.oo
Toxkan, Vd. 30, No. la, pp. 130~1306, 1992. Prroted in ß~wt Hritain .
aal-01o1~9z u.ao + ® 1992 l'cyamon P~w Ltd
EUROPEAN VIPER VENOMS: HAEMORRHAGIC AND MYOTOXIC ACTIVITIES Dnrrxtcx Mss and THOMAS L.+xaL~.~nn>~
Zentrum der Rechtsmedizin, University of Frankfurt, Kennedyallee 104, D-600 Frankfurt-70, F.R.G . (Received 12 February 1992: accepted 3 April 1992)
D. M~es and T. LANGELÜDDEKE. European viper venoms: haemorrhagic and myotoxic activities. Toxicon 30, 1303-1306, 1992 :Thirty-one venom samples from European vipers (genera Vipers and Daboia) were tested for haemorrhagic and myotoxic activity by intramuscular injection into . mice. Most venoms exhibited haemorrhagc activity and fewer had myotoxic activity, both of which are not strictly related.
and myonecrosis are serious manifestations of snakebite, causing prolonged and sometimes permanent disability . Whereas myotoxic activity is present in a wide range ofvenoms from Elapidae as well as Viperidae snakes, haemorrhagic activity is predominantly found in Viperidae venoms only (HOMMA and Tu, 1971 ; M»s and OwivsY, HABMORRHAGS
1990).
Bites of European vipers produce local swelling and discolouration, but rarely tissue damage and necrosis (GONZALE4, 1991). However, haemorrhagic activity has been detected in these viper venoms (TAx and PoxxunuxA~, 1990) and haemorrhagic factors were isolated from Vipers aspic aspis (KOMORI and SUGIIiARA, 1988) and from Vipers berus berus venom (SAMEL and SIIGUR, 1990). In the present study, 31 venoms from European vipers were tested for haemorrhagic as well as myotoxic activity. Venoms were purchased from Latoxan (Rosans, France) and from Ophidia Venin (Tavannes, Switzerland), or were a gift from Dr F. Kornalik (Prague, Czechoslovakia) (see Table 1). Venom samples were dissolved in 0.9% NaCI and 50 pl of various dilutions injected intramuscularly into the hind limb (M. biceps femoris) of mice (three per dose). After 6 hr the mice were killed by chloroform inhalation and the extent of haemorrhage in the hind leg was evaluated macroscopically after removing the skin. For histological examination muscle samples were fixed in 10% formalin and embedded in methacrylate; sections were stained with hematoxylin/eosin or azur II/methylene blue . Table 1 shows the results of testing haemorrhagic and myotoxic activities of the venoms . Using various doses (3-50 hg per mouse) moat venoms exhibited considerable haemorrhagic activity and fewer venoms had myotoxic activity . Hoth activities seem not to be strictly related. Venoms like that from Vipers aspicjrancisciredi cause extensive local myonecrosis (Fig. 1), whereas others (e.g. Vipers ursinü ursinü) are haemorrhagic, but not myotoxic at all. Variations in these activities on species level (e.g. Vipers ammodytes) are 1303
1304
Short Communications Teet,e 1 . HASl1iORRHAarC ~ e~o~roxic ecnvn~w or Eureore~v vtz~ v~oess Snake origin (source)
Vipers aspic aspic atra jratuisciredi zinnikeri
Ingyi
Vipers amrnodytes ammodytes mertdionalis montandari transcatrcasiana
ssp .
Haemorrhage
Myonecrosis
Switzerland (1) S-France (3) Italy (1) Switzerland (1) France (2) Italy (l')
-
-
Yugoslavia (1) Hulgaria (3) Bulgaria (3') Yugoslavia (3) Greece (1 ") Bulgaria (2) Bulgaria (2) Turkey (l') Turkey (1 ")
~~~
-
-
-
Vipern berus beres banrlensis
Czechoslovakia (3) Yugoslavia (1)
Vipers latarti gaditans
S-Spain (l')
Pipera seomrei cantabrica
Spain (l')
Vipcra wstnü wsinü eriwmrensis
France (I) Turkey (l')
Daboia iebetina lebetina obtusa
Turkey (1) Turkey (l') Armenia (3) Greece (2) Uzbekistan (3)
schwrizeri turanica Daboia tnarvitanica
Morocco (1)
Daboia raddei raddei
Turkey (1 ")
DoiSoia wogneri
Turkey (1 ")
Dsboia xanthlna roddel
Greece (1) Armenia (3)
-
-
-
~~~
-
~~
-
~~~
-
Mice were injected i .m . with various amounts of venom (3-SO Wg), killed after 6 hr and the extent of local haemorrhage and myonecrosis was evaluated macroscopically and microscopically. ~ ~ ~ , Haemorrhage or myonaxosis observed at a dose of 3 or 6 pg venom; ~ ~ ,venom dose 12 pg; ~ , venom dose 25 pg-no effect at a dose of 50 pg. Venom sources (number in brackets): (1) Ophidia Venin ; (2) Latoxan; (3) ih F. ICortx~tt¢; 'venom from a single specimen, all other venoms are pooled samples from several snakes .
also observed. Whether this is related to the geographical origin or an intraspecific or even an individual variation, is worth further investigations . This study supplements data obtained by TArr and PoxxunuxAl (1990) and demonstrates that European viper venoms contain potent haemorrhagins and myotoains. On the other hand, these venom effects are rarely seen in actual snakebite cases (GONZ,ALFS, 1991). This can be explained by the still moderate level of activity when compared to that of typical haemorrhagic or myotoxie venoms such as from Bibs tuietans (MsBS and
Short Communications
>;ia . 1 . LKiHT MICROCiRAPHS OF SEELETAL MUSCLE TAEEN FROM A MOUSE AFr'IIt THE 1 .m . rNIEC1iON OF 3 pg vENOM r~oM Yipero aspic jrmrcisciredi (A) AND Yipera ursinü wsinü (B), ~ECrrveLY, 6 hr. Note various stages of myonecrosis and clumped myoSbrils (A), whereas in (B) extensive haemorrhage with intact muscle cells is seen . Bar, 100 prn.
1305
1306
Short Communications
1982) or from Australian elapid snakes (M13H3 and $AMETIMA, 1980). Moreover, Européen vipers are of small size (mostly less than 1 m body length) and inject minute amounts of venom which are apparently not sufficient to produce local haemorrhage and myonecrosis . This also applies to the neurotoxic phospholipase A2, ammodytoxin A, present in Vipera ammndytes venom (RTPONJA and GuBENSexx, 1985), which acts at the presynaptic site of motor endplates (1~ et al., 1984). But neurotoxic symptoms such as ptosis, ophthahnoplegia, and paralysis of voluntary muscles are usually not observed in human cases, which is most probably due to the low amount of venom (and toxin) injected. On the other hand, European viper venoms can cause quite a number of serious effects in humans such as cardiovascular shock, extensive oedema, especially angioneurotic and even late pulmonary oedema (GONZ.ALB4, 1991). These venom actions are poorly understood and may be triggered by autopharmacologicel effects initiated by the venom. PANHO1.zElt,
REFERENCES D. (1991) Snakebite problems in Europe. In : Reptile Veno»u and Toxins ('Ib, A. T., Ed .), Handbook of Natwal Toxins, Vol. 5, pp . 687-751 . New York: Mercel Dekker. Hoe®u, M. and Ttr, A. T. (1971) Morphology of local tissue damage in experimental snake envenomation. Br. J. exp . Path. S2, 538-542. Kostoxt, Y. and Suan~iu, H. (1988) Biological study of muscle degenerating hemorrhagic factor from the venom of Vipera aspic aspic (aspic viper). Int . J. Btocltem. 20, 1417-1423. Lam, C. Y., TsN, M. C., Ct~r, Y. M., Rrrorne, A. and Guean~c, F. (1984) Mode of neuromuscular blocking action of toxic phospholipeses A= from Vipera ammodytes venom. Arclu ant. Pharmacodyn . 77rér. ?68, 313-324. Mss, D. and owrtaY, C. L. (1990) Myotoxic components of snake venoms: their biochemical and biological activities . PJiarmac. Ther. 48, 223-236. Mss, D. and Pexxot.z~, F. (1982) Isolation of a haemorrhagic principle from Bath arietans (puff adder) make venom. Toxirnn 2t1, 509-512. Mss, D. and Seea;tou, Y. (1980) Purification, from Australian elapid venoms, and properties of phospholipeses A which cause myoglobinuria in mice. Toxicon 18, 443-454. Rrrorue, A. and Gtiew9Et~, F. (1985) Ammodytoxin A, a highly lethal phospholipase Az from Vipera ommodytes amrrtodytes venom. Biochim. Biophys . Acta 828, 306-312. Sit., M. and Snoux, J. (1990) Isolation and characterization of hemorrhagic metalloproteinase from Vipera berur betas (crommon viper) venom. Comp. Biochem . P6ysiol. 97C, 209-214. Tetv, N. H. and Poxxvntttut, G . (1990) A wmparative study of the biological properties of venoms from snakes of the genus Vipera (true adders). Comp . Biochem. Pbysiol. 9tiB, 683-688. GOTiZALE4,
Toxkan, Vd. 30, No. la, pp. 130~1306, 1992. Prroted in ß~wt Hritain .
aal-01o1~9z u.ao + ® 1992 l'cyamon P~w Ltd
EUROPEAN VIPER VENOMS: HAEMORRHAGIC AND MYOTOXIC ACTIVITIES Dnrrxtcx Mss and THOMAS L.+xaL~.~nn>~
Zentrum der Rechtsmedizin, University of Frankfurt, Kennedyallee 104, D-600 Frankfurt-70, F.R.G . (Received 12 February 1992: accepted 3 April 1992)
D. M~es and T. LANGELÜDDEKE. European viper venoms: haemorrhagic and myotoxic activities. Toxicon 30, 1303-1306, 1992 :Thirty-one venom samples from European vipers (genera Vipers and Daboia) were tested for haemorrhagic and myotoxic activity by intramuscular injection into . mice. Most venoms exhibited haemorrhagc activity and fewer had myotoxic activity, both of which are not strictly related.
and myonecrosis are serious manifestations of snakebite, causing prolonged and sometimes permanent disability . Whereas myotoxic activity is present in a wide range ofvenoms from Elapidae as well as Viperidae snakes, haemorrhagic activity is predominantly found in Viperidae venoms only (HOMMA and Tu, 1971 ; M»s and OwivsY, HABMORRHAGS
1990).
Bites of European vipers produce local swelling and discolouration, but rarely tissue damage and necrosis (GONZALE4, 1991). However, haemorrhagic activity has been detected in these viper venoms (TAx and PoxxunuxA~, 1990) and haemorrhagic factors were isolated from Vipers aspic aspis (KOMORI and SUGIIiARA, 1988) and from Vipers berus berus venom (SAMEL and SIIGUR, 1990). In the present study, 31 venoms from European vipers were tested for haemorrhagic as well as myotoxic activity. Venoms were purchased from Latoxan (Rosans, France) and from Ophidia Venin (Tavannes, Switzerland), or were a gift from Dr F. Kornalik (Prague, Czechoslovakia) (see Table 1). Venom samples were dissolved in 0.9% NaCI and 50 pl of various dilutions injected intramuscularly into the hind limb (M. biceps femoris) of mice (three per dose). After 6 hr the mice were killed by chloroform inhalation and the extent of haemorrhage in the hind leg was evaluated macroscopically after removing the skin. For histological examination muscle samples were fixed in 10% formalin and embedded in methacrylate; sections were stained with hematoxylin/eosin or azur II/methylene blue . Table 1 shows the results of testing haemorrhagic and myotoxic activities of the venoms . Using various doses (3-50 hg per mouse) moat venoms exhibited considerable haemorrhagic activity and fewer venoms had myotoxic activity . Hoth activities seem not to be strictly related. Venoms like that from Vipers aspicjrancisciredi cause extensive local myonecrosis (Fig. 1), whereas others (e.g. Vipers ursinü ursinü) are haemorrhagic, but not myotoxic at all. Variations in these activities on species level (e.g. Vipers ammodytes) are 1303
1304
Short Communications Teet,e 1 . HASl1iORRHAarC ~ e~o~roxic ecnvn~w or Eureore~v vtz~ v~oess Snake origin (source)
Vipers aspic aspic atra jratuisciredi zinnikeri
Ingyi
Vipers amrnodytes ammodytes mertdionalis montandari transcatrcasiana
ssp .
Haemorrhage
Myonecrosis
Switzerland (1) S-France (3) Italy (1) Switzerland (1) France (2) Italy (l')
-
-
Yugoslavia (1) Hulgaria (3) Bulgaria (3') Yugoslavia (3) Greece (1 ") Bulgaria (2) Bulgaria (2) Turkey (l') Turkey (1 ")
~~~
-
-
-
Vipern berus beres banrlensis
Czechoslovakia (3) Yugoslavia (1)
Vipers latarti gaditans
S-Spain (l')
Pipera seomrei cantabrica
Spain (l')
Vipcra wstnü wsinü eriwmrensis
France (I) Turkey (l')
Daboia iebetina lebetina obtusa
Turkey (1) Turkey (l') Armenia (3) Greece (2) Uzbekistan (3)
schwrizeri turanica Daboia tnarvitanica
Morocco (1)
Daboia raddei raddei
Turkey (1 ")
DoiSoia wogneri
Turkey (1 ")
Dsboia xanthlna roddel
Greece (1) Armenia (3)
-
-
-
~~~
-
~~
-
~~~
-
Mice were injected i .m . with various amounts of venom (3-SO Wg), killed after 6 hr and the extent of local haemorrhage and myonecrosis was evaluated macroscopically and microscopically. ~ ~ ~ , Haemorrhage or myonaxosis observed at a dose of 3 or 6 pg venom; ~ ~ ,venom dose 12 pg; ~ , venom dose 25 pg-no effect at a dose of 50 pg. Venom sources (number in brackets): (1) Ophidia Venin ; (2) Latoxan; (3) ih F. ICortx~tt¢; 'venom from a single specimen, all other venoms are pooled samples from several snakes .
also observed. Whether this is related to the geographical origin or an intraspecific or even an individual variation, is worth further investigations . This study supplements data obtained by TArr and PoxxunuxAl (1990) and demonstrates that European viper venoms contain potent haemorrhagins and myotoains. On the other hand, these venom effects are rarely seen in actual snakebite cases (GONZ,ALFS, 1991). This can be explained by the still moderate level of activity when compared to that of typical haemorrhagic or myotoxie venoms such as from Bibs tuietans (MsBS and
Short Communications
>;ia . 1 . LKiHT MICROCiRAPHS OF SEELETAL MUSCLE TAEEN FROM A MOUSE AFr'IIt THE 1 .m . rNIEC1iON OF 3 pg vENOM r~oM Yipero aspic jrmrcisciredi (A) AND Yipera ursinü wsinü (B), ~ECrrveLY, 6 hr. Note various stages of myonecrosis and clumped myoSbrils (A), whereas in (B) extensive haemorrhage with intact muscle cells is seen . Bar, 100 prn.
1305
1306
Short Communications
1982) or from Australian elapid snakes (M13H3 and $AMETIMA, 1980). Moreover, Européen vipers are of small size (mostly less than 1 m body length) and inject minute amounts of venom which are apparently not sufficient to produce local haemorrhage and myonecrosis . This also applies to the neurotoxic phospholipase A2, ammodytoxin A, present in Vipera ammndytes venom (RTPONJA and GuBENSexx, 1985), which acts at the presynaptic site of motor endplates (1~ et al., 1984). But neurotoxic symptoms such as ptosis, ophthahnoplegia, and paralysis of voluntary muscles are usually not observed in human cases, which is most probably due to the low amount of venom (and toxin) injected. On the other hand, European viper venoms can cause quite a number of serious effects in humans such as cardiovascular shock, extensive oedema, especially angioneurotic and even late pulmonary oedema (GONZ.ALB4, 1991). These venom actions are poorly understood and may be triggered by autopharmacologicel effects initiated by the venom. PANHO1.zElt,
REFERENCES D. (1991) Snakebite problems in Europe. In : Reptile Veno»u and Toxins ('Ib, A. T., Ed .), Handbook of Natwal Toxins, Vol. 5, pp . 687-751 . New York: Mercel Dekker. Hoe®u, M. and Ttr, A. T. (1971) Morphology of local tissue damage in experimental snake envenomation. Br. J. exp . Path. S2, 538-542. Kostoxt, Y. and Suan~iu, H. (1988) Biological study of muscle degenerating hemorrhagic factor from the venom of Vipera aspic aspic (aspic viper). Int . J. Btocltem. 20, 1417-1423. Lam, C. Y., TsN, M. C., Ct~r, Y. M., Rrrorne, A. and Guean~c, F. (1984) Mode of neuromuscular blocking action of toxic phospholipeses A= from Vipera ammodytes venom. Arclu ant. Pharmacodyn . 77rér. ?68, 313-324. Mss, D. and owrtaY, C. L. (1990) Myotoxic components of snake venoms: their biochemical and biological activities . PJiarmac. Ther. 48, 223-236. Mss, D. and Pexxot.z~, F. (1982) Isolation of a haemorrhagic principle from Bath arietans (puff adder) make venom. Toxirnn 2t1, 509-512. Mss, D. and Seea;tou, Y. (1980) Purification, from Australian elapid venoms, and properties of phospholipeses A which cause myoglobinuria in mice. Toxicon 18, 443-454. Rrrorue, A. and Gtiew9Et~, F. (1985) Ammodytoxin A, a highly lethal phospholipase Az from Vipera ommodytes amrrtodytes venom. Biochim. Biophys . Acta 828, 306-312. Sit., M. and Snoux, J. (1990) Isolation and characterization of hemorrhagic metalloproteinase from Vipera berur betas (crommon viper) venom. Comp. Biochem . P6ysiol. 97C, 209-214. Tetv, N. H. and Poxxvntttut, G . (1990) A wmparative study of the biological properties of venoms from snakes of the genus Vipera (true adders). Comp . Biochem. Pbysiol. 9tiB, 683-688. GOTiZALE4,
