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

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 toxic effects of puff adder (Bitis arietans) venom, and their neutralization by antivenom.

This study investigated the in vitro toxic effects of Bitis arietans venom and the ability of antivenom produced by the South African Institute of Medical Research (SAIMR) to neutralize these effects. The venom (50 µg/mL) reduced nerve-mediated twitches of the chick biventer muscle to 19% ± 2% of initial magnitude (n = 4) within 2 h. This inhibitory effect of the venom was significantly attenuated by prior incubation of tissues with SAIMR antivenom (0.864 µg/µL; 67% ± 4%; P < 0.05; n = 3-5, unpaired t-test). Addition of antivenom at t50 failed to prevent further inhibition or reverse the inhibition of twitches and responses to agonists. The myotoxic action of the venom (50 µg/mL) was evidenced by a decrease in direct twitches (30% ± 6% of the initial twitch magnitude) and increase in baseline tension (by 0.7 ± 0.3 g within 3 h) of the chick biventer. Antivenom failed to block these effects. Antivenom however prevented the venom induced cytotoxic effects on L6 skeletal muscle cells. Venom induced a marginal but significant reduction in plasma clotting times at concentrations above 7.8 µg/100 µL of plasma, indicating poor procoagulant effects. In addition, the results of western immunoblotting indicate strong immunoreactivity with venom proteins, thus warranting further detailed studies on the neutralization of the effects of individual venom toxins by antivenom.

Chironex fleckeri (box jellyfish) venom proteins: expansion of a cnidarian toxin family that elicits variable cytolytic and cardiovascular effects.

The box jellyfish Chironex fleckeri produces extremely potent and rapid-acting venom that is harmful to humans and lethal to prey. Here, we describe the characterization of two C. fleckeri venom proteins, CfTX-A (∼40 kDa) and CfTX-B (∼42 kDa), which were isolated from C. fleckeri venom using size exclusion chromatography and cation exchange chromatography. Full-length cDNA sequences encoding CfTX-A and -B and a third putative toxin, CfTX-Bt, were subsequently retrieved from a C. fleckeri tentacle cDNA library. Bioinformatic analyses revealed that the new toxins belong to a small family of potent cnidarian pore-forming toxins that includes two other C. fleckeri toxins, CfTX-1 and CfTX-2. Phylogenetic inferences from amino acid sequences of the toxin family grouped CfTX-A, -B, and -Bt in a separate clade from CfTX-1 and -2, suggesting that the C. fleckeri toxins have diversified structurally and functionally during evolution. Comparative bioactivity assays revealed that CfTX-1/2 (25 µg kg(-1)) caused profound effects on the cardiovascular system of anesthetized rats, whereas CfTX-A/B elicited only minor effects at the same dose. Conversely, the hemolytic activity of CfTX-A/B (HU50 = 5 ng ml(-1)) was at least 30 times greater than that of CfTX-1/2. Structural homology between the cubozoan toxins and insecticidal three-domain Cry toxins (δ-endotoxins) suggests that the toxins have a similar pore-forming mechanism of action involving α-helices of the N-terminal domain, whereas structural diversification among toxin members may modulate target specificity. Expansion of the cnidarian toxin family therefore provides new insights into the evolutionary diversification of box jellyfish toxins from a structural and functional perspective.

An examination of cardiovascular collapse induced by eastern brown snake (Pseudonaja textilis) venom.

The Pseudonaja genus (Brown snakes) is widely distributed across Australia and bites account for significant mortality. Venom-induced consumption coagulopathy (VICC) and, less often, early cardiovascular collapse occur following envenoming by these snakes. We have previously examined possible mechanism(s) behind the early cardiovascular collapse following Papuan taipan (Oxyuranus scutellatus) envenoming. In the present study, we investigate early cardiovascular collapse in anaesthetized rats following administration of eastern brown snake (Pseudonaja textilis) venom, and prevention of this effect with prior administration of 'priming' doses (i.e. doses of venom which caused a transient hypotensive response) of venom. P. textilis venom (5-10 µg/kg, i.v.) induced cardiovascular collapse in anaesthetized rats, characterized by a rapid decrease in systolic blood pressure until non recordable. Prior administration of 'priming' doses of P. textilis venom (2 and 3 µg/kg) or, at least, 4-5 doses of O. scutellatus (2 µg/kg, i.v.) or Daboia russelii limitis (20 µg/kg, i.v.) venoms prevented cardiovascular collapse induced by P. textilis venom. Moreover, early collapse was also inhibited by prior administration of 2 discrete doses of Acanthophis rugosus venom. Prior administration of commercial polyvalent snake antivenom (500-3000 units/kg, i.v.) or heparin (300 units/kg, i.v.) also inhibited P. textilis venom-induced cardiovascular collapse. Our results indicate that P. textilis venom-induced cardiovascular collapse can be prevented by prior administration of sub-lethal doses of venom from P. textilis, O. scutellatus, A. rugosus and D. russelii limitis. This suggests that sudden cardiovascular collapse following envenoming is likely to involve a common mechanism/pathway activated by different snake venoms.

Differential myotoxic and cytotoxic activities of pre-synaptic neurotoxins from Papuan taipan (Oxyuranus scutellatus) and Irian Jayan death adder (Acanthophis rugosus) venoms.

Pre-synaptic PLA(2) neurotoxins are important components of many Australasian elapid snake venoms. These toxins disrupt neurotransmitter release. Taipoxin, a pre-synaptic neurotoxin isolated from the venom of the coastal taipan (Oxyuranus scutellatus), causes necrosis and muscle degeneration. The present study examined the myotoxic and cytotoxic activities of venoms from the Papuan taipan (O. scutellatus) and Irian Jayan death adder (Acanthophis rugosus), and also tested their pre-synaptic neurotoxins: cannitoxin and P-EPTX-Ar1a. Based on size-exclusion chromatography analysis, cannitoxin represents 16% of O. scutellatus venom, while P-EPTX-Ar1a represents 6% of A. rugosus venom. In the chick biventer cervicis nerve-muscle preparation, A. rugosus venom displayed significantly higher myotoxic activity than O. scutellatus venom as indicated by inhibition of direct twitches, and an increase in baseline tension. Both cannitoxin and P-EPTX-Ar1a displayed marked myotoxic activity. A. rugosus venom (50-300 µg/ml) produced concentration-dependent inhibition of cell proliferation in a rat skeletal muscle cell line (L6), while 300 µg/ml of O. scutellatus venom was required to inhibit cell proliferation, following 24-hr incubation. P-EPTX-Ar1a had greater cytotoxicity than cannitoxin, inhibiting cell proliferation after 24-hr incubation in L6 cells. Lactate dehydrogenase levels were increased after 1-hr incubation with A. rugosus venom (100-250 µg/ml), O. scutellatus venom (200-250 µg/ml) and P-EPTX-Ar1a (1-2 µM), but not cannitoxin (1-2 µM), suggesting venoms/toxin generated cell necrosis. Thus, A. rugosus and O. scutellatus venoms possess different myotoxic and cytotoxic activities. The proportion of pre-synaptic neurotoxin in the venoms and PLA(2) activity of the whole venoms are unlikely to be responsible for these activities.

In vivo and in vitro cardiovascular effects of Papuan taipan (Oxyuranus scutellatus) venom: Exploring "sudden collapse".

'Sudden collapse' following envenoming by some Australasian elapids is a poorly understood cause of mortality. We have previously shown that Oxyuranus scutellatus venom causes cardiovascular collapse in anaesthetized rats. Prior administration of a sub lethal dose of venom attenuated the response to subsequent administration of higher (lethal) venom doses. In this study, we investigated the possible mechanisms mediating this 'protective effect'. Papuan taipan venom (5µg/kg, i.v.) produced a small transient hypotension in anaesthetized rats, while 10µg/kg resulted in a 73±12% decrease in arterial pressure. Venom (20µg/kg or 50µg/kg) produced cardiovascular collapse in all animals tested (n=12). Cardiovascular collapse by 50µg/kg venom was prevented by prior administration of 'priming' doses of venom (5, 10 and 20µg/kg). Also, prior administration of indomethacin (30mg/kg, i.v.) or heparin (300units/kg, i.v.) prevented sudden collapse induced by venom (20µg/kg). Venom was without effect in isolated hearts indicating that a direct cardiac effect was unlikely to be responsible for 'sudden collapse'. Venom induced endothelium-dependent and -independent relaxation in pre-contracted rat mesenteric artery rings which was inhibited by indomethacin, IbTx and Rp-8-CPT-cAMPs. This relaxation was markedly reduced upon second exposure. Our results indicate that cardiovascular collapse induced by O. scutellatus venom may be due to a combination of release of dilator autacoids and to direct relaxation of vascular smooth muscle involving the cAMP/protein kinase A cascade. Further work will involve identification of the venom component(s) responsible for this action and may provide insight into the management of envenomed patients.

The in vitro toxicity of venoms from South Asian hump-nosed pit vipers (Viperidae: Hypnale).

Hump-nosed pit vipers (Genus Hypnale) are venomous snakes from South India and Sri Lanka. Envenoming by Hypnale species may cause significant morbidity and is characterized by local envenoming and less commonly coagulopathy and acute renal failure. Currently there are three nominal species of this genus: H. hypnale, H. zara and H. nepa. This study investigates the biochemical and pharmacological properties of the venoms from the three Hypnale species in Sri Lanka. The three Hypnale venoms had similar chromatographic profiles using reverse phase high performance liquid chromatography and fractions with procoagulant activity were identified. Hypnale venoms had potent cytotoxicity in cultured rat aorta smooth muscle cells with similar IC(50) values. The venoms had weak neurotoxic and myotoxic activity in the isolated chick biventer muscle preparation. They had mild procoagulant activity with close MCC(5) values and also phospholipase activity. Locally available polyvalent antivenom did not neutralise any venom effects. The study demonstrates that the three Hypnale venoms are similar and cytotoxicity appears to be the most potent effect, although they have mild procoagulant activity. These findings are consistent with clinical reports.

Validation of a cell-based assay to differentiate between the cytotoxic effects of elapid snake venoms.

INTRODUCTION: Acanthophis genus (i.e. death adders) and the Naja genus (i.e. cobras) belong to the family elapidae. The current study compared the in vitro cytotoxicity of venoms from four Acanthophis spp. and three Naja spp. on rat aortic smooth muscle cells, A7r5, and rat skeletal muscle cells, L6. The ability of CSL death adder antivenom and SAIMR antivenom, for Acanthophis spp. and Naja spp. venom respectively, to negate the cytotoxicity was also examined. METHODS: A cell proliferation assay was used to determine cell viability following treatment with venom in the presence or absence of antivenom. Sigmoidal growth curves were obtained, and IC(50) values were determined. RESULTS: Acanthophis spp. and Naja spp. venoms produced concentration-dependent inhibition of cell proliferation in both cell lines. Naja spp. venoms were significantly more cytotoxic than the most potent Acanthophis venom (i.e. A. antarcticus) in both cell lines. Naja spp. venoms also displayed higher sensitivity in L6 cells. SAIMR antivenom significantly inhibited the cytotoxic actions of all Naja spp. venoms in both A7r5 and L6 cells. However, death adder antivenom (CSL Ltd) was unable to negate the cytotoxic effects of Acanthophis spp. venoms. DISCUSSION: Concentrations of the predominantly cytotoxic Naja spp. venoms used were approximately three times less than the predominantly neurotoxic Acanthophis spp. venoms. SAIMR antivenom was partially effective in neutralising the effects of Naja spp. venoms. Death adder antivenom (CSL Ltd) was not effective in negating the cytotoxic effects of venom from Acanthophis spp. These results indicate that the cell-based assay is suited to the examination of cytotoxic snake venoms and may be used in conjunction with organ bath experiments to pharmacologically characterise snake venoms. Furthermore, the results suggest that the use of a skeletal muscle cell line is likely to be more clinically relevant for the examination of cytotoxic snake venoms.

Isolation and characterisation of P-EPTX-Ap1a and P-EPTX-Ar1a: pre-synaptic neurotoxins from the venom of the northern (Acanthophis praelongus) and Irian Jayan (Acanthophis rugosus) death adders.

The neurotoxicity observed following death adder envenoming has been thought to be solely due to the presence of potent post-synaptic neurotoxins. Clinically, these effects are often poorly reversed by death adder antivenom or anticholinesterase, particularly when patients present with established paralysis. This suggests that either the post-synaptic neurotoxins are irreversible/'pseudo' irreversible, or the venom contains pre-synaptic neurotoxins that do not respond to antivenom. To support the later hypothesis, a pre-synaptic neurotoxin (P-EPTX-Aa1a) has recently been isolated from the venom of Acanthophis antarcticus. We examined Acanthophis praelongus and Acanthophis rugosus venoms for the presence of pre-synaptic neurotoxins. P-EPTX-Ap1a (40,719Da) and P-EPTX-Ar1a (40,879Da) were isolated from A. praelongus and A. rugosus venoms, respectively. P-EPTX-Ap1a and P-EPTX-Ar1a are comprised of three different subunits, alpha, beta1 and beta2. The two toxins displayed similar levels of PLA(2) activity which was almost solely attributed to the alpha subunit in both toxins. P-EPTX-Ap1a (20-100nM) and P-EPTX-Ar1a (20-100nM) caused inhibition of indirect twitches of the skeletal muscle preparation without affecting contractile responses to nicotinic receptor agonists. Interestingly, only the alpha subunit of both toxins (300nM) displayed neurotoxic activity. Inhibition of PLA(2) activity markedly reduced the effect of the toxins on muscle twitch height. These results confirm that P-EPTX-Ap1a and P-EPTX-Ar1a are pre-synaptic neurotoxins and represent the second and third such toxins to be isolated from death adder venom. The presence of pre-synaptic neurotoxins in Acanthophis sp. venoms indicates that treatment strategies for envenoming by these snakes needs to be reassessed given the likelihood of irreversible neurotoxicity.

Presence of presynaptic neurotoxin complexes in the venoms of Australo-Papuan death adders (Acanthophis spp.).

Australo-papuan death adders (Acanthophis spp.) are a cause of serious envenomations in Papua New Guinea and northern Australia often resulting in neurotoxic paralysis. Furthermore, victims occasionally present with delayed-onset neurotoxicity that sometimes responds poorly to antivenom or anticholinesterase treatment. This clinical outcome could be explained by the presence of potent snake presynaptic phospholipase A(2) neurotoxin (SPAN) complexes and monomers, in addition to long- and short-chain postsynaptic alpha-neurotoxins, that bind irreversibly, block neurotransmitter release and result in degeneration of the nerve terminal. The present study therefore aimed to determine within-genus variations in expression of high molecular mass SPAN complexes in the venoms of six major species of Acanthophis, four geographic variants of Acanthophis antarcticus. Venoms were separated by size-exclusion liquid chromatography under non-denaturing conditions and fractions corresponding to proteins in the range of 22 to >60 kDa were subjected to pharmacological characterization using the isolated chick biventer cervicis nerve-muscle (CBCNM) preparation. All venoms, except Acanthophis wellsi and Acanthophis pyrrhus, contained high mass fractions with phospholipase A(2) activity that inhibited twitch contractions of the CBCNM preparation. This inhibition was of slow onset, and responses to exogenous nicotinic agonists were not blocked, consistent with the presence of SPAN complexes. The results of the present study indicate that clinicians may need to be aware of possible prejunctional neurotoxicity following envenomations from A. antarcticus (all geographic variants except perhaps South Australia), Acanthophis praelongus, Acanthophis rugosus and Acanthophis. laevis species, and that early antivenom intervention is important in preventing further development of toxicity.

A pharmacological and biochemical examination of the geographical variation of Chironex fleckeri venom.

Chironex fleckeri (box jellyfish) are found in the northern tropical waters of Australia. Although C. fleckeri have a wide geographical distribution and are able to swim large distances, adults tend to stay in small restricted areas. Clinical data shows that deaths from envenoming have not been recorded in Western Australia, yet numerous fatalities have occurred in Northern Territory and Queensland waters. One explanation for this discrepancy is a geographical variation in venom composition. This study examined the pharmacological and biochemical profiles of C. fleckeri venom from different geographical locations and seasons. Venoms were screened for cytotoxicity using a rat aortic smooth muscle cell line (A7r5). While all venoms caused concentration-dependent cytotoxicity, differences were seen in the potency of venoms from Mission Beach and Weipa, when collected in different seasons, as indicated by IC(50) values. Similarly venoms collected within the same season, from different locations around Australia, displayed marked differences in venom composition as shown by size exclusion HPLC and SDS-PAGE profiles which indicated the absence or reduced quantity of 'peaks' in some venoms. Based on IC(50) data obtained from the cell assay, the effects of the most potent (i.e. from Weipa in 2006) and the least potent (i.e. from Broome in 2007) venoms were examined in anesthetised rats. Both venoms at 10 microg/kg (i.v.) caused a transient hypertensive phase followed by cardiovascular collapse. However, at 4 microg/kg (i.v.) venom from Weipa 2006 caused a transient hypertensive phase followed by a transient decrease in MAP while venom from Broome 2007 only caused a small transient increase in MAP. This study demonstrates that there is considerable geographical variation in the composition of C. fleckeri venoms which is most distinct between specimens from western and eastern Australia and may explain the geographical variation in reported deaths.

A cell-based assay for screening of antidotes to, and antivenom against Chironex fleckeri (box jellyfish) venom.

INTRODUCTION: Chironex fleckeri is a large box jellyfish that has been labelled the 'most venomous animal' in the world. We have recently shown that the primary effect of C. fleckeri venom in vivo is cardiovascular collapse. This study utilised a cell-based assay to examine the effects of C. fleckeri venom on the proliferation of a rat aortic smooth muscle cell line. In addition, the ability of CSL box jellyfish antivenom and/or various potential treatment strategies to neutralise the effects of the venom was examined. METHODS: A7r5 cells were cultured in media containing venom. The effect of CSL box jellyfish antivenom (5 U/mL), CSL polyvalent snake antivenom (5 U/mL), lanthanum (5 microM), MgSO(4) (50 mM), verapamil (5 microM) or felodipine (5 microM) was examined. Cell viability was determined using a Cell titer 96 AQueous One Solution cell proliferation assay. RESULTS: Incubation of A7r5 cells with serially diluted venom (2-0.004 microg/mL) caused a concentration-dependent inhibition of cell proliferation with an IC(50) value of 0.056 microg/mL. This response was not affected by the absence of calcium or the presence of lanthanum in the media. Box jellyfish antivenom (5 U/mL) prevented the inhibition of cell proliferation caused by the venom. Verapamil (5 microM) had no significant effect on the inhibition. In contrast, felodipine (5 microM) or MgSO(4) (50 mM) potentiated the effects of the venom and partially negated the protective effect of the antivenom. DISCUSSION: This study displayed the ability to utilise a cell-based assay to determine the effects of C. fleckeri venom on vascular cell viability. It showed that CSL box jellyfish can neutralise the effects of the venom but only if added prior to the venom. In addition, potential adjunct therapies verapamil, felodipine and MgSO(4) were found to be ineffective, with felodipine and MgSO(4) potentiating the detrimental effects of the venom.

An examination of the cardiovascular effects of an 'Irukandji' jellyfish, Alatina nr mordens.

Irukandji syndrome is usually characterized by delayed severe abdominal, back and chest pain associated with autonomic effects including diaphoresis, hypertension and, in severe cases, myocardial injury and pulmonary oedema. It is most often associated with envenoming by the jellyfish Carukia barnesi, but a number of other jellyfish, including Alatina mordens, are now known to produce Irukandji syndrome. In the present study, nematocyst-derived venom from A. nr mordens (150-250 microg/kg, i.v.) produced a long-lasting pressor effect in anaesthetised rats. This pressor response (250 microg/kg, i.v.) was significantly inhibited by prior administration of the alpha-adrenoceptor antagonist prazosin (200 microg/kg, i.v.) but not by CSL box jellyfish antivenom (300 U/kg, i.v.). A. nr mordens venom 250 microg/kg (i.v.) caused marked increases in plasma adrenaline and noradrenaline concentrations following administration in anaesthetised rats. The venom did not contain appreciable amounts of either adrenaline or noradrenaline. A. nr mordens venom (25 microg/ml) produced a contractile response in rat electrically stimulated vas deferens which was markedly reduced in tissues pre-treated with reserpine (0.1mM) or guanethidine (0.1mM). Sodium dodecyl sulphate (SDS)-PAGE analysis showed that A. nr mordens venom is comprised of multiple protein bands ranging from 10 to 200 kDa. Western blot analysis using CSL box jellyfish antivenom indicated several antigenic proteins in A. nr mordens venom, however, it did not detect all proteins present in the venom. This study characterizes the in vitro and in vivo effects of A. nr mordens venom and indicates that the cardiovascular effects are at least partially mediated by endogenous catecholamine release.