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

Toxicon 45 (2005) 233–242 www.elsevier.com/locate/toxicon

A functional comparison of the venom of three Australian jellyfish—Chironex fleckeri, Chiropsalmus sp., and Carybdea xaymacana—on cytosolic Ca2C, haemolysis and Artemia sp. lethality* Paul M. Baileya,e, Anthony J. Bakkerb, Jamie E. Seymourc, Jacqueline A. Wilcea,d,* a

Department of Biochemistry and Molecular Biology, School of Biomedical and Chemical Sciences, The University of Western Australia, Perth, WA 6009, Australia b Department of Physiology, School of Biomedical and Chemical Sciences, The University of Western Australia, Perth, WA 6009, Australia c Department of Tropical Biology, James Cook University, McGregor Road, Cairns, Qld 4878, Australia d Department of Chemistry, School of Biomedical and Chemical Sciences, The University of Western Australia, Perth, WA 6009, Australia e Department of Emergency Medicine, School of Primary, Aboriginal and Rural Health Care, The University of Western Australia, Perth, WA 6009, Australia Received 15 June 2004; revised 15 October 2004; accepted 18 October 2004

Abstract 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 Ca2C, haemolysis and Artemia sp. lethality are compared for the first time. All three venoms caused a large, irreversible elevation of cytosolic Ca2C in myocytes as measured using the Ca2C sensitive fluorescent probe Indo-1. The L-type Ca2C channel antagonist verapamil had no effect on Ca2C influx whilst La3C, 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. q 2004 Elsevier Ltd. All rights reserved. Keywords: Cnidarian venoms; Jellyfish; Chironex fleckeri; Chiropsalmus sp.; Carybdea xaymacana; Calcium; Verapamil; Haemolysis; Lethality

1. Introduction

*

None of the authors has a conflict of interest. * Corresponding author. Address: Department of Chemistry, School of Biomedical and Chemical Sciences, The University of Western Australia, Perth, WA 6009, Australia. Tel.: C61 8 9380 3337; fax: C61 8 9380 1148. 0041-0101/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2004.10.013

Cnidarian venoms produce greatly differing effects on humans that do not correlate with jellyfish tentacle size or jellyfish morphology. From the irritation of stings by Chiropsalmus sp. to the lethal effects of Chironex fleckeri and the Irukandji syndrome resulting from Carukia barnesi envenoming, jellyfish venoms clearly vary in activity

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and composition. Comparative studies have been hindered by variations in venom preparation and by the poor availability of a range of venoms to single research groups. A superior method for venom extraction from isolated nematocysts has only recently been described (Bloom et al., 1998). This and efforts to conduct functional experiments in parallel (Ramasamy et al., 2003) will greatly assist the investigation of Cnidarian venoms. In particular, in vitro functional effects of venom will be able to be correlated with their in vivo potencies and thus aid in the identification of the as yet unknown lethal factors in the venoms. The current study compares, for the first time, the functional effects of identically prepared venoms from three Australian jellyfish with varying human toxicities— C. fleckeri, Chiropsalmus sp., and Carybdea xaymacana. Found in coastal waters of Northern Australia, C. fleckeri is the most lethal animal on the planet (Endean and Sizemore, 1988). In humans, contact with C. fleckeri tentacles results in dermonecrosis, pain and in severe cases, death (O’Reilly et al., 2001). The causes of both human and experimental animal lethality in C. fleckeri envenoming remain unclear, but a combination of cardiac and respiratory decompensation are thought to be the key events (Currie, 1994; Tibballs et al., 1998). C. fleckeri venom has been documented to possess haemolytic, myotoxic, dermonecrotic and lethal activities (Baxter and Marr, 1969; Endean and Henderson, 1969; Turner and Freeman, 1969; Endean, 1987; Collins et al., 1993). Chiropsalmus sp. is found over a narrower geographic range than C. fleckeri—from Innisfail to Cooktown on the Queensland coast (Carrette et al., 2002). It is similar, but distinct from Chiropsalmus quadrigatus and has not yet been attributed a species name. Chiropsalmus sp. is also morphologically similar to C. fleckeri. There have, however, been no deaths recorded due to envenoming by this species in Australian waters. The stings of this jellyfish are noted for local symptoms only. Experimental animal models of Chiropsalmus sp. envenoming have revealed both dermonecrotic and lethal activities(Williamson et al., 1996). A small carybdeid jellyfish found along the coastal waters of Western Australia during the summer months has been tentatively identified as C. xaymacana (L. Gershwin, pers. comm.). It is structurally distinct from Carybdea rastonii, which is found along Australia’s southern coastline. Far smaller than either C. fleckeri or Chiropsalmus sp., the sting is noted for local irritation only. To date, nothing has been reported about the effects or mechanism of action of the venom of C. xaymacana. Fundamental knowledge of the mechanisms of toxicity of jellyfish venom remains in its infancy. C. fleckeri venom has been reported to cause contraction of arterial smooth muscle in isolated vessel preparations (Freeman, 1974), and reduced coronary artery blood flow, bradycardia and negative inotropic effects in isolated perfused hearts (Turner and Freeman, 1969). When administered intravenously to animals in experimental models of envenoming, C. fleckeri

venom produces profound hypotension, cardiac arrhythmias, neurotoxic and myotoxic effects, red cell haemolysis and death within seconds to minutes (Tibballs et al., 1998). Studies of the venom have suggested the presence of large protein toxins that could underlie the myotoxic and neurotoxic effects (Endean, 1987; Endean et al., 1993). More recent studies confirm that the toxins present are likely to be proteinaceous since antivenom is able to neutralize the activity in vitro (Ramasamy et al., 2003). The specific actions of the toxins, though, have yet to be discerned. Ca2C plays a central role as an intracellular second messenger in all cells, and in cardiac muscle it plays a vital role in the excitation contraction coupling process. After stimulation of the cardiac myocyte, Ca2C influx through L-type Ca2C channels triggers Ca2C release from the intracellular Ca2C store (a process known as Ca2C-induced Ca2C release, or CICR) and the ensuing rise in cytosolic Ca2C activates the contractile response. It has therefore been suggested that disturbances to cardiac myocyte Ca2C handling may underlie the cardiac dysrhythmias and sudden cardiac standstill observed in experimentally envenomed animals and envenomed humans (Mustafa et al., 1995). In vitro haemolytic activity has been reported in a variety of jellyfish venoms (Crone, 1971; Cariello et al., 1988; Bloom et al., 2001; Chung et al., 2001; Radwan et al., 2001), and against erythrocytes from a number of different species, including human. In three species of jellyfish, this activity has been attributed to a 43 kDa proteinaceous toxin, recently isolated and sequenced (Nagai et al., 2000a,b; Chung et al., 2001; Nagai et al., 2002). The haemolytic toxins, isolated from the carybdeid jellyfish Carybdea rastonii, Chiropsalmus quadrigatus and Carybdea alata demonstrate significant homology with each other, but not with other known proteins, suggesting that these toxins represent a novel class of bioactive protein. It is thought that they form pore like structures in target membranes causing rapid cell lysis (Edwards et al., 2002). Electron microscopy of cell membranes treated with Physalia physalis venom revealed the presence of such pores with a diameter of 10–80 nm (Edwards et al., 2002). Jellyfish venom has also been reported as being toxic to a wide variety of cells other than erythrocytes (Carli et al., 1996; Cao et al., 1998; Bloom et al., 2001; Sun et al., 2002). In the current study we have compared the in vitro toxic effects of venom from three Australian jellyfish with widely varying human toxicity—C. fleckeri, Chiropsalmus sp., and C. xaymacana—on intracellular Ca2C concentration in cultured rat ventricular myocytes, and haemolysis in both human and sheep erythrocytes. We report a surprising contrast in the cellular toxicities observed for the three species, with the known differences in human lethality and our determined lethality of these venoms to brine shrimp (Artemia sp.). We also report the pore forming activity of C. fleckeri venom, and suggest that this activity is present in all three cnidarian venoms, and can account for the lytic effects of the venoms.

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2. Materials and methods 2.1. Venom preparation C. fleckeri and Chiropsalmus sp. were captured in waters near Cairns, Queensland, Australia. C. xaymacana were captured in waters near Perth, Western Australia. Following capture, whole tentacles were promptly cut from the bell, suspended in sea water, and stored at 4 8C. Nematocysts were separated from tentacular debris by filtration following release by autolysis of the tentacle over a 3-day period. The resultant nematocyst containing sediment was lyophilized and stored at K80 8C. When required, approximately 0.2 g of freeze dried nematocysts were resuspended in 2 mL of double distilled water, and then subjected to 8!30 s sonication cycles at 4 8C with 1 min rest between cycles (Bloom et al., 1998). The nematocyst suspension was then subjected to two freeze thaw cycles with liquid nitrogen. Nematocyst rupture was found to exceed 95% when formally assessed by light microscopy. The nematocyst preparation was then ultracentrifuged at 50,000 rpm for 30 min to clear cellular debris from the venom solution. Relative ‘venom concentrations’ were based upon the total protein concentration in solution. This was determined using a modification of the Bradford (1976) technique (Biorad). 2.2. Myocyte preparation Cardiac ventricular myocytes were isolated from 1-day old Harlin Sprague Dawley rats by Mr Dominic Ng (UWA) as previously reported (Bogoyevitch et al., 1995). In brief, ventricular cells were isolated by collagenase digestion, and subsequently pre plated to deplete cardiac fibroblast populations. Cardiac myocytes were then plated onto laminin coated glass cover slips in modified Eagles medium and incubated at 37 8C with 0.5% CO2 until required. 2.3. Measurement of cytosolic free Ca2C flux using the fluorescent probe Indo-1 Indo-1 is a UV-excitable fluorescent Ca2C indicator that undergoes a large blue shift in its emission ratio from 485 to 405 nm upon Ca2C binding, allowing for measurement of intracellular [Ca2C]. Cardiac ventricular myocytes were loaded with the acetoxymethyl ester of Indo-1 (Indo-1 AM) (Teflabs) for 30 min at 37 8C. Pluronic F127 (0.1%)(Teflabs), a surfactant, was used to aid the entry of Indo1-AM into the cells. Indo1-AM is rapidly hydrolysed within the cell by non-specific esterases to form free, Ca2Csensitive Indo-1, formaldehyde and acetic acid. Indo-1 loaded cells were placed in a heated 1 ml bath chamber with a replaceable coverglass bottom on the stage of an inverted microscope (Nikon) configured for epifluorescence. The cells were superfused with Krebs solution

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(NaCl 120 mM, KCl 5.4 mM, CaCl2 2.5 mM, MgSO4 1.2 mM, NaHCO3 25 mM, glucose 11 mM, Na-HEPES 5 mM, pH 7.3 after gassing with 5% CO2) and maintained at 37 8C. The excitation wavelength of 340 nm was delivered by a variable monochromator system, and the emission spectra were measured at 405 and 485 nm using a spectrophotometer (Cairn, UK). The appearance and contractile activity of the myocytes could be observed during the experiments via a red light/CCD camera system integrated into the fluorescence detector system. Addition of venom to the bath chamber was made by use of a needle and syringe. The ratio (R) of emission intensities from Indo-1 was converted to an estimation of cytosolic [Ca2C] according to the equation (Grynkiewicz et al., 1985): ½Ca2CZ Kd bðRK Rmin Þ=Rmax K R. Kd (230 nM) is the dissociation constant of Indo-1, and b is the ratio of 405 nm emission spectra under zero and maximal [Ca2C]. Rmin and Rmax are the Indo-1 emission ratios under Ca2C free and maximal Ca2C conditions, respectively. Rmin, Rmax and b were measured by performing in vitro calibration experiments in high and low Ca2C solutions that mimicked the intracellular environment with respect to ionic strength, osmolarity and pH (Bakker et al., 1998), and contained either maximal or close to zero free Ca2C concentrations. The membrane impermeable, Ca2C-sensitive form of Indo-1 (Indo-1 pentapotassium, Teflabs) was used in the calibration experiments. When examining the effects of the inhibitors lanthanum chloride (3 mM) and verapamil (1 mM), cells were incubated in a 1 mL bath of Krebs solution containing the inhibitor at 37 8C for 30 min prior to venom addition. 2.4. Measurement of cytosolic Mn2C flux using the FURA-2 quench reaction Fura-2 is a UV-excitable fluorescent probe that is routinely employed as a Ca2C indicator. Fura-2 also binds Mn2C with high affinity, and under certain conditions Mn2C binding quenches Fura-2 fluorescence (Merritt et al., 1989). Many of the pathways involved in Ca2C influx from the extracellular fluid in mammalian cells are also permeable to Mn2C (Merritt et al., 1989). Because Mn2C is not stored intracellularly, the rate of quenching of the Fura-2 signal after exposure to extracellularly applied Mn2C is indicative of the relative Mn2C permeability of the sarcolemma at that time, and by implication the Ca2C permeability. In these experiments, the cells were loaded with the acetoxymethyl ester of Fura-2 (Fura-2-AM) (Teflabs) and 0.1% pluronic F127 for 30 min at 37 8C. Fura-2-AM is rapidly hydrolysed by non-specific esterases to form free Fura-2, formaldehyde and acetic acid. Fura-2 loaded cells were exposed to an excitation wavelength of 360 nm, the ‘isobestic’ wavelength for Fura-2. At this wavelength, Fura-2 is insensitive to changes in [Ca2C] but sensitive to quenching by Mn2C. Fura-2 fluorescence from the myocytes was initially monitored in standard, Mn2C free Krebs solution. MnCl2 was

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then added to the bath, bringing the final concentration of Mn2C to 50 mM. Myocytes were found to have a small initial quench reaction that was observed as a steady decline in the fluorescence signal. Finally, venom was added to the organ bath. Any change in the slope of the decline of the fluorescence trace is indicative of a change in the rate of Mn2C influx from extracellular sources. Results are expressed as the rate of decline of the fluorescence signal. All results were measured immediately following changes in cell conditions where the rate of Fura-2 quenching is linear. The effect of the toxins on intracellular Ca2C levels was analysed using a two way analysis of variance using the statistical software package STATISTICA (Statsoft, Inc. USA). The effect of channel blockers on attenuation of the toxin-induced increase in cytosolic Ca2C was examined using a paired t-test. 2.5. Haemolysis experiments Haemolysis experiments were performed using both sheep and human erythrocytes. Human erythrocytes were obtained by peripheral venepuncture from a volunteer (JAW); the sheep erythrocytes were obtained by peripheral venous puncture of a sheep housed at the Large Animal Facility, UWA. Following venepuncture, the blood was placed into a heparinized tube and stored at 4 8C until required. To obtain a pure suspension of erythrocytes, 1 mL of whole blood was then made up to 20 mL in phosphate buffered saline (PBS), and centrifuged at 1500g for 5 min at 4 8C. The supernatant and buffy coat were then removed by gentle aspiration, and the process repeated two times. Erythrocytes were finally resuspended in PBS to make a 1% solution for haemolysis experiments. A series of venom dilutions were applied to the erythrocyte suspensions and then incubated at 37 8C for 30 min. The tubes were then centrifuged at 2000g for 10 min, and the concentration of released haemoglobin measured at 545 nm. For all experiments, a positive control was obtained by the addition of 1% triton to the erythrocyte suspension. To enable comparison of haemolysis experiments performed on different days, the volume of erythrocyte suspension used was adjusted such that 100% haemolysis corresponded to an OD545 of 0.8. All results are expressed as a percentage of complete haemolysis. The haemolysis measurement for each dilution was repeated eight times, and the experiments were repeated (nZ3) with different venom preparations. 2.6. Artemia lethality assay Artemia sp. were utilized in order to compare the ability of the venom of jellyfish to cause death in a crustacean model of jellyfish envenoming. Artemia sp. were purchased from a local aquarium supplier. A small number of animals (6–22) were added to 5 mL of filtered fresh sea water obtained from a local beach. Whole jellyfish venom was

added to the seawater/Artemia sp. suspension and left at room temperature overnight. Control experiments were performed with distilled water in place of venom. Lethality was assessed at 24 h post venom addition. A range of jellyfish venom dilutions were assessed (nZ4). 2.7. Electron microscopy Neonatal rat cardiac myocytes were isolated and cultured in 24-well plates in modified Eagles medium at 37 8C and 0.5% CO2 for 48 h. In a method similar to that described by Edwards et al. (2002), the cells were rinsed twice with Krebs solution, and then exposed to C. fleckeri venom in Krebs solution for 10 min at 37 8C. Following this, the cells were scraped from the wells and then centrifuged for 10 min at 10,000g. The resulting pellet was separated from the supernatant and then resuspended in a small volume of Krebs solution. A drop of the treated cell solution was placed on a Formvar, carbon coated grid and allowed to air dry. The cells were then negatively stained with 2% ammonium molybdate to enable visualization by transmission electron microscopy (JEOL 2000FX II TEM).

3. Results 3.1. The effect of jellyfish venom on intracellular [Ca2C] in cardiac myocytes In order to investigate the direct effect of the jellyfish venom on cardiac cells, and to compare the in vitro activities of the jellyfish venom from three disparate jellyfish species, we examined the effect of venom exposure on cultured cardiac myocytes loaded with the fluorecent Ca2C indicator Indo-1. Prior to venom application, myocyte baseline [Ca2C] was 106G14.9 nM (nZ10). Periodic brief Ca2C transients that corresponded to the frequency of the observed spontaneous contractions were observed in many of the myocytes, indicating the cultures had reached a stage of differentiation where normal excitation–contraction coupling had developed. Application of venom (bath venom protein concentrations: Chiropsalmus sp. 45 mg/mL, C. xaymacana 71 mg/mL, C. fleckeri 55 mg/mL) resulted in a short ‘latent’ period of little change in basal cytosolic Ca2C, during which Ca2C transients and spontaneous contractions ceased. After this latent period of between 5 and 30 s, a rapid and large, sigmoidal elevation in intracellular [Ca2C] occurred in the toxin-exposed myocytes, which reached a steady state level between 0.9 and 1.8 mM (effect of venom, F2,1Z57.6, p!0.0001) (Fig. 1A; Table 1). Venom treated cells did not appear to recover from exposure to venom. Cells observed for 10 min after venom application did not resume beating and assumed a constant contracted state. Approximately 150–180 s after venom exposure, a slow decline in both the 410 and 490 nm fluorescence emission

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jellyfish) is not more potent than that of Chiropsalmus sp. (its relatively harmless cousin). 3.2. A significant proportion of Ca2C enters venom treated cardiac myocytes from the extracellular fluid

Fig. 1. Measurement of cytosolic free Ca2C in cultured cardiac myocytes in response to C. fleckeri jellyfish venom. (A) Application of venom results in a short latent period followed by cessation of beating and a rapid sustained elevation of intracellular [Ca2C]. (B) Preincubation of cells with 1 mM verapamil did not affect the venom Ca2C response. (C) Preincubation of cells with 3 mM La3C resulted in a marked attenuation of the venom Ca2C response. Similar responses were measured for Chiropsalmus sp. and Carybdea xaymacana venoms.

was noted. This could indicate that the cells are becoming permeable to Indo-1, or alternatively that the cells are gradually swelling, leading to a decrease in the absolute concentration of Indo-1 in the intracellular environment. Remarkably, there was a similar Ca2C influx upon venom addition from all three jellyfish species (comparison of venom effect, F1,2Z3.72, pZ0.06, the interaction of the ANOVA was also not significant, pZ0.11), in contrast to their vastly differing human toxicities (Table 1). Even when the slight differences in venom concentration are taken into consideration, the venom of C. fleckeri (the Deadly box

To further investigate the major source of the observed rise in intracellular [Ca2C], a series of Mn2C Fura-2 quench reactions with whole C. fleckeri venom were performed. Mn2C is able to enter cells via the same pathways as Ca2C, but unlike Ca2C is not stored intracellularly to any significant extent. Hence, any quench of the Fura-2 signal must be due to the influx of extracellular Mn2C (Merritt et al., 1989) (Fig. 2). The experiment, as described above (Section 2), is performed in three stages. Stage 1, in which Mn2C has not been added, produced a decline in the Fura-2 signal of 0.98G0.29 units/s (nZ4), reflecting normal Fura-2 photo bleaching. Following addition of Mn2C (bath concentration 50 mM), the rate of Fura-2 signal decline was 9.8G 6.1 units/s (nZ3) reflecting entry of Mn2C ions into the cell, and indicating a small but significant permeability to Ca2C in the myocytes under resting conditions. Addition of C. fleckeri venom (bath venom concentration 55 mg/mL) resulted in a large increase in the rate of quenching of the Fura-2 signal to 31.4G7.6 units/s (nZ5) (comparison of pre and post venom addition Fura-2 quenching with paired t-test pZ0.048). This result shows that venom addition dramatically increases the rate of extracellular Mn2C influx, and by implication Ca2C permeability in venom treated myocytes. Therefore, the observed rise in intracellular [Ca2C] detected in earlier experiments in this study in the presence of the toxins is likely to be substantially due to the entry of Ca2C from the extracellular fluid. Unlike Indo-1, the fluorescence output of Fura-2 in quench mode is not ratiometric. This means that it is possible that the decline in Fura-2 fluorescence observed following venom exposure was due to the indicator leaking from the venom treated cells, rather than Fura-2 quench. This is unlikely, however, as the Mn2C induced effect in the presence of the toxins occurred within a matter of seconds, whereas in the Indo-experiments the raw emission fluorescence signal (410 and 490 nm) was stable for at least

Table 1 Response of cardiac myocyte [Ca2C] as measured by Indo-1 fluorescence to exposure to jellyfish venom

2C

Baseline [Ca ] (nM) Whole venom (mM) Whole venomC1 mM Verapamil (mM) Whole venomC3 mM La3C (nM)

Chironex fleckeri

Carybdea xaymacana

Chiropsalmus sp.

101G22 1.2G0.3 1.0G0.3 365G38

124G30 (nZ4) 1.9G0.4 (nZ4)* 1.5G0.05 (nZ2, pZ0.3643)a 460G39 (nZ3, pZ0.0071)a

83G12.1 (nZ4) 0.9G0.07 (nZ4)* – –

(nZ4) (nZ4)* (nZ2, pZ0.4344)a (nZ2, p!0.0001)a

*p!0.0001, two way analysis of variance. a Unpaired t-test comparing results in the presence of ‘inhibitors’ to whole venom results.

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a significant attenuation of the response to venom of C. fleckeri and C. xaymacana (bath venom protein concentrations: C. xaymacana 71 mg/mL, C. fleckeri 55 mg/mL) (Fig. 1C, Table 1). This is consistent with a highly effective blocking of specific or non-specific Ca2C channels other than L-type Ca2C channels. 3.5. The venom of C. fleckeri, Chiropsalmus sp. and C xaymacana is potently haemolytic to human and sheep erythrocytes Fig. 2. Measurement of cytosolic Mn2C flux using the Fura-2 quench reaction. Mn2C added to the extracellular fluid causes a slow rate of signal quenching. Application of C. fleckeri venom increases Fura-2 quenching, implying an increase in the rate of Mn2C entry into the cell.

2.5 min after venom application, indicating that the cells were not permeable to the dye during this time period. 3.3. The organic Ca2C channel blocker, verapamil, does not alter venom induced Ca2C influx Considering that L-type voltage gated Ca2C channels allow a large influx of Ca2C to occur during the excitation– contraction coupling process in myocytes, they make a likely candidate route for Ca2C entry into the myocytes after toxin application. Verapamil, a phenylalkamine organic L-type Ca2C channel antagonist, has been proposed as a treatment for humans envenomed by C. fleckeri (Burnett, 1990). Preincubation of cells with 1 mM verapamil for 30 min prior to venom addition produced no significant alteration in elevation of intracellular [Ca2C] seen in response to C. fleckeri (nZ2), and C. xaymacana (nZ2) (bath venom protein concentrations: C. xaymacana 71 mg/ mL, C. fleckeri 55 mg/mL) (Fig. 1B, Table 1). Whist these results do not provide evidence to dismiss the utility of verapamil in envenomed humans, they demonstrate that Ca2C is entering the cytoplasm via a mechanism other than verapamil inhibitable L-type voltage gated Ca2C channels.

Haemolysis measurements provide a rapid, easily reproducible and quantifiable method for comparison of the cytolytic properties of different jellyfish venoms. Haemolysis assays were performed with both human and ovine erythrocytes across a wide range of venom concentrations. The venom of all three jellyfish were potently haemolytic to both sheep and human erythrocytes (Fig. 3). Haemolytic activity was complete (100%) in undiluted venom (venom well concentrations: C. fleckeri 137.5 mg/ mL, Chiropsalmus sp. 112.5 mg/mL, C. xaymacana 177.5 mg/mL) from all three species within minutes. Differences in haemolytic activity between venoms could be detected at lower venom concentrations. Upon venom dilution, the haemolytic activity of the three venoms dropped off at different rates. The venom of C. xaymacana was found to be the most potently haemolytic, retaining greater than 90% haemolytic activity after 104 dilution. This is in great contrast to its low propensity for human toxicity. C. fleckeri venom displayed the next greatest potency and Chiropsalmus sp. was the least haemolytically active.

3.4. The rise in intracellular [Ca2C] is attenuated by lanthanum Transition metal cations, such as La3C, Ni2C, Mn2C and Zn , are known to non-specifically block many types of Ca2C channels. The effect may be due to competitive antagonism of Ca2C binding sites by the metal cations (Mlinar and Enyeart, 1993). They are also known to be potent inhibitors of cytolytic toxins such as a-latrotoxin (Rosenthal et al., 1990), and mellitin (McGahan et al., 1986) via as yet unknown mechanisms. To assess the ability of metal cations to inhibit the Ca2C influx due to venom, we examined the effect of La3C. Preincubation of the cultured neonatal rat cardiac myocytes with 3 mM La 3C for 30 min resulted in 2C

Fig. 3. Haemolysis of erythrocytes. A comparison of the haemolytic activity of the three jellyfish venoms at different dilutions against sheep and human erythrocytes (undiluted venom concentrations: Chironex fleckeri 137.5 mg/mL; Chiropsalmus sp. 112.5 mg/mL; Carybdea xaymacana 177.5 mg/mL). The haemolytic activity of C. xaymacana is sustained at high dilutions. Mean resultGstd error.

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Fig. 4. Comparison of jellyfish venom lethality to Artemia sp. Across a range of venom concentrations (undiluted venom concentrations: Chironex fleckeri 137.5 mg/mL; Chiropsalmus sp. 112.5 mg/mL; Carybdea xaymacana 177.5 mg/mL). C. fleckeri venom displayed the highest potency upon dilution. Mean resultGstd error.

No significant difference in haemolytic activity was noted for the venoms between sheep and human erythrocytes. 3.6. The venom of C. xaymacana, C. fleckeri and Chiropsalmus sp. is lethal to Artemia sp. Given the surprising results of the haemolysis assays, in which the venom of the jellyfish with the least human toxicity demonstrated the greatest haemolytic activity, lethality testing was performed to examine the lethal potential of each of the jellyfish venoms. Undiluted (final bath concentrations: C. fleckeri 11 mg/mL; Chiropsalmus sp. 9 mg/mL and C. xaymacana 14 mg/mL) the venom of all three jellyfish caused significant Artemia sp. lethality (Fig. 4). The venom of C. fleckeri resulted in significantly greater Artemia sp. death at low concentrations when compared with the venom of the other two jellyfish. This is in keeping with the known human toxicities of the animals and is in stark contrast to the haemolytic profiles of the jellyfish venoms. This may indicate that the toxin responsible for red cell haemolysis is not the dominant lethal component. 3.7. Electron microscopy Transmission electron microscopy of neonatal rat ventricular myocyte membranes that had been exposed to the venom of C. fleckeri demonstrated large numbers of circular lesions with an internal diameter of approximately 50–80 nm (Fig. 5). These structures were absent in control preparations that had not been exposed to C. fleckeri venom and confirm the presence of a pore forming component in the venom.

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Fig. 5. Electron micrographs of membrane fragments of cultured neonatal rat cardiac myocytes following exposure to (A) Krebs solution only and (B) C. fleckeri venom for 10 min at 37 8C. Cell membranes were centrifuged and resuspended in Krebs solution and then examined by transmission electron microscopy following staining by ammonium molybdate. Scale bars represent 200 nm.

4. Discussion The present study compares, for the first time, the venom of three different jellyfish from Australian waters—C. fleckeri (the Deadly box-jellyfish), Chiropsalmus sp., and C. xaymacana (which are relatively harmless) and finds striking similarities in the effect of all of these venoms on intracellular Ca2C flux in cultured neonatal rat myocytes. All three venoms caused a prompt sustained increase in intracellular [Ca2C] followed by apparent cell death. All three venoms also caused rapid haemolysis of erythrocytes, though in this case some differentiation between the venom potencies could be made. The Mn2C influx experiments indicated that influx of 2C Ca from the extracellular fluid contributes substantially to the marked increase in cytosolic Ca2C observed in response to application of the jellyfish venom in the cardiac myocytes. However, activation of Ca2C release from intracellular stores of the SR may also play a role, as extracellular Ca2C influx would be expected to subsequently trigger additional Ca2C release from the SR via CICR. The pathway of extracellular Ca2C influx was also investigated. Possible modes of Ca2C entry are via L-type or T-type voltage gated Ca2C channels, the NaC/Ca2C exchanger (working in reverse), via usually inactive, nonspecific cation channels, or via the formation of toxin induced membrane pores. The toxin induced extracellular Ca2C entry is not likely to be via L-type Ca2C channels, since verapamil did not produce any effect on the elevation in cytosolic Ca2C following venom administration. This is consistent with the study by Edwards and Hessinger (2000) who showed that the calcium influx induced in embryonic chick heart cells by Portuguese man-of-war venom is not blocked by organic calcium channel blockers. Ca2C influx was, however significantly inhibited in the presence of La3C

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which is an agent known to inhibit pore forming toxins (Rosenthal et al., 1990; Edwards and Hessinger, 2000). Thus whilst verapamil has been proposed by some as a treatment in severe C. fleckeri stings, there is currently no evidence that the venom of C. fleckeri exerts its effects via L-type Ca2C channels. Specific investigation of C. fleckeri induced Ca2C flux have been minimal, with a single report of Ca2C flux due to C. fleckeri venom in chicken papillary muscle and cultured chicken ventricular myocyte models (Mustafa et al., 1995). No causative agent of Ca2C or other ion-flux irregularities has been identified. Mustafa et al. examined the effect of C. fleckeri venom on isolated chicken papillary muscles and cultured chicken ventricular myocytes. They also observed a large increase in intracellular [Ca2C], though the influx was not as prompt as that observed in our study (taking minutes to occur) and showed different kinetics (the rate of influx began slowly and increased with time). They showed that the Ca2C influx was not inhibited by specific blockers of Ca2C or NaC channels or by inhibitors of the SR, NaC/KC ATPase or NaC/HC exchanger. The toxin response was, however, blocked by bathing the cells in a sodium free solution. Mustafa et al. concluded that the toxin acted primarily by increasing NaC influx into the cell, and that the increase in intracellular Ca2C occurred via the NaC/Ca2C exchange mechanism leading to intracellular Ca2C overload. Whilst this mechanism cannot be ruled out, our data suggests that the venom results in indiscriminant entry of ions from the extracellular fluid. Haemolytic activity has been reported in a wide variety of cnidarian venoms against erythrocytes from many different species (Bloom et al., 2001; Torres et al., 2001; Nagai et al., 2002). We have again confirmed the presence of haemolytic activity in C. fleckeri and Chiropsalmus sp. venom, and for the first time demonstrated haemolytic activity against both sheep and human erythrocytes in the venom of C. xaymacana. Interestingly, in our bioassay, the venom of C. xaymacana demonstrated the greatest haemolytic potency, in contrast to its human toxicity. Recently, the amino acid sequence of haemolytic proteins from Carybdea rastonii, Carybdea alata and Chiropsalmus quadrigatus have been published (Nagai et al., 2000a,b, 2002; Chung et al., 2001). Haemolytic activities have been attributed to proteinaceous toxins w43 kDa that bear sequence similarity to each other, but not to other known proteins. Given the widespread presence of haemolytic activity in a number of jellyfish, a homologous protein is likely to exist in all cnidarian species including those in the current study. The haemolytic factor is, however, unlikely to be a lethal factor in the cnidarian venoms. Not only does our current study show that haemolytic activity does not correlate with lethality, but monoclonal antibodies capable of neutralizing C. fleckeri induced haemolysis do not protect against the lethal effects of the venom in an experimental animal model of envenoming (Collins et al., 1993).

It is also of note that haemolysis, a constant feature of in vitro (Comis et al., 1989; Othman and Burnett, 1990) and experimental animal jellyfish envenoming (Azila et al., 1991; Collins et al., 1993; Endean et al., 1993) has not been demonstrated in envenomed humans. This suggests that either there are fundamental differences between jellyfish venom prepared for laboratory studies and that injected into victims, or that there are specific mechanisms present in envenomed humans but not present in experimental animals that inhibit the toxic haemolytic proteins in jellyfish venom. The haemolytic, pore forming toxin equinatoxin III (a cardiotoxic protein from the sea anemone Actina equina) has demonstrated the ability to pass through a large tissue mass in an animal model of envenoming (Suput et al., 2001). It may be, however, that cnidarian haemolytic toxins sequestered at the site of a human jellyfish sting are not capable of reaching the systemic circulation. Together the evidence points towards the presence of a pore forming toxin that is not the lethal factor in the jellyfish venom. The presence of such a toxin in the venom of C. fleckeri has been demonstrated, in the current study, using transmission electron microscopy of treated myocytes. Pores of 50–80 nm are clearly visible in the cellular membrane, and are not unlike those observed for Physalia physalis venom treated cells (Edwards et al., 2002). We propose that pore formation may underlie both the Ca2C influx and haemolysis caused by the three jellyfish venoms in the current study. The rapidity and enormity of the Ca2C influx in myocytes upon venom treatment, as well as its inhibition in the presence of Ln3C, is consistent with such a mechanism as is the haemolysis of erythrocytes. Lytic venom activity does not, however, appear to underlie the lethal effects of the cnidarian venoms as it does not correlate with the lethality of the venoms from the three species. This raises the question of why the toxin is present and why it does not appear to manifest clinically to any large degree. It may be that pore formation only occurs locally to the site of envenoming—possibly serving to aid the entry of other venom components into the envenomed victim. The lethal components remain the subject of future investigation.

Acknowledgements This work was funded by the Raine Medical Research Foundation of Western Australia (JAW), and an Athelstan and Amy Saw Medical Fellowship, Faculty of Medicine and Dentistry, University of Western Australia (PMB). JAW is an Australian Research Council Fellow. The authors would like to acknowledge the assistance of Mr Dominic Ng, Department of Biochemistry, University of Western Australia in the preparation of myocytes and Prof John Kuo, Centre for Microscopy and Microanalysis, University of Western Australia for assistance with electron micrographs.

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References Azila, N., Siao, F.K., Othman, I., 1991. Haemolytic, oedema and haemorrhage inducing activities of tentacular extract of the blubber jellyfish (Catostylus mosaicus). Comp. Biochem. Physiol. C 99, 153–156. Bakker, A.J., Head, S.I., Wareham, A.C., Stephenson, D.G., 1998. Effect of clenbuterol on sarcoplasmic reticulum function in single skinned mammalian skeletal muscle fibers. Am. J. Physiol. 274, C1718–C1726. Baxter, E.H., Marr, A.G., 1969. Sea wasp (Chironex fleckeri) venom: lethal, haemolytic and dermonecrotic properties. Toxicon 7, 195–210. Bloom, D.A., Burnett, J.W., Alderslade, P., 1998. Partial purification of box jellyfish (Chironex fleckeri) nematocyst venom isolated at the beachside. Toxicon 36, 1075–1085. Bloom, D.A., Radwan, F.F., Burnett, J.W., 2001. Toxinological and immunological studies of capillary electrophoresis fractionated Chrysaora quinquecirrha (Desor) fishing tentacle and Chironex fleckeri Southcott nematocyst venoms. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 128, 75–90. Bogoyevitch, M.A., Ketterman, A.J., Sugden, P.H., 1995. Cellular stresses differentially activate c-Jun N-terminal protein kinases and extracellular signal-regulated protein kinases in cultured ventricular myocytes. J. Biol. Chem. 270, 29710–29717. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Burnett, J.W., 1990. The use of verapamil to treat box-jellyfish stings. Med. J. Aust. 153, 363. Cao, C.J., et al., 1998. Toxicity of sea nettle toxin to human hepatocytes and the protective effects of phosphorylating and alkylating agents. Toxicon 36, 269–281. Cariello, L., Romano, G., Spagnuolo, A., Zanetti, L., 1988. Isolation and partial characterization of rhizolysin, a high molecular weight protein with hemolytic activity, from the jellyfish Rhizostoma pulmo. Toxicon 26, 1057–1065. Carli, A., Bussotti, S., Mariottini, G.L., Robbiano, L., 1996. Toxicity of jellyfish and sea-anemone venoms on cultured V79 cells. Toxicon 34, 496–500. Carrette, T., Alderslade, P., Seymour, J., 2002. Nematocyst ratio and prey in two Australian cubomedusans, Chironex fleckeri and Chiropsalmus sp. Toxicon 40, 1547–1551. Chung, J.J., Ratnapala, L.A., Cooke, I.M., Yanagihara, A.A., 2001. Partial purification and characterization of a hemolysin (CAH1) from Hawaiian box jellyfish (Carybdea alata) venom. Toxicon 39, 981–990. Collins, S.P., Comis, A., Marshall, M., Hartwick, R.F., Howden, M.E., 1993. Monoclonal antibodies neutralizing the haemolytic activity of box jellyfish (Chironex fleckeri) tentacle extracts. Comp. Biochem. Physiol. B 106, 67–70. Comis, A., Hartwick, R.F., Howden, M.E., 1989. Stabilization of lethal and hemolytic activities of box jellyfish (Chironex fleckeri) venom. Toxicon 27, 439–447. Crone, H.D., 1971. The toxic proteins of an Australian jellyfish Chironex fleckeri. Biochem. J. 121, 28P. Currie, B., 1994. Clinical implications of research on the boxjellyfish Chironex fleckeri. Toxicon 32, 1305–1313. Edwards, L., Hessinger, D.A., 2000. Portuguese Man-of-war (Physalia physalis) venom induces calcium influx into cells by permeabilizing plasma membranes. Toxicon 38, 1015–1028.

241

Edwards, L.P., Whitter, E., Hessinger, D.A., 2002. Apparent membrane pore-formation by Portuguese Man-of-war (Physalia physalis) venom in intact cultured cells. Toxicon 40, 1299–1305. Endean, R., 1987. Separation of two myotoxins from nematocysts of the box jellyfish (Chironex fleckeri). Toxicon 25, 483–492. Endean, R., Henderson, L., 1969. Further studies of toxic material from nematocysts of the cubomedusan Chironex fleckeri Southcott. Toxicon 7, 303–314. Endean, R., Sizemore, D.J., 1988. The effectiveness of antivenom in countering the actions of box-jellyfish (Chironex fleckeri) nematocyst toxins in mice. Toxicon 26, 425–431. Endean, R., Monks, S.A., Cameron, A.M., 1993. Toxins from the box-jellyfish Chironex fleckeri. Toxicon 31, 397–410. Freeman, S.E., 1974. Actions of Chironex fleckeri toxins on cardiac transmembrane potentials. Toxicon 12, 395–404. Grynkiewicz, G., Poenie, M., Tsien, R.Y., 1985. A new generation of Ca2C indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3450. McGahan, M.C., Matthews, D.O., Bentley, P.J., 1986. Effects of melittin on a model renal epithelium, the toad urinary bladder. Biochim. Biophys. Acta 855, 63–67. Merritt, J.E., Jacob, R., Hallam, T.J., 1989. Use of manganese to discriminate between calcium influx and mobilization from internal stores in stimulated human neutrophils. J. Biol. Chem. 264, 1522–1527. Mlinar, B., Enyeart, J.J., 1993. Block of current through T-type calcium channels by trivalent metal cations and nickel in neural rat and human cells. J. Physiol. 469, 639–652. Mustafa, M.R., White, E., Hongo, K., Othman, I., Orchard, C.H., 1995. The mechanism underlying the cardiotoxic effect of the toxin from the jellyfish Chironex fleckeri. Toxicol. Appl. Pharmacol. 133, 196–206. Nagai, H., et al., 2000a. Novel proteinaceous toxins from the box jellyfish (sea wasp) Carybdea rastoni. Biochem. Biophys. Res. Commun. 275, 582–588. Nagai, H., Takuwa, K., Nakao, M., Sakamoto, B., Crow, G.L., Nakajima, T., 2000b. Isolation and characterization of a novel protein toxin from the Hawaiian box jellyfish (sea wasp) Carybdea alata. Biochem. Biophys. Res. Commun. 275, 589–594. Nagai, H., Takuwa-Kuroda, K., Nakao, M., Oshiro, N., Iwanaga, S., Nakajima, T., 2002. A novel protein toxin from the deadly box jellyfish (Sea Wasp, Habu-kurage) Chiropsalmus quadrigatus. Biosci. Biotechnol. Biochem. 66, 97–102. O’Reilly, G.M., Isbister, G.K., Lawrie, P.M., Treston, G.T., Currie, B.J., 2001. Prospective study of jellyfish stings from tropical Australia, including the major box jellyfish Chironex fleckeri. Med. J. Aust. 175, 652–655. Othman, I., Burnett, J.W., 1990. Techniques applicable for purifying Chironex fleckeri (box-jellyfish) venom. Toxicon 28, 821–835. Radwan, F.F., et al., 2001. A comparison of the toxinological characteristics of two Cassiopea and Aurelia species. Toxicon 39, 245–257. Ramasamy, S., Isbister, G.K., Seymour, J.E., Hodgson, W.C., 2003. The in vitro effects of two chirodropid (Chironex fleckeri and Chiropsalmus sp.) venoms: efficacy of box jellyfish antivenom. Toxicon 41, 703–711. Rosenthal, L., Zacchetti, D., Madeddu, L., Meldolesi, J., 1990. Mode of action of alpha-latrotoxin: role of divalent cations in Ca2(C)-dependent and Ca2(C)-independent effects mediated by the toxin. Mol. Pharmacol. 38, 917–923.

242

P.M. Bailey et al. / Toxicon 45 (2005) 233–242

Sun, L.K., et al., 2002. Apoptosis induced by box jellyfish (Chiropsalmus quadrigatus) toxin in glioma and vascular endothelial cell lines. Toxicon 40, 441–446. Suput, D., Frangez, R., Bunc, M., 2001. Cardiovascular effects of equinatoxin III from the sea anemone Actinia equina (L.). Toxicon 39, 1421–1427. Tibballs, J., Williams, D., Sutherland, S.K., 1998. The effects of antivenom and verapamil on the haemodynamic actions of Chironex fleckeri (box jellyfish) venom. Anaesth. Intens. Care 26, 40–45.

Torres, M., et al., 2001. Electrophysiological and hemolytic activity elicited by the venom of the jellyfish Cassiopea xamachana. Toxicon 39, 1297–1307. Turner, R.J., Freeman, S.E., 1969. Effects of Chironex fleckeri toxin on the isolated perfused guinea pig heart. Toxicon 7, 277–286. Williamson, J., Fenner, P., Burnett, H.W., 1996. Venomous and Poisonous Marine Animals—A Medical and Biological Handbook. University of New South Wales Press (p. 504).