* Monash Venom Group, Department of Pharmacology, Monash University, Victoria 3800, Australia; and
Australian Venom Research Unit, Department of Pharmacology, University of Melbourne, Victoria 3010, Australia
Received February 26, 2003; accepted April 30, 2003
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ABSTRACT |
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Key Words: Acanthophis; A. antarcticus; antivenom; death adder; myotoxic; phospholipase A2; A. praelongus; rhabdomyolysis; A. rugosus; venom.
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INTRODUCTION |
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Acanthophis antarcticus venom has previously been examined for lethality, neurotoxicity, myotoxicity, and its effects on blood coagulation, both experimentally and clinically (Broad et al., 1979;Campbell, 1966
;Kellaway, 1929a
,b
;Mebs and Samejima, 1980
;Sutherland et al., 1981
;Wickramaratna and Hodgson, 2001
). In addition, five postsynaptic neurotoxins and four phospholipase A2 (PLA2) components have been isolated and sequenced from A. antarcticus venom (Chow et al., 1998
;Kim and Tamiya, 1981a
,b
;Sheumack et al., 1979
,1990
;Tyler et al., 1997
;van der Weyden et al., 1997
). 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., 2001). All venoms (110 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., 2001
). 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., 2002
). 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., 1981). A. antarcticus venom displayed no myotoxic activity in vivo in Rhesus monkeys (Macaca fascicularis; Sutherland et al., 1981
). In another study, Mebs and Samejima (1980)
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., 1996
). 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., 1996
). This is suggestive of rhabdomyolysis and the possible presence of myotoxic activity in the venom (Sutherland et al., 1981
). 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., 2003
). 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.
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MATERIALS AND METHODS |
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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., 1977;Bell et al., 1998
;Crachi et al., 1999b
). 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., 1994) 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 (1050 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., 1994
). Where indicated, CSL death adder antivenom (5 U/ml) was added 10 min prior (Barfaraz and Harvey, 1994
;Crachi et al., 1999a
;Fry et al., 2001
;Wickramaratna and Hodgson, 2001
) 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, Mayers 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 2b 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 Students 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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RESULTS |
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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 = 47; Figs. 3a,b). 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 = 47; Fig. 3a
; 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 = 47; Fig. 3b
; one-way ANOVA, p = 0.40).
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DISCUSSION |
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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, 1980;Sutherland et al., 1981
;Wickramaratna and Hodgson, 2001
). Liquid chromatographymass spectrometry studies have shown variations in venom composition among the venoms of A. antarcticus regional variants (Fry et al., 2001
,2002
). Furthermore, functional studies have shown variations in neurotoxicity among the venoms of A. antarcticus regional variants (Fry et al., 2001
).
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., 1994). 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., 2003
), 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, 2001
). However, in that study the venom did cause a significant increase in baseline tension (Wickramaratna and Hodgson, 2001
). 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, 1991
).
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, 2000;Currie et al., 1991
;Lalloo et al., 1995,
1996
;Sutherland, 1992
). CSL death adder antivenom is the principal therapy for envenomation by any death adder species (AMH, 2003
;White, 1998
). 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., 2003
).
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., 1988). Indeed, several clinicians have used anticholinesterases successfully to reduce the amount of antivenom administered (Currie et al., 1988
;Lalloo et al., 1996
;Little and Pereira, 2000
). 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, 2000
). 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., 2003). Studies have also shown that myotoxic fractions from other Australian elapid venoms contain PLA2 activity (Harris and MacDonell, 1981
;Mebs and Samejima, 1980
). Liquid chromatographymass spectrometry studies have shown that death adder venoms contain numerous components with molecular weights representative of PLA2s (Fry et al., 2002
). 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., 1977
;Volwerk et al., 1974
). 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., 1998
;Sim, 1998
). 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.
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ACKNOWLEDGMENTS |
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NOTES |
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