J BIOCHEM MOLECULAR TOXICOLOGY Volume 28, Number 9, 2014
Effects of Echis pyramidum Snake Venom on Hepatic and Renal Antioxidant Enzymes and Lipid Peroxidation in Rats Abdulrahman K. Al Asmari,1 Haseeb A. Khan,2 Rajamohammed A. Manthiri,1 Khalid M. Al Yahya,3 and Kitab E. Al Otaibi4 1 Research
Center, Prince Sultan Military Medical City, Riyadh, Saudi Arabia; E-mail: [email protected]
2 Department
of Biochemistry, College of Science, King Saud University, Riyadh, Saudi Arabia
3 Department
of Pharmacy, Prince Sultan Military Medical City, Riyadh, Saudi Arabia
4 Medical
Services Department, Prince Sultan Military Medical City, Riyadh, Saudi Arabia
Received 17 March 2014; revised 27 April 2014; accepted 8 May 2014
ABSTRACT: The effects of Echis pyramidum venom (EPV) (0.25, 0.50, and 1.00 mg/kg) on activities of superoxide dismutase (SOD) and catalase (CAT) and levels of thiobarbituric acid reactive substances (TBARS) and total thiols (T-SH) in liver and kidneys of rats were investigated. EPV significantly and dose dependently decreased the activities of SOD and CAT in livers. Although the kidney SOD and CAT activities were not affected by low and medium doses of EPV, the high dose significantly reduced the activities of these enzymes. Liver and kidney TBARS levels were not affected by the low and medium doses of EPV, whereas the high dose significantly increased the TBARS after 6 h postdosing. There was a significant depletion of TSH in liver and kidneys of rats exposed to a high dose of EPV. The acute phase oxidative stress due to an EPV injection points toward the importance of an early antioxidant therapy for the management of snake bites. C 2014 Wiley Periodicals, Inc. J. Biochem. Mol. Toxicol. 28:407–412, 2014; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.21578
KEYWORDS: Echis pyramidum; Snake Venom; Antioxidant Enzymes; Lipid Peroxidation; Rat
INTRODUCTION The genus Echis comprises venomous vipers found in the dry regions of Africa, Middle East, and Indian subcontinent. Three species of Echis, E. pyra-
C
Correspondence to: Abdulrahman Al-Asmari. 2014 Wiley Periodicals, Inc.
midum, E. colorotus, and E. carinatus sochureki, are distributed throughout the Arabian Peninsula, and these snakes have been responsible for most cases of envenomation in this region [1, 2]. The lethality of snake venom is mainly attributed to its highly active enzymatic component, phospholipase A2 (PLA2 ) that hydrolyzes cellular phospholipids, resulting in the release of arachidonic acid. Oxidative metabolism of arachidonic acid generates potentially toxic reactive oxygen species (ROS) including superoxide and hydroxyl free radicals [3, 4]. An imbalance between the excessive generation and poor removal of ROS causes lipid peroxidation leading to cellular damage [5, 6]. PLA2 from snake venom has been implicated in multiple pathologies including hepatotoxicity [7] and nephrotoxicity [8]. Cells are equipped with natural antioxidant defense to counteract potentially toxic ROS. The enzymes superoxide dismutase (SOD) and catalase (CAT) are important components of the cellular antioxidant machinery. These enzymes work in tandem to neutralize superoxide radical. SOD catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide (H2 O2 ). Hydrogen peroxide is itself a harmful by-product and must be quickly detoxified into nontoxic substances. The enzyme CAT catalyzes the decomposition of H2 O2 into water and oxygen. It has been shown earlier that E. pyramidum venom (EPV) produces significant oxidative stress in different organs of mice, suggesting an important role of lipid peroxidation in cytotoxicity of EPV [9]. However, the effect of EPV on tissue antioxidant enzymes was never studied. We therefore investigated the effects of EPV on SOD and CAT activities in liver and kidneys of rats. In addition, we also analyzed nonenzymatic biomarkers of oxidative stress in these organs. 407
408
AL ASMARI ET AL.
MATERIALS AND METHODS Venom Collection E. pyramidum snakes were collected from the wild locations by professional hunters and kept in a serpentarium. The venom was collected from these snakes and was filtered, lyophilized, and stored at 4°C. A stock solution of EPV (10 mg/ml) was prepared in sterile saline and used for this study.
Animals and Dosing Adult male Sprague Dawley rats weighing 200– 220 g were kept in polycarbonate cages with sawdust bedding and steel mesh cover. They were maintained at 23 ± 1°C with 12-h light–dark cycles and free access to standard laboratory food and tap water. The experimental protocol was approved by the Institutional Research and Ethics Committee of the University. The animals were randomly divided into four groups of six animals in each group. One of these groups served as control and received intraperitoneal (IP) injection of saline. The animals in the remaining three groups were treated with EPV in the doses of 0.25, 0.50, and 1.00 mg/kg bodyweight, respectively. The rats were sacrificed after 6 h, and specimens of livers and kidneys were collected for the analysis of antioxidant enzymes (SOD and CAT), thiobarbituric acid reactive substances (TBARS), and total thiols (T-SH).
Analysis of SOD Activity The activity of SOD in liver and kidney tissues was measured by the method of Marklund and Marklund [10]. The procedure is based on auto-oxidation of pyrogallol in an aqueous solution and its inhibition by SOD; the extent of superoxide radicals is directly proportional to the concentration of SOD in the reaction mixture. An aliquot (50 μL) of clear supernatant from the tissue homogenate was mixed with 850 μL of 50 mM Tris–buffer (pH 8.2) containing 1.0 mM ethylenediaminetetraacetic acid (EDTA). The reaction was started by the addition of 100 μL of 0.8 mM pyrogallol solution, and the change in absorbance was recorded for 3 min (linear phase) at 420 nm. One unit of SOD activity was defined as the amount of SOD required for the inhibition of pyrogallol autoxidation by 50% per min.
Analysis of CAT Activity This enzyme activity was measured according to the method of Clairborne [11]. This procedure is based
Volume 28, Number 9, 2014
on decomposition of H2 O2 to water and oxygen in the presence of CAT. An aliquot (50 μL) of clear supernatant from the tissue homogenate was mixed with 1.95 mL of 50 mM potassium phosphate buffer (pH 7.0) and 1.0 mL of 20 mM H2 O2 . The change in the absorbance at 240 nm was recorded immediately and after every 30 s for 3 min. CAT activity was determined using the rate of decomposition of H2 O2 , which is proportional to the reduction of the absorbance at 240 nm. One unit of CAT activity was defined as the amount of CAT decomposing 1.0 μM H2 O2 per min and was calculated using the molar extinction coefficient of H2 O2 (43.6 M−1 cm−1 at 240 nm).
Determination of TBARS The levels of TBARS, a marker of lipid peroxidation [12–14], were estimated in liver and kidney tissues using the method of Nichans and Samuelson [15]. Tissues were homogenized in Tris–HCl buffer (pH 7.5) followed by centrifugation to remove the cellular debris. An aliquot (100 μL) of supernatant from the tissue homogenate was mixed with 2 mL of TBA–TCA–HCl (1:1:1) reagent (0.37% TBA, 15% TCA, and 0.25 N HCl), and the tubes were placed in a water bath at 90°C for 10 min. The tubes were allowed to cool at room temperature, and then the absorbance of the colored solution was measured against a reagent blank at 535 nm.
Determination of T-SH The levels of T-SH, a marker of antioxidant capacity [16, 17], were determined in liver and kidney tissues by the method of Sedlak and Lindsay [18]. An aliquot (0.4 mL) of supernatant from the tissue homogenate was mixed with 2.1 mL of 0.1 M Tris–HCl (pH 8.2), 0.5 mL of 10% SDS, and 0.3 mL of 0.1 M EDTA, and the tubes were incubated in a boiling water bath for 5 min. The mixture was cooled down to room temperature. Then, 0.1 mL 5,5-dithiobis-2-nitrobenzoic acid (40 mg/100 mL methanol) was added, and the absorbance was recorded at 412 nm. A calibration curve was constructed using different amounts of cysteine (20–160 nM) to calculate T-SH levels in the samples.
Statistical Analysis The data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. Pearson’s test was used for correlation studies. A P value of
Effects of Echis pyramidum Snake Venom on Hepatic and Renal Antioxidant Enzymes and Lipid Peroxidation in Rats Abdulrahman K. Al Asmari,1 Haseeb A. Khan,2 Rajamohammed A. Manthiri,1 Khalid M. Al Yahya,3 and Kitab E. Al Otaibi4 1 Research
Center, Prince Sultan Military Medical City, Riyadh, Saudi Arabia; E-mail: [email protected]
2 Department
of Biochemistry, College of Science, King Saud University, Riyadh, Saudi Arabia
3 Department
of Pharmacy, Prince Sultan Military Medical City, Riyadh, Saudi Arabia
4 Medical
Services Department, Prince Sultan Military Medical City, Riyadh, Saudi Arabia
Received 17 March 2014; revised 27 April 2014; accepted 8 May 2014
ABSTRACT: The effects of Echis pyramidum venom (EPV) (0.25, 0.50, and 1.00 mg/kg) on activities of superoxide dismutase (SOD) and catalase (CAT) and levels of thiobarbituric acid reactive substances (TBARS) and total thiols (T-SH) in liver and kidneys of rats were investigated. EPV significantly and dose dependently decreased the activities of SOD and CAT in livers. Although the kidney SOD and CAT activities were not affected by low and medium doses of EPV, the high dose significantly reduced the activities of these enzymes. Liver and kidney TBARS levels were not affected by the low and medium doses of EPV, whereas the high dose significantly increased the TBARS after 6 h postdosing. There was a significant depletion of TSH in liver and kidneys of rats exposed to a high dose of EPV. The acute phase oxidative stress due to an EPV injection points toward the importance of an early antioxidant therapy for the management of snake bites. C 2014 Wiley Periodicals, Inc. J. Biochem. Mol. Toxicol. 28:407–412, 2014; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.21578
KEYWORDS: Echis pyramidum; Snake Venom; Antioxidant Enzymes; Lipid Peroxidation; Rat
INTRODUCTION The genus Echis comprises venomous vipers found in the dry regions of Africa, Middle East, and Indian subcontinent. Three species of Echis, E. pyra-
C
Correspondence to: Abdulrahman Al-Asmari. 2014 Wiley Periodicals, Inc.
midum, E. colorotus, and E. carinatus sochureki, are distributed throughout the Arabian Peninsula, and these snakes have been responsible for most cases of envenomation in this region [1, 2]. The lethality of snake venom is mainly attributed to its highly active enzymatic component, phospholipase A2 (PLA2 ) that hydrolyzes cellular phospholipids, resulting in the release of arachidonic acid. Oxidative metabolism of arachidonic acid generates potentially toxic reactive oxygen species (ROS) including superoxide and hydroxyl free radicals [3, 4]. An imbalance between the excessive generation and poor removal of ROS causes lipid peroxidation leading to cellular damage [5, 6]. PLA2 from snake venom has been implicated in multiple pathologies including hepatotoxicity [7] and nephrotoxicity [8]. Cells are equipped with natural antioxidant defense to counteract potentially toxic ROS. The enzymes superoxide dismutase (SOD) and catalase (CAT) are important components of the cellular antioxidant machinery. These enzymes work in tandem to neutralize superoxide radical. SOD catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide (H2 O2 ). Hydrogen peroxide is itself a harmful by-product and must be quickly detoxified into nontoxic substances. The enzyme CAT catalyzes the decomposition of H2 O2 into water and oxygen. It has been shown earlier that E. pyramidum venom (EPV) produces significant oxidative stress in different organs of mice, suggesting an important role of lipid peroxidation in cytotoxicity of EPV [9]. However, the effect of EPV on tissue antioxidant enzymes was never studied. We therefore investigated the effects of EPV on SOD and CAT activities in liver and kidneys of rats. In addition, we also analyzed nonenzymatic biomarkers of oxidative stress in these organs. 407
408
AL ASMARI ET AL.
MATERIALS AND METHODS Venom Collection E. pyramidum snakes were collected from the wild locations by professional hunters and kept in a serpentarium. The venom was collected from these snakes and was filtered, lyophilized, and stored at 4°C. A stock solution of EPV (10 mg/ml) was prepared in sterile saline and used for this study.
Animals and Dosing Adult male Sprague Dawley rats weighing 200– 220 g were kept in polycarbonate cages with sawdust bedding and steel mesh cover. They were maintained at 23 ± 1°C with 12-h light–dark cycles and free access to standard laboratory food and tap water. The experimental protocol was approved by the Institutional Research and Ethics Committee of the University. The animals were randomly divided into four groups of six animals in each group. One of these groups served as control and received intraperitoneal (IP) injection of saline. The animals in the remaining three groups were treated with EPV in the doses of 0.25, 0.50, and 1.00 mg/kg bodyweight, respectively. The rats were sacrificed after 6 h, and specimens of livers and kidneys were collected for the analysis of antioxidant enzymes (SOD and CAT), thiobarbituric acid reactive substances (TBARS), and total thiols (T-SH).
Analysis of SOD Activity The activity of SOD in liver and kidney tissues was measured by the method of Marklund and Marklund [10]. The procedure is based on auto-oxidation of pyrogallol in an aqueous solution and its inhibition by SOD; the extent of superoxide radicals is directly proportional to the concentration of SOD in the reaction mixture. An aliquot (50 μL) of clear supernatant from the tissue homogenate was mixed with 850 μL of 50 mM Tris–buffer (pH 8.2) containing 1.0 mM ethylenediaminetetraacetic acid (EDTA). The reaction was started by the addition of 100 μL of 0.8 mM pyrogallol solution, and the change in absorbance was recorded for 3 min (linear phase) at 420 nm. One unit of SOD activity was defined as the amount of SOD required for the inhibition of pyrogallol autoxidation by 50% per min.
Analysis of CAT Activity This enzyme activity was measured according to the method of Clairborne [11]. This procedure is based
Volume 28, Number 9, 2014
on decomposition of H2 O2 to water and oxygen in the presence of CAT. An aliquot (50 μL) of clear supernatant from the tissue homogenate was mixed with 1.95 mL of 50 mM potassium phosphate buffer (pH 7.0) and 1.0 mL of 20 mM H2 O2 . The change in the absorbance at 240 nm was recorded immediately and after every 30 s for 3 min. CAT activity was determined using the rate of decomposition of H2 O2 , which is proportional to the reduction of the absorbance at 240 nm. One unit of CAT activity was defined as the amount of CAT decomposing 1.0 μM H2 O2 per min and was calculated using the molar extinction coefficient of H2 O2 (43.6 M−1 cm−1 at 240 nm).
Determination of TBARS The levels of TBARS, a marker of lipid peroxidation [12–14], were estimated in liver and kidney tissues using the method of Nichans and Samuelson [15]. Tissues were homogenized in Tris–HCl buffer (pH 7.5) followed by centrifugation to remove the cellular debris. An aliquot (100 μL) of supernatant from the tissue homogenate was mixed with 2 mL of TBA–TCA–HCl (1:1:1) reagent (0.37% TBA, 15% TCA, and 0.25 N HCl), and the tubes were placed in a water bath at 90°C for 10 min. The tubes were allowed to cool at room temperature, and then the absorbance of the colored solution was measured against a reagent blank at 535 nm.
Determination of T-SH The levels of T-SH, a marker of antioxidant capacity [16, 17], were determined in liver and kidney tissues by the method of Sedlak and Lindsay [18]. An aliquot (0.4 mL) of supernatant from the tissue homogenate was mixed with 2.1 mL of 0.1 M Tris–HCl (pH 8.2), 0.5 mL of 10% SDS, and 0.3 mL of 0.1 M EDTA, and the tubes were incubated in a boiling water bath for 5 min. The mixture was cooled down to room temperature. Then, 0.1 mL 5,5-dithiobis-2-nitrobenzoic acid (40 mg/100 mL methanol) was added, and the absorbance was recorded at 412 nm. A calibration curve was constructed using different amounts of cysteine (20–160 nM) to calculate T-SH levels in the samples.
Statistical Analysis The data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. Pearson’s test was used for correlation studies. A P value of
