Bull. Egypt. Soc. Physiol. Sci. 29 (2) 2009 Abdel.Zaher Short Communication Notes on the role of Echis coloratus and Naja nigricollis Snake venoms on neuronal cell death Ahmed M. Abdel.Zaher Zagazig University - Faculty of Science – Department of Chemistry- Biochemistry division King Saud University- College of science and Arts in Shagra- Kingdom of Saudi Arabia ABSTRACT In the present study murine hippocampal HT22 cells were employed to investigate the role of Echis coloratus and Naja nigricollis snake venom on cell death. Monitoring of the release of the cytoplasmic enzyme lactate dehydrogenase (LDH) in the culture medium after treatment of the cells with different concentrations (50ug/ml, 100 μg/ml) from four fractions (F1, F2, F3 and F4) that obtained from each venom after purification. The time variation (6, 12, 18 and 24 hours) of the LDH concentration in the medium was used to indicate the total amount of lysed and, hence, the specific rates of cell death. In the first 6h from treatment of cells with F2( 50 μg/ml ) from Naja nigricollis venom( which is the most effective fraction in both venoms), LDH released into cell culture media more than treatment of cells with F3 and crude venom. Treatment of cells with a concentration of 50ug/ml and 100 μg/ml F3 Naja nigricollis (after 12h and 24h) snake venom, LDH elevated more than F2 and crude venom. Otherwise, lactate dehydrogenase (LDH) was released into cell culture media by treatment of the cell by 50 μg/ml F3 more than treatment of cells by F4 and crude venom from Echis coloratus. Key words: Echis coloratus - Naja nigricollis - HT22 cells- LDH INTRODUCTION systemic effects (neurotoxicity, myotoxicity, cardiotoxicity, and Snake venoms are complex alterations in hematological (4,5) mixtures of protein and non-protein systems) . The latter depends on the components(1,2). Snake envenomation concentration, efficiency, and rate of constitutes a medical hazard in most diffusion of target-specific toxins regions of the world(3). It is considered from the site of the bite into the as a subcutaneous/ intradermal general circulation. injection of venom into the prey or It has been reported that venoms human victims. The pathophysiology from the snake families Elapidae, of envenomation may include only Viperidae, and Crotalidae caused lysis (6) local effects (hemorrhage, edema, of different cells . Echis Coloratus is myonecrosis, and extracellular matrix a snake species that belongs to the (ECM) degradation) or may include Viperidae family that have common 83 Bull. Egypt. Soc. Physiol. Sci. 29 (2) 2009 Abdel.Zaher clinical features on the other hand, LDH assays have been used to Naja nigricollis belong to Elapiae quantify cell-mediated cytotoxicity family. Naja nigricollis snake is able induced by cytotoxic T cells, natural to propel the venom through the air to killer cells, lymphokine-activated hit the eyes of the prey. As example, killer cells, and monocytes(10,11), as the neurological disturbances well as to identify mediators that observed in E. coloratus envenomated induce cytolysis(11). animals in the absence of bleeding in The present study aimed to the brain(7). Neurotoxic activity of investigate the most effective fraction Echis Coloratus venom in dogs and in both snake venoms on cell death. guinea pigs was observed with histochemical methods showing a MATERIALS & METHODS diffuse breakdown of the blood-brain (8) barrier . Chemicals: Lactate dehydrogenase (LDH) is L-Glutamate, Triton X-100, a cytosolic enzyme present within all BAPTA-AM and all cell culture mammalian cells. The normal plasma media and reagents were obtained membrane is impermeable to LDH, from InVitrogen (Paisley, UK). but damage to the cell membrane Lactate dehydrogenase (LDH) results in a change in the membrane detection kit was obtained from Roche permeability and subsequent leakage (Lewes, UK). (9) of LDH into the extracellular fluid . Cell culture HT22 cells were In vitro release of LDH from cells cultured(14) in DMEM containing 10% provides an accurate measure of cell FBS, 2 mM L-glutamine and 50 U/ml membrane integrity and cell viability. penicillin and 50 μg/ml streptomycin As a result, the release of lactate under a humidified atmosphere (5% dehydrogenase has proved to be a CO2, 37°C). Unless indicated popular and reliable test for otherwise, 8×103 cells (counted by cytotoxicity in both immunological hemocytometer) were seeded into 24- studies, where it has superseded the well plates and incubated overnight, radioactive chromium release test as prior to media renewal and treatment (10,11) an assay for cellular cytotoxicity , of the cells with the compounds of and in biocompatibility studies, where interest in Dulbecco’s modified it has now become an important in Eagle’s medium (DMEM) containing (12,13) vitro screening test . In order to 2% FBS. For the measurement of cell assess the effects of a biomaterial on viability, 100 µl from cell culture mammalian cell cultures, cells are were used and aliquots of the media exposed to varying concentrations of were removed at the indicated time test material over a period of days. points and assayed for lactate The release of LDH into culture dehydrogenase (LDH) release, supernatant correlates with the amount according to the manufacturer's of cell death and membrane damage, instructions (Roche, Lewes, UK). providing an accurate measure of the Each sample of LDH was compared to cellular toxicity induced by the test the total, Triton X-100 releasable substance. 84 Bull. Egypt. Soc. Physiol. Sci. 29 (2) 2009 Abdel.Zaher LDH from the same well at the end of Separation and purification of each experiment. both snake venoms were carried out as The crude venoms. It has been described under Materials and milked from Naja nigricollis, and methods section. Echis coloratus snakes. Freeze-dried The intracellular LDH content of snake venoms were obtained from living attached cells is checked to be Faculty of Science- Department of constant during the culture. However, Zoology, King Saud university-KSA cells detached from the microcarriers, and Faculty of Medicine - Ain-shams and counted dead, have only released university- Egypt. part of their intracellular LDH. Purfficatlon of venoms fractions. Lactate dehydrogenase has proved to Naja nigricollis crud venom an d be an extremely useful in vitro marker Echis coloratus crude venom were for cellular toxicity. LDH assays are fractiated and partially purified by gel of great value both in immunology for filtration on sephadex G-70 and the quantification of cytotoxicity, and sephadex G-75 respectively, using in the evaluation of biomaterial 0.03M sodium phosphate buffer pH biocompatibility. 7.4, at flow rate of 20mm/hr, 3 Previous studies done on that ml/tube for 80 tubes. The protein species (E. coloratus) were focused on concentration was estimated at OD of serum and tissue (in vitro) profile of 280 nm. It was noticed that 4 fractions experimental animals. Snake venom have been obtained which were components, especially those of named F1, F2, F3 and F4 from each vipers, either activate, inhibit or venom. liberate enzymes by cellular Protein concentrations were organelles destruction(15,16). The measured by Bio-RAD Dc protein different toxic effects exhibited by assay and by Lowry method in 96 venoms of vipers were due to their well plates. All fractions (both contents of proteolytic and lipolytic venoms) were dissolved in sodium enzymes(17). phosphate buffer (pH 7.4). The protein concentration in crouds and all fractions were RESULTS & DISCUSSION measured (Table 1 and Table 2). The highest protein contents were in both Experimental models have been crude venoms. The lowest protein attempted in order to clarify the role content was F2 in Echis coloratus of Echis coloratus and Naja venom. Taking advantage of a nigricollis snake venom on cell death commonly used procedure, we by monitoring of the release of the detected the cell death by measuring cytoplasmic enzyme lactate the release of lactate dehydrogenase dehydrogenase (LDH) in the culture (LDH) into cell culture media. The medium under different time variation of the LDH concentrations of two different concentration in the medium was used venoms by incubation of HT22 cells to calculate the total amount of lysed to different levels snake venoms. and actually produced cells in the reactors, hence, the actual specific 85 Bull. Egypt. Soc. Physiol. Sci. 29 (2) 2009 Abdel.Zaher rates of cell growth and death. LDH incubation in case of F3 (100 μg\ml) release measured at different time after 24h all cells died and all LDH points following of venom incubation. elevated compare with the effect of F2 As shown in all figures, venom and crude venom. Previous study was incubation induced a time-dependent carried out by exposed epithelial increase in LDH release compared MDCK cells and mesangial cells to control, indicating injury of HT22 viper’s venom concentrations of 100– cells. 1000 µg/ml for 5 min up to 48 h, As shown in Tables 1.1, 1.2 and resulting in cells injuries(20). 1.3, the release of LDH increases as a A concentration of 50 μg\ml of function Naja nigricollis venom F1, F3 and F4 from Echis coloratus fraction concentration and time of venom with 6, 12, 18 and 24 h of incubation. Moreover, LDH released incubation period caused cell death in the presence of 50 μg\ml from F2 in and increase in LDH release (Figures the first 6 hours from incubation of and Tables 2.1, 2.2, 2.3 and 2.4) Naja nigricollis venom was more than compared with concentration of 100 that released by crude venom and μg\ml i.e. low concentration of other fractions. These results agree separated fractions from Echis with those of Isac de Castroa(18) who coloratus venom is increase of rate of funnd that the most intense effects cell death and increase than the high were obtained with venom concentration.