The in vivo cardiovascular effects of box jellyfish Chironex fleckeri venom in rats: efficacy of pre-treatment with antivenom, verapamil and magnesium sulphate.

Using a new technique to extract venom from the nematocysts, the efficacy of CSL box jellyfish antivenom (AV) and adjunct therapies, verapamil and magnesium sulfate (MgSO(4)), were investigated against the in vivo cardiovascular effects of Chironex fleckeri venom in anaesthetised rats. C. fleckeri venom (30 microg/kg; i.v.) produced a transient hypertensive response followed by hypotension and cardiovascular collapse within 4 min of administration. Prophylactic treatment of anaesthetised rats with CSL box jellyfish AV (3000 U/kg; i.v.) did not have any effect on the venom-induced pressor response, but prevented cardiovascular collapse in four out of 10 animals. Administration of verapamil (20mM@0.25 ml/min; i.v.) either alone or in combination with AV, did not have any effect on the C. fleckeri venom-induced pressor response nor the consequent hypotension or cardiovascular collapse of animals. However, the administration of verapamil negated the partially protective effects of AV. Concurrent artificial respiration of animals with the above treatments did not attenuate the C. fleckeri venom-induced cardiovascular effects. MgSO(4) (0.05-0.07M@0.25 ml/min; i.v.) alone did not have any effect on the venom-induced pressor response nor the consequent cardiovascular collapse of animals. However, although combined AV and MgSO(4) administration could not inhibit the transient pressor effect following the administration of C. fleckeri venom, it prevented cardiovascular collapse in all animals. We show for the first time, the cardiovascular effects of a C. fleckeri venom sample free of tentacular contamination and the potential of MgSO(4) as an adjunct therapy for the treatment of potentially fatal C. fleckeri envenomings.     

Partial purification of cytolytic venom proteins from the box jellyfish, Chironex fleckeri

Venom proteins from the nematocysts of Chironex fleckeri were fractionated by size-exclusion and cation-exchange chromatography. Using sheep erythrocyte haemolysis as an indicator of cytolytic activity, two major cytolysins, with native molecular masses of approximately 370 and 145kDa, and one minor cytolysin ( approximately 70kDa) were isolated. SDS-PAGE and western blot protein profiles revealed that the 370kDa haemolysin is composed of CfTX-1 and CfTX-2 subunits ( approximately 43 and 45kDa, respectively); the most abundant proteins found in C. fleckeri nematocyst extracts. The 145kDa haemolysin predominately contains two other major proteins ( approximately 39 and 41kDa), which are not antigenic towards commercially available box jellyfish antivenom or rabbit polyclonal antibodies raised against whole C. fleckeri nematocyst extracts or CfTX-1 and -2. The kinetics of CfTX-1 and -2 haemolytic activities are temperature dependent and characterised by a pre-lytic lag phase ( approximately 6-7min) prior to initiation of haemolysis. Significant amino acid sequence homology between the CfTX proteins and other box jellyfish toxins suggest that CfTX-1 and -2 may also be lethal and dermonecrotic. Therefore, further in vivo and in vitro studies are required to investigate the potential roles of CfTX-1 and -2 in the lethal effects of C. fleckeri venom.     

Identification, cloning and sequencing of two major venom proteins from the box jellyfish, Chironex fleckeri.

Two of the most abundant proteins found in the nematocysts of the box jellyfish Chironex fleckeri have been identified as C. fleckeri toxin-1 (CfTX-1) and toxin-2 (CfTX-2). The molecular masses of CfTX-1 and CfTX-2, as determined by SDS-PAGE, are approximately 43 and 45 kDa, respectively, and both proteins are strongly antigenic to commercially available box jellyfish antivenom and rabbit polyclonal antibodies raised against C. fleckeri nematocyst extracts. The amino acid sequences of mature CfTX-1 and CfTX-2 (436 and 445 residues, respectively) share significant homology with three known proteins: CqTX-A from Chiropsalmus quadrigatus, CrTXs from Carybdea rastoni and CaTX-A from Carybdea alata, all of which are lethal, haemolytic box jellyfish toxins. Multiple sequence alignment of the five jellyfish proteins has identified several short, but highly conserved regions of amino acids that coincide with a predicted transmembrane spanning region, referred to as TSR1, which may be involved in a pore-forming mechanism of action. Furthermore, remote protein homology predictions for CfTX-2 and CaTX-A suggest weak structural similarities to pore-forming insecticidal delta-endotoxins Cry1Aa, Cry3Bb and Cry3A.     

A pharmacological investigation of the venom extract of the Australian box jellyfish, Chironex fleckeri, in cardiac and vascular tissues

The pharmacology of Australian box jellyfish, Chironex fleckeri, unpurified (crude) nematocyst venom extract (CVE) was investigated in rat isolated cardiac and vascular tissues and in anaesthetised rats.In small mesenteric arteries CVE (0.01-30 μg/ml) caused contractions (EC₅₀ 1.15 ± 0.19 μg/ml) that were unaffected by prazosin (0.1 μM), bosentan (10 μM), CGRP_8-37 (1 μM) or tetrodotoxin (1 μM). Box jellyfish antivenom (5-92.6 units/ml) caused rightward shifts of the CVE concentration-response curve with no change in the maximum. In the presence of l-NAME (100 μM) the sensitivity and maximum response to CVE were increased, whilst MgSO₄ (6 mM) decreased both parameters. CVE (1-10 μg/ml) caused inhibition of the contractile response to electrical sympathetic nerve stimulation.Left atrial responses to CVE (0.001-30 μg/ml) were bi-phasic, composed of an initial positive inotropy followed by a marked negative inotropy and atrial standstill. CVE (0.3 μg/ml) elicited a marked decrease in right atrial rate followed by atrial standstill at 3 μg/ml. These responses were unaffected by 1 μM of propranolol, atropine or CGRP_8-37. Antivenom (54 and 73 units/ml) caused rightward shifts of the CVE concentration-response curve and prevented atrial standstill in left and right atria.The effects of CVE do not appear to involve autonomic nerves, post-synaptic α₁- or β₁-adrenoceptors, or muscarinic, endothelin or CGRP receptors, but may occur through direct effects on the cardiac and vascular muscle. Box jellyfish antivenom was effective in attenuating CVE-induced responses in isolated cardiac and vascular tissues.     

Cardiotoxic effects of venom fractions from the Australian box jellyfish Chironex fleckeri on human myocardiocytes

An investigation into the cardiotoxic effects in human cardiomyocytes of different fractions (as produced from an FPLC) of the venom from Chironex fleckeri showed that whole venom caused cardiac cell death in minutes, measured as cell detachment using xCELLigence technology. However, only one fraction of the venom was responsible for this effect. When all extracted venoms were recombined a similar result was seen for the toxic fraction, however these effects were slower than unfractionated venom alone even though the concentrations were similar. The difference in the results between fractioned and unfractionated venom may have been caused by compounds remaining in the FPLC column, which may interact with the toxic fraction to cause rapid cell detachment or death.     

A rapid and repeatable method for venom extraction from cubozoan nematocysts.

Various comparative studies into the biological activity and relative toxicity of cubozoan venoms have been investigated, in particular the venom from the potentially lethal cubozoan Chironex fleckeri. Efficient and reliable extraction of venom from nematocysts is essential before any research into venom toxicity can be conducted and previous cited methods of extraction have varied greatly, each with their own associated problems. A new standardised technique for the recovery of venom from nematocysts of cubozoans is investigated to decrease the variation displayed between authors due to differing extraction techniques. The use of a mini bead mill beater, as investigated in this trial, allows for the rapid extraction of venom from nematocysts and is devoid of the previously isolated problems experienced with other methods of venom isolation, such as excessive heat build up.     

The in vitro effects of two chirodropid (Chironex fleckeri and Chiropsalmus sp.) venoms: efficacy of box jellyfish antivenom.

The pharmacological and biochemical isolation of cnidarian venoms has been hindered by difficulties with both extracting pure venom from nematocysts and venom stability. The development of a new technique to extract active, pure venom of Chironex fleckeri and Chiropsalmus sp. has enabled identify both neurotoxic and myotoxic activity in their venoms. These activities are similar, but not identical in each species. Venom (50 micro g/ml) from both species significantly inhibited indirect and direct twitches of the chick biventer nerve-muscle preparation. Pre-incubation with 1U/ml box jellyfish antivenom did not have any significant effect on venom-induced reductions of indirect twitches. However, this activity was markedly attenuated by prior addition of 5U/ml antivenom, albeit to a lesser degree for Chiropsalmus sp. In contrast, prior addition of 5U/ml box jellyfish antivenom did not neutralise the myotoxic activity of C. fleckeri venom (50 micro g/ml), although it did inhibit the myotoxicity produced by Chiropsalmus sp. venom (50 micro g/ml). Antivenom (5U/ml) added 1h after the addition of C. fleckeri venom (50 micro g/ml) had no effect on the indirect or direct twitches of the skeletal muscle preparation. However, it partially restored the reduction in indirect twitch height caused by Chiropsalmus sp. venom (50 micro g/ml). Myotoxicity was confirmed in muscle preparations stained with hematoxylin and eosin.Therefore, although antivenom was able to neutralize the neurotoxic effects of both species, and the myotoxic effects of Chiropsalmus sp., when added prior to venom, it was unable to reverse the effects after venom addition. This suggests that antivenom is unlikely to be useful in the treatment of neurotoxic or myotoxic effects in patients, although these effects are rarely seen clinically.     

The in vitro vascular effects of two chirodropid (Chironex fleckeri and Chiropsella bronzie) venoms

Clinical observations suggest a primary cardiotoxic role in fatal Chironex fleckeri stings. The limited research available indicates that Chiropsella bronzie venom acts in a similar manner although appears to be less potent. The aim of the present study was to elucidate the vascular effects of C. fleckeri and C. bronzie venoms using rat isolated aorta. Both venoms produced a sustained contraction of endothelium-denuded aorta which was not significantly affected by prazosin or box jellyfish antivenom. Felodipine significantly reduced the contractile response to C. fleckeri venom but not C. bronzie venom. Both venoms produced an initial relaxation (Phase 1), followed by a sustained contraction (Phase 2), in pre-contracted endothelium-intact aorta. Removal of the endothelium significantly inhibited both phases of the response. NOLA significantly inhibited Phase 1, but not Phase 2, of the response to both venoms. Atropine, HOE 140 or BQ 123 did not have any significant inhibitory effect on either phase. In conclusion, neither C. fleckeri nor C. bronzie venoms appear to contain components with activity at alpha(1)-adrenoceptors. Antivenom was ineffective in reversing the effects of the venom suggesting it is incapable of completely neutralising nematocyst-derived venom. Determining the mechanism of action of these venoms will allow for the development of better treatment strategies.     

The in vivo cardiovascular effects of an Australasian box jellyfish (Chiropsalmus sp.) venom in rats.

Using a new technique to extract venom from the nematocysts of jellyfish, the in vivo cardiovascular effects of Chiropsalmus sp. venom were investigated in anaesthetized rats. Chiropsalmus sp. venom (150 microg/kg, i.v.) produced a transient hypertensive response (44+/-4 mmHg; n=6) followed by hypotension and cardiovascular collapse. Concurrent artificial respiration or pretreatment with Chironex fleckeri antivenom (AV, 3000 U/kg, i.v.) did not have any effect on the venom-induced hypertensive response nor the subsequent cardiovascular collapse. The cardiovascular response of animals receiving venom after the infusion of MgSO4 (50-70 mM @ 0.25 ml/min, i.v.; n=5) alone, or in combination with AV (n=5), was not significantly different from rats receiving venom alone. Prior administration of prazosin (50 microg/kg, i.v.; n=4) or ketanserin (1 mg/kg, i.v.; n=4) did not significantly attenuate the hypertensive response nor prevent the cardiovascular collapse induced by venom (50 microg/kg, i.v.). In contrast to previous work examining C. fleckeri venom, administration of AV alone, or in combination with MgSO4, was not effective in preventing cardiovascular collapse following the administration of Chiropsalmus sp. venom. This indicates that the venom of the two related box jellyfish contain different lethal components and highlights the importance of species identification prior to initiating treatment regimes following jellyfish envenoming.     

A functional comparison of the venom of three Australian jellyfish--Chironex fleckeri, Chiropsalmus sp., and Carybdea xaymacana--on cytosolic Ca2+, haemolysis and Artemia sp. lethality.

Cnidarian venoms produce a wide spectrum of envenoming syndromes in humans ranging from minor local irritation to death. Here, the effects of Chironex fleckeri, Chiropsalmus sp., and Carybdea xaymacana venoms on ventricular myocyte cytosolic Ca2+, haemolysis and Artemia sp. lethality are compared for the first time. All three venoms caused a large, irreversible elevation of cytosolic Ca2+ in myocytes as measured using the Ca2+ sensitive fluorescent probe Indo-1. The L-type Ca2+ channel antagonist verapamil had no effect on Ca2+ influx whilst La3+, a non-specific channel and pore blocker, inhibited the effect. Haemolytic activity was observed for all venoms, with C. xaymacana venom displaying the greatest activity. These activities are consistent with the presence of a pore-forming toxin existing in the venoms which has been demonstrated by transmission electron microscopy in the case of C. fleckeri. The venom of C. fleckeri was found to be more lethal against Artemia sp. than the venom of the other species, consistent with the order of known human toxicities. This suggests that the observed lytic effects may not underlie the lethal effects of the venom, and raises the question of how such potent activities are dealt with by envenomed humans.     

Variation in lethality and effects of two Australian chirodropid jellyfish venoms in fish.

The North Queensland chirodropid box jellyfish Chironex fleckeri and Chiropsalmus sp. share similar nematocyst composition and the same prey of Acetes australis shrimps in their early medusa stages; however, as C. fleckeri individuals reach larger size, the animals add fish to their diet and their complement of nematocyst types changes, allowing larger doses of venom to be delivered to prey. This study demonstrated that the venoms of the two species differ as well: despite similar effects previously documented in crustacean prey models, the two had widely different cardiac and lethal effects in fish, with C. fleckeri being substantially more potent in its ability to cause death. Comparisons between the venom delivery abilities of the two species showed that the change in nematocysts of C. fleckeri cannot alone account for its ontogenetic shift to prey fish; instead, its prey ecology clearly necessitates it having venom capable of acting efficiently to cause death in fish. Although this venom is almost certainly produced at greater metabolic cost to the animal than the less-lethal venom of Chiropsalmus sp., owing to its greater molecular protein complexity, it confers the advantage of increased caloric intake from fish prey, facilitating larger size and potentially greater reproductive output of C. fleckeri over Chiropsalmus sp.     

Factors influencing hemolytic activity of venom from the jellyfish Rhopilema esculentum Kishinouye.

In this study, hemolytic activity of venom from the jellyfish Rhopilema esculentum Kishinouye and some factors affecting it were assayed. The HU(50) of R. esculentum full venom (RFV) against chicken erythrocytes was 3.40 microg/ml and a Hill coefficient value was 1.73 suggesting at least two molecules participated in hemolytic activity. The hemolytic activity of RFV was affected by some chemical and physical factors such as divalent cations, EDTA, (NH(4))(2)SO(4), pH and temperature. In the presence of Mg(2+), Cu(2+), Zn(2+), Fe(2+), Ca(2+) (>or=2 mM), Mn(2+) ((>or=1 mM), EDTA ((>or=2 mM) and (NH(4))(2)SO(4), the hemolytic activity of RFV was reduced. RFV had strong hemolytic activity at the pH 6-10 and the hemolytic ratios were 0.95-1.19. Hemolytic activity was temperature-sensitive and when RFV was pre-incubated at temperatures over 40 degrees C, it was sharply reduced.     

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.     

An in vivo comparison of the efficacy of CSL box jellyfish antivenom with antibodies raised against nematocyst-derived Chironex fleckeri venom

Although CSL box jellyfish antivenom (AV) remains the primary treatment for Chironex fleckeri envenoming, there has been considerable debate regarding its clinical effectiveness. Animal studies have shown that AV is largely ineffective in preventing C. fleckeri-induced cardiovascular collapse. This study examined the effectiveness of CSL box jellyfish AV (ovine IgG), raised against ‘milked’ venom, and polyclonal rabbit IgG antibodies (Ab) raised against nematocyst-derived venom. A venom dose of 30 μg/kg, i.v., which causes an initial presser response (34 ± 5 mmHg; n = 7) followed by cardiovascular collapse, was used in all experiments. A bolus dose of AV (3000 U/kg, i.v.) or Ab (12 mg; i.e. an equivalent protein ‘load’ to 3000 U/kg AV), administered 15 min prior to a bolus dose of venom, did not significantly attenuate the effects of venom. The venom response was also not significantly attenuated when AV (3000 U/kg) was given as a bolus dose 10–60 min prior to venom infusion. However, when the venom was incubated with either AV (3000 U/kg) or Ab (12 mg) for 3 h prior to infusion, the effect of the venom was almost abolished. The results of this study demonstrate that antibodies raised against both ‘milked’ and nematocyst-derived venom are able to neutralise the cardiovascular collapse produced by the venom. However, large amounts of AV are required and must be preincubated with the venom to be protective. This indicates a very rapid action of the toxin(s) and that AV is unlikely to be clinically effective because it cannot be administered early enough.     

Separation of two myotoxins from nematocysts of the box jellyfish (Chironex fleckeri)

Two myotoxins, both lethal to mice by i.v. injection, were obtained by chromatography on Sephadex G-200 of material released from isolated microbasic mastigophores of C. fleckeri. Both toxins elicit contractures of skeletal (diaphragm) musculature of the rat and of smooth (ileum and vas deferens) and atrial musculature of the guinea-pig, although consistent differences in the parameters of the contractures of each muscle type elicited by the two toxins are shown. Moreover, one toxin, with a molecular weight of approximately 150,000, also elicits activity on crustacean (barnacle) musculature, whilst the other toxin, with a molecular weight of approximately 600,000, elicits no activity in barnacle musculature at the concentrations tested. The toxins are labile when released from nematocysts and they lose all myotoxic activity within 3 days at 5 degrees C. They can also be isolated chromatographically from crude extracts of the contents of mixed nematocysts of C. fleckeri. They are considered to be the principal toxins injected by C. fleckeri during nematocyst discharge and appear to be different from the C. fleckeri toxins described by other workers.     

Scyphozoan jellyfish venom metalloproteinases and their role in the cytotoxicity

The present study, for the first time, comparatively investigated the enzymatic activities (proteases and hyaluronidases) in the venoms of four Scyphozoan jellyfish species, including Nemopilema nomurai, Rhopilema esculenta, Cyanea nozakii, and Aurelia aurita. For this, various zymographic analyses were performed using assay specific substrates. Interestingly, all the four jellyfish venoms showed gelatinolytic, caseinolytic, and fibrinolytic activities, each of which contains a multitude of enzyme components with molecular weights between 17 and 130 kDa. These four jellyfish venoms demonstrated a huge variation in their proteolytic activities in quantitative and qualitative manner depending on the species. Most of these enzymatic activities were disappeared by the treatment of 1,10-phenanthroline, suggesting they might be belonged to metalloproteinases. Toxicological significance of these venom proteases was examined by comparing their proteolytic activity and the cytotoxicity in NIH 3T3 cells. The relative cytotoxic potency was C. nozakii > N. nomurai > A. aurita > R. esculenta. The cytotoxicity of jellyfish venom shows a positive correlation with its overall proteolytic activity. The metalloproteinases appear to play an important role in the induction of jellyfish venom toxicities. In conclusion, the present report proposes a novel finding of Scyphozoan jellyfish venom metalloproteinases and their potential role in the cytotoxicity.     

Pharmacologically distinct cardiovascular effects of box jellyfish (Chironex fleckeri) venom and a tentacle-only extract in rats

Using a recently developed technique to extract jellyfish venom from nematocysts, the present study investigated the in vivo cardiovascular effects of Chironex fleckeri venom and tentacle extract (devoid of nematocysts). In anaesthetised rats, venom (10 microg/kg, i.v.) produced a transient pressor response (23+/-4 mmHg) followed, in two of five animals, by cardiovascular collapse. Tentacle extract (100 microg/kg, i.v.) produced a more prolonged hypertensive effect (31+/-3 mmHg) without cardiovascular collapse. Prazosin (50 microg/kg, i.v.) did not have any significant effect on the cardiovascular effects produced by venom. However, prazosin significantly attenuated the pressor response produced by tentacle extract. Ketanserin (1 mg/kg, i.v.) did not have any significant effect on the cardiovascular response of the anaesthetised rat to venom (10 microg/kg, i.v.; 25+/-1 mmHg). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to compare the two jellyfish samples used in the present study. In addition to ensuring reproducibility of future studies and allow comparison with previous research. We show, for the first time, that a pure venom sample extracted from C. fleckeri nematocysts and a tentacle extract have cardiovascular effects in the anaesthetised rat which are different and pharmacologically distinct.     

The pharmacology of Malo maxima jellyfish venom extract in isolated cardiovascular tissues: A probable cause of the Irukandji syndrome in Western Australia

The in vitro cardiac and vascular pharmacology of Malo maxima, a newly described jellyfish suspected of causing Irukandji syndrome in the Broome region of Western Australia, was investigated in rat tissues. In left atria, M. maxima crude venom extract (CVE; 1-100 μg/mL) caused concentration-dependent inotropic responses which were unaffected by atropine (1 μM), but significantly attenuated by tetrodotoxin (TTX; 0.1 μM), propranolol (1 μM), Mg²⁺ (6 mM) or calcitonin gene-related peptide antagonist (CGRP_8-37; 1 μM). CVE caused no change in right atrial rate until 100 μg/mL, which elicited bradycardia. This was unaffected by atropine, TTX, propranolol or CGRP_8-37. In the presence of Mg²⁺, CVE 30-100 μg/mL caused tachycardia. In small mesenteric arteries CVE caused concentration-dependent contractions (pEC₅₀ 1.03 ± 0.07 μg/mL) that were unaffected by prazosin (0.3 μM), ω-conotoxin GVIA (0.1 μM) or Mg²⁺ (6 mM). There was a 2-fold increase in sensitivity in the presence of CGRP_8-37 (3 μM). TTX (0.1 μM), box jellyfish Chironex fleckeri antivenom (92.6 U/mL) and benextramine (3 μM) decreased sensitivity by 2.6, 1.9 and 2.1-fold, respectively. CVE-induced maximum contractions were attenuated by C. fleckeri antivenom (−22%) or benextramine (−49%). M. maxima CVE appears to activate the sympathetic, but not parasympathetic, nervous system and to stimulate sensory nerve CGRP release in left atria and resistance arteries. These effects are consistent with the catecholamine excess thought to cause Irukandji syndrome, with additional actions of CGRP release.     

The lethality of tentacle-only extract from jellyfish Cyanea capillata is primarily attributed to cardiotoxicity in anaesthetized SD rats

Previous studies in our laboratory have shown that tentacle-only extract (TOE) has similar hypotensive effects with nematocyst venom from jellyfish Cyanea capillata, and the experimental studies on the in vivo cardiovascular effects of TOE were further performed to explore the leading cause of death and analyze the basic physiopathologic change in anaesthztized SD rats. Plots of TOE dose versus time to death showed dose-dependent curvilinear relationship. ECG changed in a dose- and time-dependent manner. Haemodynamic parameters, including the heart rate, mean femoral arterial pressure, left ventricular developed pressure and the first derivative of left ventricular pressures, decreased, but left ventricular end-diastolic pressure did not increase. Arterial partial pressure of oxygen and oxygen saturation did not change. Lactate dehydrogenase, creatine kinase and MB isoenzyme of creatine kinase increased significantly. Histopathological examination showed congestion, haemorrhage, edema and denaturation in the heart; congestion, haemorrhage in the lung and acute congestion in the liver. Transmission electron microscopy examination found that parts of sarcomeric filaments disrupted, dissolved and disappeared, and parts of mitochondria swelled in cardiocytes. Laser scanning confocal microscope examination found that ventricular myocytes from adult rat were deformed and ultimately died within 30 min after TOE treatment. Our results reveal that cardiodepressive effect of C. capillata TOE is the leading cause of death and acute total heart failure is the basic physiopathologic change in anaesthetized SD rats.