Pakistan J. Zool., vol. 45(6), pp. 1741-1749, 2013.

Biochemical Characterization of Pyramid Viper, pyramidum, Venom

Ahmed M. Al-Shammari,1 Sami Ullah Khan,2 Mohammad Khalid Al-Sadoon3 and Saad S.M. Al-Saleh2* 1 Department of Biology, College of Sciences, University of Hail, P.O. Box 2440 Hail 81451, . 2 Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University P.O. Box 10219, Riyadh 11433, Saudi Arabia. 3 Department of Zoology, College of Sciences, King Saud University P.O. Box 2455, Riyadh 1145, Saudi Arabia.

Abstract.- This study was conducted to investigate the toxicological effects of Echis pyramidum crude venom and its purified protein fractions in male mice. The crude venom was fractionated and purified into eight fractions by 8% preparative native polyacrylamide gel electrophoresis. The mice were injected interaperitoneally with 50% of the lethal dose of crude venom and its purified fractions to keep 50% population of the injected alive to observe the different changes in the blood parameters. A significant increase was in plasma ALT level in animals injected with crude venom and fractions F1, F6 as compared to control group. A significant increase was in plasma AST level by crude venom and fraction F7. Glucose level increased significantly with crude venom whereas decreased significantly by fractions F4 and F5. There was up to 50% decrease in total plasma protein with crude venom and less than 50% decrease with fraction F7. Low plasma cholesterol levels were observed in group injected with crude venom and fraction F7. While fractions F2, F3 and F6 caused significant increase in plasma cholesterol level. Fraction F1 caused significant decrease in plasma triglyceride level whereas there was significant increase by crude venom. Blood urea level was significantly increased by crude venom fractions F2, F3 and F6. The significant increase in plasma creatinine level was produced by crude venom and fractions F1, F5, F7 and F8. Crude venom and fractions F1, F3, F5, F6 and F7 showed significant increase in uric acid level. The results of this study propose that E. pyramidum crude venom and its purified protein fractions exhibited potent toxic effects on some biochemical markers in male mice.

Keywords: Echis pyramidum, alanine aminotransferase, plasma cholesterol, plasma triglyceride, aspartate aminotransferase.

INTRODUCTION (24.5%), low blood pressure (14.3%), painful bitten site (10.2%), sweating (10.2%), bleeding in the area of bite (8.16%), nose bleeding (2.0%), fever (2.0%) bites are the source of fatality and and unconsciousness (2.0%) (Al-Shammari, 2007). morbidity and pose a serious socio-medical dilemma Snake venoms are mixtures of proteins, peptides, world wide. The pyramid viper (E. Pyramidum) is carbohydrates, lipids, metal ions and organic one of the venomous found in Saudi Arabia. compounds. The proteins and peptides account for It is widely spread in Jazan which according to approximately 90% of the dry weight (Bieber, 1979; surveillance, extends from A-Darb city north to Al- Bougis et al., 1986; Ownby and Colberg, 1987). The Mousam city in the south, near the Saudi-Yemeni non-protein fraction of snake venom is composed of borders and from Jazan city west to Fifa and Al- sodium, potassium, phosphorous, chloride, calcium, Da’r cities in the east. magnesium, manganese, zinc and copper. It also The pyramid viper has been related with the contains riboflavin, nucleosides, peptides, amino majority of envenomation cases in the region. The acids, amides, lipids and some carbohydrates victims of bites who were examined in the local (Bieber, 1979). The snake venom proteins are hospitals were suffering from symptoms of nausea enzymes, toxins or nerve growth factors. The and vomiting (26.53%), swelling at bitten place biological activities are predominantly found in the ______protein fractions rather than in the non-protein * Corresponding author: [email protected], fractions (Tu, 1977). [email protected] Efforts have been made to correlate the 0030-9923/2013/0006-1741 $ 8.00/0 Copyright 2013 Zoological Society of Pakistan toxicity of snake venoms to their enzymatic 1742 A.M. AL-SHAMMARI ET AL. activities. Owing to the diverse character of the (SDS) and Tris (hydroxymethyl) methylamine were venom and the antagonistic behavior of its different from BDH Chemicals, Poole, UK. Standard proteins components, it is advantageous to utilize purified and ampholine (pH 3.5–10.0) were from Pharmacia venom fractions in place of crude venom for diverse Uppsala, Sweden. Glycerol from Win Lab, Ltd., toxicological and pharmacological studies. Maidenhead, UK and glycin and N,N,N,N Relatively little work has been done on the Tetramethylethylene-diamine were from Riedel- toxicological effects of E. pyramidum crude venom dekhen AG, Wunstrofer StraBe, Germany. Protein and purified protein fractions. Okuda et al. (2001) molecular mass standards (Protein Marker II (6.5- purified novel disintegrins, the platelet aggregation 200) pre-stained) were from AppliChem GmbH, inhibitors pyramidin A and B from the venom of E. Ottoweg 4, D-64291 Darmstadt, Germany. All other pyramidum. Al-Asmari et al. (2006) studied the chemicals used were of analytical grade. effect of crude venom on time-course of lipid peroxidation in different organs of mice. Adel Al- Fractionation and purification of crude venom Missery et al. (2010) investigated the effect of crude Crude venom of E. pyramidum (10 mg) was venom on the activities of certain serum enzyme of fractionated using a native polyacrylamide gel mice. Wahby et al. (2012) isolated purified electrophoresis (PAGE); 8% acrylamide separating hemorrhagic metalloproteinase enzyme from E. slab gel of pH 8.8 as described by Laemmli (1970). pyramidum venom and studied its hemorrhagic The separated venom fractions were eluted from the activities in the skin of rabbit. Most recently, gel according to the procedure described by Walker Conlon et al. (2013) isolated [Ser49] phospholipase et al. (1982). The eluted protein fractions were A2 from the venom of the saw-scaled vipers E. dialyzed against distilled water, lyophilized and ocellatus, E. pyramidum leakeyi, E. carinatus stored at 4°C. The concentration of protein in each sochureki, and E. coloratus and observed their fraction thus obtained was determined by the cytotoxic activity against human non-small cell lung method of Lowry et al. (1951). Each fraction was adenocarcinoma A549 cells. So there is a potential analyzed for purity on analytical native (non-SDS) to fractionate and purify all the protein fractions of PAGE as described under the fractionation E. pyramidum venom and study toxicological procedure. effects of these purified venom fractions. We carried out this study with the aim to have better Determination of molecular weight by SDS-PAGE understanding of the role played by different The molecular weights of purified venom components of venom in envenomation by using the fractions were determined by running the purified crude venom and its purified protein fractions. venom fractions (20µg) along with crude venom (200µg) and standard proteins on SDS-PAGE at 8% MATERIALS AND METHODS acrylamide concentration according to the method of Laemmli (1970). The samples were boiled with Venom sample buffer in the presence of 0.1% SDS. The Specimens of E. pyramidum were captured in proteins used as molecular mass markers were the Saudi Arabian desert and maintained at 28±2oC myosin (200 kDa), β-galactosidase b (116 kDa), on a diurnal cycle of 7 h light/17 h dark. Each snake bovine serum albumin (68 kDa), ovalbumin (43 was fed one 20-30 g mouse every two weeks with kDa), carbonic anhydrase (30 kDa), trypsin inhibitor water ad libitum. Venom was milked from the (20 kDa) and lysozyme (14.4 kDa) and aprotinin snakes as described by Al-Saleh et al. (1994) and (6.5 kDa). stored at -20oC until used. Acrylamide was obtained from Merck, Isoelectric focusing (IEF) München, Germany. N,N-methylene bisacrylamide The isoelectric pH values (pI values) of the and coomassie brilliant blue R250 were purchased purified venom fractions were determined by using from Fluka Chemie AG, Buchs, Switzerland. an LKB 2117 Multiphor system according to the Ammonium persulphate, sodium dodecyl sulphate instructions of the manufacturer. EFFECTS OF E. PYRAMIDUM VENOM ON BIOCHEMICAL PARAMETERS 1743

Determination of median lethal dose (LD50) of crude blood cholesterol (CHO), triglycerides (TG), venom creatinine (Crea), blood urea and uric acid were The lethality of crude venom was assessed in determined by using EKTA Chem. 250 Analyzer, Swiss male mice (30±2 g) obtained from the Kodak, NY, USA and kits, Vitros Products College house and maintained at 28±2oC Chemistry (Cat. Ref: 145-0261) from Ortho-Clinical with free access to a commercial pellet diet and Diagnostics, Gohnson and Gohnson Company, NY, water ad libitum. They were divided into six groups USA. and there were 10 mice per group. The mice were injected interaperitoneally with gradually increased Statistical analysis doses of crude venom (1.25, 2.5, 5.0, 7.5, 10.0 and In order to calculate certain statistical 20.0 mg/kg body weight, in a fixed volume of 200 measurements such as the average concentration, µl and the LD was calculated according to the 50 changes in blood parameters and standard deviation arithmetic method of Karber (1931). Control mice SPSS program was used. The t-test was also used were injected with 0.9% NaCl. The observation time for comparison between the groups injected with limit used was 24 h, post-injection. These crude venom and purified fractions below the lethal experiments were approved by an institutional ethics concentration. Also to check the assumption saying committee for animal care and use. that no changes will occur between the crude venom

group, its purified fraction group and control group. Effect of crude venom and its fractions on some The final decision about results was based on p- blood parameters of normal male mice values. When p-values ≤ α, then we used alternative In this experiment ten groups, each of 10 assumption (5%), if p-values < α, we used null male mice; one group for crude venom, another for hypothesis, it means there are no significant values. control, and 8 for 8 purified venom fractions were used. The mice were injected interaperitoneally with 50% of the lethal dose of crude venom and its RESULTS AND DISCUSSION purified fractions to keep 50% population of the injected animals alive to observe different changes E. pyramidum crude venom was fractionated in the blood parameters. Blood was drawn after one into eight fractions by preparative native PAGE. hour of the injection because epidemic studies The recovery of the protein was 60%. The purity of showed that victims of snake bite reached the all fractions was checked by analytical native hospital or healthcare center in half-one hour after PAGE. On native PAGE all fractions appeared as a the bite time. The blood was drawn from eye single band (Fig. 1). However, on SDS-PAGE all capillary tube because the quantity of blood from fractions appeared as a single band except fraction eye is more than tail. It is difficult to draw blood F2 which showed two bands (Fig. 2). The from heart and there is also the risk of appearance of more than one band with fraction F2 contamination of the blood sample. could be explained by assuming that either fraction The blood was collected in EDTA tubes, F2 was oligomeric protein that was dissociated into mixed and stored in refrigerator for a couple of its subunits by the denaturing agent or that fraction minutes. Then blood samples were centrifuged at F2 on native PAGE composed of more than one 3000 rpm/minute for 10 minutes, the plasma was protein, with the same mobility on native PAGE. All separated and kept in freezer at −4°C. The analysis fractions exhibited acidic pI except fraction F8 was carried out within one hour of the sampling. which showed basic pI (Table I). The four parameters; blood glucose, total The median lethal dose for E. pyramidum protein, aspartate amino transferase (AST) and crude venom determined by Karber method was alanine amino transferase (ALT) were determined 5.06mg/kg (Table II). Recently Al-Missiry et al. by using Bayer Health Care, Express Plus Germany, (2010) reported LD50 1.4mg/Kg for an Egyptian and Kits; Reagents CK, Ref: 114978, Bayer Health of E. pyramidum which is almost 3.5 times Care, NY, USA. The other five parameters viz., less as compared to our result. 1744 A.M. AL-SHAMMARI ET AL.

Fig. 2. SDS-PAGE of E. pyramidum venom along with purified fractions (F1-F8) and protein molecular mass markers : (lane 1: protein molecular mass markers), (lane 2: 30 µg F8), (lane 3: 30 µg F7), (lane 4: 30 µg F6), (lane

5: 30 µg F5), (lane 6: 30 µg F4), (lane 7: 30 µg Fig. 1. Analytical native PAGE of E. F3), (lane 8: 30 µg F2) (lane 9, 30 µg F1) and pyramidum venom along with purified fractions crude venom (lane 10, 200 µg). (F1-F8); (lane 1: 30 µg F1), (lane 2: 30 µg F2), The proteins used as molecular mass markers (lane 3: 30 µg F3) (lane 4: 30 µg F4), (lane 5: were myosin (200 kDa), β-galactosidase (116 30 µg F5), (lane 6: 30 µg F6), (lane 7: 30 µg kDa), bovine serum albumin (68 kDa), F7), (lane 8: 30 µg F8) and crude venom (lane ovalbumin (43 kDa), carbonic anhydrase (30 9, 200 µg). kDa), trypsin inhibitor (20 kDa), lysozyme (14.4 kDa) and aprotinin (6.5 kDa).

Table I:- Molecular weight and isoelectric point of purified fractions of E. pyramidum crude was noticed in plasma ALT level in the animals venom. injected with crude venom (p<0.05, 132±2.28) compared to non-envenomated control group Fraction No. Mol. Weight (K Da) Isoelectric pH(pI) (13.4±0.73) as mentioned in Table III. There was significant but comparatively less increase in F1 105 6.3, 4.8 F2 138, 42 5.7 plasma. F3 28 5.4 ALT level by the fractions F1 (43.2±1.5, F4 83 6.3 p<0.05) and F6 (55.4±1.67, p<0.05). In case of F5 20 5.4 AST, there was significant increase in plasma level F6 12 3.5, 4.6 F7 10.5 5.7 in group injected with crude venom (189±3.75, F8 15 7.1 p<0.05) about four times as compared to control group (45.7±2.49). The increase produced by fraction F7 was about three times in comparison to The study assessed the impact of E. control (133.5±2.02, p<0.05) (Table III). The results pyramidum crude venom and its purified protein are concurrent with the preceding reports. Sant et al. fractions on the effects induced by the venom on (1974) and Tembe et al. (1975) by using E. some biochemical markers. A significant increase carinatus snake venom found that there were EFFECTS OF E. PYRAMIDUM VENOM ON BIOCHEMICAL PARAMETERS 1745

Table II.- Determination of LD50 of E. pyramidum crude venom.

Group No. Dose (mg/kg) I.P. Dose difference Mortality Mortality (Mean X) Dose difference X Mortality mean 1 1.25 0 2 2.5 1.25 1 0.5 1.25x0.5 = 0.625 3 5 2.5 5 3 2.5x3 = 7.5 4 7.5 2.5 9 7 2.5x7 = 17.5 5 10 2.5 10 9.5 2.5x9.5 = 23.75 6 20 10 10 Total 49.375

LD50 = Minimum dose that kills all animals − (Total of (dose difference x Mortality mean ) The number of a single group LD50 = 10 − (49.375)/10 LD50 = 5.06mg/Kg

Table III.- Effect of E. pyramidum crude venom and its purified protein fractions on the level of plasma aspirate aminotransferase (AST) and alanine aminotransferase (ALT).

Enzyme activity Crude Cont. F1 F2 F3 F4 F5 F6 F7 F8 (U/L) (Mean ± S.E) venom

ALT 13.4± 132.5± 43.2± 25.1± 15.8± 16.6± 9.7± 55.4± 9.2 ± 11.8± 0.73 2.28b 1.5b 1.71b 1.5 1.2 0.7 1.67b 0.57 0.77 AST 45.7± 189.6± 43.2± 57.2± 36.9± 53.9± 55.9± 47.8± 133.5± 53.8± 2.49 3.75b 0.97 1.1a 1.43 0.91a 2.14a 2.23b 2.02 2.72a

Data are expressed as means ± S.E. (n = 8). aP< 0.05; bP< 0.001. elevations in the serum concentration of AST, ALT action of the venom as reported by Abdel Nabi and ALP in animals four hours post envenomation (1993). The elevation in AST and ALT, gives compared with the control group. Al-Saleh et al. evidence about the destruction of the liver and heart (2002) reported that incubation of cultured tissues as a result of venom injection. fibroblast with E. coloratus crude venom and some The current effort described the effects of E. of its purified protein fractions resulted in similar pyramidum venom on some biochemical significant stimulatory effects on AST and ALT parameters. It was observed that glucose level activity. It is also reported by Abdel-Nabi and increased in test group injected with crude venom Rahmy (1992) using E. carinatus venom, the (p<0.05) as compared to control group. Whereas injection of sub-lethal dose caused a significant rise there was decrease in glucose level in the test in serum AST, ALT and ALP in rats accompanied groups injected with fractions F1, F3, F4, F5, F7 with disturbances in the hepatic and renal functions and F8 but most significant (p<0.05) decrease was of the envenomated animals through severe caused by fractions F4 and F5. Fractions F2 and F6 hepatocellular injuries, necrosis of hepatocytes and had no significant effect on plasma glucose level kidney tubules as well as nephrotoxic action. (p>0.1) (Table IV). Al-Saleh (2002) also reported Recently El-Missiry et al. (2010) reported the that E. carinatus crude venom caused noteworthy similar results for the elevation of serum AST and hyperglyce-mia which started as early as 15 minutes ALT level by E. pyramidum crude venom in mice. after envenomation and remained evident after 24 The fast increase in ALT and AST activities hours. It is suggested that hyperglycemic effect recorded in the present study one hour after venom produced is due to mobilization of glycogen in the envenomation may be ascribed to brutal injuries and liver which may be due to direct inhibition of necrosis of hepatocytes as well as to a nephrotoxic glucokinase or indirectly by stimulating the release 1746 A.M. AL-SHAMMARI ET AL.

Table IV.- Effect of E. pyramidum crude venom and its purified protein fractions on some blood parameters of male mice.

Parameter Crude Cont. F1 F2 F3 F4 F5 F6 F7 F8 (mg/dl) venom

Glucose 63±1.9 72±3.0a 29± 67± 40±1.3b 53± 56±2a 63± 35± 42± 1.5b 1.5 2.3a 2.9 1.0b 1.3b Total protein 1.4±0.24 0.7±0.08a 1.8± 2.1± 1.5± 1.9± 2.2± 1.5± 0.9± 1.3± 0.16 0.18 0.16 0.22 0.31 0.27 0.17 0.14 Cholesterol 47.2±1.1 8.7± 0.37 b 48.8± 62.8± 59.5± 39.4±2. 48± 49.1± 23.2± 61± 1.9 2.6b 1.3b 5a 1.95 1.6 1.2b 2.25b Triglycerides 92±2.2 119±4.9b 67± 45± 68± 50± 51± 58± 38± 32± 2.1b 2.8b 3.4b 1.1b 0.7b 4.3b 2.3b 0.7b Urea 6±0.7 12±1.0b 7±0.7 8±0.7 8±0.7 7±0.7 6±0.7 9±1.0a 7±0.7 5±0.7 Creatinine 0.1±0.02 1.4±0.09b 1.3± 0.9± 0.16± 0.1± 0.27± 0.23± 0.18± 0.8± 0.13b 0.05b 0.03 0.02 0.02b 0.01b 0.02b 0.05b Uric acid 0.2±0.01 0.3±0.02b 2.4± 0.2± 0.3± 0.2± 0.5± 0.5± 0.4± 0.2± 0.18b 0.02 0.02a 0.02 0.08b 0.08b 0.04b 0.02

Data are expressed as means ± S.E. (n = 8). * P< 0.05; * * P< 0.001. of adrenaline (Mohammad et al., 1980; Al-Saleh, previous report about the viper Naja haje venom. 2002). It is also mentioned by Al-Saleh (2002) that Abd El-Aal and Ezzat (1997) found that a sub-lethal the purified fractions F2 and F6 of E. carinatus dose of Naja haje venom decreased serum total exhibited the highest increase in plasma glucose cholesterol in rabbits. These authors explained their level after 12 hours of envenomation which is findings that by the activation of the pituitary- contrary in our report. adrenal axis resulting in increased circulatory levels There was up to 50% decrease in total plasma of ACTH and cortisol. ACTH induces specific protein with crude venom (p<0.05, 0.7±0.08) and membrane receptors in the cells of the adrenal less than 50% decrease with fraction F7 (p>0.1, cortex to increase serum cholesterol uptake. The 0.9±0.17) as compared to control group (1.4±0.24) increased level of T4 from the thyroid gland (Table IV). The decrease in total plasma protein following administration of Naja haje venom may be attributed to the presence proteolytic fractions may also explain the reason for lowering enzymes (proteases) in viper venoms. Al-Saleh serum cholesterol level (Ezzat, 1993). An inverse (1997) reported that the crude Bitis arietans and correlation between serum thyroid hormones and three of its purified protein fractions showed cholesterol levels has been reported by several caseinolytic activity. Most recently Wahby et al. workers (Devlin, 1993; Ganong, 1991). On the other (2012) isolated and purified hemorrhagic hand fractions F2, F3 and F6 caused significant metalloproteinase from three viper venom including (p<0.05) increase in plasma cholesterol level (Table E. pyramidum showing strong proteolytic activity. IV). Muhammad (2009) reported that i.p. injection The reduced levels of total protein could be due to of E. coloratus crude venom induced a significant disturbances in renal functions as well as increase in plasma cholesterol level in rabbits after haemorrhages in some internal organs. In addition, 1, 2 and 4 hours. Most recently Onyeama et al. increasing in vascular permeability and (2012) reported that the Carpet viper (Echis haemorrhages in vital organs due to the toxic action ocellatus) crude venom resulted in significant of various snake venoms (Meier and Stocker 1991; increase (p<0.05) in the serum total cholesterol. Marsh et al., 1997). The fraction F1 caused significant decrease Plasma cholesterol low level were observed (p<0.05) in plasma triglyceride level whereas there in the group injected with crude venom (8.7±0.37, was significant increase (119±4.85, p<0.05) in p<0.05) and fraction F7 (23.2±1.15, p<0.05) as plasma triglyceride level in animals injected with compared to control group. This agrees with a crude venom (Table IV). El-Aal and Ezzat (1997) EFFECTS OF E. PYRAMIDUM VENOM ON BIOCHEMICAL PARAMETERS 1747 reported that F1 of viper Naja haji venom normal control. Also, Tu (1991) and Merchant et al. significantly decreased the levels of serum (1989) reported that renal diseases caused by triglycerides after 1 and 2 hours, while F2 various snake venoms were characterized by raised significantly increased the level of serum urea, creatinine and potassium in oliguric patients. triglycerides after 3 and 4 hours. F3 induced an The increase in these values is used as an indicator initial significant increase after 1 hour followed by a of renal failure. This significant rise was ascribed to significant decrease after 2, 3 and 4 hours. The the nephrotoxic effect of venom. This was in decrease in serum triglyceride levels by some agreement with the results of Rahmy et al. (1992, fractions whereas the opposite effect by crude 1995b) and Yaguchi et al. (1996). As they venom or other fractions might be related to the mentioned that grave renal complications in case of relative action of each fraction on the hepatic Naja haje and Cerastes cerastes envenomation synthesis of triglycerides or to the activity of escort to impairment of the excretory function of the lipoprotein lipase which regulates tissue uptake of kidney. triglycerides (Devlin, 1993). Moreover the increases It may be concluded from the present study in serum cholesterol and triglycerides levels in that the E. pyramidum crude venom and its purified envenomated animals observed in the present study protein fractions exerted potent toxic effects on (Table IV) could be due to the hepatocytes damage some biochemical markers and parameters in male rendering them unable to phosphorylate the mice. There is a potential to characterize the increasing amounts of fatty acids, hence leading to purified protein fractions completely using fatty liver and alteration of cell membranes of advanced techniques, then to correlate their effects tissues (El-Asmar et al., 1979). to their structure and further manipulation of these There was significant increase (12±1.0, proteins to find their potential use in diagnostics, p<0.05) in blood urea level in the group of animals therapeutics and biological research. injected with crude venom as compared with the control group. There was significant increase in ACKNOWLEDGMENT blood urea level by fractions F2, F3 and F6 but less than caused by crude venom (Table IV). There was Funding for this work was provided by the an increase in plasma Creatinine level produced by Research Center, College of Applied Medical crude venom and some venom fractions (Table IV). Sciences, King Saud University. The highest increase was caused by fractions F1, F5, F7 and F8. The crude venom and all fractions except There is no conflict of interest to disclose. fractions F2, F4 and F8 exhibited significant increase in uric acid level. The highest increase was REFERENCES exhibited by fraction F1 (2.4±0.18, p<0.05) as compared to control group (0.2±0.01) (Table IV). EL-AAL, ABD. AND EZZAT, A.R., 1997. Effects of three Abdel Nabi (1993) has reported that a sub-lethal fractions obtained from Naja haje venom on hemolysis and lipid metabolism in rabbits. J. Venom. Anim. dose of both crude viper Cerastes cerastes venom Toxins, 3: 1-5. and its B fraction showed a significant rise in blood ABDEL-NABI, I.M. AND RAHMY, T.R., 1992. Influence of urea nitrogen and this was parallel to a significant the venom of snake Echis carinatus on the structure and increase in serum creatinine levels as well. Shaban function of the hepatic tissues of white rat. J. . and Hafez (2003) reported that intra-peritoneal Ger. Soc. Zool (C)., 171-187. injection of a sub-lethal dose of viper Naja haje ABDEL-NABI, I.M., 1993. Effect of crude Cerastes cerastes venom (0.2mg/kg) in rats induced a significant venom and fraction B on the clinical parameters of white rat. J. Egypt. Ger. Soc. Zool., 10(A):315-326. elevation in the activities of urea and creatinine as compared to normal control. Recently El-Missiry et AL-ASMARI, A., AL-MOUTAERY, K., MANTHARI, R.A AND KHAN, H. A., 2006. Time-course of lipid al. (2010) reported that LD50 of Echis pyramidum peroxidation in different organs of mice treated with venom induced a highly significant increase in urea Echis pyramidum snake venom. J. Biochem. molec. and creatinine concentrations compared to the Toxicol., 20: 93-95. 1748 A.M. AL-SHAMMARI ET AL.

AL-SALEH, S.S., NAYYAR, R., AL-SADOON, M.K., AL- Naja haje venom. J. Egypt. Soc. Toxicol., 11: 31-37. JAFARI, A. AND DUHIMAN, A.S., 1994. A rapid GANONG, W.F., 1991. Review of medical physiology. 15eds. fractionation method for the desert cobra venom Com: Appleton & Lange, Norwalk, pp. 296-311. (Walterinnesia aegyptia). Med. Sci. Res., 22: 659-660. GWYNNE, J.T. AND HESS, B., 1978. Binding and AL-SALEH, S.S., GHNEIM, H.K., HADDAD, H.Y. AND degradation of human HDL by rat adrenocortical cells. KHAN, S.U., 2002. Separation and purification of Metabolism, 27: 1593-1600. Echis coloratus venom and some biological and biochemical effects of the proteins. Cell Biochem. GWYNNE, J.T., HUGHES, T., ROUNDTREE, R. AND HESS, Funct., 20: 153-162. B., 1979. ACTH regulation of the HDL receptors of the rat adrenal cortex. In: Contemporary endocrinology (ed. Al-SALEH, S.S., 2002. The effect of Echis carinatus crude S.H. INGBAR), Plenum Medical Book, New York, pp. venom and protein fractions on carbohydrate 196-203 metabolism in rats. Cell Biochem. Funct., 20: 1-10. KARBER, G., 1931. Beitrag zur kollektiven behandlung Al-SALEH, S.S., 1997. Fractionation and purification of Bitis pharmakologischer Reihenvershuche. Arch. exp. Pathol. arietans crude venom by gel electrophoresis, and Pharmakol., 162: 480-483. characterisation of some enzymatic and biological activities. Med. Sci. Res., 25: 85-87. LAEMMLI, U.K., 1970. Cleavage of structural protein during the assembly of the head of the bacteriophage T4. Al-SHAMMARI, A. M., 2007. Ecological, eco-physiological Nature, 227: 680-685. and toxicological aspects of the pyramid viper (Echis pyramidum) in Jazan province, Saudi Arabia, Ph.D. LOWRY, O.H., ROSEBROUGH, N.J., FAAR, A.L. AND thesis, King Saud University, Saudi Arabia. RANDALL, R.J., 1951. Protein measurement with the Folin phenol reagent. J. biol. Chem., 193: 265-275. BIEBER, A.L., 1979. Metal and non-protein constituents in snake venom. In: Handbook of experimental MARSH, N., GATTULLO, D., PAGLIARO, P. AND pharmacology (ed. C.Y. Lee), vol. 52. Springer-Verlag. LOSANO, G. 1997. The Gaboon viper and Bitis Berlin, pp. 295-308. gabonica: hemorrhagic, metabolic, cardiovascular and clinical effects of the venom. Life Sci., 61: 763-769. BOUGIS, P. E., MARCHOL, P. AND ROCHAT, H., 1986. Characterization of Elapidae snake venom components MERCHANT, M.R., KHANNA, U.B., ALMEDIA, A.F., using optimized reverse – phase high performance ACHARYA, V.N. AND MITTAL, B.V., 1989. liquid chromatographic conditions and screening assays Clinicopathological study of acute renal failure for neurotoxin and phospholipase A activities. following viperine snake bite. Assoc. Physicians India, 2 37: 430-433. Biochemistry, 25: 7235-7243. CONLON, J.M, ATTOUB, S, ARAFAT, H, MECHKARSKA, MEIER, J. AND STOCKER, K., 1991. Effect of snake venoms on homeostasis. Toxicology, 21: 1711-1820. M, CASEWELL, N.R, HARRISON, R.A. AND CALVETE, J.J., 2013. Cytotoxic activities of MOHAMMAD, A.H., FOUAD, S. AND ABBAS, F. 1980. [Ser49]phospholipase A2 from the venom of the saw- Metabolic studies of the Egyptian and Allied African scaled vipers , Echis pyramidum leakeyi, snake venoms. Toxicon, 18: 381. Echis carinatus sochureki and Echis coloratus. Toxicon, MUHAMMAD, M.A., 2009. Physiological effects of 71: 96–104. envenomation by two different doses of the viper Echis DEVLIN, T.M., 1993. Biochemistry with clinical correlations. coloratusis crude venom on bio-chemical parameters in In: Biochemistry of hormones I: Polypeptide hormones. serum of Guinea pigs at different times. Egypt. Acad. J. Third eds. John Wiley, New York, pp. 500-600. biol. Sci., 1: 21-31. EL-AMAR, M. F., FARAG, R. M., SHOUKRY, S. AND EL- OKUDA, D, NOZAKI, C, SEKIYA, F. AND MORITA, T., SHIMI, I. R., 1979. Effect of scorpion Leivrus 2001 Comparative biochemistry of disintegrins isolated quinquestriatus H & E venom on lipid metabolism. from snake venom: consideration of the and Toxicon, 17: 279-283. geographical distribution of snakes in the genus Echis. J. Biochem., 129:615-20. El-MISSIRY, A.G., SHABAN, E.A., MOHAMED, R.M., AHMED, A. A., ABDALLAH, N.M. AND ONYEAMA, H.P., AHMED, M.S., OFEMILE, P.Y., IBEKWE, MOUSTAFA, M.I., 2010. Influence of ionizing H.A. AND NWANKPA, P., 2012. Effects of Calliandra radiation on Echis pyramidium snake venom: portoricensis extracts on the lipid profile of wistar rats biochemical and immunological aspects. Egyt. J. Hosp. challenged with venom of carpet viper. J. med. Sci., 3: Med., 40: 314-334. 674-678. EZZAT, A.R. AND ABD EL-AALA, A., 1989. Effect of cobra OWNBY, C.L. AND COLBERG, T.R., 1987. Characterization Naja haje venom on the adrenal activity in rabbits. of the biological and immunological properties of Qatar Univ. Bull., 9: 169-76. fractions of Prairie rattle snake (Crotalus viridis viridis) venom. Toxicon, 25: 329-1342. EZZAT, A.R., 1993. Changes in rabbit serum glucose, insulin, T4 and T3 in response to three ionopheretic fractions of RAHMY, T.R., TU, A.T., EL-BANHAWY, M., EL-ASMAR, EFFECTS OF E. PYRAMIDUM VENOM ON BIOCHEMICAL PARAMETERS 1749

M.F. AND HASSAN, F.M., 1992. Cytopathological (ed. A. T. Tu), Wiley & Sons, New York, pp. 2-400. effect of Cerastes cerastes (Egyptian sand viper) venom TU, A.T., 1991. Tissue damaging effects by snake venom and its hemorrhagic toxin on liver and kidney. An hemorrhage and myonecrosis. In: Handbook of natural electron microscopic study. J. Nat. Toxins, 1: 45-58. toxins, 5, (ed. A. T. Tu,), Dekker., New York, pp. 297- RAHMY, T.R., RAMADAN, R.A., FARID, T.M. AND El- 347. ASMAR, M.F., 1995b. Renal lesions induced by cobra WAHBY, A.F., ABDEL-ATY, A.M. AND EL-KADY, E.M., envenomation. J. Egypt. Ger. Soc. Zool., 17: 251-271. 2012. Purification of hemorrhagic SVMPs from venoms SANT, S.M., TEMBE, V.S., SALGANOKAR, D.S. AND of three vipers of Egypt. Toxicon, 59: 329–337. PURANDARE, N.M., 1974. Lesions produced by WALKER, J.E., AUFFERT, A.C. AND CARNE, A., 1982. Echis carinatus venom in experimental animals. Solid phase sequence analysis of polypeptides eluted Postgrad. Med., 21: 36-47. from polyacrylamide gels. Eur. J. Biochem., 123: 253- SHABAN, E.A. AND HAFEZ, M.N., 2003. Ability of gamma- 260. irradiated polyvalent antivenin to neutralize the toxicity YAGUCHI, T, UEDA, Y. AND ISHITOBI, F., 1996. of the Egyptian Cobra (Naja haje) venom. Egypt. J. Clincopathological study of the renal damage caused Hosp. Med., 3: 135–152. by mamushi snake (Agkistrodan biomhoffi) venom and TEMBE, V.S., SANT, S.M. AND PURANDARE, N.M., 1975. effects of cepharanthin against it in mice. Dokkyo. A clino-pathologic study of snake bite cases. Postgrad. Igakks Zasshi., 11: 127-141. Med., 21: 36-47. TU, A.T., 1977. In: Venoms: Chemistry and molecular biology (Received 4 October 2013, revised 27 October 2013)