An Examination of the Neutralization of In Vitro Toxicity of Chinese Cobra (Naja atra) Venom by Different Antivenoms

Abstract

The Chinese Cobra (Naja atra) is an elapid snake of major medical importance in southern China. We describe the in vitro neurotoxic, myotoxic, and cytotoxic effects of N. atra venom, as well as examining the efficacy of three Chinese monovalent antivenoms (N. atra antivenom, Gloydius brevicaudus antivenom and Deinagkistrodon acutus antivenom) and an Australian polyvalent snake antivenom. In the chick biventer cervicis nerve-muscle preparation, N. atra venom (1–10 µg/mL) abolished indirect twitches in a concentration-dependent manner, as well as abolishing contractile responses to exogenous acetylcholine chloride (ACh) and carbamylcholine chloride (CCh), indicative of post-synaptic neurotoxicity. Contractile responses to potassium chloride (KCl) were also significantly inhibited by venom indicating myotoxicity. The prior addition of Chinese N. atra antivenom (0.75 U/mL) or Australian polyvalent snake antivenom (3 U/mL), markedly attenuated the neurotoxic actions of venom (3 µg/mL) and prevented the inhibition of contractile responses to ACh, CCh, and KCl. The addition of Chinese antivenom (0.75 U/mL) or Australian polyvalent antivenom (3 U/mL) at the t90 time point after the addition of venom (3 µg/mL), partially reversed the inhibition of twitches and significantly reversed the venom-induced inhibition of responses to ACh and CCh, but had no significant effect on the response to KCl. Venom (30 µg/mL) also abolished direct twitches in the chick biventer cervicis nerve-muscle preparation and caused a significant increase in baseline tension, further indicative of myotoxicity. N. atra antivenom (4 U/mL) prevented the myotoxic effects of venom (30 µg/mL). However, G. brevicaudus antivenom (24 U/mL), D. acutus antivenom (8 U/mL) and Australian polyvalent snake antivenom (33 U/mL) were unable to prevent venom (30 µg/mL) induced myotoxicity. In the L6 rat skeletal muscle myoblast cell line, N. atra venom caused concentration-dependent inhibition of cell viability, with a half maximal inhibitory concentration (IC50) of 2.8 ± 0.48 μg/mL. N. atra antivenom significantly attenuated the cytotoxic effect of the venom, whereas Australian polyvalent snake antivenom was less effective but still attenuated the cytotoxic effects at lower venom concentrations. Neither G. brevicaudus antivenom or D. acutus antivenom were able to prevent the cytotoxicity. This study indicates that Chinese N. atra monovalent antivenom is efficacious against the neurotoxic, myotoxic and cytotoxic effects of N. atra venom but the clinical effectiveness of the antivenom is likely to be diminished, even if given early after envenoming. The use of Chinese viper antivenoms (i.e., G. brevicaudus and D. acutus antivenoms) in cases of envenoming by the Chinese cobra is not supported by the results of the current study.

In Vitro Neurotoxicity of Chinese Krait (Bungarus multicinctus) Venom and Neutralization by Antivenoms

“…Therefore, it seems as though both antivenoms recognize the post- and pre-synaptic neurotoxins found in B. multicinctus venom. This is not surprising as we have previously shown cross neutralization between Australian polyvalent snake antivenom and non-Australian snake species including the Chinese cobra, New Guinea small-eyed snake, monocled cobra, banded krait, common krait and king cobra [ 16 , 17 , 18 ]. The ability of other heterologous antivenoms to neutralize the neuromuscular effects of Bungarus genus venoms and toxins has also been reported previously [ 19 , 20 , 21 ].…”

“…As previously described by us [ 15 , 16 , 18 ], male chickens (aged 4–10 days) were euthanized by exsanguination following CO 2 inhalation. Biventer cervicis nerve-muscle preparations were dissected and then mounted on wire tissue holders under 1 g resting tension in 5 mL organ baths.…”

“…As previously described by us [ 15 , 16 , 18 ], the height of electrically evoked twitches in the chick biventer cervicis preparation were measured at regular time intervals and expressed as a percentage of the pre-venom twitch height. The time taken for 90% inhibition of the twitch response (t 90 values) was used to compare the neurotoxic effect of B. multicinctus venom following the addition of antivenom at different post-venom time points or repeat washing.…”

Clinical study of anti-snake venom blockade in the treatment of local tissue necrosis caused by Chinese cobra (Naja atra) bites

“…Commonly used drugs are chymotrypsin and pancreatic enzymes. It is important to note that patients bitten by Chinese cobra must use the corresponding anti-snake venom, not geographically diverse snake anti-snake venoms since antigenicities of anti-snake venoms have been found to vary from one species to another, whose titer may be very low or even ineffective [26][27][28][29].…”

Animal Venoms—Curse or Cure?

“…In these studies, cutting-edge nanofractionation analytics are employed to determine the effects of Varespladib and other small molecules on the coagulopathic effects of various crotalid and viperid snake venoms [ 61 , 62 ]. The Hodgson lab examined the neutralising abilities of different antivenoms against the effects of venom from the Chinese cobra by using the chick biventer nerve muscle preparation [ 63 ]. Another study on Australian snake venoms by Isbister et al found that phospholipase A2 levels in human snakebite victims could be used as an early indicator of envenomation by Australian elapids (with exception of brown snakes) [ 64 ].…”