Studies on physiological effects, toxinokinetics and biological characteristics of the venom of Bungarus candidus from different parts of Thailand

Abstract

The studies in four groups of Bungarus candidus from northeastern, southern and eastern Thailand, and captive born group were carried out. The determinations of morphology of Bungarus candidus from different localities of Thailand and a captive-born group revealed color variation of the typical black and white bands from individual to individual. The same total length of snakes was shown the difference in the body weight without the correlation to the sexes. The liquid venom yield per individual snake was mainly correlated to its body weight. The biological characteristics of all venom groups demonstrated the different enzymatic activities of the high activities of phospholipase A2, acetylcholinesterase, L-amino acid oxidase and hyaluronidase; the moderate activity of alkaline phosphomonoesterase and the low activities of phosphodiesterase and protease. The SDS-PAGE profiles for protein compositions showed the quantitative differences of the protein bands at the molecular weight from 7.1 to 41.3 kDa in all venom groups and from 18.1 – 41.3 kDa between the wild-caught and a captive-born venom groups. All major venom proteins on RP-HPLC were subjected to individual variation within each geographic population. In vitro studies for the effect of venom on hemolysis and the osmotic fragility of rabbit red blood cells revealed the significant difference of MCF values between venom-treated heparinized blood (0.73 – 0.74% NaCl) and venom-treated EDTA blood (0.48% NaCl). The study for neurotoxic effect in mouse phrenic nerve diaphragm preparation demonstrated that at the venom concentration of 3.5 and 7 µg/ml, all wild-caught venom groups were significantly more potent in inhibited indirectly evoked twitches blockade than that of a captive-born venom group. For the studies of neutralization, the neurotoxicity of B. candidus venom was effectively attenuated by administration of antivenom promptly (t0) with venom or by pre-incubation of venom and antivenom (1:4.8 w/v). The administration of antivenom at t50 blockade produced the different percentage of reversal of the twitch blockade. In vivo studies in rabbits for the effect of venom on cardiovascular functions, renal hemodynamics and toxinokinetics demonstrated an immediate drop in systemic arterial blood pressure (MAP) and heart rate. The reduction of MAP persisted for a short duration and then gradually improved to approach the control level within 30 min. The falls in renal blood flow and glomerular filtration rate with an increase in renal vascular resistance (RVR) accompanied with hypotension and the reduction of cardiac output were apparent until 150 min after envenomation. The renal fraction tended to decrease, but not significantly. The venom kinetic study in rabbits after intravenous injection of B. candidus venom at the doses of 50 and 150 µg/kg showed that venom kinetics were fitted in bi-compartmental open model without significant differences between two venom groups for the rate constants (α and β), the half-life (T1/2), the mean residence time (MRT), the volume of distribution (Vd) of α phase and β phase, the volume of distribution at steady state (Vdss) and total body clearance (CL T). The significant differences revealed in the values of A and B intercepts and the area under the curve (AUC). The venom in urine was detected by ELISA technique at the given times between 5 – 120 min after envenomation. These findings demonstrate that body responses to envenomation by B. candidus bite not only affect to cell injury directly but also neurotoxic effect and systemic effects, particularly on cardiovascular and renal functions.