Acrylamide (ACR) is simple compound, highly soluble in water, α, β-unsaturated carbonyl chemical compound with many advantages as well as so many disadvantages. ACR is used in many fields including in laboratories, and it is absorbed during occupational exposure [1,2]. ACR is a synthetic monomer, found in a variety of deep fried foods at high temperature. ACR can found in foods that are daily consumed, such as chips/French fries, crisps, and bread, biscuits, crackers, and breakfast cereals . Mottram
Lipids are easily susceptible to both reactive oxygen and reactive nitrogen species (ROS/RNS) than the other biomolecules . Dotan
Nitric oxide (NO) is a chemical mediator, which is an integral part in the maintenance of physiological homeostasis because of its both regulatory and protective functions.NO that produced by many cells including cells of immune system which shows systematic action on various organs, tissues and tumor cellls [15,16]. In general, NO has played numerous roles in the brain that includes soluble guanylate cyclase activation  as well as modulation of synaptic vesicle exocytosis [18,19]. In 2003, Boehning and Snyder  reported that NO action as neurotransmission modulator is an important role in the nervous system. In general, alteration in the NO production that leads to the pathological lesions of brain disorders like Alzheimer’s disease .
Glutathione (GSH) is a tripeptide (γ-Gln-Cys-Gly) that serves as a major antioxidant, ACR causes GSH depletion, and this depletion leads to redox imbalance. Due to reactions with hydrogen peroxide [22,23], conjugation reaction with ACR, and its metabolite glycidamide catalyzed by glutathione S-transferase (GST) , GSH consumption occurs in high level. The systematic increase of TBARS concentration was observed when ACR given orally to rats . The non-enzymatic antioxidants such as reduced GSH play a key role in neutralization of free radicals by donating their electrons . GSH is the thiol compound that abundants in cells of all organs, and it plays a major role in protection from oxidative stress in the brain . Glutathione reductase (GR) main function is recycling of oxidized GSH to reduced GSH, i.e., antioxidant form and it is upregulated in oxidative stress [27,28].
GPx catalyze the reduction of hydrogen peroxide as well as organic hydroperoxides in the presence of GSH, and it was extensively studied from the time of discovery . The essential role of GPx about the defensive response to oxidative stress was intensively demonstrated [30,31]. Most GPx isoenzymes of mammalian contain selenocysteine but not epididymal secretary GPx . GPx is present in cytosol and mitochondria; it is major antioxidant enzyme in the brain, predominantly expressed in microglia . GPx family isoenzymes are catalyzing the reduction H2O2 and lipid peroxides using GSH [34,35]. Ursini
GSTs are one of the versatile detoxification enzymes among Phase II enzymes, which are involved in the xenobiotic metabolism and play a major role in cellular protection against oxidative stress. GSTs (EC 220.127.116.11) are widely distributed in prokaryotes and eukaryotes, but in eukaryotes, they are Phase II detoxification enzymes , which protect cellular macromolecules from ROS, environmental carcinogens and chemotherapeutic drugs , which catalyse the nucleophilic addition of GSH to numerous toxic chemical agents (xenobiotics) and electrophilic and carcinogenic metabolites those generated by phase I enzymes [39-41].
Acetylcholine is a neurotransmitter which involved in various functions of the brain such as reward, learning, memory formation, or neuronal development . Acetylcholine esterase (AChE) (EC 18.104.22.168) is an enzyme that catalyzes the hydrolysis of acetylcholine . That the AChE activity which associates with the release of acetylcholine into the synoptic clefts . In addition to that, AChE activity may alter by free radicals . The present study is aimed to study the evaluation of biomarkers in ACR-induced neurotoxicity and brain degeneration in rat.
2. MATERIALS AND METHODS
ACR (98% purity), thiobarbituric acid (TBA), GSH, 5, 5’-dithiobis (2-nitrobenzoic acid) (DNTB), n-butanol, pyridine, and nicotinamide adenine dinucleotide phosphate (reduced) tetrasodium salt (NADPH) were purchased from HiMedia Laboratories Pvt. Ltd. Mumbai, India. Griess reagent was obtained from SRL, Mumbai, India. All other chemicals (analytical grade) were purchased from Standard Chemical Company (India).
2.2. Maintenance of Rats
Rats were allowed for acclimatized for about 1 week, housed in plastic cages and maintained them under standard conditions as per the Institutional Ethics Committee, S. V. University, through the experimental period. They were housed in 12:12 light:dark photoperiod at 23°C ± 2°C and fed them with
2.3. ACR Administration
Male Wistar rats weighing about 200 g were selected for experimentation, and each group has six animals (
|Figure 1: Exposure of acrylamide through drinking water|
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2.4. Determination of Protein
Control and ACR-treated brain sample protein content was measured by the method of Lowry
2.5. TBA Test for LPO
According to Ohkawa
2.6. Estimation of NO
NO was estimated in terms total nitrites at 540 nm according to Jablonsk
2.7. Determination of GSH
According to Kurtel
2.8. GPx Activity Assay
According to Wendel , GPx activity assay was done. Assay buffer:0.25 mM phosphate buffer (pH 7.0) containing 2.5 mM EDTA and 2.5 mM sodium azide (NaN3). The reaction mixture contains assay buffer 1.8 ml, 100 μl GR, 100 μl GSH, 100 μl NADPH, and 250 μg of enzyme source. The reaction was initiated by the addition of 100 μl CHP/H2O2 and a linear decrease in NADPH absorption at 340 nm was measured for 3 min. A blank was maintained without enzyme source.
2.9. GST Activity Assay
Rat brain GST activity assay was done by the method of Habig
2.10. Activity of AChE
According to the method of Ellman
According to Humason , brain tissue histological examinations were conducted. Briefly, collected brain tissues from both control and experimental rats were washed with physiological saline (0.9% NaCl) to remove blood and fat debris adheres to the brain. After fixation in 10% of formalin, the tissues were allowed to process. In the first step, the tissues were washed under running tap water to remove the fixative. In the second step, tissues were allowed for dehydration by a graded series of alcohol and the tissues were allowed to clear using methyl benzoate and subjected to embed in paraffin wax. In the third step, the tissue was subjected to cut with 6 μ thickness and such sections allowed for staining with hematoxylin and eosin (H and E). In the fourth step, the sections were mounted with Canada balsam and observed under light microscope.
2.12. Statistical Analysis
All the data related to this study and their results were calculated from three experiments and presented as the mean ± standard deviation. Student
3. RESULTS AND DISCUSSION
3.1. Chemostress Markers
In this study, as shown in Table 1 and Figure 2a & b that the LPO, NO and GSH levels as well as Table 1 and Figure 3a & b that the GPx, GST and AChE levels disturbances were observed in ACR-administered rat brain, at the 13th and 27th day post-ingestion.
|Table 1: ACR-induced non-enzymatic and enzymatic antioxidant biomarkers modulation|
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|Figure 2: (a and b) Adverse alterations of non-enzymatic molecules by the administration of acrylamide|
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|Figure 3: (a and b) Adverse alterations of enzymes activity by the administration of acrylamide|
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LPO levels were significantly increased (
With the substrate H2O2, GPx activity was significantly increased (
3.2. Histopathology Study
In control rat brain section, normal glial cells and pyramidal cells were noticed as shown in Figure 4. But in ACR administered (50 mg/300 ml water up to 27 days in alternative days), rat brain that histological variations were observed like degeneration of pyramidal cells, degeneration of glial cells, mild vacuolation of pyramidal cells, spongiosis in glia cells and spongiosis, on 13th day post-ingestion as shown in Figure 5, and necrosis and pyknosis, necrosis of neurons and neurophagia, focal gliosis and demyelination of nerve fibers, on 27th day post-ingestion as shown in Figure 6.
|Figure 4: Control rat brain section shows normal glial cells and pyramidal cells (H and E stain) (×10)|
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|Figure 5: Rat cerebellum section shows that degeneration of pyramidal cells, degeneration of glial cells, mild vacuolation of pyramidal cells, spongiosis in glial cells, and spongiosis on 13th day post-ingestion of acrylamide (H and E stain) (×10)|
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|Figure 6: Rat cerebellum section shows that pyknosis, necrosis of neurons and neurophagia, focal gliosis, and demyelination of nerve fibers on 27th day post-ingestion of acrylamide (H and E stain) (×10)|
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The brain is a vital, most complex organ, it serves as the center of the nervous system, and functionally, it is a coordinating and regulatory system of the body. Production of cellular energy that mediated by aerobic metabolism which generates toxic oxygen intermediates, in terms of ROS, excessive production of them causes a significant threat to cellular homeostasis . ROS is the main reason for activation or deactivation of cellular signaling pathways, in the stress condition . The cellular defense system that mediated by enzymatic and non-enzymatic antioxidants, which necessary to maintain normal cellular function . That the detoxification systems which necessary for the survival of organisms, certainly when they exposed to various stressful conditions .
In the present study, as shown in Figure 1, ACR was administered in alternative days in drinking water (50 mg/300 ml) to rat and evaluate the modulation of the non-enzymatic and enzymatic chemo-stress markers in the brain, as shown in Table 1 and Figures 2a and b, 3a and b and observed brain tissue derangements, as shown in Figures 5 and 6. ACR can enter into the body through occupation, cigarette smoke, food, water, and breast milk and distribute entire the body. Zhu
Neurons, endothelial cells, platelets, and neutrophils can release NO in response to homeostatic and pathologic stimuli . The NO produced from inducible nitric oxide synthase (iNOS) is apparently very important in host defence and chronic inflammatory response . The ACR-induced neurotoxicity leads to NO homeostasis disturbance by the contrast expression of neuronal form NO synthase and iNOS . In rat brain, the chronic mild stress has induced NO levels . Administration of high-dose ACR (50 mg/kg bwt) has enhanced NO production in terms of total nitrite (NO2–) found in rat liver . As shown in Table 1 and Figures 2a and b and 3a and b, NO level in terms of total nitrite was significantly increased (
GSH is abundant in cells of all organs, playing a key role in the protection of brain from oxidative stress [27,65]. The GSH is involved in the disposal of peroxides by brain cells and hence protects from ROS, and that astroglial GSH system is an oxidative stress marker in neurological disorders . GSH is involved in antioxidant reactions in two ways, non-enzymatically can react with ROS like O2- and OH·, hence remove them . The GSH depletion due to toxicity has increased the susceptibility to oxidative stress  and susceptibility of animals to oxidative stress . As shown in Table 1 and Figures 2a and b and 3a and b, GSH levels were significantly decreased (
The GPx family of isoenzymes are catalyzing the reduction of H2O2 and lipid peroxides with GSH [35,67]. There are five selenium-dependent GPxs, but non-selenium GPx contains selenocysteine , several studies were suggested that the GPx upregulation is the protective response in case of neuronal injury , and it exists in both cytosol and mitochondria. The lower GPx activity has associated to higher levels of ROS and cellular damage . GST-specific activity with the substrate CHP is the presence of peroxidase activity that associated with GST . Both peroxidase system and GSH impairment that lead to immune cells dysfunction, and lower survival rate of animal when exposed to a pro-oxidant . As shown in Table 1 and Figures 2a and b, 3a and b, with the substrate H2O2, GPx activity was significantly increased (
Usually, GST that detoxify the various environmental pollutants, carcinogens and endogenous toxic agents (e.g., oxidative stress products) which have electrophilic functional groups, hence produce neutralized, more water-soluble compound, and finally, remove them from the cell . GST that have many functions such as xenobiotic detoxification, removal of oxidative stress products, transport of protein, modulation of cell proliferation, and induction of the apoptosis signaling pathway . GSTs have additional functions such as peroxidase, isomerase, and thiol transferases . It detoxify the wide range of hazardous substances through transferase activity and GST-associated peroxidase activity . GST activity assay has high importance to develop the efficient therapeutics and screening of new anticancer drugs that substrate CDNB is the efficient probe for GST activity . GSTs can protect cells through detoxification from reactive electrophile that reduces cell metabolizing ability . As shown in Table 1 and Figures 2a and b and 3a and b, with the substrate CDNB, GST activity was significantly increased (
AChE is essential for normal functioning of both the central and peripheral nervous system . This enzyme is distributed in both the neural and non-neural tissues . Many studies proved that the drugs can alter the AChE activity of rat brain [82-84]. AChE activity has declined in ACR-treated mice brain . As shown in Table 1 and Figures 2a and b and 3a and b, in this study, we observed the similar activity of AChE as stated by Kopanska
In the present study, ACR-administered rat brain histopathological changes such as degeneration of pyramidal cells, degeneration of glial cells, mild vacuolation of pyramidal cells, spongiosis in glia cells, and spongiosis were observed by 13th day, as shown in Figure 5 as well as necrosis and pyknosis, necrosis of neurons and neurophagia, focal gliosis, and demyelination of nerve fibers were observed by 27th day, as shown in Figure 6 when compared to control, i.e., as shown in Figure 4. Cells such as astrocytes, microglia, neurons, and oligodendrocytes of central nervous system have various functions . Communication disturbances between the brain cells lead to the development of neurodegeneration disorders as well as initiation and progression of neurotoxicity that induced by xenobiotics [86,87]. Jangir
This study suggested that the increase of LPO, depletion of GSH, upregulation and downregulation of NO levels as well as GPx and GST, and dropped AChE activities revealed that ACR (50 mg/300 ml water, up to 27 days in alternative days) shows significant (
I am thankful to University Grants Commission, New Delhi, India (PDFSS-2011-12-SC-AND-3355), for inancial assistance as Postdoctoral Fellow.
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