International Journal of Scientific and Research Publications, Volume 5, Issue 9, September 2015 1 ISSN 2250-3153 Evaluation of the effect of gamma rays on Echis Coloratus snake venom through toxicological, immunological and biological studies. Esraa M. Samy**, Esmat A. Shaaban**, Sanaa A. Kenawy*, May A. Galal* and Walaa H. Salama*** *Department of Pharmacology and Toxicology – Faculty of Pharmacy- Cairo University **Department of Drug Radiation Research – National Center for Radiation Research and Technology – Atomic Energy Authority, Egypt ***Department of Molecular Biology, National Research Centre, Egypt Abstract- The present study was performed to evaluate the effectiveness of gamma radiation in detoxification of Echis I. INTRODUCTION Coloratus snake venom without affecting their immunogenic nake bites are a public health problem that deserves attention properties. This was carried out by studying the toxicological, S from health authorities (De Souza et al., 2012). Venomous immunological and biological properties of the crude venom. snakes are capable of inject venom, modified saliva, into another Data revealed that the toxicity of venom after (1.5 and 3 KGy) creature for the purpose of prey immobilization or self-defense gamma irradiation was reduced 6.4 and 7.2 times, respectively, (Meier and White, 2007). compared to the native venom. Immunogenicity was evaluated Echis coloratus belongs to the family Viperidae; it is the most by performing the double immunodiffusion test; same number of familiar family in Egypt (Mallow et al., 2003). Its venom visible lines joined smoothly at the corner was observed with the induces complex biological alterations and multisystem affection non-irradiated, as well as, the two dose levels gamma irradiated that may result in a death. Vipers’ venoms typically contain an (1.5 and 3 KGy) snake venom; this result indicating that there abundance of protein-degrading enzymes, called proteases, was no change in antigenic reactivity. The effect of gamma which produce symptoms such as pain, strong local swelling and radiation on some venom enzymes was studied; the necrosis. Another important enzyme in Echis Coloratus snake phospholipase A2 specific activity of native, 1.5 and 3 KGy venom is phospholipase A2. It is a small enzyme which causes gamma irradiated venom was 6.25, 4.8 and 2.7 U/mg; havoc by interfering in the normal physiological processes of the respectively. The proteolytic specific activity of native, 1.5 and 3 victim and inducing a variety of pharmacological effects (Kini, KGy gamma irradiated venom was 13, 7.5 and 5.5 U/mg; 2003). respectively. This means that proteolytic and phospholipase A2 Generally, the crude venom of viper Echis Coloratus causes activities were inhibited by irradiation. The study confirmed that swelling, pain, respiratory failure, arrhythmia, hypotension, native Echis Coloratus snake venom showed high edema circulatory collapse which leads to acute renal failure (Osman forming activity accompanied by apparent hematoma; peak and Gumma, 1974; Boviatsis et al., 2003). Liver is severely volume was achieved after 30 min and percent edema reached affected by Echis coloratus envenomation; elevation of serum 41.8 % from the initial diameter of the rat paw. This criteria levels of glucose, hepatocytes glycogen depletion and elevation greatly reduced by radiation as gamma irradiated (1.5 KGy and 3 of AST, ALT and ALP indicates hepatocellular damage (Al- KGy) venoms induced edema with peak volume achieved after Jammaz, 2003). Antivenins are the only effective treatment for 30 min and percent edema reached 29.7 and 21 % respectively, snakebites and envenomations by other venomous animals from the initial diameter of the rat paw. Echis Coloratus snake (Morais and Massaldi, 2009). In order to improve antivenin venom showed a high concentration dependant coagulant production, extend the lifespan of the immunized animals and activity; The clotting time of native, 1.5 and 3 KGy gamma decreasing the production cost, several trials have been used to irradiated venom at 200 μg/ml was 4.8, 16.8 and 26.6 seconds. detoxify venoms and maintaining its immunogenicity. One So, gamma irradiation decreased the coagulant activity of the method that has been shown to be effective for attenuating venom. These results indicate that gamma irradiation of venoms venom toxicity and maintaining immunogenicity is gamma offer an effective method for reducing the chronic toxic effect of radiation (Shabaan, 2003). venom in immunized animals for preparing the best toxoids and The present study was performed to evaluate the effectiveness vaccines, facilitating antivenin production and extending the of gamma radiation (1.5 and 3 KGy) in detoxification of Echis lifespan of immunized animals. Coloratus snake venom without affecting their immunogenic properties. This was carried out by studying the toxicological, Index Terms- Echis Coloratus, gamma radiation, LD50, immunological and biological properties of the crude venom double immunodiffusion, edema. before and after exposure to radiation. II. MATERIALS AND METHODS Animals Animals used in the present study included Swiss albino male www.ijsrp.org International Journal of Scientific and Research Publications, Volume 5, Issue 9, September 2015 2 ISSN 2250-3153 mice (20-25 g) and Wistar albino male rats (150-180 g). Animals 0.9% NaCl solution, sodium azide was added in a concentration were obtained from institute of ophthalmology (Giza, Egypt). of 0.05%, to retard bacterial growth. The wells were filled with The animals were acclimatized in the animal facilities of the 20 µl volumes. The venom samples in concentration of (20 National Center for Radiation Research and Technology under mg/ml) were added in the peripheral wells, while the antivenin appropriate conditions of temperature, humidity and light. They was added in the central well. After developing of the were allowed free access to food consisting of standard pellet precipitation bands (48 h), the plates were washed for 24 h in obtained from El-Nasr chemical company (Cairo, Egypt) and saline, dried and stained using 0.5 % Amidoschwartz stain (0.5 water ad libitum. Study was conducted in accordance with the % in 5 % acetic acid) for 7 min, washed with methanol acetic regulations approved by the ethics committee at Faculty of acid (9:1) dried in air and photographed. Pharmacy, Cairo University. Animals were purchased from the National Research Center (Giza, Egypt). Determination of phospholipase A2 (PLA2) activity of native and (1.5 KGy and 3 KGy) gamma irradiated venom Venom Venom phospholipase A2 activity of native and (1.5 KGy and The crude venom was obtained from Echis Coloratus snakes 3 KGy) gamma irradiated Echis Coloratus snake venom was kept in laboratory unit of Medical Research Centre, Faculty of determined according to the method of Marinetti (1965). A stock Medicine, Ain Shams University. The venom was pooled by of egg yolk suspension was prepared by shaking one egg yolk in milking healthy snakes collected from Sinai, Egypt and it was 0.9% NaCl to give a final volume of 100 ml suspension, stable dried and kept in the refrigerator at 4°C until used. for at least 2 weeks when stored at 0 °C. The working suspension of egg yolk was prepared by making 5-fold dilution of this stock Antivenin suspension in 0.9% NaCl. To 1 ml of the working egg yolk Egyptian polyvalent antivenin, produced in horses, was suspension, 4.9 ml of isotonic saline was added. This suspension obtained from the Holding Company for Biological Products and was mixed, and equilibrated to a temperature of 41°C for 5 Vaccines (VACSERA), Agouza, Giza, Egypt. The polyvalent minutes. 0.1 ml of the sample in saline was added to this antivenin was kept at 4°C till used. suspension. Then, the contents were rapidly mixed and the absorbance at 925 nm was recorded each 5 min for 10 min. The Irradiated Facilities control was prepared by adding 1 ml of working solution to 5 ml The venom was irradiated with gamma rays in the National 0.9% NaCl and was adjusted to give an absorbance of 0.7 (at 925 Center of Radiation and Research Technology, Cairo, Egypt, nm). One unit of enzyme is defined as the amount of venom in using (cobalt-60 gamma cell 220, Atomic Energy of Canady µg that will cause 50% turbidity reduction per 10 min. Limited (AECL), Canada). The radiation dose rate was 1.2 Gy per second. In this study, a saline solution of Echis Coloratus Determination of proteolytic activity of native and (1.5 KGy snake venom was subjected to radiation dose level of 1.5 KGy or and 3 KGy) gamma irradiated venom 3 KGy. Proteolytic activity of native and (1.5 KGy and 3 KGy) gamma irradiated Echis Coloratus snake venom was measured Determination of lethal dose fifty (LD50) of native and (1.5 using azocasein as substrate according to the method of Lemos et KGy and 3 KGy) gamma irradiated venom al. (1990). In a test tube, 2.5 % azocasein substrate solution Toxicity of Echis Coloratus snake venom was studied before dissolved in distilled water (w/v) was added to reaction mixture and after exposure to 1.5 KGy or 3 KGy gamma radiation. The containing 0.05M tris Hcl Bufer (PH=8.5) and different LD50 was determined according to the method of Spearman and concentrations of native venom and (1.5 and 3 KGy irradiated) Karber (Finney, 1964) by intraperitonial (i.p) injection of the venom, The mixtures were incubated at 37 °C for 60 minutes. venom in different doses into swiss albino mice. Six mice were Trichloroacetic acid (15%, 0.5ml) was added to the mixtures. used for each dosage. The LD50 was determined from the The reaction mixtures were cooled in an ice bath for 10 minutes; formula: the mixtures were clarified by centrifugation. The absorbance of supernatants was determined at 366 nm against blank, using M=Xk + d-dr1/N quartz cuvette.