Neurotoxic, cytotoxic and cardiovascular effects of some Australasian elapid venoms

A range of toxic components found in Australasian elapid venoms have been postulated to account for the clinical outcomes of envenoming. Depending on the species of snake, these outcomes may include neurotoxicity with flaccid paralysis, coagulopathy with spontaneous systemic hemorrhage, sudden cardiovascular collapse, and myotoxicity with skeletal muscle breakdown. In the present study, we investigated the toxins and mechanisms behind these often life-threatening symptoms from the venom of four species of Australasian elapid snakes (Acanthophis praelongus, Acanthophis rugosus, Oxyuranus scutellatus and Pseudonaja textilis) with emphasis on their neurotoxic, cytotoxic and cardiovascular activities. Previously, post-synaptic neurotoxins were postulated to be the primary components responsible for neurotoxicity following death adder envenoming in humans. However, neurotoxicity is often poorly reversed by antivenom or anticholinesterase suggesting that death adder venoms may contain pre-synaptic neurotoxins that do not respond, as well as post-synaptic neurotoxins, to antivenom. In this study, phospholipase A2 (PLA2) neurotoxins, P-EPTX-Ap1a and P-EPTX-Ar1a, were isolated from the venoms of Acanthophis praelongus (Northern death adder) and Acanthophis rugosus (Irian Jayan death adder), respectively. P-EPTX-Ap1a (20-100 nM) and P-EPTX-Ar1a (20-100 nM) inhibited indirect twitches of the chick biventer cervicis nerve-muscle preparation without affecting contractile responses to nicotinic receptor agonists. Pre-incubation of 4-bromophenacyl bromide (1.8 mM) markedly reduced the effect of both toxins on twitch height suggesting that PLA2 activity plays an important role to induce pre-synaptic neurotoxicity. Some snake pre-synaptic PLA2 neurotoxins have been reported to also possess myotoxic activity and promote cell death. Therefore, pre-synaptic neurotoxins from Oxyuranus scutellatus (Papuan taipan) and A. rugosus venoms, cannitoxin and P-EPTX-Ar1a, respectively, as well as the whole venoms, were examined for myotoxic and cytotoxic activities. Based on size-exclusion high performance liquid chromatography (HPLC) analysis, cannitoxin represents 16% of O. scutellatus venom, while P-EPTX-Ar1a represents 6% of A. rugosus venom. A. rugosus venom induced significantly higher myotoxic activity than that of O. scutellatus venom in the chick biventer cervicis nerve-muscle preparation. In a rat skeletal muscle cell line (L6), A. rugosus venom and P-EPTX-Ar1a induced significantly greater cytotoxicity than O. scutellatus venom and cannitoxin. Thus, A. rugosus and O. scutellatus venoms possess different myotoxic and cytotoxic activities. These activities were independent of the proportion of pre-synaptic neurotoxin and PLA2 activity in the whole venoms. The cytotoxicity and disruption in neurotransmitter (i.e. acetylcholine) release induced by snake pre-synaptic neurotoxins have been postulated to be due to an increase in intracellular Ca2+. In the current study, P-EPTX-Ar1a, a pre-synaptic neurotoxin from A. rugosus venom, caused an increase in cytoplasmic calcium and membrane depolarization in primary dorsal root ganglion (DRG) neurons. Influx of Ca2+ did not occur in Ca2+-free Hank’s solution with EGTA. Ca2+ influx was also significantly reduced in the presence of nifedipine or agatoxin indicating that L-type and P/Q-type voltage-gated calcium channels, respectively, are involved in Ca2+ influx. Patch clamp studies in whole cell mode demonstrated that P-EPTX-Ar1a evoked inward currents in DRG neurons which were blocked by SKF96365, a cationic channel blocker, suggesting that P-EPTX-Ar1a induced-Ca2+ influx may be related to membrane depolarization through the activation of cationic channels. Sudden cardiovascular collapse following envenoming by some Australasian elapids (e.g. Oxyuranus spp. or Pseudonaja spp.) is a poorly understood cause of mortality and morbidity. Previous studies showed that administration of O. scutellatus or Pseudonaja textilis (eastern brown snake) venoms cause sudden cardiovascular collapse in anaesthetized animals. In the current study, O. scutellatus venom failed to affect force of contraction and conductivity in rat isolated heart preparations. In anaesthetized rats, sub-lethal doses of O. scutellatus venom (5-10 µg/kg, i.v.) produced transient hypotension while 20 or 50 µg/kg (i.v.) of venom produced cardiovascular collapse in all animals tested. The administration of P. textilis venom (10 or 20 µg/kg, i.v.) to anaesthetized rats also induced sudden collapse. Interestingly, cardiovascular collapse induced by O. scutellatus or P. textilis venoms was attenuated by prior administration of small ‘priming’ doses of some Australasian elapid venoms (i.e. O. scutellatus, P. textilis or A. rugosus venoms) or a venom from an exotic snake i.e. Daboia russelii limitis. Prior administration of polyvalent snake antivenom or heparin also protected against sudden collapse induced by O. scutellatus or P. textilis venoms. A prothrombin activator-like compound, PTV3, was partially purified from P. textilis venom. Protein bands of PTV3 displayed homology to catalytic and non-catalytic subunits of prothrombin activator ‘pseutarin C’ of P. textilis venom. Administration of PTV3 (10 and 20 µg/kg, i.v.) induced rapid cardiovascular collapse which was abolished by prior administration of small priming doses of PTV3 (2 and 5 µg/kg, i.v.) or heparin. This indicates that the prothrombin activator-like compound, PTV3 may contribute to sudden cardiovascular collapse in anaesthetized rats. In isolated rat mesenteric arteries, P. textilis venom but not PTV3 induced endothelium-dependent relaxation. O. scutellatus venom also induced both endothelium-dependent and -independent relaxation in pre-contracted rat mesenteric arteries which were inhibited by indomethacin, IbTX or Rp-8-CPT-cAMPs suggesting that vascular relaxation induced by the venom may be due to a combination of release of dilator autacoids and a direct relaxing effect on vascular smooth muscle involving the cAMP/protein kinase A (PKA) cascade. We subsequently isolated a PLA2 fraction (OSC3) from O. scutellatus venom and examined the hypotensive and vascular relaxant responses. OSC3 displayed high PLA2 activity and caused endothelium-dependent and -independent relaxation in pre-contracted rat mesenteric artery rings. Indomethacin and Rp-8-CPT-cAMPs markedly attenuated vascular relaxation induced by OSC3 on endothelium-denuded mesenteric arteries. Reverse-phase HPLC analysis of OSC3 indicated the presence of 2 major components, i.e. OSC3a and OSC3b. Both components induced a hypotensive effect in anaesthetized rats which was attenuated by prior administration of indomethacin. The amino acid sequencing indicated that the active components of OSC3 showed homology to PLA2 toxins from O. scutellatus (coastal taipan) venom. This finding indicates that PLA2 of O. scutellatus venom contains indirect relaxant and hypotensive effects that involve other vasoactive compounds e.g. PGI2 or PKA. It can be concluded that Australasian elapid induced-early cardiovascular collapse involves a combination of mediator-induced relaxation and prothrombin activator-like compound. In conclusion, this study examined the mechanisms behind snake pre-synaptic neurotoxin-induced neurotoxic and cytotoxic activities and the contributing factors to early cardiovascular collapse following Australasian elapid envenoming. These data provide useful insights for the clinical management of snake envenomed patients.