Research Article
Pharmacological Changes after Black Scorpion Heterometrus fastigiousus Couzijn Envenomation in Mice
Department of Zoology, Mahatma Gandhi Post Graduate College, 273009 Gorakhpur, Uttar Pradesh, India
LiveDNA: 91.16671
Scorpion sting is a major health problem for poor communities in tropical and subtropical countries. About 1750 scorpion species reported throughout the world, only 25 scorpion species have been reported for their lethality in human1,2. The signs and symptoms of scorpion sting depend on scorpion species, its venom composition, age of the scorpion and the victims physiological response3. Scorpion sting may induce severe local skin reactions, neurological, respiratory and cardiovascular collapse. Buthidae is the largest scorpion family and includes most toxic scorpion species4-7. Being rich source of various polypeptides, scorpion venoms have diverse pharmacological and physiological activities. These venoms exert their effects by targeting ion channels8.
Scorpions belonging to genus Heterometrus (Family: Scorpionidae) are the largest scorpions living in Southeast Asian regions and are responsible for the most of the accidental stings after their equivalents of family Buthidae. The effect of this scorpion venom and mechanism by which envenomation exerts its effects has not been clearly known. However, different scientific groups have reported effects of some black scorpion species venoms9-15. Prolong and sharp burning sensation occurs around the site of sting by H. scaber9. Palamneus gravimanus envenomation results in localized irritation, edema and itching10. The H. fulvipes venom induces hemotoxicity and inhibits acetylcholine esterase activity11. The antigenic protein (Hb) of H. bengalensis venom produces irreversible nerve blockage12. H. longimanus and H. spinifer venom produce contractile responses in rat anococcygeus muscle13. Black scorpion venoms contain high concentration of acetylcholine and noradrenaline and cause reversible contraction of chick biventer cervicis muscle by cholineregic and adrenergic actions14. An increase in glucose, creatinine, blood urea nitrogen, alanine aminotransferase, creatine phosphokinase and lactic dehydrogenase and decrease in total protein, uric acid, cholesterol, calcium and phosphate in serum of albino mice was observed in experimental Palamneus gravimanus envenomation15. The present investigation, the effect of black scorpion H. fastigiousus venom on biochemical and enzymatic parameters in serum of albino mice after experimental envenomation have been studied to understand the mode of action of envenomation.
Isolation of H. fastigiousus venom: Living H. fastigiousus scorpions were purchased from Eastern Scientific Emporium, Gorakhpur, UP, India. Their venom was obtained by electric stimulation of the telson, dissolved in phosphate buffer (50 mM, pH 7.2) and centrifuged (MP01, Tarson Co., India) at 3,000 rpm and 4°C for 5 min. The supernatant was collected, lyophilized and stored at -4°C until use. The protein content in venom was estimated by Lowry et al.16 method. This study was done during January-April, 2006.
Toxicity determination: The toxicity of H. fastigiousus venom was determined in male albino mice weighing 25±5 g5. LD50 was determined by injecting 0.1 mL of scorpion venom (10, 15, 20, 25 mg kg1 b.wt.) subcutaneously. For each dose, four mice were used.
Experimental protocol for biochemical assays: Three sets of albino mice weighing 25±5 g were used to study the effect of H. fastigiousus venom. The animals in first set consisted of 12 albino mice injected with 40% of 24 h LD50 and those in the second set, also consisted of 12 albino mice injected with 80% of 24 h LD50 of H. fastigiousus venom subcutaneously. Mice of the first two sets were divided into two groups: Group I and Group II including six animals each. Group I and Group II mice from the first and second sets were used at 4 and 8 h of envenomation, respectively, for biochemical analysis. Mice from the third set consisting of six mice served as control received only phosphate buffer (50 mM, pH 7.2).
Determination of serum biochemical and enzymatic parameters: At the end of each experimental period, mice were anesthetized using vapours of ether. Blood was collected by cardiac puncture, allowed to clot and clear serum was obtained for further analysis.
Determination of glucose: Serum pyruvic acid level was determined according to Mendel et al.17 method and expressed as mg/100 mL of serum.
Determination of pyruvic acid: Serum lactic acid level was determined according to Friedeman and Haugen method and expressed as mg/100 mL of serum18.
Determination of lactic acid: Serum glucose level was determined according to Shang et al.19 method and expressed as mg/100 mL of serum.
Determination of creatinine: Serum creatinine was determined according to Bonsnes and Taussky method and expressed as mg/100 mL of serum20.
Determination of creatine kinase (CK) activity: The CK activity in serum was measured using the method of Duncan as modified by Duncan et al.21 and Saha22. CK activity was expressed as Mmol h1 mL1.
Determination of lactic dehydrogenase (LDH) activity: LDH activity in serum was measured by Annon method and expressed as micromoles of reduced pyruvate 45 min1 mg1 of protein23.
Determination of glycogen content: Glycogen content in liver and muscle tissues was determined in by DuBois et al.24 method and expressed as g/100 g tissue.
Statistical analysis: Results were expressed as Mean±SE of six replicates. Students t-test was used to verify significant differences (p<0.05) relative to controls and between sub-lethal doses and exposure periods25. F-test was performed to verify the regression coefficient equality26.
Toxicity determination: The median lethal dose (LD50) of H. fastigiousus venom was 18.6 mg kg1 b.wt., of albino mice.
Effect of H. fastigiousus venom on serum biochemical level: Glucose level was increased to 152.04% of the control, after 4 h of treatment with 80% of 24 h LD50 of scorpion venom. This level increased to 206.23% of control after 8 h of the same treatment (Table 1). Pyruvic acid level was increased to 174.07% of the control after 4 h of treatment with 80% of 24 h LD50 of scorpion venom and increased to maximum (237.04%) after 8 h of treatment with 80% of 24 h LD50 of scorpion venom (Table 1). Serum lactic acid level was 191.45% of the control after 4 h of treatment with 80% of 24 h LD50 of scorpion venom. This level was increased to 228.35% of the control after 8 h of treatment with 80% of 24 h LD50 of scorpion venom (Table 1). A maximum increase in creatinine level (175%) was found after 8 h of treatment with 80% of 24 h LD50 of scorpion venom (Table 1). All these changes in different biochemical parameters were time and dose-dependent (Table 1, p<0.05, Students t-test, F-test).
Effect of H. fastigiousus venom on serum enzyme activity: The increase in circulating CK and LDH activity was 199.1 and 153.03% respectively after 4 h of treatment with 40% of 24 h LD50 of scorpion venom (Table 2). The CK and LDH activity further increased to 232.75 and 183.95%, respectively after 8 h of treatment with 80% of 24 h LD50 of scorpion venom (Table 2). The increase in the activity of these circulating enzymes was time and dose-dependent (Table 2, p<0.05, Students t-test, F-test).
Effect of H. fastigiousus venom on glycogen level: Glycogen level in the liver and gastrocnemius tissues of albino mice was decreased to 65.85 and 60.49%, respectively after 4 h of treatment with 40% of 24 h LD50 of scorpion venom, respectively (Table 3). This level in liver and gastrocnemius tissues was decreased to 49.59 and 38.27% of the control after 8 h of treatment with 80% of 24 h LD50 of scorpion venom, respectively (Table 3). The decrease in glycogen level in the liver and gastrocnemius muscle of albino mice was time and dose-dependent (Table 3, p<0.05, Students t-test, F-test).
In the present study, venom from Asian black scorpion H. fastigiousus was isolated by electric stimulation and its median lethal dose (LD50) was 18.6 mg kg1 body weight of albino mice.
Table 1: | Effect of 40 and 80% of 24 h LD50 of H. fastigiousus venom on glucose, pyruvic acid, lactic acid and creatinine levels in the serum of albino mice |
*mg/100 mL serum, Values in parentheses indicate percent change with respect to control taken as 100%, Results were expressed as mean±SE |
Table 2: | Effect of 40 and 80% of 24 h LD50 of H. fastigiousus venom on creatine kinase (CK) and lactic dehydrogenase levels in the serum of albino mice |
*Mmol h1 mL1, **μmol of pyruvate reduced 45 min1 mg1 protein, Values in parentheses indicate percent change with respect to control taken as 100%, Results were expressed as mean±SE |
Table 3: | Effect of 40 and 80% of 24 h LD50 of H. fastigiousus venom on glycogen levels in liver and gastrocnemius muscle of albino mice |
*Values represent glycogen g/100 g tissue, Values in parentheses indicate percent change with respect to control taken as 100%, Results were expressed as Mean±SE |
The effect of sub-lethal doses of this scorpion venom on certain biochemical and enzymatic parameters was studied. Serum glucose level increased after H. fastigiousus envenomation resulting in hyperglycemia. This could be due to reduced insulin secretion, excessive release of catecholamines, decreased thyroid hormone levels and increased cortisol and glucagon levels4,27-29. Reduced insulin and increased glucagon secretion causes a sustained fall in glucose clearance and promotes glycogenolysis and gluconeogenesis, thereby increasing blood glucose levels30. Increased free fatty acid level after Mesobuthus tumulus envenomation inhibits glycogen synthatase and pyruvate dehydrogenase activity and decreases glucose transport resulting in elevated serum pyruvic acid31. Increased secretion of glucagon, cortisol and catecholamines along with insulin resistance or reduced insulin level stimulate glycogenolysis in the muscles, thus, promoting lactate formation32. Thus, during scorpion envenomation, lactate is produced but not utilized, leading to lactate acidosis32. When oxygen level is low, carbohydrates break down to produce energy and makes lactic acid. Lactic acid level becomes higher during strenuous condition, heart failure or when liver is seriously damaged. Serum creatinine is an important indicator of renal health33. It is a byproduct of muscle metabolism and is excreted unchanged by the kidney through glomerular filtration. Increased serum creatinine level indicates kidney filtration impairment. Similar results were reported in albino mice when experimented Palamneus gravimanus15.
Creatine kinase (CK) is an enzyme found predominant in the skeletal and cardiac muscle. It plays an important role in providing energy for contraction in skeletal and cardiac muscles34. The CK phosphorylates creatine in muscle cells to high-energy molecule, phosphocreatine. Phosphocreatine is used as immediate source of energy in the muscle cells35. During the muscle degeneration, muscle cells break and release their contents into the circulation. Since most of the CK exists in muscle, a rise in the circulating level of CK indicates muscle damage36. Elevated activity of CK and LDH in the blood serum after H. fastigiousus venom administration, indicates myocardial and skeletal muscle damage. The main causes of altered permeability of myocardial and skeletal muscle are circulatory hypoxia, metabolic disorders and inflammation37. Similarly, Murthy et al.38 reported an increase in circulating LDH levels during scorpion venom administration. The venom of almost all lethal scorpion species exerts similar pathological abnormalities in experimental animals15,39.
Liver glycogen is largely related to storage and export of hexose units for the maintenance of blood glucose, whereas muscle glycogen acts as a readily available source of hexose units for glycolysis within the muscle itself40. Increased glucagon, corticosteroid and catecholamine levels during scorpion envenomation function synergistically and stimulate hepatic glucose production38,40. Under stress conditions carbohydrate reserves are depleted to meet the energy demand41. These changes provide ample stimulus for glycogenolysis in the liver and muscles, which indicates rapid utilization of glycogen in response to stress caused by envenomation. Since glycogen depletion is more prevalent under hypoxia condition, a situation similar to hypoxia may occur in the tissues of envenomed mice42.
The findings of this study helps in understanding the mode of black scorpion, H. fastigiousus venom lethality. This will help the pharmacologists to design drugs for the treatment of accidental H. fastigiousus envenomation.
This study established the lethal nature of Asian black scorpion, H. fastigiousus. The outcome of this study helps in understanding the mode of action of H. fastigiousus venom and the pharmacologists in drug designing for the treatment.