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Local effects induced by coral snake venoms: Evidence of myonecrosis after experimental inoculations of venoms from five species
Toxicon, Vol . 2l, No . 6, pp . 777-783, 1983. Printed in Gtnt Brit~in.
0041-OI01/83 53 .00+ .00 ® 1983 Per~mon Preu I.td.
LOCAL EFFECTS INDUCED BY CORAL SNAKE VENOMS: EVIDENCE OF MYONECROSIS AFTER EXPERIMENTAL INOCULATIONS OF VENOMS FROM FIVE SPECIES JOS$ MARfA GUTIÉRREZ,' BRUNO LOMONTE,' ELSA PORTILLA,~ LUIS CERDAS I
and
ERMILA ROJAS' 'Instituto Clodomiro 1?icado and 'Departamento de Parasitologia, Facultad de Microbiologia, Universidad de Costa Rica, San José, Costa Rica
(Amepted for publication 19 April 1983)
J . M . Ovr»SnnEZ, B . LOMONiZ, E . PORTILIA, L. Ceanes and E. Roles . Local effaxs induced by coral snake venons : evidence of myonaxosis after experimental inoculations of venons from five species . Taxirnn 21, 777-783, 1983 . - The local effects induced by intramuscular inoculations of venons from six species of coral snakes were studied in mice. Venons of Mkrurus nlgrocinctus nigrocinctus, M. n . maspuitensis, M. alleni, M. frontalis, M. aarinkauda and M. surtrtamensis induced prominent myonaxosis which was observal histologically. FYom a morphological point of view all these venons induced a similar pattern of myonecrosis, characterved by a conspiwoua alteration of the intracellular structure . This myotoxic activity was corroboratal by an increase in plasma creative kinase levels 3 hr after i .m . igjection of M. n. nlgnvcinch4,s, M. n . mosrluitensis, M. frontalis and M. carinicvuda venons . M, mipartitus venom did not induce myonaxosis. None of the venons induced edema or hemorrhage at the site of i>ltijection .
INTRODUCTION
from many elapds induce prominent myonecrosis in experimental animals . Several potent myotoxins have been isolated from venoms of Australian elapid snakes such as Notenhis scutatus (KARLSSON et al., 1972), Oxyuranus scutellatus (FOHLMAN et al., 1976), Pseudechis coletti, P. australis and P. porphyriacus (LEONARDI et al., 1979; MEBS and SAMEJIMA, 1980). The effects of two of these toxins (notexin and taipoxin) on skeletal muscle have been studied histologically and ultrastructurally (HARRIS et al., 1975 ; HARRIS and MALTIN, 1982). Studies with venoms from other elapds, such as Ntlja rtt~ja, Bungarus fasciatus and Dendroaspisjamesoni, have demonstrated that they induce myonecrosis in mice (HOMMA and Tu, 1971 ; STRINGER et al., 1971). Clearly, myotoxins are present in a variety of elapid venoms . Coral snakes, the only elapds in America, comprise more than SO species classified in the genera Micrurus, Leptomicrurus, and Micruroides (ROZE, 1970). Their venoms induce a neurotoxic effect in humans and laboratory animals (PARRISH and KHAN, 1967 ; RUSSELL, 1967; ROSENFELD, 1971 ; Jn~rrEZ-PORRAS et al., 1973 ; MOUSSATCH$ and MELZ~NDEZ, 1979). Cardiovascular shock has also been observed in animals injected with the venoms of Micrurus fulvius (RAMSEY et al., 1972) and M. frontalis (BRAZIL et al., 1978). The venom of Micrurus nigrocinctus has a strong myotoxic action in mice after i.m . inoculation (GUTI>3RREZ et al., 1980), causing histological evidence of muscle damage and a significant increase in the plasma levels of creative kinase . These observations prompted VENOMS
777
778
JOS$ MARfA GUTI$RREZ et al.
us to perform an investigation on the myotoxic effects of venoms from several species of coral snakes collected from different geographic regions of America. In the present communication we studied the local effects (myonecrosis, hemorrhage and edema) induced by the venoms of Micrurus nigrocinctus nigrocinctus, M. n. mosquitensis, M. alleni, M. mipartitus, M. frontalis, M. carinicauda and M. surinamensis. MATERIALS AND METHODS Venoms Veaoms from Mir.~urus nigrocinctus nigrocinctus, M. n. mos~quitensis, M. alleni and M. mipartitus were obtained from specimens collected in several locations in Costa Rica and kept at the Instituto Clodomiro Picado . The venoms of M, n. nlgrocinctus and M. n. masquitenszs were studied independently because, although they belong to the same species, they exhibit karyotypic differences (GvnERRF7 and Bowvos, 1981). The venom of M. sur6~arnensiswasobtained at the Instituto Clodomiro Picado from one specimen originally collected in Colombia and donated to us by Dr . Juan Manuel Renjifo. The venom of M. frontalis was obtained in Argentina and was a gift from Dr. Jorge w. Ababa. The venom of M. carinicauda was obtained from specimens originating in Colombia. The venoms were frozen, lyophilized and stored at -70°C. Quantitation of edema The method of Y~ntnxnwn et al. (1976) was used . Groups of six mice (18-20 g.) were injected with 30 ~1 of venom solution (containing 30 hg of venom in phosphate buffered saline, pH 7.2) in the right foot pad and with 30 ld of buffered saline solution in the left foot pad. After 24 hr, mice were sacxificed by chloroform inhalation and both feet were cut at the proximal joint and weighed in an analytical balance. Edema was expressed as the per cent increase in weight of the envenomated foot relative to the weight of the saline-injected foot . Since some mice died with this dose, several experiments were performed injecting only 13 Ng of venom (in 30 pl of buffered saline) . Quantitation of hemorrhage The method of Korroo et al. (1960) was used, with several modifications. Mice (18-24 g) were injected intradermally with 10 Ng of venom (in 0.1 ml). After 4 hr they were sacrificed by chloroform inhalation and their skins were removed and observed for the presence of hemorrhage . Venoms were also inoculated i.m . in the thigh at doses of 30 Ng and 1 S Ng. After 4 hr, tissue samples were obtained from skeletal muscle of the thigh for microscopic evaluation of hemorrhage. Control acperiments wen performed by injecting buffered saline solution under the same experimental conditions . Quantttation of myonerrosir Each venom was injected i.m . into groups of four mice; two mice received a dose of 13 Ng and two mice were inoculated with 30 Pg . As controls, four mice received an i.m. injection of buffered saline solution (pH 7.2). All inoculations were in the thigh. Three hours afar venom M+n;~rnr+ pn a blood sample, obtained by cutting the tip of the tail, was collected into heparinized capillary tubes. Plasma was obtained after centrifugation and the creative kinase activity was determined (Sigma Technical Bulletin No . 320) . One unit of CK activity results in the phosphorylation of 1 nmole creative/min at 25°C . Four hours after venom injection, mice were sacrificed and tissue samples from skeletal muscle of the thigh wen obtained and immediately placed in a formaldehyde fvuuive solution. Tissues were processed routinely, embedded in paraffin, sectioned, and stained with hematoxylin-eosin. In these hiatalogical studies, myonxrosis was classified semiquantitatively based on the relative amount of necrotic fibers: absence of myonecrosis (-); mild myonecrosis (+); moderate myonecrosis (+ +) ; seven myonecrosis (+ + +). Some of the mice injected with the venoms of M. alleni and M, surinamensis died before blood samples wen taken. In these cases tissue samples were obtained at the time of death. RESULTS
Table 1 shows that the venoms of Micrurus nigrocinctus nigrocinctus, M. n. mosquitensis, M. carinicauda, M. frontalis, M. alleni and M. surinamensis induced conspicuous myonecrosis in mice in contrast to the venom of M. mipartitus which did not show myotoxic activity. Histologically, the venoms of M. n. nigrocinctus, M. n. mosquitensis, M. frontalis, M. carinicauda, M. alleni and M. surinamensis induced a qualitatively similar necrotic
pattern, with a prominent disorganization of the intracellular material (Fig. f), while quantitatively M. surinamensis induced less myonecrosis (Table 1).
779
Pto. 1. S~~.srwi, ~+usci.a ~e~eoven xaoM tinca 4 hr ~~ t.M. uvmcnoN wrrH vaxotNS. A. Physiologic saline solution . The structure of muscle fibers is nonmal 4 hr after inocilation . B . 30 Pg of Mkrurrrs nigriocinctus nlarocbectas venom . There is severe naaosis with most of the muscle fibers affected .
~eo
Pta. 1 continued . C . 13 Frg of Mknu'us Jrontalis venom. Nxrotic fibers are intermixed with normal fibers . D . 30 kg of M. carJnicauda venom . 13xtensive muscular necrosis is evident, with polymorphonuclear leukocyte inï~ltrate in some areas .
Myonecrosis Induced by Coral Snake Venoms
78 1
TABLE 1 . MYOTOXIC EFFECT INDUCED BY CORAL SNAKE VENOMS IN MICE
Venom M. n . nigrocinctus M. n, mosglritensis M. alleni M. mipanitus M. svrinamensis M. frontalis M. carinicauda
L)ose
(hB) 1S 30 1S 30 1S 30 1S 30 1S 30 13 30 13 30
HIStOIOgy" ++ +++ + + +++ ++ +++ + + + ++ + + +
Plasma Cfeatlne kinase levels (U/ml) 620f34 916f106 736f45 892f60 33f10 37ß 226f23 606ß3 S60f34 800f23 37f7
.Sceline solution "Abscence of necrosis (-); mild necrosis (+); moderate necrosis (+ +) ; severe vxrosis (+ + +) . Expressed as units (LJ)/ml f S .D . ; lU of creative kinase activity reaults iv the phosphorylatiov of 1 uncle of creative/min at 2S°C. A dash indicates that the mice died before the third hour and blood samples were vot obtained . The myonecrotic effect was corroborated by an increase in plasma creative kinase activity 3 hr after injection of M. n. nigrocinctus, M. n. mosquitensis, M. frontalis and M. carinicauda venoms (Table 1) . Mice inoculated with M. mipartitus venom showed creative kinase activity within normal ranges . Plasma creative kinase determination was not performed in mice injected with M. alleni and M. surinamensis venoms, since animals died before the third hour . However, in these cases the myotoxic effects were clearly observed histologically . Mice injected with buffered saline solution did not show evidence of myonecrosis, since plasma creative kilLase levels did nôt increase above normal ranges and the morphology of muscle fibers was normal . (Fig . lA ; Table 1) . None of the venoms induced hemorrhagic effects, either by intradermal or by i.m . injections . Histological analysis of skeletal muscle confirmed the lack of hemorrhagic activity or other vascular alterations at the site of i>~jection. The venovls did not induce local edema, since there was no increase in weight of the feet inoculated with venom, as compared to the feet injected with buffered saline solution . DISCUSSION Traditionally, coral snake venoms have been said to be neurotoxic and to cause cardiovascular problems (PARRISH and KHAN, 1%7; RoSENFELD, 1971 ; JLML~NEZ-PORRAS et al ., 1973 ; RAMSEY et al., 1972 ; BRAZIL, et al ., 1978). The present study shows that the venoms from five species of coral snakes induce myonecrosis in mice and confirms an earlier report that described myotoxicity after inoculations of M. nigtvcinctus venom (GLrcL>~RAF.9 et al., 1980). WEIS and McIsAAC (1971) demonstrated a direct action of M. fulvius venom on skeletal muscle membranes. In their experiments, muscle cells showed a hyaline degeneration after being incubated with this venom and a rapid decrease in their membrane potential. Morphologically, the necrotic fibers show a very consistent pattern in which the all is filled with masses of amorphous and disorganized material . Similar histological alterations
782
JOSÉ MAR1A GUTI$RREZ et al.
have been observed in skeletal muscle after inoculations of the venoms of the elapids Ngja ngja, Bungarusfasciatus, Uendroaspisjamesoni (HOMMA and Tu, 1971) and Oxyuranus scutellatus (HARRIS and IVIALTIN, 1982). In the case of coral snake venoms, myotoxic effects develop very rapidly after inoculation. By 3 - 4 hr, creative kinase levels are significantly increased in plasma and tissue damage is prominent. Previously, GUTIÉRREz et al. (1980) demonstrated that necrotic fibers can be found as early as 15 min after the i.m. inoculation of M. nigrocinctus venom in mice. These venoms obviously contain potent myotoxins. In regard to other local effects, our results show that neither hemorrhage nor edema develop after coral snake venom injection. EMERY and RUSSELL (1963) had previously described the lack of hemorrhaggc activity in the venom of M. fulvius. Furthermore, in our study, other vascular alterations such as angionecrosis and thrombosis were absent in the histological preparations examined . This is in clear contrast to the effects induced by pit viper venoms, which drastically affect the vasculature (HOMMA and Tu, 1971) . From a clinical point of view, myotoxicity seems to be a significant factor in envenomations due to bites by elapids such as Notechis scutatus (SUTHERLAND and COULTER, 1977) and Pseudechis australis (ROWLANDS et al., 1969). Local necrosis has also been demonstrated after cobra bites (CAMPBELL, 1979). Although there have been no clinical reports on myotoxic effects due to coral snake bites, it is possible that this effect is overlooked in humans due to the lack of macroscopically prominent local effects and to the fact that laboratory tests such as creative kinase determination and urinalysis are not performed on a routine basis. In this regard, it is interesting that a review of information concerning clinical reports of coral snake-bite cases revealed that several patients showed severe muscular pain, suggestive of myonecrosis (BOLANOS, 1982). It is important that the role of myotoxicity following envenomation induced by coral snake bites be clarified. This problem may be approached by systematically evaluating the plasma creative kinase and urinary myoglobin levels in victims of coral snake bites. Acknowledgements - The authors acknowledge the excellent technical assistance of Ar .vwxo FIAass, JAVIER Nthvez and ALFREDO Vwaaws . We also thank Dr . ~ L . Owrtsx for her valuable suggestions in preparing the manuscript. Tlu~ study was supported by the Vicerrectorla de Investigacibn, Universidad de Costa Rica, project 02-07-10-65 .
REFERENCES BOLwN06, R . (1982) Las serpientes venenosas de Centroamérica y et problems dal ofidismo . Primera parte . Aspectos zoolbgicos, epidemiol8gicos y biomédicos . Revta Costa Rirn Gent. m6d. 3, 165 . Bxwut., O . V ., Par i nAn rt , F . A . and Dus Foxrwxw, M. (1978) PaWophysiology of the envenomation produced by Micrurus jrontalis venom . In : Toxins: Animal, Plant and Mkrobial, p. 437 (RasavaeRO, P ., Ed.) Oxford : Pergamon Press . Cw~rneeLL, C. H . (1979) Symptomatology, pathology and treatment of the bites of elapid snakes . In: Handbook of Experimental Phannarology, Vol. 52, Snake Venoms, p. 898 (Lri?, C . Y ., Ed .) . Berlin : Springer Verlag . E~xx, J . A. and RUSSBLL, F . E . (1%3) Lethal and hemorrhagic properties of some North American snake venoms. In: Venomous and Poisonous Animals and Noxious Plants of the P~jfu Aroa, p. 409 (KEAOAN, H . L . and MACFARLANE, W . V ., Eds .) . Oxford : Pergamon Press . Fotn.nRwiv, J ., EwxeR, D ., Kwtet .ssoN, E . and Tfrnsr t=.rs, S . (1976) Taipoxin, an extremely potent presynaptic neurotoxin from the venom of the Australien snake Taipan (Oxyumrrus s. scutellatus) . Isolation, characterization, quaternary structure and ~harmacological properties . Eur. J. Blochem. 68, 457. GvnEttxez, J . M . and Bot .wr~os, R . (1981) Polimorfismo cromos6mico intraespecifico en la serpiente de coral Microns nigrocinctus (Ophidia : Elapidae) . Revta viol. trop. 29, 115 . GtmEaxEZ, J : M ., CHAVES, F ., RoJws, E . and Bot.wr~os, R. (1980) Efectos locales inducidos por et veneno de la serpiente coral Microns nigroclnctus en ratdn blanco . Toxirnn 18, 633 .
Myonecrosis Induced by Coral Snake Venoms
78 3
HAaa~s, J. B. and MwtTnv, C. A. (1982) Myotoxic activity of the crude venom and the principal neurotoxin, taipoxin, of the Australian taipan, OXyunanus scnteUatus. Br. 1. Pharmar. 76, 61 . Hwweni, J. B., JoEnvsox, M. A. and Kwatssox, E. (1975) Pathological responses of rat akdetal muscle to a single subcutaneous igjection of a toxin isolated from the venom of the Australian tiger snake, Notenhis srutatus scutatus. Clln. exp. Pharntar. Physiol. 2, 383. HowKw, M. and Tu, A. T. (1971) Morphology of local tissue damage in experimental snake envcnomation . Br. J. exp. Path. 52, 583. Jn~xez-Poaaws, J. M., G6~Laww, M. A., Ronatauez-BwaQuEao, J. A., Mtxrox, S. A., Ja ., GawYnox, J. J. and AMwawL, A. do . (1973) Reptile toxins . In: Biology Data Book, Vol. II, p. 697. Federation of American Societies for Experimental Biology (FASEH). Kwatssox, E., Bws~e, D. and RYnsrr, L. (1972) Purification of a preaynaptic neurotoxin from the venom of the Australian tiger snake, Notenhis srutatus scvtatus. Toxioon 1", 403. Koxno, H., Koxno, S., I~zwww, L, Muawrw, R. and Oxswew, A. (1960) Studies on the quantitative method for the determination of hemorrhagic activity of habu snake venom. lap. 1. mad. Sci. Blot. 13, 43 . Ltioxwant, T. M., Hownw, M. E. and Sr~vce, I. (1979) A lethal myotoxin isolated from the venom of the Australian king brown make (Psruderhis austrolis). TaYicon 17, 549. Mss, D. and Swt~tmtw, Y. (1980) Purifxation, from Australian dapid veaoms, sect properties of phosphoGpases A which cause myoglobinuria in mice. Toxkon 18, 443. Mousswn.~é, H. and Mm .~vne2, T. (1979) Some pharmacological observations with Elapidae and Crotalidae make venoma in the guinea-pig denervated diaphragm. On the spxificity of the cholinergic blockade by their venons . Revta brndl. Blot. 3!, 605. Pwwttsx, H. M. and Kxwx, M. S. (1%7) Bites by coral snakes: report of 11 representative cases. Am. 1. mud. ScJ. 253, 561 . Rwt~r, H. W., TAYIAR, J. W., Hoawcxow, I. B. and Sxmm, G. K. (1972) Mechanism of shock produced by an elapid snake (Micnrnrsf. ,julvhrs) venom in dogs . Am. I. Phystol. 222, 782. Ros~.n, G. (1971) Symptomatology, pathology and treatment of make bites in South America. In: Venomous AnlmaLs and 77u'r Venom, Vol. II . Venomous Vatebnates, p. 343 (BOcHt~., W. and HUCKLEY, E., Eds.). New York : Academic Press. Rowuxns, J. B., Mwsrwatrw, F. L., Kwxu~ws, H. A. and HwnrswoarH, D. (1969) Clinical and pathological expects of a fatal case of mulga (Pseuderhis australis) snakebite. Mad. !. Aunt. 2, 226. Rote, J. (1970) Mlcrurus. In : Catalogue of the Neotropical Squamata. Part I, Snakes, p. 1% (PErexs, J. A. end Onatws-Mtawrrow, B., Eda.). Washington D.C.: Smithsonian Institute Press. RUSSm.[., F. E. (1%7) Bites by the Sonoran coral snake, Micruroides euryxanthus. Toxirnn S, 39 . STacxamt, J. M., Kwnvtfle, R. A. and TU, A. T. (1971) LJltraatructural studies on myonecrosis induced by cobra venom in mice . Toxk. appl. Pharmac. 18, 442. Su~euxo, S. K. and COIJLTEa, A. R. (1977) Three in .~n~~+ ve cas~a of tiger snake (Notenhis srutatus) envmomadon aced how a radioimmunoassay proved the diagnosis. Mcd. !. Aunt. 2, 177. Wens, R. aced Mclsnwc, R. J. (1971) Cardiovascular and muscular effects of venom from coral snake, Mlcrurus Julvhis. Taxirnn 9, 219. Ywwiwrwww, M., Noznn, M. and HoxwMw, Z. (1976) Fractionation of saldshima-habu (Trlmursunrs degans) venom and lethal, hemorrha~c and edema-forming activities of the fractions. In: Animal, Plant andMicrobial Toudns, Vol. I, p. 97 (Oxswrw, A., HwYwset, K. and Swwwi, Y., Eda.). New York : Plenum Press.
0041-OI01/83 53 .00+ .00 ® 1983 Per~mon Preu I.td.
LOCAL EFFECTS INDUCED BY CORAL SNAKE VENOMS: EVIDENCE OF MYONECROSIS AFTER EXPERIMENTAL INOCULATIONS OF VENOMS FROM FIVE SPECIES JOS$ MARfA GUTIÉRREZ,' BRUNO LOMONTE,' ELSA PORTILLA,~ LUIS CERDAS I
and
ERMILA ROJAS' 'Instituto Clodomiro 1?icado and 'Departamento de Parasitologia, Facultad de Microbiologia, Universidad de Costa Rica, San José, Costa Rica
(Amepted for publication 19 April 1983)
J . M . Ovr»SnnEZ, B . LOMONiZ, E . PORTILIA, L. Ceanes and E. Roles . Local effaxs induced by coral snake venons : evidence of myonaxosis after experimental inoculations of venons from five species . Taxirnn 21, 777-783, 1983 . - The local effects induced by intramuscular inoculations of venons from six species of coral snakes were studied in mice. Venons of Mkrurus nlgrocinctus nigrocinctus, M. n . maspuitensis, M. alleni, M. frontalis, M. aarinkauda and M. surtrtamensis induced prominent myonaxosis which was observal histologically. FYom a morphological point of view all these venons induced a similar pattern of myonecrosis, characterved by a conspiwoua alteration of the intracellular structure . This myotoxic activity was corroboratal by an increase in plasma creative kinase levels 3 hr after i .m . igjection of M. n. nlgnvcinch4,s, M. n . mosrluitensis, M. frontalis and M. carinicvuda venons . M, mipartitus venom did not induce myonaxosis. None of the venons induced edema or hemorrhage at the site of i>ltijection .
INTRODUCTION
from many elapds induce prominent myonecrosis in experimental animals . Several potent myotoxins have been isolated from venoms of Australian elapid snakes such as Notenhis scutatus (KARLSSON et al., 1972), Oxyuranus scutellatus (FOHLMAN et al., 1976), Pseudechis coletti, P. australis and P. porphyriacus (LEONARDI et al., 1979; MEBS and SAMEJIMA, 1980). The effects of two of these toxins (notexin and taipoxin) on skeletal muscle have been studied histologically and ultrastructurally (HARRIS et al., 1975 ; HARRIS and MALTIN, 1982). Studies with venoms from other elapds, such as Ntlja rtt~ja, Bungarus fasciatus and Dendroaspisjamesoni, have demonstrated that they induce myonecrosis in mice (HOMMA and Tu, 1971 ; STRINGER et al., 1971). Clearly, myotoxins are present in a variety of elapid venoms . Coral snakes, the only elapds in America, comprise more than SO species classified in the genera Micrurus, Leptomicrurus, and Micruroides (ROZE, 1970). Their venoms induce a neurotoxic effect in humans and laboratory animals (PARRISH and KHAN, 1967 ; RUSSELL, 1967; ROSENFELD, 1971 ; Jn~rrEZ-PORRAS et al., 1973 ; MOUSSATCH$ and MELZ~NDEZ, 1979). Cardiovascular shock has also been observed in animals injected with the venoms of Micrurus fulvius (RAMSEY et al., 1972) and M. frontalis (BRAZIL et al., 1978). The venom of Micrurus nigrocinctus has a strong myotoxic action in mice after i.m . inoculation (GUTI>3RREZ et al., 1980), causing histological evidence of muscle damage and a significant increase in the plasma levels of creative kinase . These observations prompted VENOMS
777
778
JOS$ MARfA GUTI$RREZ et al.
us to perform an investigation on the myotoxic effects of venoms from several species of coral snakes collected from different geographic regions of America. In the present communication we studied the local effects (myonecrosis, hemorrhage and edema) induced by the venoms of Micrurus nigrocinctus nigrocinctus, M. n. mosquitensis, M. alleni, M. mipartitus, M. frontalis, M. carinicauda and M. surinamensis. MATERIALS AND METHODS Venoms Veaoms from Mir.~urus nigrocinctus nigrocinctus, M. n. mos~quitensis, M. alleni and M. mipartitus were obtained from specimens collected in several locations in Costa Rica and kept at the Instituto Clodomiro Picado . The venoms of M, n. nlgrocinctus and M. n. masquitenszs were studied independently because, although they belong to the same species, they exhibit karyotypic differences (GvnERRF7 and Bowvos, 1981). The venom of M. sur6~arnensiswasobtained at the Instituto Clodomiro Picado from one specimen originally collected in Colombia and donated to us by Dr . Juan Manuel Renjifo. The venom of M. frontalis was obtained in Argentina and was a gift from Dr. Jorge w. Ababa. The venom of M. carinicauda was obtained from specimens originating in Colombia. The venoms were frozen, lyophilized and stored at -70°C. Quantitation of edema The method of Y~ntnxnwn et al. (1976) was used . Groups of six mice (18-20 g.) were injected with 30 ~1 of venom solution (containing 30 hg of venom in phosphate buffered saline, pH 7.2) in the right foot pad and with 30 ld of buffered saline solution in the left foot pad. After 24 hr, mice were sacxificed by chloroform inhalation and both feet were cut at the proximal joint and weighed in an analytical balance. Edema was expressed as the per cent increase in weight of the envenomated foot relative to the weight of the saline-injected foot . Since some mice died with this dose, several experiments were performed injecting only 13 Ng of venom (in 30 pl of buffered saline) . Quantitation of hemorrhage The method of Korroo et al. (1960) was used, with several modifications. Mice (18-24 g) were injected intradermally with 10 Ng of venom (in 0.1 ml). After 4 hr they were sacrificed by chloroform inhalation and their skins were removed and observed for the presence of hemorrhage . Venoms were also inoculated i.m . in the thigh at doses of 30 Ng and 1 S Ng. After 4 hr, tissue samples were obtained from skeletal muscle of the thigh for microscopic evaluation of hemorrhage. Control acperiments wen performed by injecting buffered saline solution under the same experimental conditions . Quantttation of myonerrosir Each venom was injected i.m . into groups of four mice; two mice received a dose of 13 Ng and two mice were inoculated with 30 Pg . As controls, four mice received an i.m. injection of buffered saline solution (pH 7.2). All inoculations were in the thigh. Three hours afar venom M+n;~rnr+ pn a blood sample, obtained by cutting the tip of the tail, was collected into heparinized capillary tubes. Plasma was obtained after centrifugation and the creative kinase activity was determined (Sigma Technical Bulletin No . 320) . One unit of CK activity results in the phosphorylation of 1 nmole creative/min at 25°C . Four hours after venom injection, mice were sacrificed and tissue samples from skeletal muscle of the thigh wen obtained and immediately placed in a formaldehyde fvuuive solution. Tissues were processed routinely, embedded in paraffin, sectioned, and stained with hematoxylin-eosin. In these hiatalogical studies, myonxrosis was classified semiquantitatively based on the relative amount of necrotic fibers: absence of myonecrosis (-); mild myonecrosis (+); moderate myonecrosis (+ +) ; seven myonecrosis (+ + +). Some of the mice injected with the venoms of M. alleni and M, surinamensis died before blood samples wen taken. In these cases tissue samples were obtained at the time of death. RESULTS
Table 1 shows that the venoms of Micrurus nigrocinctus nigrocinctus, M. n. mosquitensis, M. carinicauda, M. frontalis, M. alleni and M. surinamensis induced conspicuous myonecrosis in mice in contrast to the venom of M. mipartitus which did not show myotoxic activity. Histologically, the venoms of M. n. nigrocinctus, M. n. mosquitensis, M. frontalis, M. carinicauda, M. alleni and M. surinamensis induced a qualitatively similar necrotic
pattern, with a prominent disorganization of the intracellular material (Fig. f), while quantitatively M. surinamensis induced less myonecrosis (Table 1).
779
Pto. 1. S~~.srwi, ~+usci.a ~e~eoven xaoM tinca 4 hr ~~ t.M. uvmcnoN wrrH vaxotNS. A. Physiologic saline solution . The structure of muscle fibers is nonmal 4 hr after inocilation . B . 30 Pg of Mkrurrrs nigriocinctus nlarocbectas venom . There is severe naaosis with most of the muscle fibers affected .
~eo
Pta. 1 continued . C . 13 Frg of Mknu'us Jrontalis venom. Nxrotic fibers are intermixed with normal fibers . D . 30 kg of M. carJnicauda venom . 13xtensive muscular necrosis is evident, with polymorphonuclear leukocyte inï~ltrate in some areas .
Myonecrosis Induced by Coral Snake Venoms
78 1
TABLE 1 . MYOTOXIC EFFECT INDUCED BY CORAL SNAKE VENOMS IN MICE
Venom M. n . nigrocinctus M. n, mosglritensis M. alleni M. mipanitus M. svrinamensis M. frontalis M. carinicauda
L)ose
(hB) 1S 30 1S 30 1S 30 1S 30 1S 30 13 30 13 30
HIStOIOgy" ++ +++ + + +++ ++ +++ + + + ++ + + +
Plasma Cfeatlne kinase levels (U/ml) 620f34 916f106 736f45 892f60 33f10 37ß 226f23 606ß3 S60f34 800f23 37f7
.Sceline solution "Abscence of necrosis (-); mild necrosis (+); moderate necrosis (+ +) ; severe vxrosis (+ + +) . Expressed as units (LJ)/ml f S .D . ; lU of creative kinase activity reaults iv the phosphorylatiov of 1 uncle of creative/min at 2S°C. A dash indicates that the mice died before the third hour and blood samples were vot obtained . The myonecrotic effect was corroborated by an increase in plasma creative kinase activity 3 hr after injection of M. n. nigrocinctus, M. n. mosquitensis, M. frontalis and M. carinicauda venoms (Table 1) . Mice inoculated with M. mipartitus venom showed creative kinase activity within normal ranges . Plasma creative kinase determination was not performed in mice injected with M. alleni and M. surinamensis venoms, since animals died before the third hour . However, in these cases the myotoxic effects were clearly observed histologically . Mice injected with buffered saline solution did not show evidence of myonecrosis, since plasma creative kilLase levels did nôt increase above normal ranges and the morphology of muscle fibers was normal . (Fig . lA ; Table 1) . None of the venoms induced hemorrhagic effects, either by intradermal or by i.m . injections . Histological analysis of skeletal muscle confirmed the lack of hemorrhagic activity or other vascular alterations at the site of i>~jection. The venovls did not induce local edema, since there was no increase in weight of the feet inoculated with venom, as compared to the feet injected with buffered saline solution . DISCUSSION Traditionally, coral snake venoms have been said to be neurotoxic and to cause cardiovascular problems (PARRISH and KHAN, 1%7; RoSENFELD, 1971 ; JLML~NEZ-PORRAS et al ., 1973 ; RAMSEY et al., 1972 ; BRAZIL, et al ., 1978). The present study shows that the venoms from five species of coral snakes induce myonecrosis in mice and confirms an earlier report that described myotoxicity after inoculations of M. nigtvcinctus venom (GLrcL>~RAF.9 et al., 1980). WEIS and McIsAAC (1971) demonstrated a direct action of M. fulvius venom on skeletal muscle membranes. In their experiments, muscle cells showed a hyaline degeneration after being incubated with this venom and a rapid decrease in their membrane potential. Morphologically, the necrotic fibers show a very consistent pattern in which the all is filled with masses of amorphous and disorganized material . Similar histological alterations
782
JOSÉ MAR1A GUTI$RREZ et al.
have been observed in skeletal muscle after inoculations of the venoms of the elapids Ngja ngja, Bungarusfasciatus, Uendroaspisjamesoni (HOMMA and Tu, 1971) and Oxyuranus scutellatus (HARRIS and IVIALTIN, 1982). In the case of coral snake venoms, myotoxic effects develop very rapidly after inoculation. By 3 - 4 hr, creative kinase levels are significantly increased in plasma and tissue damage is prominent. Previously, GUTIÉRREz et al. (1980) demonstrated that necrotic fibers can be found as early as 15 min after the i.m. inoculation of M. nigrocinctus venom in mice. These venoms obviously contain potent myotoxins. In regard to other local effects, our results show that neither hemorrhage nor edema develop after coral snake venom injection. EMERY and RUSSELL (1963) had previously described the lack of hemorrhaggc activity in the venom of M. fulvius. Furthermore, in our study, other vascular alterations such as angionecrosis and thrombosis were absent in the histological preparations examined . This is in clear contrast to the effects induced by pit viper venoms, which drastically affect the vasculature (HOMMA and Tu, 1971) . From a clinical point of view, myotoxicity seems to be a significant factor in envenomations due to bites by elapids such as Notechis scutatus (SUTHERLAND and COULTER, 1977) and Pseudechis australis (ROWLANDS et al., 1969). Local necrosis has also been demonstrated after cobra bites (CAMPBELL, 1979). Although there have been no clinical reports on myotoxic effects due to coral snake bites, it is possible that this effect is overlooked in humans due to the lack of macroscopically prominent local effects and to the fact that laboratory tests such as creative kinase determination and urinalysis are not performed on a routine basis. In this regard, it is interesting that a review of information concerning clinical reports of coral snake-bite cases revealed that several patients showed severe muscular pain, suggestive of myonecrosis (BOLANOS, 1982). It is important that the role of myotoxicity following envenomation induced by coral snake bites be clarified. This problem may be approached by systematically evaluating the plasma creative kinase and urinary myoglobin levels in victims of coral snake bites. Acknowledgements - The authors acknowledge the excellent technical assistance of Ar .vwxo FIAass, JAVIER Nthvez and ALFREDO Vwaaws . We also thank Dr . ~ L . Owrtsx for her valuable suggestions in preparing the manuscript. Tlu~ study was supported by the Vicerrectorla de Investigacibn, Universidad de Costa Rica, project 02-07-10-65 .
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HAaa~s, J. B. and MwtTnv, C. A. (1982) Myotoxic activity of the crude venom and the principal neurotoxin, taipoxin, of the Australian taipan, OXyunanus scnteUatus. Br. 1. Pharmar. 76, 61 . Hwweni, J. B., JoEnvsox, M. A. and Kwatssox, E. (1975) Pathological responses of rat akdetal muscle to a single subcutaneous igjection of a toxin isolated from the venom of the Australian tiger snake, Notenhis srutatus scutatus. Clln. exp. Pharntar. Physiol. 2, 383. HowKw, M. and Tu, A. T. (1971) Morphology of local tissue damage in experimental snake envcnomation . Br. J. exp. Path. 52, 583. Jn~xez-Poaaws, J. M., G6~Laww, M. A., Ronatauez-BwaQuEao, J. A., Mtxrox, S. A., Ja ., GawYnox, J. J. and AMwawL, A. do . (1973) Reptile toxins . In: Biology Data Book, Vol. II, p. 697. Federation of American Societies for Experimental Biology (FASEH). Kwatssox, E., Bws~e, D. and RYnsrr, L. (1972) Purification of a preaynaptic neurotoxin from the venom of the Australian tiger snake, Notenhis srutatus scvtatus. Toxioon 1", 403. Koxno, H., Koxno, S., I~zwww, L, Muawrw, R. and Oxswew, A. (1960) Studies on the quantitative method for the determination of hemorrhagic activity of habu snake venom. lap. 1. mad. Sci. Blot. 13, 43 . Ltioxwant, T. M., Hownw, M. E. and Sr~vce, I. (1979) A lethal myotoxin isolated from the venom of the Australian king brown make (Psruderhis austrolis). TaYicon 17, 549. Mss, D. and Swt~tmtw, Y. (1980) Purifxation, from Australian dapid veaoms, sect properties of phosphoGpases A which cause myoglobinuria in mice. Toxkon 18, 443. Mousswn.~é, H. and Mm .~vne2, T. (1979) Some pharmacological observations with Elapidae and Crotalidae make venoma in the guinea-pig denervated diaphragm. On the spxificity of the cholinergic blockade by their venons . Revta brndl. Blot. 3!, 605. Pwwttsx, H. M. and Kxwx, M. S. (1%7) Bites by coral snakes: report of 11 representative cases. Am. 1. mud. ScJ. 253, 561 . Rwt~r, H. W., TAYIAR, J. W., Hoawcxow, I. B. and Sxmm, G. K. (1972) Mechanism of shock produced by an elapid snake (Micnrnrsf. ,julvhrs) venom in dogs . Am. I. Phystol. 222, 782. Ros~.n, G. (1971) Symptomatology, pathology and treatment of make bites in South America. In: Venomous AnlmaLs and 77u'r Venom, Vol. II . Venomous Vatebnates, p. 343 (BOcHt~., W. and HUCKLEY, E., Eds.). New York : Academic Press. Rowuxns, J. B., Mwsrwatrw, F. L., Kwxu~ws, H. A. and HwnrswoarH, D. (1969) Clinical and pathological expects of a fatal case of mulga (Pseuderhis australis) snakebite. Mad. !. Aunt. 2, 226. Rote, J. (1970) Mlcrurus. In : Catalogue of the Neotropical Squamata. Part I, Snakes, p. 1% (PErexs, J. A. end Onatws-Mtawrrow, B., Eda.). Washington D.C.: Smithsonian Institute Press. RUSSm.[., F. E. (1%7) Bites by the Sonoran coral snake, Micruroides euryxanthus. Toxirnn S, 39 . STacxamt, J. M., Kwnvtfle, R. A. and TU, A. T. (1971) LJltraatructural studies on myonecrosis induced by cobra venom in mice . Toxk. appl. Pharmac. 18, 442. Su~euxo, S. K. and COIJLTEa, A. R. (1977) Three in .~n~~+ ve cas~a of tiger snake (Notenhis srutatus) envmomadon aced how a radioimmunoassay proved the diagnosis. Mcd. !. Aunt. 2, 177. Wens, R. aced Mclsnwc, R. J. (1971) Cardiovascular and muscular effects of venom from coral snake, Mlcrurus Julvhis. Taxirnn 9, 219. Ywwiwrwww, M., Noznn, M. and HoxwMw, Z. (1976) Fractionation of saldshima-habu (Trlmursunrs degans) venom and lethal, hemorrha~c and edema-forming activities of the fractions. In: Animal, Plant andMicrobial Toudns, Vol. I, p. 97 (Oxswrw, A., HwYwset, K. and Swwwi, Y., Eda.). New York : Plenum Press.