Oxidative stress markers in relation to acute sodium arsenate (Na3AsO4) exposure in buffalo red blood cells in vitro

Introduction

Contamination of arsenic in the environment can be due to its natural existence and manmade industrial activities (Dash et al. 2016) [3] . Arsenic contaminates the water, air, feed and soil globally (Rana et al. 2010) [11] . Buffalo health is under threat due to arsenic exposure as well as human health due to consumption of contaminated milk. Acute arsenic contamination causes imbalances in oxidant-antioxidant system leading to different health issues in buffaloes (Flora et al. 2007) [6] . Red blood cells (RBC) have high oxygen carrying capacity. Hence, different biomolecules in RBCs are vulnerable to oxidative damage. Documented studies on sodium arsenate induced changes in oxidant-antioxidant status in buffalo RBCs is scanty. Keeping in view the above facts, changes in oxidant-antioxidant markers were studied in buffalo erythrocytes exposed to sodium arsenate in vitro.

Materials and Methods

Preparation of erythrocytes

Female healthy buffalo red blood cells were obtained from Innovative Research Inc. biotechnology company. Erythrocytes were washed thrice with phosphate-buffered saline (PBS), pH 7.4 and resuspended in PBS. Counting of erythrocytes was conducted using hemocytometer to adjust cell number to 2×10 6 cells/ml.

Exposure of Erythrocytes to sodium arsenate:

Erythrocytes (2×10 6 cells/ml) were incubated with PBS (Control) or sodium arsenate (0.01 µg to 0.05 µg) for 2 hrs (acute exposure) at 37 °C as follows:

Control group: Erythrocyte suspension incubated with PBS.

Treatment groups: Consisted of Group T 1 to T 5 in which erythrocyte suspensions were incubated with 0.01, 0.02, 0.03, 0.04 and 0.05 µg Na 3 AsO 4 respectively for 2 hrs.

[Maximum Permissible Limit of arsenic in drinking water for animal health is 0.01 µg/ml (WHO)].

Estimation of Oxidative stress Markers:

Concentration of malondialdehyde, was estimated by the method of Placer et al. 1966 [9] . Assay of superoxide dismutase was carried out as per the method of Nishikimi et al. 1972 [8] . Glutathione peroxidase assay was carried out as per the method of Flohe and Gunzler 1984 [5] . Estimation of catalase activity was conducted as per the method of Aebi 1984 [1] . UV/VIS spectrophotometer was used for all estimations.

Statistical analysis

Independent t-test was used for the analysis of data.

Results and Discussion

Buffalo RBCs challenged with sodium arsenate (0.01-0.5 µg for 2 hrs.) showed enhanced (p<0.05) generation of malondialdehyde, in a concentration dependent manner. Peak level of malondialdehyde production was at 0.5 µg sodium arsenate concentration. Increased antioxidant enzyme response e.g. superoxide dismutase (T 1 and T 2 ), glutathione peroxidase (in group T 1 ) and catalase (T 1 ) was noticed during initial concentrations of sodium arsenate exposure compared to the corresponding controls. However, the activities of superoxide dismutase (T 3 -T 5 ), catalase (T 2 -T 5 ) and glutathione peroxidase (T 2 -T 5 ) significantly (p<0.05) decreased in a concentration dependent manner with increasing dose of sodium arsenate treatment to buffalo RBCs. (Tab. 1).

Acute Na 3 AsO 4 exposure increased the lipid peroxidation and declined the level of antioxidant enzymes e.g. superoxide dismutase, glutathione peroxidase and catalase in buffalo erythrocytes as observed in this study. This creates the oxidative stress environment inside the RBCs. Different arsenic compounds disturbs the redox homeostasis inside the cell producing oxidative stress (Flora et al. 2007) [6] . Na 3 AsO 4 can generate reactive oxygen species (ROS) causing oxidative stress and apoptosis in various animal cells (Lynn et al. 2000, Felix et al. 2005) [7,4] . Exposure to arsenic at very low concentration for short duration also produces superoxide anion radical, singlet oxygen, hydroxyl radical, hydrogen peroxide etc. in various animal cells (Qian et al. 2003, Bashir et al. 2006; Flora et al. 2007) [10,2,6] causing lipid peroxidation, membrane damage and cell death. The results of this study are in line with above reports. Cellular antioxidants modulate the oxidative stress response in the cells. Elimination of ROS was carried out by the combined action of superoxide dismutase and catalase. Superoxide radicals were converted to hydrogen peroxide and molecular oxygen by superoxide dismutase. Catalase converts hydrogen peroxide to water. In this study, decline in activities of superoxide dismutase and catalase in Na 3 AsO 4 exposed buffalo erythrocytes can attributed to increased ROS production by arsenic or decrease action and synthesis of antioxidant enzymes due to toxic insult (Flora et al. 2007, Sinha et al. 2008) [6,12] .

Conclusion

Short duration exposure of sodium arsenate to buffalo RBCs creates oxidative stress as observed from the enhanced cellular lipid peroxidation and decreased enzymatic antioxidants.

References

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