammation even without substantial
*Address correspondence to this author at the Departments of Pharmacology and Therapeutics, United Arab Emirates University, P.O. Box # 17666,Al Ain, Abu Dhabi, United Arab Emirates; Tel: 009713-7137524;
Fax: 009713-7672033; E-mail. [email protected]
AChE decrement. For instance, chronic exposure of sarin has been reported to raise cytokines in rat brain irrespective of AChE diminution [5]. In spite of the no effect on serum cholinesterase, chronic administration of chlorpyrifos increases glial fibrillary acidic protein (GFAP) expression in the rat’s hippocampus [6]. Whereas macrophage function and mast cells degranulation was found to be enhanced in mice after low levels exposure of malathion [7]. The redox- inflammatory responses are involved in maintaining homeostasis but overt, uncontrolled activity leads to deleterious effects [8]. Overproduction of proinflammatory cytokines [i.e. interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α)] and excessive inflammation in the brain is characteristic of many neurodegenerative diseases including Parkinson, Alzheimer, depression, dementia and multiple sclerosis (Tables 1 and 2).
Neurodegenerative diseases are manifestations of long-term, low-grade inflammation which invariably becomes pathogenic and contributes to neuronal injury and cell death (Tables 1 and 2). Convincingly it has been demonstrated that that proinflammatory cytokines exacerbate neuroinflammat- ion and sustain neurodegenerative processes. The most extensively studied proinflammatory cytokines are IL-1β and TNF-α which are produced during damage within the CNS.
Increases in IL-1β and TNF-α have been observed before neuronal death occurs [9, 10]. The damage resulting from the
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exposure of neurotoxicants within CNS surges both anti- inflammatory and pro-inflammatory cytokines [11-13].
The acute toxicity of OPCs is well recognized and established. However, the toxicity of long term exposure is still in its infancy for many of the highly toxic compounds which are promoted for use and considered relatively safe at the no-observable adverse effect level (NOAEL) doses.
Therefore, the current study was undertaken to examine the status of proinflammatory cytokines and oxidative stress markers in rat brain employing sub-chronic exposure of the pharmacologically and toxicologically safe dose. This is relevant to the NOAEL dose of TBS which is a highly toxic OPC by WHO classification of OPC insecticides and pesticides. Currently, TBS is the fourth leading OPC pesticide used in the USA [14] and reported to be one of the highest killers of fish and birds in the USA (http:
//www.abcbirds.org/abcprograms/policy/toxins/profiles/terbu fos.html last accessed on 25.3.14). Several epidemiological studies [15-18] have revealed a link between the chronic
exposure of OPC and neurodegenerative diseases but the evidence-based studies are scanty and do not represent the entire diversified groups of OPC. The majority of studies were carried out on moderate or mildly toxic OPC (Class II and III) except Sarin, a nerve gas, and parathion, an extremely toxic OPC (class Ia). TBS belongs to the highly toxic class (Ib) of OPC. Schematic representation of the tentative mechanism of TBS-induced neuroinflammation is depicted in Fig. (1).
Furthermore, emerging evidence is available to demonstrate that diabetes mellitus pathophysiology involves a low grade chronic inflammation which is characterized by the up-regulation of proinflammatory cytokines [19]. The risk of exposure to TBS in normal as well as in diabetic rats was evaluated to demonstrate whether NOAEL exposure of TBS could exacerbate neuroinflammation and subsequent neurodegeneration. It has been shown that peripherally- derived proinflammatory cytokines could also influence pathogenesis in the CNS [20]. Hence, diabetic rats exposed
Fig. (1). Schematic representation of the mechanism of terbufos sulfone (TBS)-induced neuroinflammation and subsequent neurodegeneration. IL-1β: Interleukin-1β, IL-6: interleukin-6 and TNF-α: tumor necrosis factor-α.
Non-Observable Adverse Effect Dose of Terbufos Sulfone CNS & Neurological Disorders - Drug Targets, 2014, Vol. 13, No. 8 1399
to TBS could develop neurodegeneration faster. Our study investigated the status of proinflammatory cytokines and the oxidative markers lactate dehydrogenase (LDH), reduced glutathione (GSH) and nitric oxide (NO) under diabetic conditions in rats exposed to TBS and the influence of AChE inhibition on the status of IL-1β, IL-6 and TNF-α.
MATERIALS AND METHODS Experimental Animals
Wistar, male, albino rats (weighing 200-250 g) bred at the Animal Research Facility, College of Medicine and Health Sciences, United Arab Emirates University, United Arab Emirates from the original stock were used in the present study. The animals were cage-housed in the polypropylene cages (43×22.5×20.5 cm) at standard laboratory conditions (temperature; 23 ± 1°C; humidity; 50 ± 4% day/night cycle; 12h/12h) with not more than 4 rats in
each cage and appropriately acclimatized before inclusion in the study. The animals were fed commercial rat chow diet containing standard diet (Emirates Feed Factory, Abu Dhabi, UAE) and tap water ad libitum. All the experimental procedures were performed in strict compliance with the rules and regulations of the institutional animal ethics committee for the animal care, welfare and use of laboratory animals.
Induction of Experimental Diabetes Mellitus
Diabetes in rats was produced by a single i.p. injection of streptozotocin (60 mg/kg body weight) in a freshly-prepared citrate buffer solution of 0.1 M and pH 4.5. The occurrence of hyperglycemia in rats were confirmed after two weeks of streptozotocin (STZ) injection and diabetes was confirmed by measuring blood glucose values using the One - Touch Glucometer (Lifescan Inc., Milpitas, CA, USA). The rats were four weeks diabetic at the termination of the Table 1. Significance of studied proinflammatory cytokines and their cellular sources and biological activities.
Proinflammatory Cytokine Cellular Source Biological Effects Neurodegenerative Effect
IL-1β Activated monocytes/macro-
phages, endothelial cells and microglia
− An endogenous pyrogen
− Stimulates other proinflammatory cytokines
Neuroinflammation leading to disruption of neuron dendrites development and outgrowth
IL-6 Activated
monocytes/macrophages, T- cells, astrocytes etc.
− An endogenous pyrogen
− Stimulates liver secretion of acute phase proteins
− Stimulates B-lymphocytes to produce antibodies and in concert with IL-1β causes T- cell activation
Neuroinflammation leading to disruption of neuron dendrites development and outgrowth, decreasing survival of fetal serotonin neurons
TNF-α Activated
monocytes/macrophages, endothelial cells microglia, T cells and natural killer cells
− An endogenous pyrogen
− Initiates a cascade of cytokines which mediate an inflammatory response.
− Regulates the expression of many genes for the host response to infection& induces apoptosis
Neuronal apoptosis, disruption of neuron dendrites development and outgrowth
IL-1β: Interleukin-1β, IL-6: Interleukin-6 and TNF- α: Tumor Necrosis Factor-α.
Table 2. Up-regulation of some of the proinflammatory cytokines in neuronal disease and psychotic disorders.
S. No. Disease Status of Main Proinflammatory Cytokines References
1 Multiple Sclerosis TNF-α ↑, IL-6↑ and IL-1β ↑ [21]
2 Alzheimer TNF-α ↑, IL-6↑ and IL-1β ↑ [22]
3 Parkinson TNF-α ↑, IL-6↑ and IL-1β ↑ [23]
4 Schizophrenia TNF-α ↑, IL-6↑ and IL-1β ↑ [24]
5 Ischemic stroke TNF-α ↑, IL-6↑ and IL-1β ↑ [25]
6 Motor disability IL-1β ↑ [26]
7 Depression TNF-α ↑, IL-6↑ and IL-1β ↑ [27]
8 Mood disorder TNF-α ↑, IL-6↑ and IL-1β ↑ [28]
9 Bipolar disorder TNF-α ↑, IL-6↑ [29]
10 Psychotic disorder TNF-α ↑, IL-6↑ and IL-1β ↑ [30]
11 Neuronal cell death TNF-α ↑, IL-6↑ and IL-1β ↑ [31]
12 Neuronal injury TNF-α ↑, IL-6↑ and IL-1β ↑ [32]
IL-1β: Interleukin-1β, IL-6: Interleukin-6 and TNF-α: Tumor Necrosis Factor-α.
experiments. The rats with blood glucose levels above 250 mg/dl were only included in the experiments.
Experimental Study Design
The rats were divided in a total of four groups each containing six rats. TBS was given intraperitoneally at a dose of 100nmol/500µL every day to each rat for a total of fifteen days. The selected dose of TBS in this study was NOAEL dose. (http://www.fao.org/docrep/006/Y5221E/y5221e0r. htm) and did not produce substantial inhibition of activity of red blood cell acetylcholinesterase (RBC-AChE) in the normal rats. The control rats were intraperitoneally injected 500 µL saline.
Group 1: Saline control group.
Group 2: Non-diabetes TBS treated group.
Group 3: Diabetes control group.
Group 4: Diabetes TBS treated group.
Kits, Chemicals and Reagents
The stock solution of TBS (100 mM) was prepared in dry acetone. The working solution for intraperitoneal injection was prepared immediately before the use in experiment by diluting the stock solution with saline. TBS and STZ were purchased from Sigma-Aldrich Chemie (Sigma-Aldrich Chemie GmbH, Steinheim, Germany).
Blood Glucose Measurement
The blood glucose levels were checked on day 8 of each week before giving the 8th injection of TBS. The blood samples were collected from the tail vein. One Touch Ultra- Glucometer (Lifescan Inc., Milpitas, CA, USA) was used to check the glucose level.
RBC-AChE Activity
For the measurement of RBC-AChE, the blood samples were collected from the tail vein of the rats. The activity of RBC-AChE in diluted whole-blood samples was measured using a selective butyrylcholinesterase inhibitor, Ethoprop- azine (Sigma-Aldrich Chemie, Steinheim, Germany), as described previously [33]. The freshly collected venous blood sample was diluted 1: 100 in a solution (0.1M phosphate buffer + Triton-X) and frozen immediately at - 20°C until analysis. The measurement of RBC-AChE enzyme and total hemoglobin contents were carried out using a Milton Roy Spectronics 301 spectrophotometer (Milton Roy, Ivyland, PA, USA). The assay of RBC-AChE activity, based on Ellman method, estimates the reduction of dithiobisnitrobenzoic acid (DTNB) to nitrobenzoate by thiocholine, which is the product of acetylthiocholine hydrolysis [34]. The collected samples for processing were diluted in phosphate buffer (0.1 M; pH 7.4) and further incubated with DTNB (10 mM) and ethopropazine (6 mM) for the duration of 20 min at the temperature of 37°C before adding acetylthiocholine. The changes in the absorbance of mixture was measured spectrophotometrically at 436 nm and calculated using an absorption coefficient of TNB at 436 nm (ε = 10.6 mM-1cm1). The values obtained were further norm-
alized to the hemoglobin (Hb) content (determined as cyan- methemoglobin) and expressed as mU µmol-1Hb-1 [35].
Blood and Brain Collection for Biochemical Tests
After two weeks of the treatment, blood samples were collected from rats after decapitation and centrifuged at 3000 rpm for 10 min. The brain was excised and snaps frozen in aluminum foil. The serum and brain samples were then kept frozen at -80°C. The brain was divided into two equal halves sagitally, one half (right) was weighed and homogenized with homogenizing buffer containing tris hydrochloric acid (10 mM; PH 8), sodium chloride (140 mM), potassium chloride (300 mM), ethylene ditetra acetic acid (1 mM), Triton-X (0.5%), sodium deoxycholate (0.5%) in 1:5 ratio weight by volume (w/v). The samples were centrifuged at 15000g for 30 min. The obtained clear supernatant was stored at -80°C for further analysis of proinflammatory cytokines, LDH, GSH and NO.
IL-1β, IL-6 and TNF-α Determination
ELISA kits for the determination of IL-1β, IL-6 and TNF-α in whole brain samples were obtained from R&D systems Inc. (Minneapolis, MN). The absorbance in the plates was measured at 450 nm wavelength and 620 as reference wavelength by Magellan 6 software using ELISA reader (Tecan, Hungary).
LDH, NO and GSH Determination
The levels of LDH in both serum and brain were estimated by auto-analyzer, COBAS INTEGRA 400 PLUS (Roche Diagnostics, Germany). The level of NO was measured according to the procedure used by Cabezudo et al. [36], based on Griess method. Further, 100 µl of supernatant/serum was mixed with an equal volume of Griess reagent and incubated at room temperature for 10 to 15 min. The absorbance was measured in an automated microplate reader (Tecan, Hungary) at the wavelength of 492 nm. The nitrite concentration was quantitated using NaNO2 as standard and was expressed as micro molar (µM). The level of GSH was measured following the manufacturer’s protocols for the assay kit (Sigma Chemical Co, MO, USA).
Statistical Analysis
Statistical analysis was performed on all the data obtained from the experiments using SPSS 21.0 (SPSS Inc., Chicago, IL, USA). The criterion of statistical significance was set at p ≤ 0.05. The enzyme activity over the time was compared to control values following the Mann-Whitney rank order test.
RESULTS
Body Weight of the Animals
The body weight of the animals was recorded at the end of each week in all four groups. Fig. (2) shows the average body weight during the two weeks of treatment. Animals in the control and TBS-treated groups showed a steady increase in body weight. The percentage change in body weight was
Non-Observable Adverse Effect Dose of Terbufos Sulfone CNS & Neurological Disorders - Drug Targets, 2014, Vol. 13, No. 8 1401
not statistically significant when compared with the corresponding controls.
Fig. (2). Body weight over two weeks of treatment.
Blood Glucose Level
The blood glucose levels are depicted in Fig. (3). The three week average blood glucose levels in normal control and non-diabetes TBS-treated groups were 104±7 mg/dL and 100±8 mg/dL respectively. The average blood glucose levels in diabetes control and diabetes TBS-treated rats were 460±72 mg/dL and 511±29 mg/dL respectively. The average levels of blood glucose in the group of diabetic rats treated with TBS increased markedly over time but were not statistically different from the same measurements collected from the diabetes control group.
Fig. (3). Glucose level (mean±sd) in two weeks of treatment.
RBC-AChE Activity
The activity of RBC-AChE enzyme was determined as a percentage of the baseline levels and the results are shown in Fig. (4). The values from the animals in the saline control group were 93±8% in week 1 and 103±9 in week 2. The average enzyme levels in the non-diabetes, TBS-treated
group were102±14 and 94±15% of the baseline levels, respectively. The average RBC-AChE levels in the diabetes control group were 88±20 in week 1 and 84±30 in week 2.
The average levels for the diabetic rats treated with TBS exhibited a sharp and significant decrease in RBC-AChE activity over time. The average enzyme activity levels were 41±18 in week 1 and 12±2% in week 2.
Fig. (4). RBC-AChE activity. The values represent Mean±SD of 6 rats. *p > 0.05 diabetes control vs diabetes TBS treated. RBC- AChE: Red blood cells-acetylcholinesterase.
LDH Levels in Serum and Brain Homogenates
The average LDH levels in serum and brain are shown in Fig. (5). The LDH levels in serum and brain of the animals in the saline control group were 708±182 U/L and 1581±498 U/L respectively. The average LDH levels in serum and brain of the animals in the non-diabetes TBS-treated group were1021±287 U/L and 3333±1077 U/L, respectively. The average level of LDH in the brain of non-diabetes TBS-
Fig. (5). LDH levels in serum and brain. The values represent Mean±SD of 6 rats.*p > 0.05 control vs TBS treated. LDH: Lactate dehydrogenase.
50 100 150 200 250 300 350 400 450
Week 0 Week 1 Week 2
Weight in grams
Saline control Non- diabetes TBS treated Diabetes control Diabetes TBS treated
0 100 200 300 400 500 600 700 800
Saline control Non-diabetes TBS treated
Diabetes control Diabetes TBS treated
Glucose mg/dl
Week 0 Week 1 Week 2
0 20 40 60 80 100 120
Saline control Non-Diabetes TBS treated
Diabetes control Diabetes TBS treated
Percent of baseline
Week 1 Week 2
*
*
0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000
Saline control Non-diabetes TBS treated Diabetes control Diabetes TBS treated Saline control Non-diabetes TBS treated Diabetes control Diabetes TBS treated
Serum LDH Brain LDH
U / L
* *
treated group was statistically significant (p-value 0.037) from the values of saline control group but it was not significant when compared with the values from diabetic rats treated with TBS (p-value 0.754). The average values for LDH activity in serum from diabetic control and diabetes TBS-treated rats was 829±174U/L and 1283±574 U/L, respectively. The brain samples showed values of 1620±464U/L and 3589±1038 U/L, respectively for diabetes control and diabetes TBS-treated groups which represents a statistically significant difference.
Level of Proinflammatory Cytokines (IL-1β, IL-6 and TNF- α)
Table 3 represents the average levels of proinflammatory cytokines; IL-1β, IL-6 and TNF-α in both control and TBS treated brain homogenates. All the proinflammatory cytokines measured in this study were markedly increased in non-diabetes TBS-treated group and significantly greater than the values obtained from the saline control group (p≤
0.05). However, there was no statistically significant difference in IL-1β levels in diabetes TBS treated group when compared with diabetes control.
Levels of NO and GSH in Brain Homogenates
The average levels of NO and GSH in both control and treated serum and brain are shown in Table 4. A modest but statistically insignificant decrease in GSH was observed in both TBS-treated groups when compared with the values from their corresponding control groups. However, 95% confidence interval for the mean values showed a wide range and it seems that probably some of the rats were highly tolerant towards bearing the stress and some were sensitive to coping with the stress. Interestingly, in present study NO could not be measured in the brain samples. It was found beyond the detection limits of the protocol, which was 2.5 µM.
DISCUSSION
The findings of the current study showed that sub- chronic exposure of an NOAEL dose of TBS caused a statistically significant rise in the level of proinflammatory cytokines in brain tissues. The level of LDH was augmented significantly in brain tissue (p-value 0.037 and 0.014 for non-diabetes and diabetes TBS-treated groups respectively) but not in serum (p-values 0.221 and 0.462 respectively) from treated rats. A significant reduction in RBC-AChE was observed in diabetes TBS-treated rats when compared to values obtained from animals in the diabetes control group (Fig. 4; p-value 0.027 and 0.013 in week 1 and 2 respectively) only. However, oxidative stress parameters, NO and GSH were not significantly changed (Table 4).
Evidence regarding the non-cholinergic effect of OPC, particularly with neurological disorders, has been demonstrated in the literature [37, 38] and reviews [39, 40].
The results from this current study indicated that TBS has non-canonical detrimental targets at a non-lethal dose since there was no significant effect of RBC-AChE on non- diabetes rats. In the present study, the exposure of an NOAEL dose of TBS significantly induced proinflammatory cytokines levels which indicate that repeated exposure of non-lethal doses of TBS may have the potential to induce NI and subsequent neurodegeneration.
With acute OPC poisoning, the expression of cytokines which propagates the inflammation is a normal homeostasis phenomenon meant for neuroprotection [2]. However, apoptosis plays an important role in neurotoxic cell death in the brain [41]. NI is the mark of many neuronal diseases or disorders (Table 2). The evidence indicates that NI causes and accelerates long-term neurodegenerative disease by playing a central role in the very early development of chronic conditions including dementia [42]. A number of studies [37, 43] have demonstrated axonal degeneration, neural cell death and neural loss after exposure to OPCs in
Table 3. The average levels of proinflammatory cytokines in the brain of the experimental groups.
Mean±SD (pg/ml) 95% Confidence Interval for Mean
Lower Bound Upper Bound
IL-1β
Saline control 168.88±39.81 105.53 232.22
TBS treated 236.52±41.30 (p-value 0.050) 185.23 287.80
Diabetes control 221.78±84.56 87.23 356.34
Diabetes/TBS treated 257.48±39.20 (p-value 0.624) 208.81 306.16
IL-6
Saline control 74.77±44.26 4.35 145.20
TBS treated 156.83±38.77 (p-value 0.027) 108.69 204.98
Diabetes control 68.10±10.39 51.57 84.63
Diabetes/TBS treated 111.33±30.65 (p-value 0.050) 13.71 149.38
TNF-α
Saline control 47.62±15.05 23.67 71.57
TBS treated 94.48±24.88 (p- value 0.014) 63.58 125.37
Diabetes control 57.70±3.23 52.56 62.85
Diabetes/TBS treated 107.44±22.67 (p value 0.014) 79.29 135.58
The values represent Mean±SD of 6 rats. IL-1β: Interleukin-1β, IL-6: Interleukin-6 and TNF-α: Tumor Necrosis Factor-α. P-values are in comparison to corresponding saline and diabetes controls.
Non-Observable Adverse Effect Dose of Terbufos Sulfone CNS & Neurological Disorders - Drug Targets, 2014, Vol. 13, No. 8 1403
rats, mice and other species. For instance, Kaur et al. [37]
reported that chronic low dose exposure of dichlorvos causes impaired mitochondrial bioenergetics and apoptotic neuronal degeneration. The exposure of low-level, long-term OPC causes DNA fragmentation which is a sign of neuronal apoptosis. Chronic dichlorvos exposure can induce oxidative stress, resulting in the over-expression of pro-apoptotic genes, DNA damage and finally leading to caspase- dependent apoptotic cell death in rat brain [38].
In recent years, evidence from epidemiological [2, 39, 44] and a few animal studies has suggested that pesticides, including sub lethal or non-lethal exposure to OPC pesticides, may affect many aspects of neuronal pathophysiology and may be a risk factor for Parkinson, Alzheimer, sclerosis, stroke, depression, schizophrenia etc.
For instance Binukumar et al. [45] demonstrated that sub- chronic exposure of dichlorvos causes an increase in IL-1β, IL-6 and TNF-α in the brain and could increases the risk for Parkinson disease by enhancing the vulnerability of loss of dopaminergic neurons. Astiz et al. [46] reported that sub- chronic exposure of dimethoate enhances proinflammatory status in the brain as was also observed in the present study.
Davis et al. [47] reported numerous episodes of acute and chronic OPC intoxication upon exposure in a clinical study.
The relationship between chronic exposure of OPC and resultant alterations in central cholinergic or dopaminergic activity has been suggested. Based on the toxicities, it has been speculated that the risk for the late onset of Parkinsonism exists among the agricultural workers. Freire and Koifman [15], in an epidemiological study, revealed that exposure to chlorpyrifos is strongly associated with the development of Parkinson disease. Ali and Rajini [48]
demonstrated that low levels of monochrotophos elicits the dopaminergic features of Parkinson disease in C. elegans.
Sub-chronic exposure of chlorpyrifos and methamidophos has also been shown to cause depression-like behavior in experimental animals [49-51]. Blanc-Lapierre et al. [52]
found cognitive and psychological changes in relation to OPC insecticide exposure. Cognitive disorders were found associated with acute and chronic exposures, and psychiatric disorders mostly with just poisonings. Salazar et al. [53]
reported Alzheimer-type symptoms in mice after eight months of chlorpyrifos exposure. The suggested mechanisms to cause the Parkinson and other neurodegenerative diseases
comprise inflammation, oxidative stress, mitochondrial dysfunction, promotion of α -synuclein fibrillation and interference with dopamine transporters. In the present study, GSH and NO were not significantly affected however a modest decrease over the time studied indicates that there may be a possibility that on long exposure, TBS may deplete GSH levels. Based on the results from the present study, it can be speculated that exposure for two weeks was not sufficient to induce changes in oxidative status and a profound antioxidant status has helped in maintaining the oxidative status. Although, oxidative stress has been considered a factor for neurodegeneration in the literature it must be remembered that each OPC has a unique toxicological profile [54] and TBS may be an OPC that does not affect oxidative parameters significantly. Lukaszewicz- Hussain [55] found that glutathione levels decreased in chlorfenvinphos intoxication at a dose 50% smaller than NOAEL in rat brain and suggested that sub-chronic administration of chlorfenvinphos leads to a change in the brain oxidative status. In the present study, GSH was also found to be decreased but not sufficiently to be significant.
dos Santos et al. [56] reported that oxidative stress parameters were not predisposed in different parts of animal brain when treated with malathion. Mostafalou et al. [57]
worked on rat hepatocytes treated with malathion and concluded that the main cause of cell death was mitochondrial dysfunction. The present work is limited to only two oxidative parameters. Further investigations with more non-enzymatic and enzymatic oxidative parameters are warranted. Moreover, the significant increase in LDH substantiates the evidence for elevated proinflammatory cytokines. The levels of LDH have been found to become increased in injured, apoptosis or diseased conditions [58, 59]. Daifuku et al. [60] found that oral administration of LDH to mice, stimulates immunoglobulin and cytokine production including TNF-α by lymphocytes in vivo.
Altogether, the results suggest that exposure of NOAEL doses of TBS, even for just two weeks has the potential to enhance the level of proinflammatory cytokines and promote cellular injury evidenced by increased average LDH levels.
Interestingly, the changes observed were independent of the degree of AChE inhibition (Fig. 4). This suggests that occupational exposure of TBS, even at NOAEL doses may bring risk in those who are prone to develop diabetes and can Table 4. The average levels of NO and GSH in the brain of different experimental groups.
Mean±SD (µM) 95% Confidence Interval for Mean
Lower Bound Upper Bound
NO
Saline control ≥2.5 ≥2.5 ≥2.5
TBS treated ≥2.5 ≥2.5 ≥2.5
Diabetes control ≥2.5 ≥2.5 ≥2.5
Diabetes/TBS treated ≥2.5 ≥2.5 ≥2.5
GSH
Saline control 44±4 38 50
TBS treated 37±18 14 59
Diabetes control 46±7 36 56
Diabetes/TBS treated 33±26 1 65
The values represent Mean ± SD of 6 rats. NO: Nitric Oxide, GSH: Reduced Glutathione.
worsen the diabetic condition by promoting neuroinflam- mation and subsequent neurodegeneration.
CONCLUSION
The study concludes that TBS at NOAEL dose and even with the short exposure has the potential to cause NI and subsequent neurodegeneration, which is a hallmark of many chronic neuronal and psychological disorders. The results from this study also encourage speculation that a more profound effect would develop with prolonged exposure of TBS. Further detailed investigations with prolonged exposure are suggested.
LIST OF ABBREVIATIONS AChE = Acetylcholinesterase DTNB = Dithiobisnitrobenzoic acid GSH = Reduced glutathione IL-1β = Interleukin-1beta IL-6 = Interleukin-6
LDH = Lactate dehydrogenase NO = Nitric oxide
NOAEL = No-observable adverse effect level OPCs = Organophosphorus compounds RBC = Red blood cells
TNF-α = Tumor necrosis factor- alpha TBS = Terbufos sulfone
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
The work in the author’s (Shreesh Ojha) laboratory is supported by grant (31M099) from the National Research Foundation, United Arab Emirates University, UAE.
Authors acknowledge Ms. Nima Nalin for her technical assistance.
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Received: March 30, 2014 Revised: May 23, 2014 Accepted: June 15, 2014
PMID: 25345510
