Elsevier

Toxicology Letters

Volume 209, Issue 1, 25 February 2012, Pages 11-20
Toxicology Letters

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

https://doi.org/10.1016/j.toxlet.2011.11.025Get rights and content

Abstract

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), CGRP8–37 (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 CGRP8–37. 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.

Highlights

► This study examined the pharmacology of Australian box jellyfish venom extract CVE. ► CVE caused vascular contraction unaffected by prazosin, bosentan or tetrodotoxin. ► Box jellyfish antivenom and MgSO4 inhibited CVE-induced vascular contraction. ► In cardiac tissues, CVE caused marked negative inotropy and bradycardia. ► CVE effects do not involve autonomic nerves, adrenoceptors or muscarinic receptors.

Introduction

Chironex fleckeri is a multi-tentacled box jellyfish prevalent in northern Australian and Indo-pacific during the summer months and is considered one of the most venomous creatures in the world (Pearn and Covacevich, 1988). It has been associated with about 70 fatalities in Australian waters (Currie and Jacups, 2005, Fenner and Williamson, 1996) primarily through rapid cardiorespiratory arrest. Yet, despite the potency of C. fleckeri venom, its pharmacological actions remain poorly understood.

Indeed it has been more than 50 years since Wiener first characterised the basic toxicity of this venom in animal studies; the triad of lethality through cardio-respiratory arrest, combined with dermonecrosis and haemolysis (Southcott and Kingston, 1959). By 1969 these findings were extended to show that the venom actions were due to separate protein fractions (Baxter and Marr, 1969). Thereafter, research focused on better defining the features of that cardiovascular toxicity, the chemical nature of the toxins and developing treatment options (Barnes, 1966, Barnes, 1967, Baxter and Marr, 1969, Endean et al., 1969, Endean et al., 1993, Freeman and Turner, 1969).

It was quickly established that the venom caused a biphasic change in blood pressure consisting of an initial hypertensive phase followed by potentially lethal hypotension (Freeman, 1974, Freeman and Turner, 1969, Freeman and Turner, 1972). The former appeared to be the result of direct vasoconstriction of vascular smooth muscle and the latter cardiac toxicity combined with baroreceptor stimulation. Similarly, in subsequent studies on anaesthetised piglets, the venom caused the rapid onset of severe hypotension and cardiac dysrhythmias (Tibballs et al., 1998). Recent experiments in anaesthetised rats, using a more pure nematocyst, rather than tentacle-derived venom, confirm these initial in vivo findings (Ramasamy et al., 2004). Although this research has demonstrated the cardiotoxic nature of C. fleckeri venom, its precise mechanism of action remains elusive.

The first studies on isolated mammalian cardiac tissues (perfused guinea pig hearts) demonstrated concentration-dependent differences in response to Chironex venom—low concentrations caused decreases in coronary flow, heart rate and force of contraction that were reversible, whilst higher concentrations resulted in irreversible arrhythmias and abolition of ventricular contraction (Turner and Freeman, 1969). More recently, in rat isolated aorta, nematocyst-derived venom was shown to induce a small transient relaxation followed by a sustained irreversible contraction (Winter et al., 2007) and, in rat resistance mesenteric arteries, the venom caused a concentration-dependent contraction (Winkel et al., 2005).

The molecular basis of this cardiovascular toxicity was first explored in the 1960s. It was quickly suggested that altered membrane permeability might underlie all of the observed venom effects (Freeman and Turner, 1969). Specifically, considering the cardiotoxicity, C. fleckeri venom has been shown to cause an increase in resting and active force in ferret isolated papillary muscles (Mustafa et al., 1995), as well as an irreversible increase in intracellular Ca2+ in rat cardiac myocytes that was inhibited by the non-specific channel and pore blocker, La3+ (Bailey et al., 2005). This has led to the concept that ionophore action on human cardiac muscle can lead to atrioventricular block and systolic dysfunction which may be the cause of cardiac arrest following this envenomation (Brinkman and Burnell, 2007, Freeman, 1974, Freeman and Turner, 1969, Mustafa et al., 1995, Winter et al., 2007). However, a proxy for cell permeability changes caused by cubozoan jellyfish venom, haemolysis, although reported in some human cases (Flecker, 1952) does not correlate with lethality per se (Bailey et al., 2005). Separately, as pain is also a prominent clinical feature of this envenomation, Cuypers et al. (2006) investigated the effect of this venom on the TRPV1 channel, a non-selective cation channel expressed in nociceptive neurons. They demonstrated that this venom is an allosteric modulator of such channels, suggesting that sensory nerve toxicity may also contribute to the pathogenesis of this envenomation.

Box jellyfish antivenom has been produced since 1971 and is indicated for use after a serious box jellyfish envenomation where there is risk of cardiovascular collapse (Baxter and Marr, 1969, Baxter and Marr, 1974, Winkel et al., 2003). Although antivenom use is associated with survival, there is still much debate surrounding its efficacy (Bailey et al., 2003, Ramasamy et al., 2004). A number of studies have found that box jellyfish antivenom was unable to attenuate the effects of the venom (Ramasamy et al., 2004, Winter et al., 2007) or requires prolonged pre-incubation (3 h) to do so (Winter et al., 2009). In particular, one study found that the antivenom was only able to prevent cardiovascular collapse in 40% of anaesthetised rats exposed to the venom (Ramasamy et al., 2004). In addition, there is a report of a sting victim dying despite being administered the antivenom (Currie, 2003). Some groups have expressed concern that the antivenom is unable to neutralise all components of the venom that would be injected into a victim (Endean and Sizemore, 1988, Ramasamy et al., 2003). In an effort to address this perceived therapeutic deficiency, previous investigations (Bloom and Burnett, 1999, Ramasamy et al., 2004, Tibballs et al., 1998) have explored the role of L-type Ca2+ antagonists, such as verapamil and nifedipine, as potential ancillary agents against the myocardial Ca2+ influx apparent following C. fleckeri envenomation. Despite some initially positive work by Bloom and Burnett (1999) in mice, Ramasamy et al. (2004) and Tibballs et al. (1998) found, in rats and piglets, verapamil to be not only ineffective but it actually exacerbated the cardiovascular toxicity of this venom. Subsequently, magnesium sulphate (MgSO4) has been proposed as an alternative or combination therapy for the treatment of C. fleckeri envenomation. Indeed this drug is currently indicated for use in the management of Irukandji type cubozoan jellyfish stings (Corkeron et al., 2004, Corkeron, 2003) and has also been shown to increase the efficacy of box jellyfish antivenom in anaesthetised rats (Ramasamy et al., 2004).

In light of the many remaining questions about the nature of C. fleckeri-induced pathology and its treatment, the aim of this study was to compare the cardiovascular pharmacology of this venom (as an unpurified (crude) nematocyst venom extract; CVE) in isolated small resistance arteries, left and right atria, as well as in monitored anaesthetised animals. It extends prior studies of venom mechanism and treatment modalities by studying multiple tissues from a relevant mammalian preparation (rat) over a wide range of venom and drug concentrations. It also undertakes the first study of the possible role of a sensory neuropeptide CGRP in this envenomation and provides the first direct assessment of catecholamines in Chironex venom itself.

Section snippets

Collection of specimens

C. fleckeri specimens were collected in the waters offshore from Darwin, Northern Territory, in January and February 1999–2000. Specimens were formally identified by Dr. P. Alderslade (Northern Territory Art Gallery and Museum) and tentacles were removed and frozen at −70 °C for transportation by air to The University of Melbourne. Tentacle samples were stored at −70 °C until required.

Extraction of venom and protein determination

The protocol used to obtain C. fleckeri crude venom extract (CVE) was previously described by Winkel et al. (2005)

Catecholamines

C. fleckeri CVE contained noradrenaline and DOPA (dihydroxyphenylalanine), its precursor, at levels of approximately 13 ng/ml and 3 ng/ml, respectively (data not shown; personal communication, Dr. Gavin Lambert, Baker IDI Heart and Diabetes Institute).

Mesenteric arteries

In isolated mesenteric arteries, in the presence of vehicle (PSS 6 μl) pre-treatment, C. fleckeri CVE caused a concentration-dependent increase in contraction to 62 ± 3% KPSS with an EC50 of 1.15 ± 0.19 μg/ml (n = 5; Fig. 1a; Table 1). Box jellyfish

Discussion

This study provides the first detailed analysis of the in vitro effects of C. fleckeri venom that compares isolated peripheral vascular tissues with left and right atria from a single mammalian species (Fig. 7). As such, it extends previous findings that derive from in vitro studies of mixed species and tissues (Freeman, 1974, Freeman and Turner, 1969, Freeman and Turner, 1972), single vascular tissue types (Ramasamy et al., 2003, Winter et al., 2007) or from non-mammalian species (Ramasamy et

Conflict of interest

There are no conflicts of interest.

Acknowledgements

The authors wish to thank Ms Linda Cornthwaite-Duncan for completing the anaesthetised rat experiments. We also thank Dr Gavin Lambert and Ms Reena Chopra of the Human Neurotransmitters Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, for undertaking the catecholamine assays. The Australian Venom Research Unit was funded by the Australian Government Department of Health and Ageing during the time of these studies.

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