Role of free radicals and apoptosis in arsenic toxicity in Wistar rats Singh Bandana1, Singh N.D.1,*, Devi L. Geeta1, Banga H.S.1, Mahajan V.1 1Department of Veterinary Pathology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141004, Punjab, India *Corresponding author: drndsingh@gmail.com
Abstract The present study was conducted to study the role of oxidative stress and apoptosis in induced arsenic toxicity, when given at the same level at which it is found in drinking water in different parts of India. Young male and female Wistar rats were randomly divided in four groups viz. I, II, III, and IV and were given sodium arsenite at a dose rate of 0.01mg/l, 2.5mg/l, 5mg/l and distilled water, respectively. The animals were fed arsenic for a period of 15 weeks. The tissue oxidative stress-related biochemical parameters in testes, kidneys, livers, ovary and brains, nitrotyrosine staining and apoptosis changes were recorded in male and female rats. The level of lipid peroxidation was increased and superoxide dismutase and catalase levels were decreased significantly in a dose dependent manner. The nitrotyrosine and caspase 3 immunostaining was done to assess oxidative stress and apoptosis, respectively. It was concluded that even in very minute quantity arsenic generates free radicals and induces apoptosis which may cause damage to vital organs of the body. Top Keywords Apoptosis, Arsenic, Nitrotyrosine, Oxidative stress, Wistar rats. Top |
INTRODUCTION Arsenic is metalloids which are cytotoxic and carcinogenic and are widely circulated in nature and primarily transported in the environment by water. In India, the use of tube wells for water supply in gangetic delta has led to arsenic poisoning in large number of animals and humans. The ground water of different states of India has toxic level of arsenic1,2. Arsenic concentration exceeds 0.05 mg/l in ground water in Bihar and as high as 34 mg/l in tube well water in West Bengal. Further, arsenic poisoning is one of the most important causes of heavy metal poisoning in canines and bovines3. Exposure of arsenic in human is associated with cancers, injury to organ and toxicity. Arsenic can cause oxidative injury by reducing anti-oxidative capacity4. Arsenic toxicity can damage the mitochondrial functions and morphology leading to generation of free radicals such as hydroxyl radicals (−OH), superoxide anion through chain reactions and causes severe oxidative damage to the cells5. |
The present study was conducted to assess the arsenic induced oxidative stress and apoptosis in a rat model, when given at the same level at which it is found in drinking water in different parts of India. |
Top MATERIALS AND METHODS Experimental animals Young male (40) and female (52) Wistar rats (80–90 g) were used in the study. Chemicals Sodium arsenite was obtained from LOBA CHEMIE Pvt. Ltd. Mumbai. All other reagents used were analytical-grade laboratory chemicals from standard commercial suppliers. Experimental design and dosage The protocol was as per Organization for Economic Co-operation and Development (OECD-421) guidelines. All protocols were performed in accordance with the guidelines of the Animal Care Ethics Committee of the University. Experiments were conducted in the Department of Veterinary Pathology, GADVASU. Young male (40) and female Wistar rats (52) (80–90 g) were used in the study. The rats were kept for 7 days to acclimatize in laboratory condition prior to start of experiment protocol. On day 8, the rats were divided randomly into four groupsviz, Group I (Sodium arsenite @0.01mg/litre of drinking water), Group II (Sodium arsenite @2.5mg/litre of drinking water), Gropu III (Sodiumarsenite @5mg/litre of drinking water) and Group IV (Control group/distilled water) so as to have equal weight and were kept in separate cages in a 12 hr light and 12 hr dark cycles. The entire male (n=10 in each groups) and female Wistar rats (n=13) of four experimental groups were given treatment of sodium arsenite in drinking water for 15 weeks. The dosage of arsenic in group I was the maximum permissible dose as per WHO, Group II was 1/20th of LD50 of arsenic while Group III was 1/10th of LD50 of arsenic Oxidative stress related biochemical parameters Estimations of different oxidative stress-related biochemical parameters viz., lipid peroxidation (LPO), catalase and superoxide dismutase (SOD)in testes, kidneys, livers and brains of male and female rats were carried out. Briefly, fresh tissue samples (10 mg) were homogenised in 1ml of ice-cold phosphate buffered saline (pH 7.4), using a tissue homogenizer with a teflon pestle at 4°C. The resultant tissue homogenate was used for measurements of Total protein, Lipid peroxidation (LPO), catalase and Superoxide dismutase (SOD) activity. LPO was determined in terms of MDA (malondialdehyde) production, by the thiobarbituric acid (TBA) method as described by Shafiq-ur-Rehman6 Activities of catalase enzymes were estimated as described by7,8. SOD activities were estimated as per the method described by Madesh and Balasubramanian9. Assessment of oxidative stress and apoptosis by Immunohistochemistry (IHC) For assessment of oxidative stress, detection of nitrotyrosine and for apoptosis, detection of caspase 3 was performed by using immunohistochemistry as described earlier10. Briefly 5-im paraffin sections on poly- L-lysine coated slides were rehydrated. After heat induced antigen retrieval and after endogenous peroxidase bocking, the slides were incubated with primary antibody anti mouse nitrotyrosin (abcam, UK) at 1:1000 dilutions and rabbit polyclonal caspase-3 (abcam, UK) antibody (1:200) overnight at 4°C in a humidified chamber. This was followed by incubation with secondary antibody (ABC, Universal, Vector) and colour developed using diaminobenzidine (DAB) substrate (Vector, Immpact peroxidase substrate kit) and counterstained with Gill‘s hematoxylin. In negative control, tissue section was processed without application of primary antibody. Top RESULTS Arsenic induced oxidative stress in different organ Increased level of lipid peroxidation (LPO) in arsenic group MDA levels in various organs for males and females are given in table 1 and table 2, respectively. Liver and kidney malondialdehyde (MDA) levels in males and females of group III and II was significantly higher as compared to control group. The values of MDA in brain of males and females of group III was significantly higher than control group. Significant difference was seen in MDA level of group III and control testis tissue homogenates. However, MDA levels in ovary homogenates of all the treated groups did not differ significantly. The MDA level in group I and control group did not differ significantly for any of the organ. Decreased level of Superoxide dismutase (SOD) in arsenic group SOD levels in various organs for males and females are given in table 3 and table 4, respectively. SOD activities in liver and kidney of males and females of group III and II were significantly lower as compared to control group. Brain showed low SOD value in group III as compared to that of control group. SOD level in testis of group III was lower than control group. However SOD value for ovary was comparable in group I, group II, group III and group IV. Decreased level of catalase in arsenic group Catalase levels in various organs for males and females are given in table 5 and table 6, respectively. Liver and kidney catalase level of males and females was significantly lower in group III and II as compared to control. The catalase activity in group I was comparable with control. The catalase value for brain of males and females of group III was significantly lower with respect to control group. Catalase level of testis and ovary of group III was significantly lower than control group. Increased expression of nitrotyrosinin arsenic group Liver, kidney and testes in group III showed a general distribution of nitrotyrosin. Group I (Fig. 1, 3) showed only 1–2 cells per field and control group showed no nitrotyrosin staining. There were 4–5 cells per field in group II and 10–12 cells per field in group III (Fig. 2, 4, 5). Group II showed less nitrotyrosin staining as compared to group III. Arsenic induced apoptosis in different organs Arsenic induced caspase-3 mediated apoptosis in kidney, liver, brain and testes was observed in different groups. The activity of caspase-3 in these organs in sodium arsenite treated groups was significantly higher in a dose dependent manner. In group III (Fig. 7, 9, 10) and II (Fig. 6), 10–12 and 5–6 immunoreactive(IR) cells per field respectively were observed in liver, kidney and testes. In these groups, many types of cells like hepatocytes in liver, epithelial cells of PCT in kidney, spermatogonial cells in testes showed positive staining for caspase-3. However, in group I (Fig. 8) IR cells (1–2 cells per field) were very less in liver and kidney as compared to group III and II. In group IV, no apoptotic cells were evident in liver, kidney and testes. Top DISCUSSION In the present study, rats exposed to sodium arsenite at three different doses showed oxidative stress as indicated by a dose dependent increased in LPO and decreased in catalase and SOD. Lipid peroxidation is a basic cellular deteriorating process induced by oxidative stress and occurs readily in the tissues rich in highly oxidizable polyunsaturated fatty acids11. As the free radicals count was increased due to arsenic toxicity, the more and more lipids undergo per oxidation, increasing the level of lipid peroxides like MDA in various organs. Lipid peroxide level in liver and kidney from only arsenic exposed group was significantly higher than the respective control values12.It was reported that lipid peroxidation is one of the characteristic features of augmented oxidative stress associated with arsenic toxicity13. SOD and catalase are the two most important radical scavenging enzymes and body‘s secondary defense against oxygen metabolites produced due to transitional heavy metals14. As the concentration of free radicals was increased, the concentrations of these two enzymes were reduced. Arsenic treatment nonsignificantly suppressed SOD activity in liver and kidney and catalase activity in liver, but catalase activity in kidney decreased significantly indicating damaging effects of arsenic on endogenous antioxidant enzyme in kidney14. The decrease in activity of SOD and Catalase with increase in LPO activity in the present experiment was in consonance with the findings of various workers15,16,17,18,19,20-21.The decrease in levels of SOD and catalase might be due to enhanced superoxide production during arsenic metabolism.Insufficient supply of NADH during arsenic metabolism might decrease the activity of catalase22.Arsenic induced formation of reactive oxygen species and depletion of antioxidant cell defences which can result in disruption of the pro-oxidant/antioxidant balance in mammalian tissues23,24. |
Exposure to arsenic might be stimulating the generation of reactive oxygen species (ROS) such as superoxide anion, hydroxyl radical (OH”), hydrogen peroxides (H2O2) and peroxynitrite (ONOO') in a variety of cells which might lead to damage to essential organs25,26,27,28,29-30. Oxidative stress increases the production of superoxide (O2) and NO forming peroxynitrite (ONOO). The production of ONOO” is further capable of oxidizing several lipoproteins and of nitrating tyrosine residues in many proteins leading to formation of nitrotyrosin. Thus, the presence of nitrotyrosine on proteins can be used as a marker for peroxynitrite formation. In the present study, there was increased expression of nitrotyrosine in various organs of arsenic exposed rats. Anti-nitrotyrosine immunostaining is a very helpful tool to find the extent of damage to organs due to oxidative stress31. |
In the present study there were increased apoptosis in various organs of arsenic exposed animals. Apoptosis can be initiated through intrinsic or extrinsic pathway. In the intrinsic pathway the cell kills itself because it senses cell stress, while in the extrinsic pathway the cell kills itself because of signals from other cells. Both pathways induce cell death by activating caspase. Caspase-3 is the key mediators of apoptosisnecessary for proteins cleavage and for apoptosis-associated margination of chromatin, DNA fragmentation, and nuclear collapse during apoptosis. The detection of activated caspase-3 was regarded as a valuable and specific tool for detecting apoptotic cells in tissue sections, even before all the morphological features of apoptosis occur32. In the present study, immunolocalisation of caspase-3 showed nuclear staining and mild intracytoplasmic. Nuclear accumulation of active caspase-3 during apoptosis has been observed and further showed caspase-3 translocated from the cytoplasm into the nucleus after induction of apoptosis33.The more positive staining for caspase-3 in islets from type 1 diabetics may correspond to accelerated apoptosis cascade in the islets before processing to eventual cell death34. The arsenic might be causing damage to mitochondrial wall by free radical injury which leads to release of pro-apoptotic particles. Similar results have also been reported by various workers35-36. IHC for the activated caspase-3 is a reliable technique for the early identification and quantification of apoptotic cells in routine histological sections. |
The present study showed alteration in oxidative stress markers and a caspase-3 related apoptosis in arsenic exposed rats. Thus, this indicates the role of oxidative stress and apoptosis in arsenic toxicity. This finding may provide an additional knowledge on the mechanism of toxicity of arsenic. |
Top Conflict of interest The authors do not have any conflict of interest. |
Top Figures | Fig. 1.: Hepatocytes in liver section showing presence of nitrotyrosin. 1–2 immunoreactive cells were observed per field (arrow). IHC *1000 (Group I)
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| | Fig. 2.: Hepatocytes in liver section showing intense increase in nitrotyrosin. 10–12 immunoreactive cells were observed per field (arrow). IHC *1000 (Group III)
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| | Fig. 3.: Epithelial cells of PCT/DCT in kidney giving positive signal for increased nitrotyrosin. 1–2 immuno reactive cells per field were observed. IHC *1000 (Group I)
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| | Fig. 4.: Epithelial cells of PCT/DCT in kidney giving positive signal for increased nitrotyrosin. 10–12 immuno reactive cells per field were observed. IHC *1000 (Group III)
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| | Fig. 5.: Spermatogonial cells in section of testes giving positive signal for presence of nitrotyrosin. 10–12 immuno reactive cells per fieldwereobserved.IHC *1000 (Group III)
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| | Fig. 6.: Hepatocytes in liver section showing positive signal for caspase 3 activity. 5–6 immunoreactive cells were observed per field (arrow). IHC *1000 (Group II).
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| | Fig. 7.: Hepatocytes in liver section showing positive signal for caspase 3 activity 10–12 immunoreactive cells were observed per field (arrow). IHC *1000 (Group III).
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| | Fig. 8.: Epithelial cells of PCT/DCT in kidney giving signal for increased caspase 3 activity 1–2 immuno reactive cells per field were observed. IHC *1000 (Group I)
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| | Fig. 9.: Epithelial cells of PCT/DCT in kidney giving signal for increased caspase 3 activity. 10–12 immuno reactive cells per field were observed. IHC *1000 (Group III)
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| | Fig. 10.: Spermatogonial cells in section of testes giving signal for presence of caspase 3. 10–12 immuno reactive cells per field were observed. IHC *1000 (Group III).
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Tables | Table 1.: Malon-dialdehyde levels (nM MDA/g) in different organs of males in different groups fed with Sodium arsenite.
| | Group | Liver | Kidney | Testes | Brain | | I | 30.25±1.99c | 31.12±1.23c | 27.13±1.78b | 26.08±1.34b | | II | 37.65±1.43b | 36.65±2.01b | 31.34±1.09b | 26.15±1.7b | | III | 44.11±1.54a | 48.97±1.34a | 39.32±1.06a | 32.76±0.98a | | IV | 28.70±1.23c | 27.87±1.77c | 29.15±1.20b | 25.34±1.23b |
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| *The values (Mean±S.E.) in a column having different superscript differ significantly from each other at 5% level of significance. | | | Table 2.: Malon-dialdehydelevels (nM MDA/g) in different organs of females in different groups fed with Sodium arsenite.
| | Group | Liver | Kidney | Ovary | Brain | | I | 32.25±0.87c | 32.82±1.03c | 32.11±0.88a | 25.07±1.04b | | II | 38.35±0.92b | 36.65±1.00b | 30.04±0.99a | 27.66±1.2b | | III | 43.11±1.04a | 44.57±0.87a | 29.82±1.07a | 30.54±1.02a | | IV | 30.10±0.83c | 31.15±1.07c | 31.65±0.97a | 26.11±1.43b |
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| * The values (Mean±S.E.) in a column having different superscript differ significantly from each other at 5% level of significance. | | | Table 3.: Superoxide dismutase (SOD) levels (U) in different organs of males in different groups fed with Sodium arsenite.
| | Group | Liver | Kidney | Testes | Brain | | I | 16.78±1.06a | 17.32±0.55a | 16.98±0.86a | 16.34±0.99b | | II | 13.87±1.35b | 12.13±0.63b | 15.18±0.88a | 14.33±1.02b | | III | 8.93±0.66c | 8.01±1.05c | 10.45±0.79b | 9.23±0.67b | | IV | 18.65±0.76a | 17.86±1.04a | 17.54±1.02a | 16.00±0.66a |
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| * The values (Mean±S.E.) in a column having different superscript differ significantly from each other at 5% level of significance. | | | Table 4.: Superoxide dismutase (SOD) levels (U) in different organs of females in different groups fed with Sodium arsenite.
| | Group | Liver | Kidney | Ovary | Brain | | I | 16.23±1.12a | 15.78±0.87ab | 16.33±1.09a | 15.34±1.02a | | II | 13.45±1.09b | 14.12±1.06b | 15.76±1.23a | 14.35±1.04a | | III | 9.98±2.01c | 9.05±1.03c | 14.67±0.89a | 9.45±0.57b | | IV | 16.87±0.77a | 17.88±0.54a | 17.54±0.93a | 16.98±0.76a |
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| *The values (Mean±S.E.) in a column having different superscript differ significantly from each other at 5% level of significance. | | | Table 5.: Catalase activity (KA/g) in different organs of males in different groups fed with Sodium arsenite.
| | Group | Liver | Kidney | Testes | Brain | | I | 191.12±7.60a | 188.71±5.30a | 175.45±4.69a | 182.76±5.33a | | II | 141.23±4.52b | 145.56±2.68b | 172.43±3.67a | 176.47±5.67a | | III | 103.55±4.63c | 123.06±4.41c | 140.45±7.89b | 140.23±4.34b | | IV | 195.09±5.03a | 190.23±5.41a | 174.91±4.98a | 190.89±2.87a |
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| *The values (Mean ± S.E.) in a column having different superscript differ significantly from each other at 5% level of significance. | | | Table 6.: Catalase activity (KA/g) in different organs of females in different groups fed with Sodium arsenite.
| | Group | Liver | Kidney | Ovary | Brain | | I | 178.12±8.80a | 171.45±6.29a | 176.34±3.09a | 169.09±3.89a | | II | 151.23±6.70b | 140.56±3.48b | 169.13±7.87a | 168.33±2.98a | | III | 133.55±4.63c | 120.71±6.39c | 151.99±8.00b | 145.43±4.37b | | IV | 183.09±5.03a | 170.23±9.56a | 178.19±4.32a | 171.32±5.22a |
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| *The values (Mean ± S.E.) in a column having different superscript differ significantly from each other at 5% level of significance. | |
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