AN IN VITRO STUDY OF THE EFFECTS OF VENOM OF AUSTRALIAN ELAPIDS ON MURINE SKELETAL MUSCLE AND THE PROTECTIVE EFFECT OF HOMOLOGOUS PLASMA

Original paper

AN IN VITRO STUDY OF THE EFFECTS OF VENOM OF AUSTRALIAN ELAPIDS ON MURINE SKELETAL MUSCLE AND THE PROTECTIVE EFFECT OF HOMOLOGOUS PLASMA

T. L. BUTLER, P. F. JACOBSEN , P. J. MIRTSCHIN, B. A. KAKULAS

1 Australian Neuromuscular Research Institute, Queen Elizabeth II Medical Centre, Nedlands, Western Australia, Australia, 6009; 2 Department of Neuropathology, Royal Perth Hospital, GPO Box X2213, Perth, Western Australia, Australia, 6001; 3 Venom Supplies Pty Ltd, PO Box 547, Tanunda, South Australia, Australia, 5352.

ABSTRACT: The venom of many dangerous Australian snakes has a myotoxic component and some are strongly myolytic. The myotoxicity of venom of seven Australian elapid snakes was studied to determine their relative in vitro potency in causing cell death of C2C12 cells, a myoblast cell line, and murine myotubes in mixed cell culture. The venom of Pseudechis australis proved to be the most myotoxic, Austrelaps superbus and Pseudechis porphyriacus venoms also exhibited high myotoxicity relative to the other venoms tested. The specificity of Pseudechis porphyriacus venom was tested using the human glioma cell line TC3 and was shown to exhibit a general cytotoxicity. Myotoxicity, however, was the predominant action of the venom.

It has long been known that certain animals such as the mongoose (Herpestes edwardsii) are able to survive envenomation. Some species of snakes also possess this property and the neutralising factor(s) responsible for this in P. porphyriacus has been shown to be present in the serum. The protective effect of homologous plasma from P. porphyriacus venom was also studied with reference to myotoxicity and cytotoxicity. The results of this study clearly demonstrated protection by homologous plasma using a myoblast cell line, C2C12, a primary mixed cell culture and TC3 cells. While protection was clear, particularly using high concentrations of venom, it was not absolute, and homologous plasma did not afford continued protection from the effects of the venom. In the mixed cell culture experiments venom/plasma mixtures pre-incubated for 30 min were more protective than venom/plasma mixtures which were not pre-incubated, in contrast to the results of cell culture studies, which showed little difference.

KEY WORDS: snake venom antidote, plasma, myotoxicity, murine skeletal muscle, Australian elapids.

INTRODUCTION

There are approximately 190 recognised species of snakes both terrestrial and marine in Australia (10), including about thirty terrestrial snakes which can be considered dangerous to man (11). The venom of many of these snakes has myotoxic components (14) and a number of studies have been conducted using both crude venoms and purified toxins which show strong myotoxicity for some venoms (3,8,13). None of these studies, however, utilised cultures of myoblasts or differentiated myotubes which allows determination of the specific myotoxic effect of the venoms or toxins. In order to treat envenomation more effectively, a better understanding of the mode of action of the various venoms, which will likely include a number of specific and non-specific effects, is required. Upon this basis, we studied the myotoxicity of the venoms of seven Australian elapid snakes in vitro including the Tiger snake (Notechis scutatus), Peninsula brown (Pseudonaja inframacula), Copperhead (Austrelaps superbus), Red bellied black (Pseudechis porphyriacus), Mulga or King brown (Pseudechis australis), Death adder (Acanthophis antarcticus) and the Taipan (Oxyuranus scutellatus). The venom of all of these snakes is recognised as having myolytic activity with the exception of P. inframacula(14). As an adjunct, we tested the specificity of P. porphyriacus venom which is reportedly strongly myolytic (13,14).

In 1781, Fontanna was the first to note that "the venom of the viper is not venomous to its species" (4). Kellaway in 1931 (9) later demonstrated that the majority of Australian venomous snakes had a significant degree of immunity, not only to their own venom but also to venoms of closely related species. A number of neutralising factors have now been identified. All are natural blood products and over half are from snake blood (15). Recent work has shown that snake serum contains factor(s) which are inhibitory to the action of homologous venom (12). Further, one of us has shown that serum from two Australian elapids, P. australis and P. porphyriacus, is capable of neutralising venom of other snakes (15). More specific studies have revealed that serum prevents echinocytosis of red blood cells (6) and neurotoxic effects (4). However, to the authors' best knowledge, no in vitro studies have yet been conducted to determine the protective effect of snake serum or plasma on muscle. For this reason, the protective effect of homologous plasma was studied with reference to myotoxicity.

MATERIAL AND METHODS

VENOMS: Freeze-dried snake venoms and plasma were kindly supplied by Venom Supplies Pty Ltd (PO Box 547, Tanunda, South Australia). Venom from Notechis scutatus, Pseudonaja inframacula, Austrelaps superbus, Pseudechis porphyriacus, Pseudechis australis, Acanthophis antarcticus and Oxyuranus scutellatus were used. The plasma used was taken from Pseudechis australis and Pseudechis porphyriacus. Stock solutions were prepared by reconstitution of venoms in sterile water to a concentration of 50mg/ml or 250mg/ml and stored at -20°C. These concentrations represent final concentrations in the experiments of 500µg/ml and 2.5mg/ml.

PLASMA: Freeze-dried plasma was reconstituted in sterile water and heat inactivated by incubating at 56°C for 20 min before use. This was necessary because untreated plasma was found to be toxic to all cells studied.

CELL LINES: C2C12 cells were originally obtained by Yaffe and Saxel(16) through selective serial passage of myoblasts cultured from the thigh muscle of C3H mice 70 h after a crush injury. These cells were shown to be capable of differentiation. TC3 cells were derived from a human glioblastoma multiforme. Both cell lines were maintained in tissue culture flasks at 37°C in 5%CO2 in air and fed Dulbecco-modified Eagle's medium supplemented with 10% foetal calf serum. The cells were prepared for use by trypsinizing and plating into 12-well trays (MULTIWELL Tissue Culture Plate, Falcon, Cat No. 3043) at an approximate concentration of 1 x 10 cells per well in l ml of culture medium. They were then incubated at 37°C for 1 hour to allow cells to adhere. This procedure was employed for the preparation of cells in all experiments.

EFFECT OF VENOMS ON C2C12 CELLS: C2C12 cells were treated with venom to determine the time course and percentage of cell death. 10µl of each venom was added to separate wells to give a final concentration of 500µg/ml. Additional wells not receiving venom served as controls. The wells were incubated at 37°C for 2 hours and examined at regular intervals during this time. The cells were then stained with trypan blue to determine the degree of cell death, the medium removed and fresh medium added. Following overnight incubation, the cells were examined to assess the extent of cell re-growth.

SPECIFICITY OF P. PORPHYRIACUS VENOM: The specificity of P. porphyriacus venom was tested using TC3 cells, a human glioma cell line initiated by one of us (PFJ) at the Department of Neuropathology, RPH. The protective effect of homologous plasma was tested concurrently. The protocol did not differ from that used for experiments involving C2C12 cells. Venom was used at a final concentration of 500µg/ml and control wells were included in all experiments.

MIXED TISSUE CULTURE: Tissue culture studies were performed to determine the in vitro effect of venom on differentiated muscle and confirm the results of experiments conducted using the C2C12 cell line. Hind limbs of foetal ARC Random mice were removed, cut into small pieces and placed into 12-well tissue culture trays. The tissue explants were incubated for several days until myotubes developed and began contracting. These cultures contained morphologically distinct cell types including myotubes, fibroblasts and epithelial cells. The mixed cultures were treated with 10µ1 of P. porphyriacus venom, representing a final concentration of 500µg/ml, and wells not receiving treatment served as controls.

PROTECTIVE EFFECT OF PLASMA: Plasma was tested for its ability to protect cells from the myotoxic and cytotoxic effects of snake venom. P. porphyriacus venom was used at final concentrations of 2.5mg/ml and 500µg/ml for treatment of C2C12 cells and at a final concentration of 500mg/ml for treatment of TC3 cells and cultured tissue. In each case, 10µl of venom was mixed with 100µl of P. porphyriacus heat-inactivated plasma. This was then added to separate wells with thorough mixing. Mixtures of 10µl of P. porphyriacus venom and 100µl of P. porphyriacus plasma were also pre-incubated at 37°C for 30 min before addition to wells. The dynamics of protection by homologous plasma was studied more closely using the same protocol with P. australis venom at a final concentration of 25µg/ml and 100µl homologous serum. Control wells containing venom only, plasma only and no treatment were included in all experiments.

QUANTITATIVE ANALYSIS: Morphology and number of treated cells were compared with controls to determine the effect of the venoms and venom/plasma mixtures.

However, difficulties were encountered with quantitative analysis. While trypan blue staining was performed to assess the extent of cell death, non-viable cells were also non-adherent and, thus, difficult to quantify. Furthermore, cell death continued after removal of venom and quantitative analysis was, therefore, very inaccurate and of little validity. Morphological analysis gave a good indication of venom toxicity, cell death following treatment beginning with a characteristic change in morphology and ending in a loss of adherence. Non-adherent cells were shown to be non-viable both by trypan blue staining and a lack of re-growth following overnight incubation in fresh medium. The results obtained through morphological analysis, therefore, were confirmed with routine addition of fresh medium following removal of venom and analysis of the extent of cell growth following overnight incubation.

RESULTS

Preliminary experiments were conducted to determine the concentration of venom required to cause significant cell death (>50%) within a 5-hour period. These experiments revealed that high concentrations (500µg/ml) of venom were required. Subsequently, the experiments were carried out using this or higher concentrations of venom.

EFFECT OF VENOMS ON MYOBLASTS: All venoms included in the study were tested concurrently to determine their relative potency. Morphological analysis revealed characteristic changes in cells affected by venom regardless of the species from which it was derived, namely the cells exhibited a 'spiky' or stellate appearance (Figure 1).Following this, cells became progressively more rounded and finally non-adherent and non-viable, as shown by trypan blue staining. Comparison with control cells (Figure 2) revealed morphological changes within 30 min in cells treated with P. australis (Figure 3), A. superbus and P. porphyriacus venom. Within 60 min, morphological changes were apparent in cells treated with N. scutatus and A. antarcticus venom. Venom from O. scutellatus produced morphological changes within 2 h. At this time the morphology of P. inframacula venom-treated cells was relatively unchanged when compared to control cells.

Trypan blue staining was carried out at 2 h but contributed little information. Examination of adherent cells at 2 h revealed that the venom of P. australis was clearly more toxic to C2C12 cells than the other venoms tested. The characteristic morphological changes and cell death were more rapid than that observed with the other venoms. Equating non-adherence with cell death, approximately 90% cell death was observed for P. australis venom. Of the adherent cells, almost 100% exhibited a rounded morphology. Approximately 80% cell death was observed in wells treated with venom of A. superbus and adherent cells were rounded or displayed short, 'spiky' processes. Wells treated with P. porphyriacus venom suffered approximately 60% cell death. Although adherent cells were predominantly rounded, a small proportion exhibited a relatively normal appearance.

The difference in toxicity between N. scutatus and A. antarcticus venom was difficult to assess. At 2 h, approximately 20% cell death was observed in wells treated with both venoms and adherent cells of both were predominantly rounded but also occasionally exhibited an almost normal appearance. Wells treated with venom from O. scutellatus exhibited some evidence of cell death, but most adherent cells were unchanged. Finally, wells treated with P. inframacula displayed little evidence of cell death or difference in morphology when compared to controls.

After the 2-hour incubation, both treated and control cells were washed with saline and fresh medium added. The cells were then incubated overnight and the results confirmed through the extent of cell re-growth, allowing the venoms to be ranked according to decreasing lethal potency as follows: P. australis.> A. superbus > P. porphyriacus > N. scutatus > A. antarcticus > O. scutellatus > P. inframacula.

SPECIFICITY OF P. PORPHYRIACUS VENOM: Treatment of TC3 cells with P. porphyriacus venom revealed that the venom was also lethal to other cell types. Work by others has shown that P. porphyriacus venom is toxic to a variety of cell types, including human fibroblasts, mouse myeloma cells and myofibrils. Concurrent treatment of C2C12 cells and TC3 cells, however, revealed that cell death was slower and less extensive for TC3 cells than that observed for C2C12 cells.

MIXED TISSUE CULTURE: Cells grown from cultured tissue and treated with venom exhibited the same changes as those observed in the cell culture experiments, namely a rounding up of the cells and loss of adherence. Immediately following the addition of venom and venom/ plasma mixtures, myotubes ceased contracting. Myotubes in wells treated with venom retracted from the surrounding cells. While all cell types were affected by the venom, myotubes were the first to show changes.

PROTECTIVE EFFECT OF PLASMA: When venom was used at a final concentration of 500µg/ml, differences between venom-treated cells and venom/plasma-treated cells were clear within l h of initiation of the experiments using C2C12 cells. Protection was not absolute, with cell death still apparent in venom/plasma-treated cells when compared to controls, however, the effects of the venom were less marked (Figure 4). Homologous plasma afforded similar protection against P. porphyriacus venom for TC3 cells. Additionally, these experiments did not reveal any distinct difference in the level of protection between pre-incubated and non-incubated venom/plasma mixtures. Some protection against the effects of the venom was also achieved using homologous plasma in mixed tissue culture. That is, cell death was delayed. Interestingly, unlike the results of cell culture studies, pre-incubated venom/plasma mixtures appeared to be somewhat more protective than venom/plasma mixtures which were not pre-incubated.

The action of P. porphyriacus venom used at a final concentration of 2.9mg/ml was clearly inhibited by homologous plasma. Within 10-20 min of the addition of venom, 100% cell death was observed in C2C12 cells. Cells treated with a pre-incubated mixture of venom and plasma, however, showed no effects at this time.

Although short-term protection by plasma was achieved, it was apparent that homologous plasma did not afford continued protection against the effects of the venom in any of the experiments conducted. Following overnight incubation in fresh medium, venom-treated wells and venom/plasma-treated wells appeared similar. Furthermore, although some cell re-growth was observed in venom/plasma-treated wells of mixed tissue culture, growth was very slow and myotubes failed to develop.

The difference between the short-term protective effect of plasma and protection over a longer period was demonstrated by experiments using P. australis venom at a concentration of 25µg/ml and 100µl of homologous plasma and extending the experimental period to 7 hours. Short-term protection of C2C12 cells against the myotoxic action of the venom was shown when cells were assessed 4 hours after the addition of venom/plasma mixtures. Examination of the cells after 7 hours, however, revealed that the plasma was no longer protective (Table 1).

DISCUSSION

Snake venoms have a wide variety of actions, each venom unique in its combination and proportion(14). All venoms in this study, with the exception of P. inframacula, exhibited some degree of myotoxicity. Preliminary experiments revealed that high concentrations of venom were required to cause significant myoblast cell death in vitro. This, however, likely only highlights the fact that these venoms have a wide variety of actions. In vitro studies allow the researcher to concentrate on a single or limited spectrum of actions and eliminates other possible compounding factors which may lead to more significant effects in vivo.

As previously stated, venom of the Pseudechis genus is considered to be largely myotoxic. A recent study has demonstrated that several homologous phospholipases A2 isoenzymes purified from P. australis venom were predominantly myotoxic(5). The results of the current study also revealed that, of the venoms tested, Pseudechis venom displayed the highest level of myotoxicity. While a general cytotoxicity was found using P. porphyriacus venom, comparison between the rate of cell death of C2C12 cells and TC3 cells showed that cell death was much faster in C2C12 cells. A study by Bruses et al.(2) revealed that myotoxins used in vitro at high concentrations were capable of destruction of all cell types studied, while at lower concentrations they were muscle-specific. Thus, the cytotoxic effect noted in the current study may have been as a result of the high concentrations of venom used. Nevertheless, C2C12 cells in the cell culture experiments and myotubes in the mixed cell culture were preferentially affected. This offers support for a significant myotoxic component of Pseudechis venom.

The first effect seen in our study utilising mixed cell cultures was a cessation of myotube contraction. The same effect was noted immediately following the addition of both venom and venom/plasma mixtures. This supports the results reported recently by Chen et al.(3) in which isolated skeletal muscle preparations treated with venom of P. australis showed depressed muscle contractility. This, they concluded, was due to a direct myotoxic action of the venom.

Plasma afforded some protection against the effects of venom in our study but was not absolute and did not extend long-term. However, the concentrations of venom used in the study were quite high when compared to the likely systemic dose resulting from envenomation. Nevertheless, inhibition was achieved, even using venom which was diluted only 1/2 from the original concentration (neat) and this suggests that, in fact, the inhibitors present in the plasma are very powerful. Our in vitro studies are somewhat similar to the in vivo experiments of Thurn et al.(15) who found that the serum of P. porphyriacus protected 3/4 mice against subcutaneous inoculation of 4 LD50 dose of N. scutatus venom. In earlier work, Fortes-Dias et al.(7) extracted and partly purified a neutralising alpha1-globulin factor from homologous plasma of the South American rattlesnake (Crotalus durissus terrificus). The venom of Crotalus durissus terrificus acts mainly as a neurotoxin, but nevertheless, serves to highlight the fact that homologous plasma from many snakes has a protective mechanism.

Of interest was the difference between the results of cell culture and tissue culture studies in the requirement of pre-incubation of the venom/plasma mixtures to confer significant protection. Many studies to date have pre-incubated venom and serum before use(7,15). Pre-incubation did not appear to greatly increase the protective effect of plasma in the cell culture studies presented here. However, tissue culture experiments suggested that pre-incubation may be beneficial. This difference may be due to differences between the cells. The C2C12 cells used were undifferentiated myoblasts, which had adapted to growth in vitro over many years. Although useful as an experimental model, these cells probably differ in many respects from normal mouse myoblasts and, therefore, may not be affected by venom in the same manner as the myoblasts and myotubes present in the primary tissue culture. Moreover, myotubes per se may be more susceptible to the effects of myotoxins.

It was observed that apparently viable cells failed to survive after venom had been replaced by fresh medium and incubated overnight. This may have been because the venom remained bound to the cell membrane and continued to elicit damage or perhaps the cells were already damaged beyond repair (although not evident morphologically). Similarly, cells treated with venom/plasma mixtures failed to survive, although cell death was delayed. This may also have been as a result of bound venom or venom/plasma conjugate or as a result of damage elicited by the venom before removal.

Harris et al.(8) carried out in vivo experiments in which local myolysis 12-24 hours after a single subcutaneous injection of purified venom of the Australian tiger snake (Notechis scutatus scutatus) was demonstrated. Myoblasts which survived this treatment differentiated and fused to form myotubes and muscle regeneration was complete after 21 days. The results of the current study showed that no myotube reformation occurred when the mixed cell cultures were left to regenerate after treatment with venom or venom/plasma mixture. This suggests that normal mouse myoblasts as well as differentiated myotubes were also damaged. However, Harris et al. (8) point out that it is necessary for the local nerve supply to be intact for effective regeneration, a factor which was absent in our explant studies.

The ranking of myotoxicity of the venoms included in the study were compared to their mouse LD50 values(1) (Table 2). The two did not correlate, again highlighting the fact that each venom has a variety of actions, a variable portion of which may be myotoxicity.

Cell culture techniques utilising the mouse myoblast cell line C2C12 and mouse primary tissue cultures have proven useful in confirming in vitro the relative myotoxicities of a variety of venoms in this study. Furthermore, the protective effects of Pseudechis plasma against homologous venom has now been established in vitro. These preliminary studies open the way to more extensive experiments on the action of venoms toxic to mammals and perhaps more importantly on the mode of action of protection of homologous plasma.

TABLE 1.
Comparison of the short-term and long-term protective effect of P. australis serum against the effects of homologous venom. As the morphological changes associated with cell death resulted in a loss of adherence, the number of cells/field were counted in addition to the percentage of non-viable adherent cells (shown by the trypan blue exclusion method). Examination of venom/serum-treated C2C12 cells after 4 hours clearly showed protection against venom. At 7 hours, however, this group closely resembles venom-treated C2C12 cells, revealing the short-term nature of the protective effect.

TABLE 2.
Comparison of LD50 values(1) and the determined in vitro myotoxicity of venoms, highlighting the fact that venoms have a variety of actions, a variable portion of which may be myotoxicity.

FIGURE 1.
Photomicrograph of C2C12 cells 30 min after treatment with 500µg/ml of P. australis venom, affected cells showing the characteristic 'spiky' or stellate appearance (x250).

FIGURE 2.
Photomicrograph of untreated C2C12 cells at 30 min (x125).

FIGURE 3.
Photomicrograph of C2C12 cells 30 min after exposure to venom of P. australis showing many cells rounded up and the remainder exhibiting the characteristic stellate morphology of affected cells (x125).

FIGURE 4.
Photomicrograph of C2C12 cells after exposure to a mixture of 500µg/ml of P. australis venom and 100µl of homologous plasma showing a marked reduction in the extent of cell death relative to venom-treated cells (x125).

ACKNOWLEDGEMENTS

We thank Venom Supplies Pty Ltd for kindly supplying the venoms and plasmas used in these studies. T. L. Butler was supported by a summer scholarship offered by the Australian Neuromuscular Research Institute.

REFERENCES

01 BROAD AJ., SUTHERLAND SK., COULTER AR. The lethality in mice of dangerous Australian and other snake venom. Toxicon, 1979, 17, 661-64.

02 BRUSES JL., CAPASO J., KATZ E., PILAR G. Specific in vitro biological activity of snake venom miotoxins. J. Neurochem., 1993, 60, 1030-42.

03 CHEN SZJ., GOPALAKRISHNAKONE P., GWEE MCE. Pharmacological effects and pathological changes induced by the venom of Pseudechis australis in isolated skeletal muscle preparations. Toxicon, 1994, 32, 303-15.

04 DOMONT GB., PERALES J., MOUSSATCHE H. Natural anti-snake venom proteins. Toxicon, 1991, 29, 1183-94.

05 FATEHI M., ROWAN EG., HARVEY AL., HARRIS JB. The effects of five phospholipases A2 from the venom of king brown snake Pseudechis australis, on nerve and muscle. Toxicon, 1994, 32, 1559-72.

06 FLACHSENBERGER W., LEIGH C M., MIRTSCHIN PJ. Sphero-echinocytosis of the human red blood cells caused by snake, red-back spider, bee, and blue-ringed octopus venoms and its inhibition by snake sera. Toxicon, 1995, 33, 791-7.

07 FORTES-DIAS CL., FONSECA BCB., KOCHVA E., DINIZ CR. Purification and properties of an antivenom factor from the plasma of the South American rattlesnake (Crotalus durissus terrificus). Toxicon, 1991, 29, 997-1008.

08 HARRIS JB., JOHNSON MA., KARLSSON E. Pathological responses of rat skeletal muscle to a single subcutaneous injection of a toxin isolated from the venom of the Australian tiger snake, Notechis scutatus scutatus. Clin. Exp. Pharmacol. Physiol., 1975, 2, 383-404.

09 KELLAWAY CH. The immunity of Australian snakes to their own venoms. Med. J. Aust., 1931, 2, 35-52.

10 MIRTSCHIN PJ., DAVIS R. Dangerous snakes of Australia. Rev. ed. Ure Smith Sydney Press: Ure Smith Press, 1982.

11 MIRTSCHIN PJ., DAVIS R. Snakes of Australia. Dangerous and Harmless. Melbourne: Hill of Content, 1992.

12 PONRAJ D., GOPALAKRISHNAKONE P. Inhibitory effects of snake sera on several venom-induced myonecrosis, haemorrhage and oedema activities in mice. Toxicon, 1996, 34, 622-3.

13 ROWLANDS JB., MASTAGLIA FL., KAKULAS BA., HAINSWORTH D. Clinical and pathological aspects of a fatal case of Mulga (Pseudechis australis) snakebite. Med. J. Aust., 1969, 226-30.

14 SUTHERLAND SK. Australian animal toxins. The creatures, their toxins and care of the poisoned patient. Melbourne: Oxford University Press, 1983.

15 THURN MJ., BROADY KW., MIRTSCHIN PJ. Neutralization of tiger snake (Notechis scutatus) venom by serum from other Australian elapids. Toxicon, 1993, 31, 909-12.

16 YAFFE D., SAXEL D. Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature, 1977, 270, 725-7.

Received 19 March 1997

Accepted 24 July 1997

CORRESPONDENCE TO:

P.F. JACOBSEN - Department of Neuropathology, Royal Perth Hospital, GPO Box X2213, Perth, Western Australia, Australia, 6001.

Publication Dates

• Publication in this collection
  26 Nov 1998
• Date of issue
  1998

History

• Received
  19 Mar 1997
• Accepted
  24 July 1997