Species-Dependent Variations in the in Vitro Myotoxicity of Death Adder (Acanthophis) Venoms

Janith C. Wickramaratna*, Bryan G. Fry{dagger} and Wayne C. Hodgson*,1

* Monash Venom Group, Department of Pharmacology, Monash University, Victoria 3800, Australia; and {dagger} Australian Venom Research Unit, Department of Pharmacology, University of Melbourne, Victoria 3010, Australia

Received February 26, 2003; accepted April 30, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Based on early studies on Acanthophis antarcticus (common death adder) venom, it has long been thought that death adder snake venoms are devoid of myotoxicity. However, a recent clinical study reported rhabdomyolysis in patients following death adder envenomations, in Papua New Guinea, by a species thought to be different to A. antarcticus. Subsequently, a myotoxic phospholipase A2 component was isolated from A. rugosus (Irian Jayan death adder) venom. The present study examined the venoms of A. praelongus (northern), A. pyrrhus (desert), A. hawkei (Barkly Tableland), A. wellsi (black head), A. rugosus, A. sp. Seram and the regional variants of A. antarcticus for in vitro myotoxicity. Venoms (10–50 µg/ml) were examined for myotoxicity using the chick directly (0.1 Hz, 2 ms, supramaximal V) stimulated biventer cervicis nerve-muscle preparation. A significant contracture of skeletal muscle and/or inhibition of direct twitches were considered signs of myotoxicity. This was confirmed by histological examination. All venoms displayed high phospholipase A2 activity. The venoms (10–50 µg/ml) of A. sp. Seram, A. praelongus, A. rugosus ,and A. wellsi caused a significant inhibition of direct twitches and an increase in baseline tension compared to the vehicle (n = 4–6; two-way ANOVA, p < 0.05). Furthermore, these venoms caused dose-dependent morphological changes in skeletal muscle. In contrast, the venoms (10–50 µg/ml; n = 3–6) of A. hawkei, A. pyrrhus , and regional variants of A. antarcticus were devoid of myotoxicity. Prior incubation (10 min) of CSL death adder antivenom (5 U/ml) prevented the myotoxicity caused by A. sp. Seram, A. praelongus, A. rugosus , and A. wellsi venoms (50 µg/ml; n = 4–7). In conclusion, clinicians may need to be mindful of possible myotoxicity following envenomations by A. praelongus, A. rugosus, A. sp. Seram, and A. wellsi species.

Key Words: Acanthophis; A. antarcticus; antivenom; death adder; myotoxic; phospholipase A2; A. praelongus; rhabdomyolysis; A. rugosus; venom.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Death adders (genus Acanthophis) are unique among Australian snakes in both morphology and behavior. Although classified into the Elapidae family of snakes they are viper-like in appearance and habit (Campbell, 1966Go;Cogger, 2000Go). Death adders are the widest ranging of the Australian elapids being found not only in continental Australia, but north throughout the Torres Straight Islands, Papua New Guinea, Irian Jaya, and the Indonesian islands of Seram, Halmahera, Obi, and Tanimbar. Although up to 12 species and 3 subspecies of death adders have been described thus far (Hoser, 1998Go), considerable debate remains about species identification (Wuster et al., 1999Go). Of these, only the venom of the common (A. antarcticus) death adder has been studied in detail.

Acanthophis antarcticus venom has previously been examined for lethality, neurotoxicity, myotoxicity, and its effects on blood coagulation, both experimentally and clinically (Broad et al., 1979Go;Campbell, 1966Go;Kellaway, 1929aGo,bGo;Mebs and Samejima, 1980Go;Sutherland et al., 1981Go;Wickramaratna and Hodgson, 2001Go). In addition, five postsynaptic neurotoxins and four phospholipase A2 (PLA2) components have been isolated and sequenced from A. antarcticus venom (Chow et al., 1998Go;Kim and Tamiya, 1981aGo,bGo;Sheumack et al., 1979Go,1990Go;Tyler et al., 1997Go;van der Weyden et al., 1997Go). However, no myotoxic components have been isolated from this venom.

Previously, using the chick isolated biventer cervicis nerve-muscle (CBCNM) preparation, we studied the venoms of the northern (A. praelongus), desert (A. pyrrhus), Barkly Tableland (A. hawkei), black head (A. wellsi), Irian Jayan (A. rugosus), and A. sp. Seram for in vitro neurotoxicity (Fry et al., 2001Go). All venoms (1–10 µg/ml) caused dose-dependent neurotoxicity, which was postsynaptic in nature. In the same study, CSL death adder antivenom (1 U/ml), which is raised against A. antarcticus venom, prevented the neurotoxic effects of A. pyrrhus, A. praelongus, and A. hawkei venoms. However, it was markedly less effective against the venoms of A. rugosus, A. wellsi, and A. sp. Seram (Fry et al., 2001Go). At a higher concentration, antivenom (5 U/ml) was effective against all venoms. In another study, the venoms of major species and regional variants of death adders were investigated by liquid chromatography/mass spectrometry (Fry et al., 2002Go). This study revealed a great diversity in venom composition.

Based on early studies on A. antarcticus venom it was thought that death adder venoms were devoid of myotoxic activity (Sutherland et al., 1981Go). A. antarcticus venom displayed no myotoxic activity in vivo in Rhesus monkeys (Macaca fascicularis; Sutherland et al., 1981Go). In another study, Mebs and Samejima (1980)Go fractionated A. antarcticus venom by ion-exchange chromatography. None of the isolated fractions caused myoglobinuria in mice after sc injection. However, a clinical study reported myotoxic activity following death adder envenomations, in Papua New Guinea, by a species thought to be different to A. antarcticus (Lalloo et al., 1996Go). In this study one patient developed renal failure following delayed presentation and two-thirds of envenomed patients had significantly elevated creatine kinase levels (Lalloo et al., 1996Go). This is suggestive of rhabdomyolysis and the possible presence of myotoxic activity in the venom (Sutherland et al., 1981Go). Recently, we have shown that venom of the Irian Jayan death adder (A. rugosus) causes dose-dependent in vitro myotoxicity, and subsequently isolated the first myotoxic PLA2 from a death adder venom (Wickramaratna et al., 2003Go). However, no studies have been performed to determine the effectiveness of CSL death adder antivenom, which has been raised against A. antarcticus venom, in neutralizing the myotoxic activity of A. rugosus venom.

The first aim of this study was to examine the venoms of A. praelongus, A. pyrrhus, A. hawkei, A. wellsi, A. sp. Seram, and the regional variants of A. antarcticus for in vitro myotoxic activity. The second was to determine the effectiveness of CSL death adder antivenom in neutralizing the myotoxic activity of death adder venoms.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Venom preparation and storage.
A. antarcticus venoms were obtained from populations in New South Wales (NSW), Queensland (Qld), South Australia (SA), and Western Australia (WA). A. praelongus venom was from populations in Cairns, Queensland; A. pyrrhus venom from Alice Springs, Northern Territory; A. wellsi venom from the Pilbarra region of Western Australia; A. hawkei venom from the Barkly Tableland region of Northern Territory; A. rugosus venom from Irian Jaya (West Papua), and A. sp. Seram from the island of Seram, Indonesia. Venoms were either purchased from Venom Supplies Pty. Ltd., South Australia, or milked from specimens caught by Dr. Bryan Fry. For each geographic variant or species, venoms were collected and pooled to minimize the effects of individual variations (Chippaux et al., 1991Go). Freeze-dried venoms and stock solutions of venoms prepared in 0.1% bovine serum albumin (BSA) in 0.9% saline were stored at –20°C until required.

Determination of phospholipase A2 activity.
The PLA2 activity of death adder venoms was determined using a secretory PLA2 colorimetric assay kit (Cayman Chemical, Ann Arbor, MI). The assay uses the 1,2-dithio analogue of diheptanoyl phosphatidylcholine as a substrate. Free thiols generated upon hydrolysis of the thio ester bond at the sn-2 position by PLA2 are detected using DTNB (5,5'-dithiobis(2-nitrobenzoic acid)). Color changes were monitored by the CERES900C microplate reader (Bio-Tek Instruments, Winooski, VT) at 405 nm, sampling every min for a 5 min period. PLA2 activity was expressed as micromoles of phosphatidylcholine hydrolysed per min per mg of enzyme.

Inactivation of PLA2 activity with 4-bromophenacyl bromide.
The PLA2 activity of A. rugosus, A. sp. Seram, and A. praelongus venoms were inhibited by alkylation with 4-bromophenacyl bromide (4-BPB). A. rugosus, A. sp. Seram, and A. praelongus venoms (10,000 µg/ml) were made up in sodium cacodylate-HCl buffer (25 µl, 0.1 M, pH 6.0), and 4-BPB made up in acetone was added to give a final concentration of 1.8 mM (Abe et al., 1977Go;Bell et al., 1998Go;Crachi et al., 1999bGo). Each vial containing the above solution was then incubated at 30°C for 16 h. As a positive control, A. rugosus, A. sp. Seram, and A. praelongus venoms (10,000 µg/ml) made up in sodium cacodylate-HCl buffer were incubated with acetone. As a negative control, sodium cacodylate-HCl buffer was incubated with 1.8 mM 4-BPB in acetone.

Chick isolated biventer cervicis nerve-muscle preparation.
Male White leg horn chicks aged between 9 and 11 days were killed with CO2 and both biventer cervicis nerve-muscle preparations were removed. These were mounted under 1 g resting tension in organ baths (5 ml) containing Krebs solution of the following composition (mM): NaCl, 118.4; KCl, 4.7; MgSO4, 1.2; KH2PO4, 1.2; CaCl2, 2.5; NaHCO3, 25; and glucose, 11.1. The Krebs solution was bubbled with carbogen (95% O2 and 5% CO2) and maintained at 34°C. Direct twitches were evoked by stimulating the muscle directly every 10 s with pulses of 2 ms duration at a supramaximal voltage (Harvey et al., 1994Go) using a Grass S88 stimulator. After a 30-min equilibration period, to ensure selective stimulation of muscle, d-tubocurarine (10 µM) was added and left in the organ bath for the duration of the experiment. Death adder venoms (10–50 µg/ml), 4-BPB modified A. rugosus, A. sp. Seram, and A. praelongus venoms (50 µg/ml) or relevant controls were left in contact with the preparations for a 3 h period. A significant contracture of skeletal muscle (i.e., a rise in baseline) and/or inhibition of direct twitches were considered signs of myotoxicity (Harvey et al., 1994Go). Where indicated, CSL death adder antivenom (5 U/ml) was added 10 min prior (Barfaraz and Harvey, 1994Go;Crachi et al., 1999aGo;Fry et al., 2001Go;Wickramaratna and Hodgson, 2001Go) to the addition of death adder venoms (50 µg/ml).

Morphological studies.
After the conclusion of the functional myotoxic experiments, the tissues were quickly placed in Tissue Tek and frozen with liquid nitrogen. The tissues were stored at –80°C until required. Using a Leica CM1800 cryostat, tissues were cut into transverse sections (14 µm) and placed onto gelatin-coated slides. Tissue sections were post fixed for 15 min in a solution containing 4% paraformaldehyde in phosphate buffered saline (PBS; [mol/l] NaCl, 0.137; KH2PO4, 0.002; and Na2HPO4, 0.008). Tissue sections were routinely stained with haematoxylin and eosin, and examined under a light microscope (Olympus BX 51, Olympus Optical Co., Japan). Areas exhibiting typical pathological changes were photographed using an Olympus C-4040ZOOM (Olympus Optical Co., Japan) digital camera.

Chemicals and drugs.
The following drugs and chemicals were used: 4-bromophenacyl bromide (4-BPB), BSA, cacodylic acid (sodium cacodylate), d-tubocurarine chloride, eosin, Mayer’s Hematoxylin solution (Sigma Chemical Co., St. Louis, MO). Except where indicated, stock solutions were made up in distilled water. 4-BPB was made up in acetone. Death adder antivenom, which is raised against A. antarcticus venom in horses, was obtained from CSL Ltd. (Melbourne, Australia).

Analysis of results and statistics.
For isolated tissue experiments, responses were measured via a Grass force displacement transducer (FT03) and recorded on a MacLab System. The twitch height was expressed as a percentage of the initial twitch height (i.e., prior to the addition of venom or vehicle). Full data (i.e., response curves over a 3 h period) are shown for twitch height and baseline tension at 50 µg/ml venoms. However, for brevity full data are not shown at venom concentrations of 10 µg/ml and 30 µg/ml. Instead, data for all venom concentrations are summarized in Figures 2a and 2bGo using only the twitch height and baseline tension values at the 180-min time point. Statistical difference was determined by a two-way ANOVA on the twitch heights, at the 180-min time point, at different concentrations of venoms. Likewise, a two-way ANOVA was performed on the contractile responses induced by the venoms at different concentrations at the 180-min time point (i.e., only the data at the 180-min time point have been statistically analyzed). For 4-BPB modified venom studies, statistical difference was determined by a two-way ANOVA on the data at the 180-min time point. Statistical difference between the PLA2 activity of 4-BPB treated and untreated venom was determined by a Student’s unpaired t-test. All ANOVAs were followed by a Bonferroni-corrected multiple t-test. Statistical significance was indicated when p < 0.05. All statistical tests were carried out using the SigmaStat (ver. 1.0) software package.



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FIG. 2. The effect of Acanthophis venoms (50 µg/ml; n = 3–6) or vehicle (n = 6) on (a) direct twitches or (b) baseline tension of the CBCNM preparation. *p < 0.05, significantly different from vehicle, two-way ANOVA.

 

    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Phospholipase A2 Activity
High PLA2 activity was detected in all death adder venoms (Table 1Go). While there was a large variation in the PLA2 activity of death adder venoms, A. pyrrhus venom had the highest specific activity, 476.4 ± 12.4 µmol/min/mg (n = 12). The positive control, bee venom PLA2, had a specific activity of 287.5 ± 17.5 µmol/min/mg (n = 4). 4-BPB treated A. rugosus, A. sp. Seram, and A. praelongus venoms had significantly reduced PLA2 activities of 2.0 ± 0.9, 1.7 ± 1.1, and 1.8 ± 1.0 µmol/min/mg (n = 8) compared to their untreated venoms with specific activities of 140.2 ± 10.4, 420.4 ± 10.8, and 255.0 ± 8.6 µmol/min/mg, respectively (n = 6–8; Student’s unpaired t-test, p < 0.05).


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TABLE 1 Phospholipase A2 Activity of Death Adder Venoms
 
Chick Isolated Directly-Stimulated Biventer Cervicis Nerve-Muscle Preparation
Myotoxic studies.
The venoms (10–50 µg/ml) of A. antarcticus (NSW, Qld, SA, WA), A. hawkei, A. pyrrhus, and A. wellsi had no significant inhibitory effect on the direct twitches compared to the vehicle (n = 3–6; two-way ANOVA, p < 0.0001; Figs. 1a and 2aGoGo). In contrast, A. sp. Seram venom (10–50 µg/ml) caused a significant inhibition of direct twitches compared to the vehicle (n = 4–6; Figs. 1a and 2aGoGo). However, this effect was not concentration-dependent as there was no significant difference in the twitch inhibition caused by A. sp. Seram venom at 10 µg/ml and 50 µg/ml (n = 4; Fig. 2aGo). Both A. praelongus venom (30–50 µg/ml) and A. rugosus venom (30–50 µg/ml) caused a significant inhibition of direct twitches compared to the vehicle (n = 4–6; Figs. 1a and 2aGoGo). This effect was concentration-dependent with A. praelongus (50 µg/ml) and A. rugosus (50 µg/ml) venoms causing a significantly greater inhibition of direct twitches compared to A. praelongus and A. rugosus venom at 10 µg/ml, respectively (n = 5–6; Fig. 2aGo). When taking all concentrations into consideration A. sp. Seram venom was significantly more potent in causing direct twitch inhibition than either A. praelongus venom or A. rugosus venom (n = 4–6). In contrast, there was no significant difference between A. praelongus venom and A. rugosus venom (n = 5–6; Fig. 2aGo).



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FIG. 1. The effect of Acanthophis venoms (10–50 µg/ml; n = 3–6) or vehicle (n = 6) on (a) direct twitches or (b) baseline tension of the CBCNM preparation at the 180-min time point. *p < 0.05, significantly different from vehicle, two-way ANOVA.

 
The venoms (10–50 µg/ml) of A. antarcticus (NSW, Qld, SA, WA), A. hawkei, and A. pyrrhus had no significant effect on the baseline tension compared to the vehicle (n = 3–6; two-way ANOVA, p < 0.0001; Figs. 1b and 2bGoGo). While A. wellsi venom (10–30 µg/ml) had no significant effect on the baseline tension, A. wellsi venom (50 µg/ml) induced a significant increase in baseline tension compared to the vehicle (n = 4–6). The venoms (10–50 µg/ml) of A. sp. Seram, A. praelongus, and A. rugosus induced a significant increase in baseline tension compared to the vehicle (n = 4–6). However, there was no significant difference in the baseline contraction caused by A. sp. Seram venom at 10 µg/ml and 50 µg/ml (n = 4; Fig. 2bGo). This was also the case with A. praelongus and A. rugosus venoms.

Antivenom studies.
Prior incubation (10 min) of CSL death adder antivenom (5 U/ml) prevented the inhibition of direct twitches and the increase in baseline tension caused by A. sp. Seram, A. praelongus, A. rugosus, and A. wellsi venoms (50 µg/ml; n = 4–7; Figs. 3a,bGo). A. sp. Seram, A. praelongus, A. rugosus, and A. wellsi venoms (50 µg/ml) in the presence of antivenom (5 U/ml) had no significant inhibitory effect on the direct twitches compared to the antivenom control (n = 4–7; Fig. 3aGo; one-way ANOVA, p = 0.49). Furthermore, A. sp. Seram, A. praelongus, A. rugosus, and A. wellsi venoms (50 µg/ml) in the presence of antivenom (5 U/ml) had no significant effect on the baseline tension compared to the antivenom control (n = 4–7; Fig. 3bGo; one-way ANOVA, p = 0.40).



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FIG. 3. The effect of A. praelongus, A. rugosus, A. sp. Seram, and A. wellsi venoms (50 µg/ml; n = 4–7) or vehicle (BSA; n = 4) in the presence of antivenom (5 U/ml) on (a) direct twitches or (b) baseline tension of the CBCNM preparation. *p < 0.05, significantly different from antivenom control, one-way ANOVA.

 
4-BPB modified venom studies.
A. rugosus, A. sp. Seram, and A. praelongus venoms (50 µg/ml) incubated with 4-BPB had no significant inhibitory effect on direct twitches compared to 4-BPB plus vehicle (n = 4–6; two-way ANOVA, p < 0.0001; Fig. 4aGo). However, A. rugosus A. sp. Seram, and A. praelongus venoms (50 µg/ml) incubated with vehicle (acetone) significantly inhibited direct twitches compared to 4-BPB plus vehicle (sodium cacodylate; n = 4–6; Fig. 4aGo). A. rugosus, A. sp. Seram, and A. praelongus venoms (50 µg/ml) incubated with 4-BPB had no significant effect on the baseline tension compared to 4-BPB plus vehicle (n = 4–6; two-way ANOVA, p < 0.0001; Fig. 4bGo). However, A. rugosus, A. sp. Seram, and A. praelongus venoms (50 µg/ml) incubated with vehicle induced a significant increase in baseline tension compared to 4-BPB plus vehicle (n = 4–6; Fig. 4bGo).



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FIG. 4. The effect of A. rugosus, A. sp. Seram, and A. praelongus venoms (50 µg/ml; n = 4–6) or vehicle (sodium cacodylate; n = 6) incubated with 4-BPB (1.8 mM) on (a) direct twitches or (b) baseline tension of the CBCNM preparation. Positive control was A. rugosus, A. sp. Seram, and A. praelongus venoms (50 µg/ml; n = 5) incubated in vehicle (acetone). *p < 0.05, significantly different from 4-BPB plus vehicle, two-way ANOVA.

 
Morphological studies.
Light microscopy studies of tissues exposed to A. sp. Seram, A. praelongus, A. rugosus, and A. wellsi venoms (10–50 µg/ml) showed dose-dependent morphological changes in skeletal muscle compared to the vehicle control tissues (Figs. 5a–e;Go data not shown for other venoms). These changes included the appearance of necrotic cells, vacuoles, edema, and cellular infiltrate. In contrast, tissues exposed to A. antarcticus (NSW, Qld, SA, WA), A. hawkei, and A. pyrrhus venoms (10–50 µg/ml) were similar in morphology to the vehicle control tissues (Fig. 5fGo; data not shown for other venoms). Prior incubation of CSL death adder antivenom (5 U/ml) prevented most of the morphological changes from occurring due to A. sp. Seram, A. praelongus, A. rugosus, and A. wellsi venoms. In the case of A. rugosus venom (50 µg/ml) a few vacuoles were evident in some tissues even in the presence of antivenom (5 U/ml; Fig. 5gGo). There were no detectable morphological changes in tissues equilibrated with antivenom alone (data not shown). A. rugosus, A. sp. Seram, and A. praelongus venoms (50 µg/ml) incubated with vehicle (i.e., acetone) induced morphological changes similar to the corresponding venom (50 µg/ml). However, no detectable morphological changes were seen in tissues exposed to A. rugosus (Fig. 5hGo), A. sp. Seram and A. praelongus venoms (50 µg/ml) incubated with 4-BPB or vehicle incubated with 4-BPB (data not shown).



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FIG. 5. Transverse sections of CBCNM preparations exposed to (a) vehicle (BSA); (b) A. rugosus venom (10 µg/ml); (c) A. rugosus venom (50 µg/ml); (d) A. sp. Seram venom (50 µg/ml); (e) A. wellsi venom (50 µg/ml); (f) A. antarcticus venom (WA; 50 µg/ml); (g) A. rugosus venom (50 µg/ml) in the presence of antivenom (5 U/ml); (h) A. rugosus venom (50 µg/ml) incubated with 4-BPB (1.8 mM). Scale bars, 100 µm in all micrographs. Arrows indicate prominent vacuoles; arrowheads indicate necrotic cells; double arrows indicate edema; double arrowheads indicate cellular infiltrate.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Based on earlier studies on A. antarcticus venom it was thought that death adder venoms were devoid of myotoxic activity (Mebs and Samejima, 1980Go;Sutherland et al., 1981Go). However, a recent clinical study reported evidence of rhabdomyolysis in patients following death adder envenomations, in Papua New Guinea, by a species different to A. antarcticus (Lalloo et al., 1996Go). More recently, a myotoxic PLA2 from A. rugosus venom was isolated (Wickramaratna et al., 2003Go). Consequently, the present study examined the venoms of A. praelongus, A. pyrrhus, A. hawkei, A. wellsi, A. sp. Seram, and the regional variants of A. antarcticus for in vitro myotoxic activity. In addition, this study examined the effectiveness of CSL death adder antivenom in neutralizing the myotoxic activity of death adder venoms.

Death adder venoms were examined for in vitro myotoxicity using the directly stimulated CBCNM preparation. A. antarcticus (NSW, Qld, SA, WA), A. hawkei, and A. pyrrhus venoms did not cause a significant inhibition of the direct twitch height or an increase in the baseline tension. Furthermore, light microscopy studies indicated that tissues treated with these venoms had morphology similar to vehicle control tissues. Thus, these studies have shown that A. antarcticus (NSW, Qld, SA, WA), A. hawkei, and A. pyrrhus venoms are devoid of in vitro myotoxic activity. While several previous studies have shown that A. antarcticus venom is devoid of myotoxic activity none have examined the regional variations of this venom (Mebs and Samejima, 1980Go;Sutherland et al., 1981Go;Wickramaratna and Hodgson, 2001Go). Liquid chromatography–mass spectrometry studies have shown variations in venom composition among the venoms of A. antarcticus regional variants (Fry et al., 2001 Go,2002Go). Furthermore, functional studies have shown variations in neurotoxicity among the venoms of A. antarcticus regional variants (Fry et al., 2001Go).

Although A. wellsi venom had no effect on the direct twitch height it induced a dose-dependent increase in baseline tension. At the higher concentration, A. wellsi venom also caused morphological changes in skeletal muscle. Thus, suggesting that at higher concentrations this venom causes in vitro myotoxic activity. Both A. praelongus and A. rugosus venoms caused concentration-dependent inhibition of direct twitches, and an increase in baseline tension. Inhibition of direct twitches and a rise in baseline tension have been postulated to be indicative of myotoxic activity (Harvey et al., 1994Go). Light microscopy studies showed that tissues exposed to A. praelongus and A. rugosus venoms caused dose-dependent morphological changes. Although we have previously shown that A. rugosus venom causes in vitro myotoxic activity (Wickramaratna et al., 2003Go), this venom was included in the present study to allow for a comparison between venoms. In contrast to this study, a previous study showed that A. praelongus venom at 30 µg/ml did not cause a significant inhibition of direct twitches compared to the vehicle control (Wickramaratna and Hodgson, 2001Go). However, in that study the venom did cause a significant increase in baseline tension (Wickramaratna and Hodgson, 2001Go). This previous study neither examined a higher concentration of A. praelongus venom nor the morphology of exposed tissues. The use of younger chicks in the previous study may have contributed to this variability between the two studies (Harris, 1991Go).

At all concentrations tested, A. sp. Seram venom caused a significant inhibition of direct twitches and an increase in baseline tension. However, the twitch inhibition and the increase in baseline tension were not dose-dependent. Perhaps, had lower concentrations been tested, a dose-dependent effect may have been observed. Morphological studies however, showed dose-dependent skeletal muscle changes in tissues exposed to A. sp. Seram venom. Clearly, of all death adder venoms tested, A. sp. Seram venom was the most myotoxic.

While death adder envenomations have been uncommon in Australia in recent times due to habitat destruction and consequent decimation of populations, they are still significant health problem in Papua New Guinea and Irian Jaya (Currie, 2000Go;Currie et al., 1991Go;Lalloo et al., 1995, Go1996Go;Sutherland, 1992Go). CSL death adder antivenom is the principal therapy for envenomation by any death adder species (AMH, 2003Go;White, 1998Go). Since A. antarcticus venom lacks myotoxic activity, and given that death adder antivenom has been raised against A. antarcticus venom, it was of clinical relevance to examine the efficacy of death adder antivenom against the in vitro myotoxicity of A. praelongus, A. rugosus, A. sp. Seram, and A. wellsi venoms. Prior incubation of antivenom totally prevented the inhibition of direct twitches and the increase in baseline tension caused by A. praelongus, A. rugosus, A. sp. Seram, and A. wellsi venoms. In addition, antivenom prevented most of the morphological changes from occurring due to these venoms. Therefore, CSL death adder antivenom is effective in neutralizing the in vitro myotoxic activity of death adder venoms. Previously, we have shown that death adder antivenom was effective in neutralizing the in vitro myotoxic activity of acanmyotoxin-1 (Wickramaratna et al., 2003Go).

Since the most important clinical symptoms of death adder envenomations are due to postsynaptic neurotoxicity, anticholinesterase therapy has been suggested to supplement death adder antivenom (Currie et al., 1988Go). Indeed, several clinicians have used anticholinesterases successfully to reduce the amount of antivenom administered (Currie et al., 1988Go;Lalloo et al., 1996Go;Little and Pereira, 2000Go). Anticholinesterase therapy has proven especially useful in Papua New Guinea and Irian Jaya to reduce the high costs associated with the use of death adder antivenom (Currie, 2000Go). However, given the results of the present study, clinicians may need to be mindful of possible myotoxicity following envenomations from A. praelongus, A. rugosus, A. sp. Seram, and A. wellsi species. With concomitant anticholinesterase therapy the neurotoxicity of death adder envenomations may resolve, however, unchecked myotoxicity could cause myoglobinuria and then renal failure.

Previously it was shown that acanmyotoxin-1, a myotoxic component from A. rugosus venom, contained high PLA2 activity (Wickramaratna et al., 2003Go). Studies have also shown that myotoxic fractions from other Australian elapid venoms contain PLA2 activity (Harris and MacDonell, 1981Go;Mebs and Samejima, 1980Go). Liquid chromatography–mass spectrometry studies have shown that death adder venoms contain numerous components with molecular weights representative of PLA2s (Fry et al., 2002Go). Therefore, death adder venoms were examined for PLA2 activity. While high PLA2 activity was detected in all death adder venoms, A. pyrrhus venom had the highest specific activity. In order to examine whether the PLA2 activity of A. rugosus, A. sp. Seram, and A. praelongus venoms is necessary for the myotoxic action, these venoms were subjected to 4-BPB modification. Although a myotoxic PLA2 component has previously been isolated from A. rugosus venom this venom was subjected to 4-BPB modification to determine the presence of other components that may cause myotoxicity but are not mediated by PLA2 activity. Studies have shown that PLA2 activity can be inhibited by acylation using 4-BPB (Abe et al., 1977Go;Volwerk et al., 1974Go). 4-BPB treated A. rugosus, A. sp. Seram, and A. praelongus venoms had significantly reduced PLA2 activity and no myotoxic activity. Thus, suggesting that PLA2 activity is necessary for the myotoxic activity of these death adder venoms. However, no direct relationship was found between the degree of PLA2 activity and the myotoxic activity of death adder venoms. For example, while A. pyrrhus venom had the highest PLA2 activity it was devoid of myotoxic activity. This suggests the presence of other non-myotoxic PLA2 components in those non-myotoxic death adder venoms. In fact, several PLA2 components with antiplatelet activity have been isolated from A. antarcticus and A. praelongus venoms (Chow et al., 1998Go;Sim, 1998Go). Similarly, it is possible that other non-myotoxic PLA2 components may also contribute to the PLA2 activity of myotoxic death adder venoms.

In conclusion, A. sp. Seram, A. praelongus, A. rugosus, and A. wellsi venoms caused in vitro myotoxicity in the CBCNM preparation. In contrast, A. antarcticus (NSW, Qld, SA, WA), A. hawkei, and A. pyrrhus venoms were devoid of myotoxic activity. Although CSL death adder antivenom has been raised against A. antarcticus venom it is effective in neutralizing the myotoxic activity of A. praelongus, A. rugosus, A. sp. Seram, and A. wellsi venoms. Given the results of this study clinicians need to be mindful of possible myotoxicity following envenomations by A. praelongus, A. rugosus, A. sp. Seram, and A. wellsi death adder species.


    ACKNOWLEDGMENTS
 
We thank Dr. Geoff Isbister (Clinical Toxicologist and Emergency Physician, Newcastle Mater Misericordiae Hospital, Australia) for his useful comments. We are grateful to CSL Ltd. for the generous gift of death adder antivenom. This research was funded by a Monash University Small Grant and from financial assistance by the Australia and Pacific Science Foundation. We would also like to thank the relevant departments in Queensland, the Northern Territory, Victoria, and Western Australia for the granting of scientific permits necessary to undertake collection of death adder specimens.


    NOTES
 
1 To whom correspondence should be addressed. Fax: +61-3-99055851. E-mail: wayne.hodgson{at}med.monash.edu.au. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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