Open Access Research Article J Toxins May 2017 Volume 4, Issue 1 © All rights are reserved by El-Ghffar et al. Journal of Isolation and Characterization of Toxins A. Abd El - Aal1, Eman A. Abd El-Ghffar1* and a Myotoxic Fraction from Ceras- Mohamed Mostafa Soliman1,2 1Department of Zoology,Faculty of Science, University of Ain Shams, Egypt tes Vipera Snake Venom 2Biology Department,Faculty of Science, Jazan university, KSA *Address for Correspondence Eman A. Abd El-Ghffar, Department of Zoology, Faculty of Science, Ain Keywords: Phospholipase A2 - Myotoxin; Viper venom; Cerastes Shams University, Cairo, Egypt, E-mail: [email protected] vipera; Myonecrotic action Submission: 3 March, 2017 Abstract Accepted: 10 May, 2017 The aim of this study was to evaluate the biological effects of Published: 15 May, 2017 crude Cerastes vipera venom and some phospholipase A2 (PLA2) Copyright: © 2017 El - Aal AA, et al. This is an open access article dis- activity containing fraction (CV-PL-F4). Furthermore, the current study tributed under the Creative Commons Attribution License, which permits examined the antimyotoxic action of dexamethasone, vitamins (Vit.) unrestricted use, distribution, and reproduction in any medium, provided E and C and trypsin against the isolated fraction having the highest the original work is properly cited. PLA2 activity and crude venom. In the present study, a PLA2, designed CV-PL-F4, was isolated from Cerastes vipera snake venom through Sephadex G-75, and a screening for PLA2 activity (via an indirect covering a glass funnel that emptied into a glass tube in the ice. The hemolysis assay) guided the search for a myotoxin. In conclusion, venom was lyophilized and stored at 4ᵒC until used. Sephadex G-75, dexamethasone and Vit E are potentially useful in treating the phosphatidylcholine, casein (fat-free), sodiumdodecylsulfate (SDS), myotoxic effect induced by Cerastes vipera crude venom and their CV-PL-F4 fraction. 2-mercaptoethanol, acrylamide, coomassie brilliant blue R250, trypsin, bovine serum albumin dexamethasone, Vit E, vitamin C and protein molecular weight markers were obtained from Sigma Introduction Chemical Co. The bite of the viperCerastes vipera has a serious medical problem. Animals C. vipera venom contains a complex mixture of enzymes [1] including Male CD-1 White mice weighing approximately 20-25 g were phospholipase A2 [2] and proteases [3,4]. Some clinical effects housed in specially designed cages and maintained in conditions following snake bite are due to PLA2 and proteases which are among the major components of viper venoms. Although these enzymes of good ventilation, normal temperatures, and humidity ranges. Mice were housed 5 animals in each cage to avoid crowding under share a common cytotoxic activity and similar structural features with conditions of equal periods of light and dark in the animal house non-toxic mammalian pancreatic enzymes, they also promote a wide (12 hr light and 12 hr dark cycle). Mice were fed on standard pellets, variety of pathological symptoms in animals, such as cardiotoxicity, containing all nutritive elements (proteins, fats, carbohydrates, myotoxicity, anticoagulant activity and hemolysis [5-7]. Since vitamins, salts and minerals). Drinking water and food were provided phospholipase A is almost invariably the most toxic component of 2 ad libitum throughout the period of study. All experiments followed the venoms, it probably plays an important role in mortality and the ethical standards for animal experiments in toxicological research morbidity following snakebite [5,8]. The primary target of PLA2 is the recommended by the International Society of Toxinology [20]. cell membrane inducing the hydrolysis of its phospholipids [9] as the hydrolysis of erythrocyte membrane [10-12] and the damage of the Fractionation of Cerastes vipera venom muscle cell membrane inducing myonecrosis [13-15]. A few PLA2 Samples of lyophilized Cerastes vipera venom (110 mg) were of the vipers have been isolated and purified [2,9,16,17]. The study reconstituted in 5 ml 0.03 M ammonium acetate buffer, pH 4.6 and of myotoxicity of Cerastes vipera venom has received relatively little applied to a 2.5x75 cm column (Pharmacia Biotech, Piscataway, NJ, attention [18,19]. The purposes of the present study are (a) to isolate a USA) packed with Sephadex G-75 equilibrated with the same buffer fraction having a myonecrotic effect with PLA2 activity from Cerastes and eluted at a flow rate of 1 ml/min using an EP-1 Econo Pump (Bio- Vipera venom, (b) to evaluate the biological properties of the fraction, Rad Laboratory, Hercules, CA, USA). Five-milliliter fractions were and (c) to investigate the antimyotoxic action of dexamethasone, Vit collected, and elution profile was monitored at 280 nm. E and C and trypsin for the isolated fraction. Hemolytic activity of Cerastes vipera venom and its fraction Materials and Methods Hemolytic activity of the crude venom and its isolated venom Venom and chemicals fraction was determined by measuring the release of hemoglobin from rabbit erythrocytes enhanced by the addition of phosphatidylcholine. Cerastes vipera venom was extracted from adult vipers collected This indirect hemolytic protocol was performed in glass tubes. from Aswan area and maintained in the Venom Research Lab, 10,20,40 and 60 µg of the crude venom and its isolated fraction Department of Zoology, Faculty of Science, Ain Shams University. were added to a prewarm (to 37 °C) assay mixture containing 0.1 Venom was collected from the vipers every four weeks for a six- ml of phosphatidylcholine suspension (10 mg /L), 1 ml of rabbit month period by allowing the vipers to bite through a rubber glove erythrocytes (2.5% v/v) and 2.9 ml of Tris buffer (1 mM, pH 7.5) Citation: Abd El - Aal A, Abd El-Ghffar EA, Soliman MM. Isolation and Characterization of a Myotoxic Fraction from Cerastes Vipera Snake Venom J Toxins. 2017;4(1): 9 Citation: Abd El - Aal A, Abd El-Ghffar EA, Soliman MM. Isolation and Characterization of a Myotoxic Fraction from Cerastes Vipera Snake Venom J Toxins. 2017;4(1): 9 ISSN: 2328-1723 containing NaCl (100 mM), KCl (100 mM) and CaCl2 (10 mM). Protein concentrations of crude venom, column eluates, and After incubation for 1 hr, the tubes were centrifuged at 2500 g for 15 isolated fractions were measured by the method of Lowry et al. min using IEC-Centra-4R cooling centrifuge (I. E. Company, USA). with bovine serum albumin as a standard or by spectrophotometric The supernatant was used to estimate the degree of hemolysis at 540 absorption at 280 nm [25]. nm. Hemolytic unit (HU50) value is the amount venom or a fraction LD determination caused 50% hemolysis and was calculated according to the method 50 of [21]. The amount of a venom or its fractions that are sufficient to kill 50 percent of a population of animals usually within a certain Myotoxic activity of the venom and fractions time is called median lethal dose (LD50). LD50 was determined by Ten fractions obtained from the viper venom were tested for Intraperitoneal (i.p.) injection of different CV-PL-F4 doses into male their ability to induce myonecrosis by intramuscular (i.m.) injection CD-1 White mice. The survival time for each mouse was recorded up into male white CD-1 mice (n=3). After 3 hours, the mice were to 24 hr. LD50 was calculated according to the method described by anesthetized with ether and killed by cervical dislocation. Samples Meier and Theakston [26]. of serum and muscle tissue were taken and processed as described Experimental protocol by Rodrigues et al. [22]. Myotoxicity was assessed by the increase in serum Creatine Kinase (CK) activity, in addition to the determination a- Myotoxic effect of Cerastes vipera venom and CV-PL-F4: of myonecrotic index using histological analysis. Cerastes vipera venom (10 µg/ 25 g mice) or myotoxin CV-PL-F4 (30 µg/ 25 g mice) was dissolved in 0.1 ml of 0.85% NaCl. The dose of Essay of PLA2 of Cerastes vipera myotoxin Cerastes vipera venom used in this study was equal to the sub LD50, The PLA2 activity of the CV-PL-F4 myotoxin fraction was where the LD50 of the venom was equal to 15.62 µg/ 25 g mouse. determined using the method described by Joubert and Taljaard CD-1 mice (64 animals) were divided into eight groups, each of eight [23]. 1 ml of egg yolk suspension was mixed with 4.0 ml 0.1 M Tris animals. Six animal groups were i.m. injected in the dorsolateral aspect buffer, pH 8, and 0.1 ml of the enzyme and incubated at 30 °C. The of the gastrocnemius muscle with either venom or CV-PL-F4 to study decrease of the turbidity of the suspension was recorded at 740 nm. the effect of the viper venom and CV-PL-F4 for three-time intervals. The absorbance change between 5 min and 15 min intervals was taken Mice injected with the crude venom or CV-PL-F4 was sacrificed after as the relative amount of enzyme activity. One unit was defined as the 1, 3 and 24 hr. The other two animal groups were injected with 0.85% amount of enzyme which produced a decrease of 0.01 in absorbance NaCl only and were used as a control. in 10 min. b- Antimyotoxic effect of dexamethasone, vitamins E, and C Proteolytic activity assay of Cerastes vipera myotoxin and trypsin: Ten animal groups (80 mice) were used each of eight animals. Two animal groups injected i.m. with the saline solution Proteolytic activity of CV-PL-F was estimated with casein (fat- 4 containing 10 µg of Cerastes vipera venom or 30 µg CV-PL-F and free, BDH, England) using the method described by Mandelbaum et 4 were used as control groups.