Physiological effects of envenomation by two different doses of the viper Echis coloratus is crude venom on biochemical parameters in serum of Guinea pigs at different times

The eastern carpet viper Echis coloratusis widely distributed from Africa (eastern Egypt). In the present study the effects of crude venom of the viper Echis coloratusis on serum biochemical parameters of guinea pigs. Adult male guinea pigs (550 ± 25 g body weigh) were divided into three groups (15 each). In the control group, guinea pigs were interaperitoneally (i.p) injected with 50 μL saline solution. The second group was i.p. injected with 0.1μgm/g b.w. of crude venom in 50 μL saline solutions. The third group was i.p. injected with 0.2 μgm/g. b.w. of the crude venom in 50 μL saline solution. The results indicated that the injection of crude venom of the viper Echis coloratusis induced a significant decrease in total serum protein, albumin, globulin and uric acid within 1, 2 and 4 hrs. after injection. In contrast, the levels of glucose, cholesterol, triglycerides, urea and creatinine were increased significantly in envenomated Guinea pigs. Viper Echis coloratusis crude venom caused hepatic and renal dysfunction in envenomated Guinea pigs.


INTRODUCTION
Snake venom is a complex mixture of many substances, such as toxins, enzymes, growth factors, activators, and inhibitors with a wide spectrum of biological activities (Theakston, 1983 andRahmy andHemmaid, 2000).They are also known to cause different metabolic disorders by altering the cellular inclusions and enzymatic activities of different organs (Aiesenberg, 1981).The eastern carpet viper Echis coloratusis widely distributed from Africa (eastern Egypt).(Gasperetti, 1988;Cherlin, 1990 andWarrell, 1995).This species has got the highest incidence of snake bites, next to cerastes gasperettii (Al-Sadoon, 1991 andAl-Sadoon et al., 1991).The bites of the Echis genus could be response for the highest mortality level that exceeded all other snake genera, with a death rate of 7-15% of untreated victims (Moav et al., 1963 andWarrell andArnett, 1976).Clinical symptoms of Eachis envenomation are characterized by highly complex pathophysiological features of local as well as systemic nature (Warrell, 1993).Several investigators have studied the biochemical and pharmacological effects of the venom from different species that belong genus Echis (Theakston, 1983;Al-Gammaz, et al., 1999 andYamazaki andMorita, 2007).However, venom components may be altered by the geographical location and habitat of the snake (Zingali et al., 1993;Sasa, 1999 andSalazar et al., 2007).Additionally, zoological distribution and environmental condition could influence the overall biological behavior of snake venoms of the same species (Hassan et al., 1980;Werner et al., 1991;Werner, 1994;Tan and Ponnudurai, 1999;and Warrell, 1997).The present study was designed to investigate the effects of different doses of the viper Five animals of each group (I, II and III) were sacrificed at 1, 2 and 4 hours respectively post-injection of crude venom Serum analysis: Blood was collected from each animal into plain centrifuge tubes, left for 1 hr.at room temperature for clotting.Serum was separated by centrifugation at 3000g for 30 min.and analyzed, for the concentration of total protein, albumin, urea, creatinine, uric acid, glucose, cholesterol and triglycerides determination.Kits purchased from Spinreact, S. A. Ctra.Santa Coloma, Spain.All other chemicals used were of analytical reagent grade.Glucose determination was carried out according to the method Trinder (1969).Determination of total serum protein was estimated according to Peters (1968) method.Serum albumin was determined according to the method described by Doumas et al. (1971 and1972).While; serum globulin was obtained from the difference between the total serum protein and serum albumin.Cholesterol was determined by enzymatic method as described by Richmond (1973), while triglycerides were determined by the enzymatic colorimetric method as described by Young (1975).Creatinine was determined by kinetic method described by Hare (1950), while determination of urea was according to the enzymatic method of (Patton and Crouch, 1977).Serum uric acid was determined by quantitative determination method of Young (1975).

Statistical analysis:
Data are presented as standard error (S.E.) of the means and were statistically analyzed using SPSS version 8.0 for Software.Differences between moments were analyzed by Kruskal-Wallis Test and parameters of each test group were compared with control group by Mann-Whitney Test.Results were considered significant when p value was lower than 0.05.

1-Effects of the different doses (i.p) injection of viper Echis coloratusis crude venom on the levels of serum total proteins, albumin and globulin in serum.
A) Serum total protein content: The results presented in table (1) show that the i.p. injection of 0.1µgm/gm (b.w.) in the serum of Guinea pigs did not induce significant difference change of their serum total protein content from their corresponding control values after 1 hour.On the other hand, i.p. injection of 0.1µgm/gm induced significant decreases of their means of serum total protein content.These decreases were pronouncing and reached 19.82% (P<0.05) after 2 hours and 26.54% (P<0.01) after 4 hours.However, the injection of 0.2µgm/gm (b.w.) of the crude venom after 1, 2 and 4 hours of injection caused significant decreases of the means serum total protein levels from that of the corresponding control value.These decreases were pronouncing and reached 20.63% (P<0.05)after1 hour, 29.95% (P<0.01) after 2 hours and 37.09% (P<0.01) after 4 hours (Table 1).

B) Serum albumin content:
Table (1) show also that the i.p. injection of 0.1µgm/gm (b.w.) in the Guinea pigs did not induce significant difference change of their serum albumin content from their corresponding control values after 1 and 2 hour.However, i.p. injection of 0.1µgm/gm induced significant decreases of the mean of serum albumin content.These decrease was pronouncing and reached 18.02% (P<0.05) after 4 hours.On the other hand, the injection of 0.2µgm/gm (b.w.) of the crude venom after 1, 2 and 4 hours of injection caused significant decreases of the means serum albumin levels from that of the corresponding control value.These decreases

Globulin
were pronouncing and reached 10.27% (P<0.05),21.75% (P<0.05) and 32.27% (P<0.01) after 1, 2 and 4 hours of injection, respectively as shown (Table 1).C) Serum globulin content: As indicated in table (1) also that the i.p. injection of 0.1µgm/gm did not induce significant difference change of their serum globulin content from their corresponding control values after 1 hour.However, i.p. injection of 0.2µgm/gm induced significant decreases of the means of serum globulin content.These decreases were pronouncing and reached 32.66% (P<0.01) and 36.27%(P<0.01) after 2 and 4 hours, respectively.On the other hand, the injection of 0.2µgm/gm (b.w.) of the crude venom after 1, 2 and 4 hours of injection caused significant decreases of the means serum globulin levels from that of the corresponding control value.These decreases were pronouncing and reached 32.56% (P<0.01),39.73% (P<0.01) and 42.71% (P<0.01) after 1, 2 and 4 hours of injection, respectively as shown (Table 1).

2-Effects of the different doses ( i.p) injection of viper
Echis coloratusis crude venom on the levels of creatinine, urea and uric acid.A) Serum creatinine content: The results presented in table (2) show that the injection of 0.1µgm/gm to Guinea pigs caused significant increases in serum creatinine.These increases were 33.33% (P<0.05),95.56% (P<0.01) and 95.65% (P<0.01) after 1, 2 and 4 hours of injection, respectively.On the other hand, the injection of crude venom; 0.2µgm/gm caused also significant increases.These increases were 102.38% (P<0.001),211.11% (P<0.001) and 182.61% (P<0.001) after 1, 2 and 4 hours of injection, respectively as shown in (Table 2).

B) Serum urea content:
The results presented in table (2) show that the injection of 0.1µgm/gm to Guinea pigs caused significant increases in serum urea.These increases were 52.60% (P<0.01),114.21% (P<0.01) and 107.11% (P<0.01) after 1, 2 and 4 hours of injection crude venom, respectively.The injection of crude venom; 0.2µgm/gm caused also significant increases.These increases were 83.79% (P<0.01),136.81% (P<0.001) and 134.46% (P<0.01) after 1, 2 and 4 hours of injection, respectively as shown in (Table 2).2), the present study shows that the injection of 0.1µgm/gm to Guinea pigs caused non significant decrease in serum uric acid 1 hour of injection.However, after 2 and 4 hours of injection of 0.1µgm/gm the decreases were significantly.On the other hand, the injection of 0.2µgm/gm to Guinea pigs caused significant decreases in serum uric.These decreases were (20%; P<0.05, 43.75%; P<0.05 and 41.33%; P<0.05) after 1, 2 and 4 hours, respectively.

DISCUSSION
Several works dealing with the effects of snake venoms in blood cells, marrow cells and in cells from other organs of animals, like muscle, liver, kidney and skin, showed varying results, depending on the experimental concentrations, exposure time, site of injection, and type of toxin (Maria et al., 2003 andFox andSerrano, 2008).The liver is a major producer for most of serum proteins and its total level in the blood is a main liver function test.It is established that liver is the main source of plasma albumin.Decrease in plasma albumin is mainly due to the diminishing of its synthesis in hepatic cells, accompanied by losses of large amounts of albumin into the urine and gastrointestinal tract due to damage kidney and intestinal mucosa (West, 1985).It is worth mentioning that bone marrow is the main site of immunoglobulin production.Bone marrow plasma cells are derived from plasma plastic cells that have been generated in the peripheral lymphoid organs following antigen stimulation and have migrated to the bone marrow.These cells find in the bone marrow environment the survival and activation signals that allow them to generate mature plasma cells to produce high amounts of Igs (Hibi and Dosh, 1986;Liu et al., 1992 andMacMillan et al., 1994).Moreover, cytokines such as IL-6 and IL-10 control the production of Igs by non-dividing mature plasma cells (Roldan et al., 1992 a and b).
The present study revealed (Table 1) that, the injection of crude venom of viper Echis coloratusis causes a reduction in serum total proteins, albumin, globulin and uric acid in envenomated Guinea pigs at 1, 2 and 4 hours post-injection of crude venom.These findings are in agreement with other investigators who reported that, the reduction in serum total proteins, albumin, globulin and uric acid in envenomated rats was observed in laboratory animals injected with viper snake venoms (Abdul-Nabi et al., 1997;Fahim 1998and Al-Jammaz et al., 1998and 1999).It might be assumed that, the reduced levels of these serum constituents could be due to disturbances in renal functions as well as haemorrhages in some internal organs.In addtion, the increasing in vascular permeability and haemorrhages in vital organs due to the toxic action of various snake venoms were described by (Meier and Stocker 1991;March et al., 1997).
High levels creatinine indicates several disturbances in the kidney (Maxine and Benjamine, 1985).In the present study (Table 2), the rise in serum urea and creatinine levels indicates impairment of renal function.Similar observations were reported in rats following administration of various viper venoms (Abdel-Nabi, 1993;Rahmy et al., 1995;Omran et al., 1997;Abdel-Nabi et al., 1997 andSchneemann et al., 2004).Such increased vascular permeability, together with, renal damage would further aggravate the accompanying hypoproteinemia and hypoalbuminaemia.Furthermore, the rise in serum urea and creatinine associated with the reduction of serum uric acid level observed, in the present study, supports the proposed impairment of renal function.Similar observations were reported following various viper envenomation of rats (Sant and Purandare, 1972;Rahmy et al., 1995;Abdul-Nabi et al., 1997 andOmran et al., 1997).The findings that urea and creatinine acutely increased after the Bothrops jararaca venom infusion confirmed the acute renal damage in the present study experimental model and are in agreement with previous studies (Burdmann et al., 1993) in which a significant decrease in the glomerular filtration rate and diuresis were observed in anesthetized rats infused with the crude venom of snake Bothrops jararaca.
The increases in serum cholesterol and triglycerides levels in envenomated Guinea pigs observed in the present study (Table 3), could be due to the hepatocytes damage rendering them unable to phosphorylate the increasing amounts of fatty acids, hence leading to fatty liver and alteration of cell membranes of tissues (El-Asmar et al., 1979).Such disturbances of serum electrolytes were reported in rats following various snake venom injections (Mohamed et al., 1964;Al-Jammaz, 1995 andLewis andGutmann, 2004).Furthermore, Meier and Stocker (1991) suggested that, these disturbances might be due to acute nephropathy following viper bites.In addition, Mohamed et al (1980) speculate that this effect was brought about by stimulation of adrenal cortex leading to aldosterone secretion.It worthy to mention that, several studies have been made on the metabolic, cardiovascular and haematological effects of viper venoms on man and experimental animals (Tilbury et al., 1987;Soslau et al., 1988;Abu-Sinna et al., 1993;Abdul-Nabi et al., 1997 andFahim, 1998), and found that, various venoms viper cause alterations of rat metabolism (Al-Jammaz et al., 1998 and1999).Furthermore, several workers reported that, acute renal failure characterized by vascular lesions and tubular necrosis in the renal cortex following various snake bites (Tilbury et al., 1987).
In the present study, the levels of serum glucose were significantly increased after 1, 2 and 4 hours in the envenomated guinea pigs.The increases in serum glucose levels could be attributed to the effects of the venom on glycogen metabolism in the hepatocytes, muscle fibers and medullary catecholamines that stimulate glycogenolysis and gluconeogenesis in those tissues (Ohhira et al., 1991, Abdul-Nabi et al., 1997and March et al., 1997).
In conclusion, the measurements of biochemical parameters following viper Echis coloratusis crude venom injection, clearly demonstrate the disturbances of vital organs, especially liver, kidney, bone marrow and muscles.Such these disturbances are remaining for 4 hr after envenomation of Guinea pigs at least.

Table ( 2
): The Effects of the different doses ( i.p) injection of viper Echis coloratusis crude venom on the levels of serum creatinine (mg/dl), urea (mg/dl) and uric acid (mg/dl) in Guinea pigs at the 1, 2 and 4 hours after crude venom injection.

Table ( 3
): The Effects of the different doses ( i.p) injection of viper Echis coloratusis crude venom on the levels of serum glucose (mg/dl), cholesterol (mg/dl) and triglycerides (mg/dl) in Guinea pigs at the 1, 2 and 4 hours after crude venom injection.