Camp. Biorhem. F’hysiol.Vol. 102C, No. I, pp. 125-128, 1992
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Printedin Great Britain
0306-4492/92 $5.00 + 0.00 1992 PergamonPressLtd
THE INVOLVEMENT OF COLLAGENASE IN THE NECROSIS INDUCED BY THE BITES OF SOME SPIDERS RONALD K. ATKINSON
School
and
LYNETTE G. WRIGHT
of Applied Science, University College of Southern Queensland, Toowoomba, Queensland, Australia [Tel: (076) 312269; Fax: (076) 3617621 (Received 22 August 1991)
Abstract-l. The midgut extracts of 13 Australian spider species produced cellular disruption in mouse skin in tissue culture conditions. 2. Microbial collagenase and the venoms of some of these species had similar effects. 3. Five venoms also caused severe dermonecrosis in living mice. 4. Pre-mixing the venoms with r_-cysteine caused complete in uivo and partial in vitro inhibition of their effects. 5. It was concluded that collagenase is a major factor in the aetiology of necrotic arachnidism.
INTRODUCTION
Following the initial reports of cutaneous necrosis induced by the bites of the North American spider, Loxosceles reclusa (Atkins et al., 1958), many studies of the biochemical reasons for the dermonecrosis caused by bites from this and several other spider species have been performed (Russell and Gertsch, 1983). Several possible mechanisms for the phenomenon of necrotic arachnidism have now been suggested, including interference with the blood coagulation processes (Babcock et al., 1986) localized activation of leukocytes or platelets (Smith and Micks, 1970; Kurpiewski ef al., 1981), and the involvement of such degradative enzymes as collagenase (Kaiser and Raab, 1967) proteases (Schenone 1978), and sphingomyelinase D and Suarez, (Kurpiewski et al., 1981; Rekow et al., 1983). Atkinson and Wright (1990, 1991) studied the necrotic activities of the venoms of several Australian spider species, both when the venom was injected subcutaneously into living mice and when it was added to either mouse or human skin in tissue culture conditions. Their results indicated that while spider venoms do indeed have some potential to cause haemolysis, complement consumption, and interference with blood coagulation, these effects are not the main reason why the bites of certain species result in localized tissue necrosis. Instead, their results added weight to the earlier suggestion of Geren et al. (1973) and Perret (1977) that some of the toxic effects of spider venoms might be due to contamination of the venoms by digestive tract fluids voided at the same time as the venoms. By histological examination of skin that had been exposed to several of the more potent Australian spider venoms, Atkinson and Wright showed that these venoms, collected by electrical stimulation, caused a severe loss of epidermal cell-cell adhesion. However, this was not the case for venoms collected with no possibility of contamination by digestive secretions or for venom gland extracts.
It was particularly noteworthy that the venom gland extracts of some species that had been shown to possess strongly necrotic venoms lacked necrotic activity. It is well known that spiders can only ingest liquified food and normally secrete digestive enzymes into their prey to carry out the initial break-up of fibrous animal tissues (Foelix, 1982). Thus it seemed possible that a digestive enzyme common to all spiders might be the agent responsible for any necrotic activity a spider venom possesses. Wright et al. (1973) concluded that the hyaluronidase activity found in L. reclusa venom is not responsible for the necrosis caused by this venom. Atkinson and Wright (1990) were unable to demonstrate the presence of hyaluronidase in the distinctly necrogenic venom of the wolf spider, Lycosu godejiroyi. Eskafi and Norment (1976) obtained histological evidence that L. reclusu venom contains a protease. Several attempts have already been made to show that there is collagenase in spider venoms. Kaiser and Raab (1967) claimed that the venoms of both Phoneutriu feru and Lycosa erythrognutha lacked demonstrable collagenase. While Wright et al. (1973) found no collagenase activity in L. reclusa venom, they did find evidence of a protease. On the other hand, Geren et al. (1973) concluded that L. reclusu venom lacks protease activity. Despite the evidence against collagenase as the causal agent in those spider venoms that are necrogenie, it was felt that further experiments to test for the presence of this enzyme in spider midgut extracts were warranted. The ability of commercial collagenase to induce dermonecrosis and loss of cellLcel1 adhesion was also considered worth testing, as was the ability of crude venoms and midgut extracts to digest purified native collagen. Finally, since Seifter et al. (1959) have reported that the amino acid, L-cysteine, inhibits microbial collagenase, it seemed appropriate to establish whether or not L-cysteine can inhibit any of the necrotic effects produced by spider venoms or midgut extracts. 125
126
RONALD K. ATKINSON and MATERIALS
AND METHODS
Spider species tested The following Australian spider species were tested in this study: AWUX (~udrQnyc~e) jn~n~~~ Hickman, Nanlea salanitri Raven, Misgolas pul&helius(Rainbow and Pulleine), Badumna insignis (Koch), Lampona cylindrata (Koch), Pholcus ohalanaioides (Fuesslin). Astia hariola (Koch). Isopeda &a (Koch), Isipeda imkanis (Koch), Latrodectbs hasselri Thorell, Nephila edtdis (Labillardiere), Eriophora transmarina (Keyserling), and Lycosa godeffroyi Koch. All specimens used were collected in or near Toowoomba, Queensland. Only adult female spiders were used. Preparation of venoms and midgut extracts All venoms tested were collected by an electrostimulation method, as previously described (Atkinson and Wright, 1990). Since most of the glandular parts of the digestive tracts of spiders are in the abdominal midgut regions (Foelix, 1982), specimens of the species under study were anaesthetized in a CO, atmosphere and the midgut components of each spider dissected out as completely as possible. Each midgut was then immediately macerated in 0.5 ml (in the case of the small species: A. hariola, L. hasselti, P. phalangioides, and B. insignia) or 1.O ml (all of the larger species) of distilled water. and the extract was centrifuged at 8OOg for 3 min. The supernatant was then removed and freeze-dried before beinn stored at - I2,‘C. For some tests a sam$e of commercially prepared microbial collagenase (Worthington Biochemical Corp., New Jersey) was also used, its effects being compared with those of the venoms and midgut extracts.
LYNETTEG. WRIGHT
were used. One water and one L-cysteine mixture were placed immediately in a boiling water bath for 10min to inactivate any enzyme activities present then were cooled and centrifuged. The other water and L-cysteine tubes were incubated at 37°C for 6 hr before being treated in the same way. A 0.5 ml sample of each of the four resulting supernatants was then added to 1.0 ml of acetate buffer, pH 5.4, and 1.Oml of 0.1% aqueous ninhydrin solution and left in a boiling water bath for 10min. The extent of colour development was then determined by reading the A,,, of the mixture in an LKB Ultrospec (Pharmacia) spectrophotometer. To estimate the collagenase activity of the test material the value of A,,0 (6-O hr) in the presence of L-cysteine was subtracted from that in its absence and the result multiplied by 193 (since an A,,, difference of 3.6 resulted when 5 mg of commercial collagenase with a specified activity of 139 units/mg was used as test material). RESULTS
In vivo studies with nscrogenic uenoms
The venoms of N. edulis, E. transmarina, I. vasta, I. immanis, and L. godeffroyi were available in relatively larger amounts than those of the other species under examination. Hence, sets of four mice (mixed strain and sex) were given 5 ~1 scaip injections of each of these venoms, duplicate sets of mice receiving the same amount of the same venom but with L-cysteine added at the rate of 22mg/ml. After 6 hr the mice were all killed and the injection sites examined visually and later histologically. All five venoms Tests for necrosis in vivo consistently caused development of a necrotic response (the venom of When a venom or extract was to be tested for in t1IL.o lesion and inflammatory necrotic activity, the procedure followed was as previously L. g~~e~o~~ being slightly less effective than the described (Atkinson and Wright. 1990. 19911. Briefly, a others in this respect) but in all cases the presence 30 gauge needle and a syringe capable of accurately deliverof L-cysteine totally prevented the development of ing 5 microlitre volumes was used to inject the test material both necrosis and inflammation. The histological into the scalps of unanaesthetized mice, the effects being appearance of the lesions produced in the absence of examined 6 hr later by killing the mice and removing the L-cysteine was indistinguishable from that reported skin over the injection sites for subsequent histoIogical previously (Atkinson and Wright, 1990, 1991) for examination. Australian spiders as well as for L. reclusa (Atkins Necrosis in &sue culture condiiions et al., 1958; Butz et al., 1971). When 10 microlitres of a 10 mg/ml solution of Once again, previously developed methods (Atkinson and commercial collagenase in saline was injected into the Wright, 1990, 1991) were employed. Pieces of fresh mouse skin were placed in small tissue culture wells containing scalps of four mice, an extensive oedematous area TC199 medium (Commonwealth Serum Labs, Melbourne), developed subcutaneously at each injection site but this either having been diluted with half its volume of venom the epidermis remained intact. The same resuh was or used to dissolve a freeze-dried midgut extract. By keeping obtained when N. edulis midgut extract (10 microthe final volume down to 50-150 microlitres (depending litres, equivalent to a tenth of an abdomen) was on the amount of midgut residue to be dissolved) it was injected. The lack of epidermal damage as caused by possible to maximize the necrotic effects produced. After the venoms showed that these venoms also have 6 hr in a 5% CO, atmosphere at 37“C the pieces of skin were important inflammatory effects. removed and prepared for histological examination. Tests for coliagenare activity in vitro To test the abilities of the venoms and midgut extracts to digest purified collagen, a modification of the method described by Appel (1974) was used. For each individual test 5 mg of purified native collagen (Type 1, insoluble, from bovine achilles tendon, Sigma, St Louis) was suspended in 0.5 ml of Tris calcium buffer, pH 7.2, containing 5 mg commercial collagenase or 10 microlitres of crude venom or haff of the extract of one midgut, and then 0.5 ml of either distilled water or 2.2 mgjml L-cysteine (as the hydrochloride, Sigma, St Louis) solution was added. This mixture of reagents was prepared four times for each test material, the midgut extracts of two spiders being combined and then divided into four equal portions, two to be mixed with 0.5 ml of water and the other two with 0.5 ml of L-cysteine solution. When venoms were being tested, pooled samples
Efects a~veno~zs and m~dgar extracts in tissue culture conditions The venoms of N. edulis, E. transmarina, I. vasta, I. immanis, and L. godefsroyi all produced severe loss of mouse skin cell-cell adhesion after 6 hr in tissue culture conditions. Once again, the histological changes were as previously reported (Atkinson and Wright, 1990, 1991). The same was true for the midgut extracts of all 13 spider species tested, although the extent of disruption was notably less for the smaller species, presumably because relatively little midgut tissue was present to be extracted in these instances. Indeed, the loss of cellular adhesion might not have been visible but for the fact that
Collagenase and necrotic arachnidism the individual midgut extracts of A. hariolu were dissolved in only 25 ~1 of TC199 medium, compared with 50 ~1 for the P. phalungioides extracts, 100 ~1 for the B. insignis, L. cylindruta, and L. husselti extracts and 200~1 for the extracts from all of the large species tested. Commercial collagenase was tested in a similar manner at concentrations of 0.1, 0.5, 2, 10 and 50 mg/ml and produced severe tissue disruption when at least 2 mg/ml was present and visible (though much less severe) cellular detachment even at the 0.1 mg/ml concentration. However, when L-cysteine was added to the tissue culture wells at the rate of 11 mg/ml, all of the venoms and midgut extracts again caused significant tissue disruption, although the effects appeared to be somewhat reduced. This was not the case when commercial collagenase was pre-mixed with r_-cysteine, the skin samples then remaining totally unaffected even when the L-cysteine concentration was only 0.11 mg/ml. It was also noted that if the fluid in the culture well, having been acidified by the addition of the r_-cysteine solution, was not then adjusted to a slightly alkaline pH (by addition of a small volume of 14% NaHCO, solution), the extent of tissue disruption was much less than otherwise. In vitro estimations
of midgut collugenase activities
The assays of the 13 species tested for midgut extract collagenase activity are presented in Table 1. In each case two estimations were made. The collagenase activities of the crude venoms of N. edulis, E. transmarina, and I. immunis were also estimated and are included in Table 1. It can be seen that the venoms and midgut extracts of all species tested contained measurable amounts of collagenase activity. It was also apparent that the larger the species the more collagenase there is in the adult midgut. DISCUSSION
The results presented in this paper support the suggestion that when humans or other animals are bitten by spiders and subsequently develop necrotic lesions at the bite sites, the reason for the ulceration is not that the offending spiders’ venom glands secrete a necrotoxin but that their venoms have become contaminated with digestive tract fluids during voidTable
1. Assays of the collagenase actwities in the crude venoms and midgut extracts of several Australian spider species
Species N. edulis E. transmarina I. immanis I. vasta N. .wlanitri M. pulchellus A. infensus L. godeflroyi B. insignis L. hasselti L. cylindrata P. pholangioides A. horiola
Adult female body length (mm) 22 25 35 34 30 26 35 27 I8 IO I8 9 5
Units of collagenase activity in IOml One midgut (2 specimens) of venom 2100, 1472, 1310, 1196, 1352, 1184, 1500. 614, 272, 818, 140, 134, 60,
2440 2654 1388 1280 1428 1506 1250 890 520 908 606 132 44
I55 57 41
127
ing. This is to be expected, having regard to the usual prey and manner of feeding of spiders (Foehx, 1982). Strong circumstantial evidence that the digestive secretions from members of a wide range of spider families contain collagenase activity was also obtained. It therefore seems reasonable to conclude that this collagenase activity is probably the main reason for the phenomenon of necrotic arachnidism. This conclusion appears to contradict the findings of several workers who could not demonstrate the involvement of collagenase in the aetiology of the necrotic lesions and who instead obtained evidence for sphingomyelinase D as the main necrotoxin of spider venoms (Kurpiewski et al., 1981; Rekow et al., 1983). This discrepancy may be an indication that L. reclusa, the spider mostly involved when studies of necrotic arachnidism have been reported, has a different necrogenic action from that of the species tested above. However, this seems unlikely since representatives from a wide range of Araneomorph and Mygalomorph families were chosen for the necrotic studies reported here and all species appeared to produce the same effects, differing only in their relative potencies. In addition, many workers have chosen to use mechanically expressed venom or venom gland extracts rather than venom samples that could have become contaminated with digestive secretions. Kurpiewski et al. (1981) even claimed that sphingomyelinase D isolated from L. reclusa venom caused the typical necrotic lesion after 8 days and suggested that the observed dermonecrosis caused by L. reclusu venom might be partly due to an abnormal haemostatic effect involving platelet aggregation. This finding is difficult to reconcile with the results presented in this paper, and it is noteworthy that all of the necrogenic Australian spider venoms tested produced visible lesions in a matter of a few hours. Perhaps the most important observation reported above is the fact that the amino acid L-cysteine totally blocked the necrotic effects of the venoms of several different spider species when premixed with the crude venoms. A therapeutic use for this amino acid in the treatment of dermonecrosis caused by spider bites is therefore possible. The fact that L-cysteine was shown to be able to inhibit collagenase activity in vitro and is relatively specific for this enzyme (Seifter et al., 1959), provides further evidence that collagenase is of prime importance in the aetiology of the in vivo necrosis caused by many spider species. Unfortunately, L-cysteine was found to be only partially successful in blocking the in vitro necrosis caused by both raw spider venoms and midgut extracts. The explanation for this discrepancy is uncertain but appears likely to be due to instability of the L-cysteine in tissue culture conditions, which are slightly alkaline. Seifter et al. (1959) reported that collagenase is inhibited by sulphydryl-containing agents such as L-cysteine, probably because these chelate a metallic component of the enzyme. The oxidation of L-cysteine to L-cystine in alkaline conditions will thus prevent this inhibition. The fact that L-cysteine (supplied as the hydrochloride and therefore acidic) totally blocked the actions of microbial collagenase in vitro was probably because only onetenth as much L-cysteine was used on that occasion
128
RONALD
K.
ATKINSON and LYNETTE G. WRIGHT
so that no neutralization of the culture medium was necessary. The ability of spider venoms and midgut extracts to cause total dissociation of the cells of the entire epidermis in mice was somewhat surprising. Bornstein and Sage in their 1980 review showed that there is still much uncertainty about the number of types of collagen that are found in nature and also about their distributions within the mammalian body. These authors refer to “nonstructural” collagen that assists in cellular attachment to a substratum in association with fibronectin and other matrix proteins. It is therefore likely that it is this type of collagen that is being dissolved when spider venoms and midgut extracts cause the observed loss of epidermal cell adhesion.
Bornstein P. and Sage H. (1980) Structurally distinct collagen types. Ann. &v. Biochem. 49, 957-1003. Butz W. C., Stacv L. D. and Hervford N. N. (1971) Arachnid&m in rabbits. Archs. Path. 91, 97-100. Eskafi F. M. and Norment B. R. (1976) Physiolo~cal action of Loxosceles reclusa (G&M) venom on insect larvae. Toxicon 14, 7-13.
Foelix R. F. (1982) The Biology of Spiders, pp. 32-52. Harvard University Press, Massachusetts. Geren C. R., Ghan-T. K., Ward B. C., Howell D. E., Pinkston K. and Ode11 G. V. (1973) Comoosition and properties of extract of fiddleback (ioxosceies rechtsa) spider venom apparatus. Toxicon 11, 471479. Kaiser E. and Raab W. (1967) Collagenolytic activity of snake and spider venoms. Toxicon 4, 2X-255. Kurpiewski G., Forrester L. J., Barrett J. T. and Campbell B. J. (1981) Platelet aggregation and sphingomyelinase D activity of a purified toxin from the venom of Loxosceles rec&.
Ackno~,ledge~enfs-The research described in this paper was financially supported by the Darling Downs Association for Advanced Education, to whom the authors are very grateful.
REFERENCES
Appel W. (1974) Collagenases. In Methods of Enzymatic Anal~ysis(Edited by Bergmeyer H. R.), 2nd Edn, Vol. 2, 1059-1063. Academic Press, New York. Atkins J. A.. Wingo C. W., Sodeman W. A. and Flynn J. E. (19581Necrotic arachnidism. Am. /. ITOR.Med. HJX. .7, 165-184. Atkinson R. K. and Wright L. G. (1990) A study of the necrotic actions of the venom of the wolf spider, Lycosu godefioyi, on mouse skin. Comp. Biochem. Physjoi. MC, 319-325.
Atkinson R. K. and Wright L. G. (1991) Studies of the necrotic actions of the venoms of several Australian spiders, Comp. Biochrm. Physiol. 98C, 441-444. Babcock J. L., Suber R. L., Frith C. H. and Geren C. R. (1981) Systemic effect in mice of venom apparatus extract and toxin from the brown recluse spider (Loxosceles reclusa). Toxicon 19, 46347 I.
Biochim. Biophys. Acta 678, 467-476.
Perret B. A. (1977) Proteolvtic activitv of taranttda venoms due to contamination with saliva. Tox~co~ 15, 505-510. Rekow M. A., Civello D. J. and Geren C. R. (1983) Enzymatic and hemolytic properties of brown recluse spider (Loxosceles reclusa) toxins and extracts of venom apparatus, cephalothorax and abdomen. Toxicon 21, 44-444. Russell F. E. and Gertsch W. J. (1983) Letter to the editor. Toxica?i 21, 337-339. Schenone H. and Suarez G. (1978) Venoms of Scytodidae. Genus Loxosceles. In Handbook qf Experimental Pharmacology (Edited by Bettini S.), Vol. 48, pp. 247-275. Springer, Berlin. Seifter S., Gallop P. M., Klein L. and Meilman E. (1959) Studies on collagen. II Properties of purified collagenase and its inhibition. J. biof. Chem. 234, 285-293. Smith C. W. and Micks D. W. (1970) The role of polymorphonuclear leukocytes in the lesion caused by the venom of the brown spider, Loxosceles reclusa. Lab. Invest. 22, 90-93.
Wright R. P., Elgert K. D., Campbell B. J. and Barrett J. T. (1973) Hyaluronidase and esterase activities of the venom of the poisonous brown recluse spider. Arch. Biochem. Bjophys. 159, 415-426.
0
Printedin Great Britain
0306-4492/92 $5.00 + 0.00 1992 PergamonPressLtd
THE INVOLVEMENT OF COLLAGENASE IN THE NECROSIS INDUCED BY THE BITES OF SOME SPIDERS RONALD K. ATKINSON
School
and
LYNETTE G. WRIGHT
of Applied Science, University College of Southern Queensland, Toowoomba, Queensland, Australia [Tel: (076) 312269; Fax: (076) 3617621 (Received 22 August 1991)
Abstract-l. The midgut extracts of 13 Australian spider species produced cellular disruption in mouse skin in tissue culture conditions. 2. Microbial collagenase and the venoms of some of these species had similar effects. 3. Five venoms also caused severe dermonecrosis in living mice. 4. Pre-mixing the venoms with r_-cysteine caused complete in uivo and partial in vitro inhibition of their effects. 5. It was concluded that collagenase is a major factor in the aetiology of necrotic arachnidism.
INTRODUCTION
Following the initial reports of cutaneous necrosis induced by the bites of the North American spider, Loxosceles reclusa (Atkins et al., 1958), many studies of the biochemical reasons for the dermonecrosis caused by bites from this and several other spider species have been performed (Russell and Gertsch, 1983). Several possible mechanisms for the phenomenon of necrotic arachnidism have now been suggested, including interference with the blood coagulation processes (Babcock et al., 1986) localized activation of leukocytes or platelets (Smith and Micks, 1970; Kurpiewski ef al., 1981), and the involvement of such degradative enzymes as collagenase (Kaiser and Raab, 1967) proteases (Schenone 1978), and sphingomyelinase D and Suarez, (Kurpiewski et al., 1981; Rekow et al., 1983). Atkinson and Wright (1990, 1991) studied the necrotic activities of the venoms of several Australian spider species, both when the venom was injected subcutaneously into living mice and when it was added to either mouse or human skin in tissue culture conditions. Their results indicated that while spider venoms do indeed have some potential to cause haemolysis, complement consumption, and interference with blood coagulation, these effects are not the main reason why the bites of certain species result in localized tissue necrosis. Instead, their results added weight to the earlier suggestion of Geren et al. (1973) and Perret (1977) that some of the toxic effects of spider venoms might be due to contamination of the venoms by digestive tract fluids voided at the same time as the venoms. By histological examination of skin that had been exposed to several of the more potent Australian spider venoms, Atkinson and Wright showed that these venoms, collected by electrical stimulation, caused a severe loss of epidermal cell-cell adhesion. However, this was not the case for venoms collected with no possibility of contamination by digestive secretions or for venom gland extracts.
It was particularly noteworthy that the venom gland extracts of some species that had been shown to possess strongly necrotic venoms lacked necrotic activity. It is well known that spiders can only ingest liquified food and normally secrete digestive enzymes into their prey to carry out the initial break-up of fibrous animal tissues (Foelix, 1982). Thus it seemed possible that a digestive enzyme common to all spiders might be the agent responsible for any necrotic activity a spider venom possesses. Wright et al. (1973) concluded that the hyaluronidase activity found in L. reclusa venom is not responsible for the necrosis caused by this venom. Atkinson and Wright (1990) were unable to demonstrate the presence of hyaluronidase in the distinctly necrogenic venom of the wolf spider, Lycosu godejiroyi. Eskafi and Norment (1976) obtained histological evidence that L. reclusu venom contains a protease. Several attempts have already been made to show that there is collagenase in spider venoms. Kaiser and Raab (1967) claimed that the venoms of both Phoneutriu feru and Lycosa erythrognutha lacked demonstrable collagenase. While Wright et al. (1973) found no collagenase activity in L. reclusa venom, they did find evidence of a protease. On the other hand, Geren et al. (1973) concluded that L. reclusu venom lacks protease activity. Despite the evidence against collagenase as the causal agent in those spider venoms that are necrogenie, it was felt that further experiments to test for the presence of this enzyme in spider midgut extracts were warranted. The ability of commercial collagenase to induce dermonecrosis and loss of cellLcel1 adhesion was also considered worth testing, as was the ability of crude venoms and midgut extracts to digest purified native collagen. Finally, since Seifter et al. (1959) have reported that the amino acid, L-cysteine, inhibits microbial collagenase, it seemed appropriate to establish whether or not L-cysteine can inhibit any of the necrotic effects produced by spider venoms or midgut extracts. 125
126
RONALD K. ATKINSON and MATERIALS
AND METHODS
Spider species tested The following Australian spider species were tested in this study: AWUX (~udrQnyc~e) jn~n~~~ Hickman, Nanlea salanitri Raven, Misgolas pul&helius(Rainbow and Pulleine), Badumna insignis (Koch), Lampona cylindrata (Koch), Pholcus ohalanaioides (Fuesslin). Astia hariola (Koch). Isopeda &a (Koch), Isipeda imkanis (Koch), Latrodectbs hasselri Thorell, Nephila edtdis (Labillardiere), Eriophora transmarina (Keyserling), and Lycosa godeffroyi Koch. All specimens used were collected in or near Toowoomba, Queensland. Only adult female spiders were used. Preparation of venoms and midgut extracts All venoms tested were collected by an electrostimulation method, as previously described (Atkinson and Wright, 1990). Since most of the glandular parts of the digestive tracts of spiders are in the abdominal midgut regions (Foelix, 1982), specimens of the species under study were anaesthetized in a CO, atmosphere and the midgut components of each spider dissected out as completely as possible. Each midgut was then immediately macerated in 0.5 ml (in the case of the small species: A. hariola, L. hasselti, P. phalangioides, and B. insignia) or 1.O ml (all of the larger species) of distilled water. and the extract was centrifuged at 8OOg for 3 min. The supernatant was then removed and freeze-dried before beinn stored at - I2,‘C. For some tests a sam$e of commercially prepared microbial collagenase (Worthington Biochemical Corp., New Jersey) was also used, its effects being compared with those of the venoms and midgut extracts.
LYNETTEG. WRIGHT
were used. One water and one L-cysteine mixture were placed immediately in a boiling water bath for 10min to inactivate any enzyme activities present then were cooled and centrifuged. The other water and L-cysteine tubes were incubated at 37°C for 6 hr before being treated in the same way. A 0.5 ml sample of each of the four resulting supernatants was then added to 1.0 ml of acetate buffer, pH 5.4, and 1.Oml of 0.1% aqueous ninhydrin solution and left in a boiling water bath for 10min. The extent of colour development was then determined by reading the A,,, of the mixture in an LKB Ultrospec (Pharmacia) spectrophotometer. To estimate the collagenase activity of the test material the value of A,,0 (6-O hr) in the presence of L-cysteine was subtracted from that in its absence and the result multiplied by 193 (since an A,,, difference of 3.6 resulted when 5 mg of commercial collagenase with a specified activity of 139 units/mg was used as test material). RESULTS
In vivo studies with nscrogenic uenoms
The venoms of N. edulis, E. transmarina, I. vasta, I. immanis, and L. godeffroyi were available in relatively larger amounts than those of the other species under examination. Hence, sets of four mice (mixed strain and sex) were given 5 ~1 scaip injections of each of these venoms, duplicate sets of mice receiving the same amount of the same venom but with L-cysteine added at the rate of 22mg/ml. After 6 hr the mice were all killed and the injection sites examined visually and later histologically. All five venoms Tests for necrosis in vivo consistently caused development of a necrotic response (the venom of When a venom or extract was to be tested for in t1IL.o lesion and inflammatory necrotic activity, the procedure followed was as previously L. g~~e~o~~ being slightly less effective than the described (Atkinson and Wright. 1990. 19911. Briefly, a others in this respect) but in all cases the presence 30 gauge needle and a syringe capable of accurately deliverof L-cysteine totally prevented the development of ing 5 microlitre volumes was used to inject the test material both necrosis and inflammation. The histological into the scalps of unanaesthetized mice, the effects being appearance of the lesions produced in the absence of examined 6 hr later by killing the mice and removing the L-cysteine was indistinguishable from that reported skin over the injection sites for subsequent histoIogical previously (Atkinson and Wright, 1990, 1991) for examination. Australian spiders as well as for L. reclusa (Atkins Necrosis in &sue culture condiiions et al., 1958; Butz et al., 1971). When 10 microlitres of a 10 mg/ml solution of Once again, previously developed methods (Atkinson and commercial collagenase in saline was injected into the Wright, 1990, 1991) were employed. Pieces of fresh mouse skin were placed in small tissue culture wells containing scalps of four mice, an extensive oedematous area TC199 medium (Commonwealth Serum Labs, Melbourne), developed subcutaneously at each injection site but this either having been diluted with half its volume of venom the epidermis remained intact. The same resuh was or used to dissolve a freeze-dried midgut extract. By keeping obtained when N. edulis midgut extract (10 microthe final volume down to 50-150 microlitres (depending litres, equivalent to a tenth of an abdomen) was on the amount of midgut residue to be dissolved) it was injected. The lack of epidermal damage as caused by possible to maximize the necrotic effects produced. After the venoms showed that these venoms also have 6 hr in a 5% CO, atmosphere at 37“C the pieces of skin were important inflammatory effects. removed and prepared for histological examination. Tests for coliagenare activity in vitro To test the abilities of the venoms and midgut extracts to digest purified collagen, a modification of the method described by Appel (1974) was used. For each individual test 5 mg of purified native collagen (Type 1, insoluble, from bovine achilles tendon, Sigma, St Louis) was suspended in 0.5 ml of Tris calcium buffer, pH 7.2, containing 5 mg commercial collagenase or 10 microlitres of crude venom or haff of the extract of one midgut, and then 0.5 ml of either distilled water or 2.2 mgjml L-cysteine (as the hydrochloride, Sigma, St Louis) solution was added. This mixture of reagents was prepared four times for each test material, the midgut extracts of two spiders being combined and then divided into four equal portions, two to be mixed with 0.5 ml of water and the other two with 0.5 ml of L-cysteine solution. When venoms were being tested, pooled samples
Efects a~veno~zs and m~dgar extracts in tissue culture conditions The venoms of N. edulis, E. transmarina, I. vasta, I. immanis, and L. godefsroyi all produced severe loss of mouse skin cell-cell adhesion after 6 hr in tissue culture conditions. Once again, the histological changes were as previously reported (Atkinson and Wright, 1990, 1991). The same was true for the midgut extracts of all 13 spider species tested, although the extent of disruption was notably less for the smaller species, presumably because relatively little midgut tissue was present to be extracted in these instances. Indeed, the loss of cellular adhesion might not have been visible but for the fact that
Collagenase and necrotic arachnidism the individual midgut extracts of A. hariolu were dissolved in only 25 ~1 of TC199 medium, compared with 50 ~1 for the P. phalungioides extracts, 100 ~1 for the B. insignis, L. cylindruta, and L. husselti extracts and 200~1 for the extracts from all of the large species tested. Commercial collagenase was tested in a similar manner at concentrations of 0.1, 0.5, 2, 10 and 50 mg/ml and produced severe tissue disruption when at least 2 mg/ml was present and visible (though much less severe) cellular detachment even at the 0.1 mg/ml concentration. However, when L-cysteine was added to the tissue culture wells at the rate of 11 mg/ml, all of the venoms and midgut extracts again caused significant tissue disruption, although the effects appeared to be somewhat reduced. This was not the case when commercial collagenase was pre-mixed with r_-cysteine, the skin samples then remaining totally unaffected even when the L-cysteine concentration was only 0.11 mg/ml. It was also noted that if the fluid in the culture well, having been acidified by the addition of the r_-cysteine solution, was not then adjusted to a slightly alkaline pH (by addition of a small volume of 14% NaHCO, solution), the extent of tissue disruption was much less than otherwise. In vitro estimations
of midgut collugenase activities
The assays of the 13 species tested for midgut extract collagenase activity are presented in Table 1. In each case two estimations were made. The collagenase activities of the crude venoms of N. edulis, E. transmarina, and I. immunis were also estimated and are included in Table 1. It can be seen that the venoms and midgut extracts of all species tested contained measurable amounts of collagenase activity. It was also apparent that the larger the species the more collagenase there is in the adult midgut. DISCUSSION
The results presented in this paper support the suggestion that when humans or other animals are bitten by spiders and subsequently develop necrotic lesions at the bite sites, the reason for the ulceration is not that the offending spiders’ venom glands secrete a necrotoxin but that their venoms have become contaminated with digestive tract fluids during voidTable
1. Assays of the collagenase actwities in the crude venoms and midgut extracts of several Australian spider species
Species N. edulis E. transmarina I. immanis I. vasta N. .wlanitri M. pulchellus A. infensus L. godeflroyi B. insignis L. hasselti L. cylindrata P. pholangioides A. horiola
Adult female body length (mm) 22 25 35 34 30 26 35 27 I8 IO I8 9 5
Units of collagenase activity in IOml One midgut (2 specimens) of venom 2100, 1472, 1310, 1196, 1352, 1184, 1500. 614, 272, 818, 140, 134, 60,
2440 2654 1388 1280 1428 1506 1250 890 520 908 606 132 44
I55 57 41
127
ing. This is to be expected, having regard to the usual prey and manner of feeding of spiders (Foehx, 1982). Strong circumstantial evidence that the digestive secretions from members of a wide range of spider families contain collagenase activity was also obtained. It therefore seems reasonable to conclude that this collagenase activity is probably the main reason for the phenomenon of necrotic arachnidism. This conclusion appears to contradict the findings of several workers who could not demonstrate the involvement of collagenase in the aetiology of the necrotic lesions and who instead obtained evidence for sphingomyelinase D as the main necrotoxin of spider venoms (Kurpiewski et al., 1981; Rekow et al., 1983). This discrepancy may be an indication that L. reclusa, the spider mostly involved when studies of necrotic arachnidism have been reported, has a different necrogenic action from that of the species tested above. However, this seems unlikely since representatives from a wide range of Araneomorph and Mygalomorph families were chosen for the necrotic studies reported here and all species appeared to produce the same effects, differing only in their relative potencies. In addition, many workers have chosen to use mechanically expressed venom or venom gland extracts rather than venom samples that could have become contaminated with digestive secretions. Kurpiewski et al. (1981) even claimed that sphingomyelinase D isolated from L. reclusa venom caused the typical necrotic lesion after 8 days and suggested that the observed dermonecrosis caused by L. reclusu venom might be partly due to an abnormal haemostatic effect involving platelet aggregation. This finding is difficult to reconcile with the results presented in this paper, and it is noteworthy that all of the necrogenic Australian spider venoms tested produced visible lesions in a matter of a few hours. Perhaps the most important observation reported above is the fact that the amino acid L-cysteine totally blocked the necrotic effects of the venoms of several different spider species when premixed with the crude venoms. A therapeutic use for this amino acid in the treatment of dermonecrosis caused by spider bites is therefore possible. The fact that L-cysteine was shown to be able to inhibit collagenase activity in vitro and is relatively specific for this enzyme (Seifter et al., 1959), provides further evidence that collagenase is of prime importance in the aetiology of the in vivo necrosis caused by many spider species. Unfortunately, L-cysteine was found to be only partially successful in blocking the in vitro necrosis caused by both raw spider venoms and midgut extracts. The explanation for this discrepancy is uncertain but appears likely to be due to instability of the L-cysteine in tissue culture conditions, which are slightly alkaline. Seifter et al. (1959) reported that collagenase is inhibited by sulphydryl-containing agents such as L-cysteine, probably because these chelate a metallic component of the enzyme. The oxidation of L-cysteine to L-cystine in alkaline conditions will thus prevent this inhibition. The fact that L-cysteine (supplied as the hydrochloride and therefore acidic) totally blocked the actions of microbial collagenase in vitro was probably because only onetenth as much L-cysteine was used on that occasion
128
RONALD
K.
ATKINSON and LYNETTE G. WRIGHT
so that no neutralization of the culture medium was necessary. The ability of spider venoms and midgut extracts to cause total dissociation of the cells of the entire epidermis in mice was somewhat surprising. Bornstein and Sage in their 1980 review showed that there is still much uncertainty about the number of types of collagen that are found in nature and also about their distributions within the mammalian body. These authors refer to “nonstructural” collagen that assists in cellular attachment to a substratum in association with fibronectin and other matrix proteins. It is therefore likely that it is this type of collagen that is being dissolved when spider venoms and midgut extracts cause the observed loss of epidermal cell adhesion.
Bornstein P. and Sage H. (1980) Structurally distinct collagen types. Ann. &v. Biochem. 49, 957-1003. Butz W. C., Stacv L. D. and Hervford N. N. (1971) Arachnid&m in rabbits. Archs. Path. 91, 97-100. Eskafi F. M. and Norment B. R. (1976) Physiolo~cal action of Loxosceles reclusa (G&M) venom on insect larvae. Toxicon 14, 7-13.
Foelix R. F. (1982) The Biology of Spiders, pp. 32-52. Harvard University Press, Massachusetts. Geren C. R., Ghan-T. K., Ward B. C., Howell D. E., Pinkston K. and Ode11 G. V. (1973) Comoosition and properties of extract of fiddleback (ioxosceies rechtsa) spider venom apparatus. Toxicon 11, 471479. Kaiser E. and Raab W. (1967) Collagenolytic activity of snake and spider venoms. Toxicon 4, 2X-255. Kurpiewski G., Forrester L. J., Barrett J. T. and Campbell B. J. (1981) Platelet aggregation and sphingomyelinase D activity of a purified toxin from the venom of Loxosceles rec&.
Ackno~,ledge~enfs-The research described in this paper was financially supported by the Darling Downs Association for Advanced Education, to whom the authors are very grateful.
REFERENCES
Appel W. (1974) Collagenases. In Methods of Enzymatic Anal~ysis(Edited by Bergmeyer H. R.), 2nd Edn, Vol. 2, 1059-1063. Academic Press, New York. Atkins J. A.. Wingo C. W., Sodeman W. A. and Flynn J. E. (19581Necrotic arachnidism. Am. /. ITOR.Med. HJX. .7, 165-184. Atkinson R. K. and Wright L. G. (1990) A study of the necrotic actions of the venom of the wolf spider, Lycosu godefioyi, on mouse skin. Comp. Biochem. Physjoi. MC, 319-325.
Atkinson R. K. and Wright L. G. (1991) Studies of the necrotic actions of the venoms of several Australian spiders, Comp. Biochrm. Physiol. 98C, 441-444. Babcock J. L., Suber R. L., Frith C. H. and Geren C. R. (1981) Systemic effect in mice of venom apparatus extract and toxin from the brown recluse spider (Loxosceles reclusa). Toxicon 19, 46347 I.
Biochim. Biophys. Acta 678, 467-476.
Perret B. A. (1977) Proteolvtic activitv of taranttda venoms due to contamination with saliva. Tox~co~ 15, 505-510. Rekow M. A., Civello D. J. and Geren C. R. (1983) Enzymatic and hemolytic properties of brown recluse spider (Loxosceles reclusa) toxins and extracts of venom apparatus, cephalothorax and abdomen. Toxicon 21, 44-444. Russell F. E. and Gertsch W. J. (1983) Letter to the editor. Toxica?i 21, 337-339. Schenone H. and Suarez G. (1978) Venoms of Scytodidae. Genus Loxosceles. In Handbook qf Experimental Pharmacology (Edited by Bettini S.), Vol. 48, pp. 247-275. Springer, Berlin. Seifter S., Gallop P. M., Klein L. and Meilman E. (1959) Studies on collagen. II Properties of purified collagenase and its inhibition. J. biof. Chem. 234, 285-293. Smith C. W. and Micks D. W. (1970) The role of polymorphonuclear leukocytes in the lesion caused by the venom of the brown spider, Loxosceles reclusa. Lab. Invest. 22, 90-93.
Wright R. P., Elgert K. D., Campbell B. J. and Barrett J. T. (1973) Hyaluronidase and esterase activities of the venom of the poisonous brown recluse spider. Arch. Biochem. Bjophys. 159, 415-426.
