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Bothrops lanceolatus (Fer de lance) venom induces oedema formation and increases vascular permeability in the mouse hind paw
Toxicon 38 (2000) 209±221 www.elsevier.com/locate/toxicon
Bothrops lanceolatus (Fer de lance) venom induces oedema formation and increases vascular permeability in the mouse hind paw Albetiza LoÃbo de ArauÂjo a,*, Ana Olõ via de Souza a, Maria Alice da Cruz-Hoȯing b, Carlos Alberto Flores c, 1, Cassian Bon d a
Departamento de Farmacologia, Faculdade de CieÃncias MeÂdices, Universidade Estadual de Campinas, CP 6111, 13083-970 Campinas, SP, Brazil b Departamento de Histologia e Embriologia, IB, UNICAMP, 13083-970 Campinas, SP, Brazil c Departamento de Farmacologia, UFCE, Fortaleza, CE, Brazil d Unite des Venins, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris, France Received 27 January 1999; accepted 19 April 1999
Abstract The ability of snake venoms to increase vascular permeability and to induce oedema through the release of pharmacologically active substances is well known. We have studied the oedema and vascular permeability induced by Bothrops lanceolatus venom in male Swiss white mice. Paw oedema was induced by the subplantar injection of B. lanceolatus venom (125±1000 ng/paw) and was quanti®ed as the increase in paw weight. Changes in vascular permeability were assessed by measuring the amount of Evans blue dye extravasation. The oedema and the increase in vascular permeability were maximal within 2 h and had resolved after 24 h. The administration of the vasodilator iloprost (20 ng/paw) immediately after B. lanceolatus venom potentiated the oedema and the increase in vascular permeability by approximately four-fold. Pretreating the mice with indomethacin, dexamethasone, NDGA or BW A4C inhibited the venom-induced oedema and the increase in vascular permeability. In contrast, histamine, serotonin and PAF-acether antagonists (mepyramine, cyproheptadine and WEB 2086, respectively) were ineective. Histological examination showed that B. lanceolatus venom (250 ng and 500 ng/paw) caused thickening of the inner dermal layers which was accompanied by extensive intercellular spaces indicative of
* Corresponding author. Tel.: +55-19-788-7185/8173; fax: 55-19-289-2968. 1 Deceased. 0041-0101/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 9 9 ) 0 0 1 4 5 - 2
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oedema. In addition, there was a marked in®ltration of in¯ammatory cells, particularly neutrophils, into the underlying muscle layer. The latter, however, remained morphologically unaected during the 3 h of observation. Venom doses larger than 500 ng/ paw produced intense haemorrhage. These results indicate that B. lanceolatus venom induces oedema and increases vascular permeability in the mouse hind paw. The principal mediators of this in¯ammatory response are cyclooxygenase and lipoxygenase products. # 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction Most snakebites in Central and South America are caused by Bothrops species. Envenomation by this genus is characterized by a highly complex pathophysiological picture which includes local as well as systemic eects (Rosenfeld, 1971). The local manifestations caused by Bothrops venoms include oedema, pain, haemorrhage and necrosis, which may result in prolonged or permanent disability (Brazil, 1911; Moura da Silva et al., 1991; Ferreira et al., 1992). The pathogenesis of these eects has been ascribed to the action of proteases (Vital Brazil, 1982). The oedema and the increase in vascular permeability caused by these venoms is related, at least in part, to their ability to stimulate the release of pharmacologically active substances (Vargaftig et al., 1974; Rothschild and Rothschild, 1979; Lomonte, 1985). Furthermore, the ischaemia evoked by the oedema (Chapman, 1968) may aggravate the venom-induced lesion. Oedema formation is a common feature of the cutaneous in¯ammatory response and is dependent on a synergism between mediators that increase vascular permeability and those that increase blood ¯ow (Williams and Morley, 1973; Williams and Peck, 1977). One of the consequences of altered capillary permeability in local in¯ammation is the extravasation of leucocytes (Hamblin, 1994). The accumulation of these cells at in¯ammatory sites in the skin of various animals is mediated by an increase in local blood ¯ow (Issekutz and Movat, 1979; Issekutz, 1981; Buckley et al., 1991). Bothrops lanceolatus (Fer de lance) inhabits the island of Martinique where it is responsible for about 20 envenomations each year (Thomas et al., 1996). Since envenomation by this species diers in some aspects from that of mainland Bothrops, e.g. the absence of systemic bleeding (Thomas et al., 1996), we have examined some of the mechanisms involved in the in¯ammatory response induced by B. lanceolatus venom in the mouse paw.
2. Materials and methods 2.1. Reagents Glacial acetic acid, ethanol, formaldehyde, formamide, haematoxylin±eosin and
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xylol were purchased from Merck (Darmstadt, Germany). Sodium bicarbonate and Evans blue were supplied by Reagen (Rio de Janeiro, RJ, Brazil). Dexamethasone, carrageenan, cyproheptadine, platelet-activating factor (PAF), indomethacin, mepyramine and nordihydroguaiaretic acid (NDGA) were obtained from Sigma (St. Louis, MO, USA). Sodium chloride and ethyl ether were from Chemco (SaÄo Paulo, SP, Brazil). BWA4C was from Wellcome Research Laboratories (Beckenham, Kent, UK). DMSO was from Fisher Scienti®c (Pittsburgh, PA, USA). Iloprost was a gift from Schering (FRG) and WEB 2086 was from Boehringer (Mannheim, Germany). 2.2. Venom Dessicated Bothrops lanceolatus venom was from a stock held at the Unite des Venins, Institut Pasteur, France and was dissolved in saline (®nal concentration, 1 mg/ml) and immediately stored at ÿ208C until used. 2.3. Animals Adult male Swiss white mice (18±30 g) were supplied by the UNICAMP Central Animal House Services (CEMIB). 2.4. Measurement of oedema formation The oedema-inducing activity of the venom was assayed according to the method of Yamakawa et al. (1976). Mice under light ether anaesthesia were injected in the right or left hind foot pad with 50 ml of saline containing B. lanceolatus venom (125, 250, 500 or 1000 ng/paw) and the same volume of saline was injected into the contralateral paw. At various times after the injection of venom, the animals were sacri®ced with an overdose of ether and both feet were removed at the tibio-tarsal articulation and weighed. The extent of oedema was calculated as the dierence in weight between the foot injected with venom and that injected with saline. The results were expressed as the increase in weight (mg) for each dose of venom. 2.5. Measurement of vascular permeability The changes in vascular permeability following the injection of B. lanceolatus venom were determined by the Evans blue dye procedure of Griswold et al. (1986). 30 min before being sacri®ced, the mice received an i.v. injection of 0.3 ml of a 0.25% solution of Evans blue in saline. At the appropriate times thereafter, the animals were sacri®ced and the paws removed at the tibio-tarsal articulation and weighed. Subsequently, the paws were homogenized and incubated in 3 ml of formamide at 578C for 24 h after which the optical density of the resulting supernatant was read at 619 nm using a Uvikon 810 spectrophotometer (Kontron Instruments, Switzerland). The concentration of dye was determined from a
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standard curve of Evans blue in formamide as described by Bertrand et al. (1993). The changes in vascular permeability were expressed as the dierence in the amount of dye extravasation between the foot injected with venom and that injected with saline. 2.6. The eect of various drugs on B. lanceolatus venom-induced oedema and increase in vascular permeability Groups of mice (n = 5±12/group) received dexamethasone (1 mg/kg), indomethacin (5 mg/kg), nordihydroguaiaretic acid (NDGA, 100 mg/kg), BW A4C (50 mg/kg), WEB 2086 (50 mg/kg) or cyproheptadine (5 mg/kg) subcutaneously, 1 h before the administration of B. lanceolatus venom. In the case of mepyramine (6 mg/kg), the drug was administered i.p. 15 min before the venom. The resulting oedema and vascular permeability changes were then measured within 2 h of venom administration as described above. 2.7. Histopathological analysis Groups of three mice (20±30 g) anaesthetised with ethyl ether received a subcutaneous injection of 50 ml of venom (250 ng or 500 ng/paw) in the footpad. The animals were sacri®ced at various times thereafter (0.5, 1, 1.5, 2 and 3 h) and both feet were removed and ®xed in Bouin solution for 24 h followed by decalci®cation in 10% formaldehyde containing 5% acetic acid for 5 days. The tissues were subsequently dehydrated in an alcohol series (80, 90 and 100%) and embedded in paran. Sections 50 mm thick were stained with haematoxylin and eosin and examined by light microscopy. In control experiments, physiological saline solution was injected under the same conditions as described above. 2.8. Statistical analysis The values are the mean 2 S.E.M. of results obtained with the indicated number of animals. The statistical signi®cance of dierences between groups was evaluated using Student's unpaired t-test or by analysis of variance (ANOVA) followed by Tukey's multiple comparison test where appropriate. A p value <0.05 was considered to indicate signi®cance. 3. Results 3.1. Paw oedema and changes in vascular permeability caused by B. lanceolatus venom The time courses for the oedema and vascular permeability responses to 250 ng of B. lanceolatus venom are shown in Fig. 1. The maximal responses for both phenomena were observed 2 h after venom injection and corresponded to an
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increase in paw weight (oedema) of 10 2 1.6 mg (n = 6) and an Evans blue extravasation (vascular permeability) of 3.0 21.1 mg of Evans blue per g of tissue (n = 6). In both cases, the responses decreased gradually over 24 h. Fig. 2 shows the dose response curves obtained 2 h after the injection of B. lanceolatus venom. Venom doses of 500 and 1000 ng/paw caused local haemorrhage (results not shown) but no deaths occurred during the 24 h study period. The vasodilator iloprost (PGI2) alone induced oedema and increased vascular permeability and, when injected simultaneously with BLV (250 ng), PGI2 potentiated the venom-induced oedema and the increase in vascular permeability (Fig. 3). Since the responses to BLV+PGI2 were greater than the sum of the responses to the two stimuli separately, we conclude that PGI2 did indeed potentiate the action of BLV. 3.2. Eect of anti-in¯ammatory drugs on B. lanceolatus venom-induced oedema and the increase in vascular permeability Pretreating the mice with mepyramine, cyproheptadine and WEB 2086 did not reduce the oedema produced by the venom (250 ng/paw) at any of the doses tested (Fig. 4). However, drugs which interfere with arachidonate metabolism (indomethacin, BW A4C, NDGA and dexamethasone) generally inhibited the venom-induced oedema and the increase in vascular permeability, except for indomethacin which did not prevent the venom-induced changes in vascular permeability (Fig. 5) 3.3. Histopathological analysis Histological comparison between saline (Fig. 6a) and venom-treated paws showed that B. lanceolatus venom induced marked oedema (Fig. 6b), haemorrhage
Fig. 1. The time-course of B. lanceolatus venom-induced oedema and the increase in vascular permeability. Time-course of the oedema (A) and the increase in vascular permeability (B) in the mouse hind paw after a subplantar injection of saline (.) or B. lanceolatus venom (250 ng; Q). Each point represents the mean2 S.E.M. of 5±12 mice. P < 0.05 and P < 0.01 compared to the saline control.
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Fig. 2. The dose±response curves for B. lanceolatus venom-induced oedema and the increase in vascular permeability. The oedema (A) and the increase in vascular permeability (B) were measured 2 h after the subplantar injection of various doses of venom. Each column represents the mean2S.E.M. of 5±12 mice. P < 0.05 and P < 0.01 compared to the saline control.
Fig. 3. Potentiation by prostaglandin I2 (iloprost) of the oedema and increase in vascular permeability produced by B. lanceolatus venom. The potentiating eect of PGI2 (Ilo, 20 ng) on the venom-induced oedema (A) and the increase in vascular permeability (B) was determined 2 h after the administration of venom (250 ng/paw). Each column represents the mean2 S.E.M. of 5±12 mice. P < 0.05 and P < 0.01 compared to the saline control.
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Fig. 4. Mepyramine, cyproheptadine and WEB 2086 do not inhibit B. lanceolatus venom-induced oedema and the increase in vascular permeability. The mice were pretreated with mepyramine (M, 6 mg/kg), cyproheptadine (C, 5 mg/kg) or WEB 2086 (WEB 2086, 50 mg/kg) prior to the injection of B. lanceolatus venom (BLV). The paw oedema (A) and the increase in vascular permeability (B) were measured as described in Section 2. Each column represents the mean2 S.E.M. of 5±12 mice. The control response to venom 250 ng alone is shown for each treatment.
and leukocyte in®ltration (Fig. 7a and c) in the sub-epidermal layer of connective tissue. The oedema and leukocyte in®ltration (Fig. 7b) were seen at nonhaemorrhagic doses of venom. The diuse in¯ammatory in®ltrate which accumulated in the underlying muscle layer 3 h after venom injection consisted mainly of polymorphonuclear neutrophils (Fig. 7a and b). 4. Discussion B. lanceolatus venom produced oedema and altered vascular permeability in the mouse hind paw. These responses were followed by abundant leukocyte in®ltration and, at high doses of venom, by haemorrhage. The kinetics and cell composition of the in¯ammatory in®ltrate observed in the footpad were similar to those previously reported for muscle injected with Bothrops asper venom (GutieÂrrez et al.,1986; Lomonte et al., 1993). Trebien and Calixto (1989) reported that the oedema caused by B. jararaca venom in rats was maximal within 1 h and disappeared within 24 h. Studies using other snake venoms have shown that the initiation of oedema and the time required for it to reach a maximum vary considerably (GutieÂrrez et al., 1980; Lomonte, 1985; Vishwanath et al., 1987;
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Fig. 5. The eect of nordihydroguaiaretic acid, indomethacin, BW A4C and dexamethasone on B. lanceolatus venom-induced paw oedema and the increase in vascular permeability. The mice were pretreated with nordihydroguaiaretic acid (NDGA, 100 mg/kg), indomethacin (INDO, 5 mg/kg), BW A4C (50 mg/kg) or dexamethasone (DEXA, 1 mg/kg) before the injection of B. lanceolatus venom (BLV). The paw oedema (A) and the increase in vascular permeability (B) were measured as described in Section 2. Each column represents the mean2 S.E.M. of 5±12 mice. P < 0.05, P < 0.01 and P < 0.001 compared to venom alone. The control response to venom (250 ng) alone is shown for each treatment.
Selistre et al., 1990). These results may be explained by the dierent origins of the venoms, their composition and by the species of animal used. The extent of oedema observed with B. lanceolatus venom was less marked than that seen when carrageenan (100 mg/paw) was used as the in¯ammatory stimulus (results not shown). The oedema-inducing eect of B. lanceolatus venom probably resulted from the combined action of several components (GutieÂrrez and Lomonte, 1989). In the present study, mepyramine, an H1 receptor antagonist, cyproheptadine, a dual inhibitor of serotonin and histamine receptors, and WEB 2086, a PAF-acether antagonist, did not inhibit the oedema or the increase in vascular permeability induced by this venom, suggesting that histamine, serotonin and PAF-acether are not involved in these responses. Similar results were obtained by Trebien and Calixto (1989) and Perales et al. (1992) for B. jararaca venom. On the other hand, when drugs which interfered with the metabolism of arachidonic acid, including the corticosteroid dexamethasone, the lipoxygenase inhibitor BW A4C and the dual lipoxygenase and cyclooxygenase inhibitor NDGA, were tested, all of them signi®cantly inhibited the venom-induced oedema and the increase in vascular
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Fig. 6. Histological appearance of the mouse footpad three hours after a subcutaneous injection of 0.9% saline (a, 50 ml, control) or B. lanceolatus snake venom (b, 250 ng). In (a) the dermal region (D) between the epidermis and the skeletal muscle ®bers (M) has a normal appearance (100±120 mm thick). G=sebaceous glands. HE. Bar=100 mm. In (b) there is extensive thickening (400±450 mm) of the dermis (D) resulting from the oedema caused by the venom. Note also neutrophils rarefaction of the dermal connective tissue (collagen type 1 ®bers). F=hair follicles. HE. Bar=100 mm.
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Fig. 7. Haemorrhage and leucocyte in®ltration in the hindpaw dermis and muscle of mice following the subcutaneous injection of B. lanceolatus venom (500 ng). (a) Extravascular red blood cells (RB) and an in¯ammatory leucocyte in®ltration (mainly neutrophils, N) in the interstitial tissue of the paw. HE. Bar=50 mm. (b) Extensive neutrophil (N) in®ltration originating from venules in the muscle layer (M) underlying the dermis. T=tendon. HE. Bar=100 mm. (c) Detail of the dermal layer showing enlargement of the interstitial space caused by oedema. Note the branching venule with leucocytes migrating across the vessel wall. F=hair follicles. HE. Bar=50 mm.
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permeability. The cyclooxygenase inhibitor indomethacin inhibited only the oedematogenic activity of the venom, with no eect on the increase in vascular permeability. These results suggest that arachidonic acid metabolites produced via the lipoxygenase pathway participate in the pathogenesis of B. lanceolatus venominduced mouse paw oedema. The inability of indomethacin to inhibit the increase in vascular permeability may be interpreted as a lack of involvement of cyclooxygenase products in this particular phenomenon. The participation of dierent products of arachidonic acid metabolism in the oedema produced by snake venoms has been suggested by other studies (Vishwanath et al., 1987; Trebien and Calixto, 1989). Generally, mediators that increase vascular permeability and those that are vasodilators can act synergistically to produce oedema in experimental in¯ammatory reactions (Williams and Peck, 1977; Williams, 1979; Williams and JoseÂ, 1981). This was shown here by the ability of the vasodilator iloprost to potentiate the oedema produced by 250 ng of B. lanceolatus venom (Fig. 3). Since the above experiments were performed with crude BLV, we cannot rule out the possibility that the oedema and changes in vascular permeability also involved the action of haemorrhagic factors present at concentrations which did not cause haemorrhage but which were still able to produce the above vascular changes. Assessment of the contribution of these factors to the edema and alterations in vascular permeability should be possible once these proteins have been isolated from BLV. In conclusion, the oedema and the increase in vascular permeability produced by B. lanceolatus venom in the mouse hind paw are mediated mainly by cyclooxygenase and lipoxygenase products and can be potentiated by iloprost. Histamine, serotonin and PAF-acether appear to have only a minor role in these phenomena.
Acknowledgements This research was supported partially by the Fundac° aÄo de Amparo aÁ Pesquisa do Estado de SaÄo Paulo (FAPESP) and Fundo de Apoio ao Ensino e Pesquisa (FAEP-UNICAMP). A.O.S. was the recipient of a studentship from the Conselho Nacional de Desenvolvimento CientõÂ ®co e TecnoloÂgico (CNPq, Brazil). We thank Mrs. Marta Beatriz Leonardo for the histological work, Mr. Gustavo H. da Silva for photographing the plates, Dr. Stephen Hyslop for correcting the English and Ms. Alessandra Priscila Ponciano for typing the manuscript.
References Bertrand, C., Geppetti, P., Baker, J., Yamawaki, I., Wandel, A.J., 1993. Role of neurogenic in¯ammation in antigen-induced vascular extravasation in guinea pig trachea. J. Immunol. 150, 1479± 1485.
220
A. LoÃbo de ArauÂjo et al. / Toxicon 38 (2000) 209±221
Brazil, V., 1911. La DeÂfense Contre l'Ophidisme, 2nd ed. Pocai and Weis, SaÄo Paulo. Buckley, T.L., Brain, S.D., Collins, P.D., Williams, T.J., 1991. In¯ammatory edema induced by interactions between interleukin-1 and neuropeptide calcitonin gene-related peptide. J. Immunol. 146, 3424±3430. Chapman, D., 1968. The symptomatology, pathology and treatment of the bites of venomous snakes of Central and Southern Africa. In: BuÈcherl, W., Buckley, E., Deulofeu, V. (Eds.), Venomous Animals and their Venoms, vol. I. Academic Press, New York, pp. 463±527. Ferreira, M.L., Moura da Silva, A.M., Mota, I., 1992. Neutralization of dierent activities of venoms from nine species of Bothrops snakes by Bothrops jararaca antivenom. Toxicon 30, 1591±1602. Griswold, D.E., Webb, E.F., Clark, M.A., Mong, S., 1986. Phlogistic activity of leukotriene D4 in the mouse. In¯ammation 10, 1±7. GutieÂrrez, J.M., Lomonte, B., 1989. Local tissue damage induced by Bothrops snake venoms. Mem. Inst. Butantan 51, 211±223. GutieÂrrez, J.M., Arroyo, O., BolanÄos, R., 1980. Mionecrosis, hemorragia y edema inducidos por el veneno de Bothrops asper en raton blanco. Toxicon 18, 603±610. GutieÂrrez, J.M., Chaves, F., Cerdas, L., 1986. In¯ammatory in®ltrate in skeletal muscle injected with Bothrops asper venom. Rev. Biol. Trop. 34, 209±219. Hamblin, A.S., 1994. Cytokines. In: Dale, M., Foreman, J.C., Fan Tai-Ping, D. (Eds.), Textbook of Immunopharmacology, 3rd ed. Blackwell Scienti®c Publications, London, pp. 179±192. Issekutz, A.C., 1981. Eect of vasoactive agents on polymorphonuclear leukocyte emigration in vivo. Lab. Invest. 45, 234±240. Issekutz, A.C., Movat, H.Z., 1979. The eect of vasodilator prostaglandins on polymorphonuclear leukocyte in®ltration and vascular injury. Am. J. Pathol. 107, 300±309. Lomonte, B., 1985. Edema-forming activity of bushmaster (Lachesis muta stenophrys ) and Central American rattlesnake (Crotalus durissus durissus ) venoms and neutralization by a polyvalent antivenom. Toxicon 23, 173±176. Lomonte, B., Tarkowski, A., Hanson, L.A., 1993. Host response to Bothrops asper snake venom. Analysis of edema formation, in¯ammatory cells, and cyokine release in a mouse model. In¯ammation 17, 93±105. Moura da Silva, A.M., Desmond, H., Laing, G., Theakston, R.D.G., 1991. Isolation and comparison of myotoxins isolated from venoms of dierent species of Bothrops snakes. Toxicon 29, 713±723. Perales, J., Amorim, C.Z., Rocha, S.L.G., Domont, G.B., MoussatcheÂ, H., 1992. Neutralization of the oedematogenic activity of Bothrops jararaca venom on the mouse paw by an antibothropic fraction isolated from opossum (Didelphis marsupialis ) serum. Agents Actions 37, 250±259. Rosenfeld, G., 1971. Symptomatology, pathology and treatment of snake bites in South America. In: BuÈcherl, W., Buckley, E.E. (Eds.), Venomous Animals and their Venoms, vol II. Academic Press, New York, pp. 345±403. Rothschild, A.M., Rothschild, Z., 1979. Liberation of pharmacologically active substances by snake venoms. In: Lee, C.Y. (Ed.), Snake Venoms. Handbook of Experimental Pharmacology, vol. 52. Springer-Verlag, Berlin, pp. 591±628. Selistre, H.S., Queiroz, L.S., Cunha, O.A.B., De Souza, G.E.P., Giglio, J.R., 1990. Isolation and characterization of hemorrhagic, myonecrotic and edema-inducing toxins from Bothrops insularis (jararaca ilhoa) snake venom. Toxicon 28, 261±273. Research Group on Snake Bite in Martinique, Thomas, L., Tyburn, B., 1996. Bothrops lanceolatus bites in Martinique: clinical aspects and treatment. In: Bon, C., Goyon, M. (Eds.), Envenomings and their Treatments. Fondation Marcel MeÂriex, Lyon, p. 255. Trebien, H.A., Calixto, J.B., 1989. Pharmacological evaluation of rat paw edema induced by Bothrops jararaca venom. Agents Actions 26, 292±300. Vargaftig, B.B., Bhargava, N., Bonta, I.L., 1974. Haemorrhagic and permeability increasing eects of Bothrops jararaca and other Crotalidae venoms as related to amine or kinin release. Agent Actions 4, 163±168. Vishwanath, B.S., Kini, R.M., Gowda, T.V., 1987. Characterization of three edema inducing phospholipase A2 enzymes from habu (Trimeresurus ¯avoviridis ) venom and their interaction with the alkaloid aristolochic acid. Toxicon 25, 501±515.
A. LoÃbo de ArauÂjo et al. / Toxicon 38 (2000) 209±221
221
Vital Brazil, O., 1982. Pec° onhas. In: Corbett, C.E. (Ed.), FarmacodinaÃmica. Guanabara Koogan, Rio de Janeiro, pp. 1044±1074. Williams, T.J., 1979. Prostaglandin E2, prostaglandin I2 and the vascular changes in in¯ammation. Br. J. Pharmac. 65, 517±524. Williams, T.J., JoseÂ, P.J., 1981. Mediation of increased vascular permeability after complement activation (histamine-independent action of rabbit C5a). J. Exp. Med. 153, 136±153. Williams, T.J., Morley, J., 1973. Prostaglandins as potentiators of increased vascular permeability in in¯ammation. Nature 246, 215±217. Williams, T.J., Peck, M.J., 1977. Role of prostaglandin-mediated vasodilatation in in¯ammation. Nature 270, 530±532. Yamakawa, M., Nozaki, M., Hokama, Z., 1976. Fractionation of Sakishima habu (Trimeresurus elegans ) venom and lethal, hemorrhagic and edema-forming activities of the fractions. In: Ohsaka, A., Hayashi, K., Sawai, Y. (Eds.), Animal, Plant and Microbial Toxins, vol. 1. Plenum Press, New York, pp. 97±109.
Bothrops lanceolatus (Fer de lance) venom induces oedema formation and increases vascular permeability in the mouse hind paw Albetiza LoÃbo de ArauÂjo a,*, Ana Olõ via de Souza a, Maria Alice da Cruz-Hoȯing b, Carlos Alberto Flores c, 1, Cassian Bon d a
Departamento de Farmacologia, Faculdade de CieÃncias MeÂdices, Universidade Estadual de Campinas, CP 6111, 13083-970 Campinas, SP, Brazil b Departamento de Histologia e Embriologia, IB, UNICAMP, 13083-970 Campinas, SP, Brazil c Departamento de Farmacologia, UFCE, Fortaleza, CE, Brazil d Unite des Venins, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris, France Received 27 January 1999; accepted 19 April 1999
Abstract The ability of snake venoms to increase vascular permeability and to induce oedema through the release of pharmacologically active substances is well known. We have studied the oedema and vascular permeability induced by Bothrops lanceolatus venom in male Swiss white mice. Paw oedema was induced by the subplantar injection of B. lanceolatus venom (125±1000 ng/paw) and was quanti®ed as the increase in paw weight. Changes in vascular permeability were assessed by measuring the amount of Evans blue dye extravasation. The oedema and the increase in vascular permeability were maximal within 2 h and had resolved after 24 h. The administration of the vasodilator iloprost (20 ng/paw) immediately after B. lanceolatus venom potentiated the oedema and the increase in vascular permeability by approximately four-fold. Pretreating the mice with indomethacin, dexamethasone, NDGA or BW A4C inhibited the venom-induced oedema and the increase in vascular permeability. In contrast, histamine, serotonin and PAF-acether antagonists (mepyramine, cyproheptadine and WEB 2086, respectively) were ineective. Histological examination showed that B. lanceolatus venom (250 ng and 500 ng/paw) caused thickening of the inner dermal layers which was accompanied by extensive intercellular spaces indicative of
* Corresponding author. Tel.: +55-19-788-7185/8173; fax: 55-19-289-2968. 1 Deceased. 0041-0101/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 9 9 ) 0 0 1 4 5 - 2
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oedema. In addition, there was a marked in®ltration of in¯ammatory cells, particularly neutrophils, into the underlying muscle layer. The latter, however, remained morphologically unaected during the 3 h of observation. Venom doses larger than 500 ng/ paw produced intense haemorrhage. These results indicate that B. lanceolatus venom induces oedema and increases vascular permeability in the mouse hind paw. The principal mediators of this in¯ammatory response are cyclooxygenase and lipoxygenase products. # 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction Most snakebites in Central and South America are caused by Bothrops species. Envenomation by this genus is characterized by a highly complex pathophysiological picture which includes local as well as systemic eects (Rosenfeld, 1971). The local manifestations caused by Bothrops venoms include oedema, pain, haemorrhage and necrosis, which may result in prolonged or permanent disability (Brazil, 1911; Moura da Silva et al., 1991; Ferreira et al., 1992). The pathogenesis of these eects has been ascribed to the action of proteases (Vital Brazil, 1982). The oedema and the increase in vascular permeability caused by these venoms is related, at least in part, to their ability to stimulate the release of pharmacologically active substances (Vargaftig et al., 1974; Rothschild and Rothschild, 1979; Lomonte, 1985). Furthermore, the ischaemia evoked by the oedema (Chapman, 1968) may aggravate the venom-induced lesion. Oedema formation is a common feature of the cutaneous in¯ammatory response and is dependent on a synergism between mediators that increase vascular permeability and those that increase blood ¯ow (Williams and Morley, 1973; Williams and Peck, 1977). One of the consequences of altered capillary permeability in local in¯ammation is the extravasation of leucocytes (Hamblin, 1994). The accumulation of these cells at in¯ammatory sites in the skin of various animals is mediated by an increase in local blood ¯ow (Issekutz and Movat, 1979; Issekutz, 1981; Buckley et al., 1991). Bothrops lanceolatus (Fer de lance) inhabits the island of Martinique where it is responsible for about 20 envenomations each year (Thomas et al., 1996). Since envenomation by this species diers in some aspects from that of mainland Bothrops, e.g. the absence of systemic bleeding (Thomas et al., 1996), we have examined some of the mechanisms involved in the in¯ammatory response induced by B. lanceolatus venom in the mouse paw.
2. Materials and methods 2.1. Reagents Glacial acetic acid, ethanol, formaldehyde, formamide, haematoxylin±eosin and
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xylol were purchased from Merck (Darmstadt, Germany). Sodium bicarbonate and Evans blue were supplied by Reagen (Rio de Janeiro, RJ, Brazil). Dexamethasone, carrageenan, cyproheptadine, platelet-activating factor (PAF), indomethacin, mepyramine and nordihydroguaiaretic acid (NDGA) were obtained from Sigma (St. Louis, MO, USA). Sodium chloride and ethyl ether were from Chemco (SaÄo Paulo, SP, Brazil). BWA4C was from Wellcome Research Laboratories (Beckenham, Kent, UK). DMSO was from Fisher Scienti®c (Pittsburgh, PA, USA). Iloprost was a gift from Schering (FRG) and WEB 2086 was from Boehringer (Mannheim, Germany). 2.2. Venom Dessicated Bothrops lanceolatus venom was from a stock held at the Unite des Venins, Institut Pasteur, France and was dissolved in saline (®nal concentration, 1 mg/ml) and immediately stored at ÿ208C until used. 2.3. Animals Adult male Swiss white mice (18±30 g) were supplied by the UNICAMP Central Animal House Services (CEMIB). 2.4. Measurement of oedema formation The oedema-inducing activity of the venom was assayed according to the method of Yamakawa et al. (1976). Mice under light ether anaesthesia were injected in the right or left hind foot pad with 50 ml of saline containing B. lanceolatus venom (125, 250, 500 or 1000 ng/paw) and the same volume of saline was injected into the contralateral paw. At various times after the injection of venom, the animals were sacri®ced with an overdose of ether and both feet were removed at the tibio-tarsal articulation and weighed. The extent of oedema was calculated as the dierence in weight between the foot injected with venom and that injected with saline. The results were expressed as the increase in weight (mg) for each dose of venom. 2.5. Measurement of vascular permeability The changes in vascular permeability following the injection of B. lanceolatus venom were determined by the Evans blue dye procedure of Griswold et al. (1986). 30 min before being sacri®ced, the mice received an i.v. injection of 0.3 ml of a 0.25% solution of Evans blue in saline. At the appropriate times thereafter, the animals were sacri®ced and the paws removed at the tibio-tarsal articulation and weighed. Subsequently, the paws were homogenized and incubated in 3 ml of formamide at 578C for 24 h after which the optical density of the resulting supernatant was read at 619 nm using a Uvikon 810 spectrophotometer (Kontron Instruments, Switzerland). The concentration of dye was determined from a
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standard curve of Evans blue in formamide as described by Bertrand et al. (1993). The changes in vascular permeability were expressed as the dierence in the amount of dye extravasation between the foot injected with venom and that injected with saline. 2.6. The eect of various drugs on B. lanceolatus venom-induced oedema and increase in vascular permeability Groups of mice (n = 5±12/group) received dexamethasone (1 mg/kg), indomethacin (5 mg/kg), nordihydroguaiaretic acid (NDGA, 100 mg/kg), BW A4C (50 mg/kg), WEB 2086 (50 mg/kg) or cyproheptadine (5 mg/kg) subcutaneously, 1 h before the administration of B. lanceolatus venom. In the case of mepyramine (6 mg/kg), the drug was administered i.p. 15 min before the venom. The resulting oedema and vascular permeability changes were then measured within 2 h of venom administration as described above. 2.7. Histopathological analysis Groups of three mice (20±30 g) anaesthetised with ethyl ether received a subcutaneous injection of 50 ml of venom (250 ng or 500 ng/paw) in the footpad. The animals were sacri®ced at various times thereafter (0.5, 1, 1.5, 2 and 3 h) and both feet were removed and ®xed in Bouin solution for 24 h followed by decalci®cation in 10% formaldehyde containing 5% acetic acid for 5 days. The tissues were subsequently dehydrated in an alcohol series (80, 90 and 100%) and embedded in paran. Sections 50 mm thick were stained with haematoxylin and eosin and examined by light microscopy. In control experiments, physiological saline solution was injected under the same conditions as described above. 2.8. Statistical analysis The values are the mean 2 S.E.M. of results obtained with the indicated number of animals. The statistical signi®cance of dierences between groups was evaluated using Student's unpaired t-test or by analysis of variance (ANOVA) followed by Tukey's multiple comparison test where appropriate. A p value <0.05 was considered to indicate signi®cance. 3. Results 3.1. Paw oedema and changes in vascular permeability caused by B. lanceolatus venom The time courses for the oedema and vascular permeability responses to 250 ng of B. lanceolatus venom are shown in Fig. 1. The maximal responses for both phenomena were observed 2 h after venom injection and corresponded to an
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increase in paw weight (oedema) of 10 2 1.6 mg (n = 6) and an Evans blue extravasation (vascular permeability) of 3.0 21.1 mg of Evans blue per g of tissue (n = 6). In both cases, the responses decreased gradually over 24 h. Fig. 2 shows the dose response curves obtained 2 h after the injection of B. lanceolatus venom. Venom doses of 500 and 1000 ng/paw caused local haemorrhage (results not shown) but no deaths occurred during the 24 h study period. The vasodilator iloprost (PGI2) alone induced oedema and increased vascular permeability and, when injected simultaneously with BLV (250 ng), PGI2 potentiated the venom-induced oedema and the increase in vascular permeability (Fig. 3). Since the responses to BLV+PGI2 were greater than the sum of the responses to the two stimuli separately, we conclude that PGI2 did indeed potentiate the action of BLV. 3.2. Eect of anti-in¯ammatory drugs on B. lanceolatus venom-induced oedema and the increase in vascular permeability Pretreating the mice with mepyramine, cyproheptadine and WEB 2086 did not reduce the oedema produced by the venom (250 ng/paw) at any of the doses tested (Fig. 4). However, drugs which interfere with arachidonate metabolism (indomethacin, BW A4C, NDGA and dexamethasone) generally inhibited the venom-induced oedema and the increase in vascular permeability, except for indomethacin which did not prevent the venom-induced changes in vascular permeability (Fig. 5) 3.3. Histopathological analysis Histological comparison between saline (Fig. 6a) and venom-treated paws showed that B. lanceolatus venom induced marked oedema (Fig. 6b), haemorrhage
Fig. 1. The time-course of B. lanceolatus venom-induced oedema and the increase in vascular permeability. Time-course of the oedema (A) and the increase in vascular permeability (B) in the mouse hind paw after a subplantar injection of saline (.) or B. lanceolatus venom (250 ng; Q). Each point represents the mean2 S.E.M. of 5±12 mice. P < 0.05 and P < 0.01 compared to the saline control.
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Fig. 2. The dose±response curves for B. lanceolatus venom-induced oedema and the increase in vascular permeability. The oedema (A) and the increase in vascular permeability (B) were measured 2 h after the subplantar injection of various doses of venom. Each column represents the mean2S.E.M. of 5±12 mice. P < 0.05 and P < 0.01 compared to the saline control.
Fig. 3. Potentiation by prostaglandin I2 (iloprost) of the oedema and increase in vascular permeability produced by B. lanceolatus venom. The potentiating eect of PGI2 (Ilo, 20 ng) on the venom-induced oedema (A) and the increase in vascular permeability (B) was determined 2 h after the administration of venom (250 ng/paw). Each column represents the mean2 S.E.M. of 5±12 mice. P < 0.05 and P < 0.01 compared to the saline control.
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Fig. 4. Mepyramine, cyproheptadine and WEB 2086 do not inhibit B. lanceolatus venom-induced oedema and the increase in vascular permeability. The mice were pretreated with mepyramine (M, 6 mg/kg), cyproheptadine (C, 5 mg/kg) or WEB 2086 (WEB 2086, 50 mg/kg) prior to the injection of B. lanceolatus venom (BLV). The paw oedema (A) and the increase in vascular permeability (B) were measured as described in Section 2. Each column represents the mean2 S.E.M. of 5±12 mice. The control response to venom 250 ng alone is shown for each treatment.
and leukocyte in®ltration (Fig. 7a and c) in the sub-epidermal layer of connective tissue. The oedema and leukocyte in®ltration (Fig. 7b) were seen at nonhaemorrhagic doses of venom. The diuse in¯ammatory in®ltrate which accumulated in the underlying muscle layer 3 h after venom injection consisted mainly of polymorphonuclear neutrophils (Fig. 7a and b). 4. Discussion B. lanceolatus venom produced oedema and altered vascular permeability in the mouse hind paw. These responses were followed by abundant leukocyte in®ltration and, at high doses of venom, by haemorrhage. The kinetics and cell composition of the in¯ammatory in®ltrate observed in the footpad were similar to those previously reported for muscle injected with Bothrops asper venom (GutieÂrrez et al.,1986; Lomonte et al., 1993). Trebien and Calixto (1989) reported that the oedema caused by B. jararaca venom in rats was maximal within 1 h and disappeared within 24 h. Studies using other snake venoms have shown that the initiation of oedema and the time required for it to reach a maximum vary considerably (GutieÂrrez et al., 1980; Lomonte, 1985; Vishwanath et al., 1987;
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Fig. 5. The eect of nordihydroguaiaretic acid, indomethacin, BW A4C and dexamethasone on B. lanceolatus venom-induced paw oedema and the increase in vascular permeability. The mice were pretreated with nordihydroguaiaretic acid (NDGA, 100 mg/kg), indomethacin (INDO, 5 mg/kg), BW A4C (50 mg/kg) or dexamethasone (DEXA, 1 mg/kg) before the injection of B. lanceolatus venom (BLV). The paw oedema (A) and the increase in vascular permeability (B) were measured as described in Section 2. Each column represents the mean2 S.E.M. of 5±12 mice. P < 0.05, P < 0.01 and P < 0.001 compared to venom alone. The control response to venom (250 ng) alone is shown for each treatment.
Selistre et al., 1990). These results may be explained by the dierent origins of the venoms, their composition and by the species of animal used. The extent of oedema observed with B. lanceolatus venom was less marked than that seen when carrageenan (100 mg/paw) was used as the in¯ammatory stimulus (results not shown). The oedema-inducing eect of B. lanceolatus venom probably resulted from the combined action of several components (GutieÂrrez and Lomonte, 1989). In the present study, mepyramine, an H1 receptor antagonist, cyproheptadine, a dual inhibitor of serotonin and histamine receptors, and WEB 2086, a PAF-acether antagonist, did not inhibit the oedema or the increase in vascular permeability induced by this venom, suggesting that histamine, serotonin and PAF-acether are not involved in these responses. Similar results were obtained by Trebien and Calixto (1989) and Perales et al. (1992) for B. jararaca venom. On the other hand, when drugs which interfered with the metabolism of arachidonic acid, including the corticosteroid dexamethasone, the lipoxygenase inhibitor BW A4C and the dual lipoxygenase and cyclooxygenase inhibitor NDGA, were tested, all of them signi®cantly inhibited the venom-induced oedema and the increase in vascular
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Fig. 6. Histological appearance of the mouse footpad three hours after a subcutaneous injection of 0.9% saline (a, 50 ml, control) or B. lanceolatus snake venom (b, 250 ng). In (a) the dermal region (D) between the epidermis and the skeletal muscle ®bers (M) has a normal appearance (100±120 mm thick). G=sebaceous glands. HE. Bar=100 mm. In (b) there is extensive thickening (400±450 mm) of the dermis (D) resulting from the oedema caused by the venom. Note also neutrophils rarefaction of the dermal connective tissue (collagen type 1 ®bers). F=hair follicles. HE. Bar=100 mm.
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Fig. 7. Haemorrhage and leucocyte in®ltration in the hindpaw dermis and muscle of mice following the subcutaneous injection of B. lanceolatus venom (500 ng). (a) Extravascular red blood cells (RB) and an in¯ammatory leucocyte in®ltration (mainly neutrophils, N) in the interstitial tissue of the paw. HE. Bar=50 mm. (b) Extensive neutrophil (N) in®ltration originating from venules in the muscle layer (M) underlying the dermis. T=tendon. HE. Bar=100 mm. (c) Detail of the dermal layer showing enlargement of the interstitial space caused by oedema. Note the branching venule with leucocytes migrating across the vessel wall. F=hair follicles. HE. Bar=50 mm.
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permeability. The cyclooxygenase inhibitor indomethacin inhibited only the oedematogenic activity of the venom, with no eect on the increase in vascular permeability. These results suggest that arachidonic acid metabolites produced via the lipoxygenase pathway participate in the pathogenesis of B. lanceolatus venominduced mouse paw oedema. The inability of indomethacin to inhibit the increase in vascular permeability may be interpreted as a lack of involvement of cyclooxygenase products in this particular phenomenon. The participation of dierent products of arachidonic acid metabolism in the oedema produced by snake venoms has been suggested by other studies (Vishwanath et al., 1987; Trebien and Calixto, 1989). Generally, mediators that increase vascular permeability and those that are vasodilators can act synergistically to produce oedema in experimental in¯ammatory reactions (Williams and Peck, 1977; Williams, 1979; Williams and JoseÂ, 1981). This was shown here by the ability of the vasodilator iloprost to potentiate the oedema produced by 250 ng of B. lanceolatus venom (Fig. 3). Since the above experiments were performed with crude BLV, we cannot rule out the possibility that the oedema and changes in vascular permeability also involved the action of haemorrhagic factors present at concentrations which did not cause haemorrhage but which were still able to produce the above vascular changes. Assessment of the contribution of these factors to the edema and alterations in vascular permeability should be possible once these proteins have been isolated from BLV. In conclusion, the oedema and the increase in vascular permeability produced by B. lanceolatus venom in the mouse hind paw are mediated mainly by cyclooxygenase and lipoxygenase products and can be potentiated by iloprost. Histamine, serotonin and PAF-acether appear to have only a minor role in these phenomena.
Acknowledgements This research was supported partially by the Fundac° aÄo de Amparo aÁ Pesquisa do Estado de SaÄo Paulo (FAPESP) and Fundo de Apoio ao Ensino e Pesquisa (FAEP-UNICAMP). A.O.S. was the recipient of a studentship from the Conselho Nacional de Desenvolvimento CientõÂ ®co e TecnoloÂgico (CNPq, Brazil). We thank Mrs. Marta Beatriz Leonardo for the histological work, Mr. Gustavo H. da Silva for photographing the plates, Dr. Stephen Hyslop for correcting the English and Ms. Alessandra Priscila Ponciano for typing the manuscript.
References Bertrand, C., Geppetti, P., Baker, J., Yamawaki, I., Wandel, A.J., 1993. Role of neurogenic in¯ammation in antigen-induced vascular extravasation in guinea pig trachea. J. Immunol. 150, 1479± 1485.
220
A. LoÃbo de ArauÂjo et al. / Toxicon 38 (2000) 209±221
Brazil, V., 1911. La DeÂfense Contre l'Ophidisme, 2nd ed. Pocai and Weis, SaÄo Paulo. Buckley, T.L., Brain, S.D., Collins, P.D., Williams, T.J., 1991. In¯ammatory edema induced by interactions between interleukin-1 and neuropeptide calcitonin gene-related peptide. J. Immunol. 146, 3424±3430. Chapman, D., 1968. The symptomatology, pathology and treatment of the bites of venomous snakes of Central and Southern Africa. In: BuÈcherl, W., Buckley, E., Deulofeu, V. (Eds.), Venomous Animals and their Venoms, vol. I. Academic Press, New York, pp. 463±527. Ferreira, M.L., Moura da Silva, A.M., Mota, I., 1992. Neutralization of dierent activities of venoms from nine species of Bothrops snakes by Bothrops jararaca antivenom. Toxicon 30, 1591±1602. Griswold, D.E., Webb, E.F., Clark, M.A., Mong, S., 1986. Phlogistic activity of leukotriene D4 in the mouse. In¯ammation 10, 1±7. GutieÂrrez, J.M., Lomonte, B., 1989. Local tissue damage induced by Bothrops snake venoms. Mem. Inst. Butantan 51, 211±223. GutieÂrrez, J.M., Arroyo, O., BolanÄos, R., 1980. Mionecrosis, hemorragia y edema inducidos por el veneno de Bothrops asper en raton blanco. Toxicon 18, 603±610. GutieÂrrez, J.M., Chaves, F., Cerdas, L., 1986. In¯ammatory in®ltrate in skeletal muscle injected with Bothrops asper venom. Rev. Biol. Trop. 34, 209±219. Hamblin, A.S., 1994. Cytokines. In: Dale, M., Foreman, J.C., Fan Tai-Ping, D. (Eds.), Textbook of Immunopharmacology, 3rd ed. Blackwell Scienti®c Publications, London, pp. 179±192. Issekutz, A.C., 1981. Eect of vasoactive agents on polymorphonuclear leukocyte emigration in vivo. Lab. Invest. 45, 234±240. Issekutz, A.C., Movat, H.Z., 1979. The eect of vasodilator prostaglandins on polymorphonuclear leukocyte in®ltration and vascular injury. Am. J. Pathol. 107, 300±309. Lomonte, B., 1985. Edema-forming activity of bushmaster (Lachesis muta stenophrys ) and Central American rattlesnake (Crotalus durissus durissus ) venoms and neutralization by a polyvalent antivenom. Toxicon 23, 173±176. Lomonte, B., Tarkowski, A., Hanson, L.A., 1993. Host response to Bothrops asper snake venom. Analysis of edema formation, in¯ammatory cells, and cyokine release in a mouse model. In¯ammation 17, 93±105. Moura da Silva, A.M., Desmond, H., Laing, G., Theakston, R.D.G., 1991. Isolation and comparison of myotoxins isolated from venoms of dierent species of Bothrops snakes. Toxicon 29, 713±723. Perales, J., Amorim, C.Z., Rocha, S.L.G., Domont, G.B., MoussatcheÂ, H., 1992. Neutralization of the oedematogenic activity of Bothrops jararaca venom on the mouse paw by an antibothropic fraction isolated from opossum (Didelphis marsupialis ) serum. Agents Actions 37, 250±259. Rosenfeld, G., 1971. Symptomatology, pathology and treatment of snake bites in South America. In: BuÈcherl, W., Buckley, E.E. (Eds.), Venomous Animals and their Venoms, vol II. Academic Press, New York, pp. 345±403. Rothschild, A.M., Rothschild, Z., 1979. Liberation of pharmacologically active substances by snake venoms. In: Lee, C.Y. (Ed.), Snake Venoms. Handbook of Experimental Pharmacology, vol. 52. Springer-Verlag, Berlin, pp. 591±628. Selistre, H.S., Queiroz, L.S., Cunha, O.A.B., De Souza, G.E.P., Giglio, J.R., 1990. Isolation and characterization of hemorrhagic, myonecrotic and edema-inducing toxins from Bothrops insularis (jararaca ilhoa) snake venom. Toxicon 28, 261±273. Research Group on Snake Bite in Martinique, Thomas, L., Tyburn, B., 1996. Bothrops lanceolatus bites in Martinique: clinical aspects and treatment. In: Bon, C., Goyon, M. (Eds.), Envenomings and their Treatments. Fondation Marcel MeÂriex, Lyon, p. 255. Trebien, H.A., Calixto, J.B., 1989. Pharmacological evaluation of rat paw edema induced by Bothrops jararaca venom. Agents Actions 26, 292±300. Vargaftig, B.B., Bhargava, N., Bonta, I.L., 1974. Haemorrhagic and permeability increasing eects of Bothrops jararaca and other Crotalidae venoms as related to amine or kinin release. Agent Actions 4, 163±168. Vishwanath, B.S., Kini, R.M., Gowda, T.V., 1987. Characterization of three edema inducing phospholipase A2 enzymes from habu (Trimeresurus ¯avoviridis ) venom and their interaction with the alkaloid aristolochic acid. Toxicon 25, 501±515.
A. LoÃbo de ArauÂjo et al. / Toxicon 38 (2000) 209±221
221
Vital Brazil, O., 1982. Pec° onhas. In: Corbett, C.E. (Ed.), FarmacodinaÃmica. Guanabara Koogan, Rio de Janeiro, pp. 1044±1074. Williams, T.J., 1979. Prostaglandin E2, prostaglandin I2 and the vascular changes in in¯ammation. Br. J. Pharmac. 65, 517±524. Williams, T.J., JoseÂ, P.J., 1981. Mediation of increased vascular permeability after complement activation (histamine-independent action of rabbit C5a). J. Exp. Med. 153, 136±153. Williams, T.J., Morley, J., 1973. Prostaglandins as potentiators of increased vascular permeability in in¯ammation. Nature 246, 215±217. Williams, T.J., Peck, M.J., 1977. Role of prostaglandin-mediated vasodilatation in in¯ammation. Nature 270, 530±532. Yamakawa, M., Nozaki, M., Hokama, Z., 1976. Fractionation of Sakishima habu (Trimeresurus elegans ) venom and lethal, hemorrhagic and edema-forming activities of the fractions. In: Ohsaka, A., Hayashi, K., Sawai, Y. (Eds.), Animal, Plant and Microbial Toxins, vol. 1. Plenum Press, New York, pp. 97±109.