Evaluation of Approximate LD50 and pharmacological effects of Echis carinatus (Saw-scaled viper) venom from Central Punjab, Pakistan
A Feroze, S Malik, M Nawaz
ld, mice, pakistan, saw-scaled viper, toxicity
A Feroze, S Malik, M Nawaz. Evaluation of Approximate LD50 and pharmacological effects of Echis carinatus (Saw-scaled viper) venom from Central Punjab, Pakistan. The Internet Journal of Pharmacology. 2007 Volume 6 Number 1.
The assessment of toxic and pharmacological properties of snake venoms and their components is based predominantly on the animal experiments. Several modifications using fewer animals than the classical LD50 assay have been published. Using these methods an Approximate LD50 can be determined, the precision of and reproducibility of which is sufficient for most purposes of lethality testing. The pharmacological studies of the saw-scaled viper help us to locate and work on the components in the venom responsible for bringing these changes in the victims. So far in Pakistan the toxicity of snake venoms has been evaluated only through the classical LD50. The present work comprises the estimation of the toxicity of crude venom of
The total toxicity of snake venom encompasses the entire effects of all the toxic constituents. This toxicity of the venom and its other pharmacological effects on the victim are tested and studied through various animal experiments. The venom amount that kills 50% of the test animals is defined as the LD50, where LD abbreviates for Lethal Dose. Researchers have usually carried out LD50 calculations according to Reed and Muench (1958) Litchfield and Wilcoxon (1949) method. The LD50 test introduced by Trevan (1927) has gained acceptance as a measure of acute toxicity. However the LD50 figure is by no means constant with a given substance and can be affected by wide range of different factors. Nevertheless, the information on the lethality of snake venoms is required although in many cases it may result in unnecessary waste of experimental animals (Zbinden and Flurry, 1981, Brown, 1985).
Several modifications using fewer animals than the classical LD50 assay have been published. Using these methods an approximate LD50 can be determined, the precision and reproducibility of which is sufficient for most purposes of lethality testing. Meier and Theakston (1986) calculated the approximate LD50 values of some snake venoms by using the method of Baccari (1949) as modified by Molinengo (1979).
This technique is based on the results obtained by comparison of doses injected with observed survival times of experimental animals. Several researchers have studied the biochemical and pharmacological effects of venoms from different species of snakes from different localities (Al-Asmari, 2005). Meier and Theakston (1986) applied this technique and got consistent results. Some workers suggested that although statistical precision and reproducibility of an LD50 test could probably be improved by sacrificing large number of animals, its outcome is influenced by a considerable number of factors. In Pakistan some work has been done regarding the determination of toxicity of cobra venom through the LD50 (Alam and Ali, 1998), though in this work, the conventional classical LD50 has been performed.
Material and Methods
Snakes and venom
Fifteen Saw-scaled vipers,
Fifty adult albino white mice belonging to both sexes were purchased from Manawa Research Institute (MRI) Lahore, Punjab. The pooled venom was injected intraperitoneally; separately into groups of 10 mice with doses ranging from 1 to 10-mg/kg-body wt. As all the snakes used in the experiment belonged to the same species, pooled venom was suggested and preferred to the individual venom in order to have a cumulative effect of the toxins present in a particular snake species. Survival times (time between injection and death) of each animal for 24 hours were recorded. The LD50 of each snake species was determined according to the mathematical scheme adopted by Meier and Theakston (1986) some modification was however made in their method where by the maximum three-hour survival time was increased up to 24 hours for bringing convenience and authenticity to results.
No variables related to age, geographical origin, sex, and diet of snakes were controlled although variation in venom composition, even in natural conditions, may be associated with these variables. The effects of these variables on the toxicity of the venom in mice, although certainly present, were therefore not observed.
Results and Discussion
Calculation of Approximate LD:
Survival times observed after intraperitoneal (
Pharmacological effects of viper venom on mice
The pharmacological effects of the crude venom were characterized on a number of mice, however some significant effects observed in various models with visible symptoms are described.
Increased hypersensitivity with non-lethal dose
When a mouse was injected intraperitoneally with a non – lethal dose 1 mg/kg of viper venom, it grew surprisingly energetic and hypersensitive. It ate more feed than the normal and, during one of the countless attempts of escape from the finely wired jar; it sometimes excreted the fecal matter that was larger, almost double the normal fecal matter size. The mouse remained alive and active and did not by any means seem to suffer badly from the effects of the non-lethal dose. It showed no symptoms of pain or agitation.
Sloughing of skin with sub-lethal dose
When a sub lethal dose (2 mg/kg) was injected to a mouse, there was a little sloughing of the dead tissue around the injection site after a day or so. The being grew weaker, ate poorly, and appeared generally miserable for about two days before it recovered its health.
As the venom of the viper
Paralysis with partially lethal dose
When a partially lethal (4.5 mg/kg) dose was injected to a mouse, the weakness and difficulty in breathing developed and lasted for a few hours. The tissues at the site of the injection were dissolved. The animal would be obviously uncomfortable from the start; it scratched, rubbed at the injection site and was generally irritable and hyperactive and hypersensitive. Next came a quiet period during which the animal would sit huddled up but alert. The injection site was dark. There would be a short period of almost normal activity followed by another quiet stage that eventually terminated in collapse and death. The skin around the injection site grew gangrenous. The mouse became flat, immobile with its hind legs paralyzed. The survival time of the mouse was about 18 hours (table 1).
Respiratory failure with lethal dose
When a mouse was injected intraperitoneally with a lethal, but not overwhelming, dose of viper venom (5 mg/kg), it showed little immediate evidence of pain or discomfort. For a short time it behaved normally but soon it started showing signs of weakness. Its breathing got labored, and its flanks became peculiar, punched in appearance. Soon it could no longer get to its feet with the breathing becoming ever more difficult. As respiration ceased, the heart continued to beat for a few minutes. The survival time of the mouse was noted to be about 16 hours and 20 minutes (table 1).
Convulsive seizures and necrosis at higher doses
At the next lethal doses (6
Pharmacological studies clarify the clinical and pathological features of human fertility. Furthermore these help to understand specific resistance and susceptibility of various warm blooded and cold-blooded species (Perez
Two type of toxic effects were most easily observable in the all the mice, i.e. the change in color and dissolution of the skin at the site of the injection and the mice becoming prone, motionless with their hind legs paralyzed. The dissolution of the skin is an evident consequence of the myotoxic effect of envenomation. Paralysis of hind legs making the mouse flat and immobile reflects the neurotoxic effect of the venom.
In the Approximate LD50 the number of the animals sacrificed is significantly reduced i.e. up to 10 mice for one type of venom. This small number becomes negligible when compared with the use of more than 200 mice for one type of venom through the classical LD50. Moreover the use of 8-10 animals for one test gives LD50 values within the same statistical range as that obtained using the classical LD50 assay which employs much more experimental animals.
The intraperitoneal Toxicity of
As classical LD50 experiments, owing to their high level of variability, are far from satisfactory, the approximately method of LD50 suggested for scientific, economic and ethical reasons was tried and found relatively unproblematic, suitable and effective. Approximate method has thus been found a satisfactory alternative to classical LD50 determinations for animal venoms, owing to its exactitude and reproducibility for most purposes of lethality testing in animal models.
Ahsan Feroze, Zoological Sciences Division, Pakistan Museum of Natural History, Garden Avenue, Shakarparian, Islamabad 44000, Pakistan e.mail: email@example.com