Pharmacological characterization of mouse hind paw oedema induced by Bothrops insularis (jararaca ilhoa) snake venom

Toxicon 42 (2003) 515–523 www.elsevier.com/locate/toxicon

Pharmacological characterization of mouse hind paw oedema induced by Bothrops insularis (jararaca ilhoa) snake venom Ana M. Barbosaa, Renata O. do Amaralb, Catarina F.P. Teixeirab, Stephen Hyslopc, Jose´ C. Cogoa,* a

Serpenta´rio do Centro de Estudos da Natureza, Instituto de Pesquisa e Desenvolvimento, Universidade do Vale do Paraı´ba (UNIVAP), Avenida Shishima Hifumi 2911, Urbanova, Sa˜o Jose´ dos Campos 12244-000, SP, Brazil b Laborato´rio de Farmacologia, Instituto Butantan, Avenida Vital Brazil 1500, 05503-900 Sa˜o Paulo, SP, Brazil c Departamento de Farmacologia, Faculdade de Cieˆncias Me´dicas, Universidade Estadual de Campinas (UNICAMP), CP 6111, 13083-970 Campinas, SP, Brazil Received 22 April 2003; accepted 23 July 2003

Abstract Bothrops snake venoms produce marked local effects, including oedema, haemorrhage and necrosis. The ability of Bothrops insularis venom to induce oedema in mice was investigated. Venom was injected into hind paws and the change in volume over time was measured by plethysmometry. B. insularis venom (0.01– 2.5 mg/paw) induced paw oedema which, at high doses ($0.5 mg/paw), was accompanied by haemorrhage. The peak oedematogenic response occurred 3 h after venom injection with all doses and decreased gradually thereafter, but was still elevated with high doses after 24 h. Pretreating the mice with cyproheptadine (histamine H1 and serotonin 5-HT2 receptor antagonist), mepyramine (histamine H1 receptor antagonist), L NAME (inhibitor of nitric oxide synthase), indomethacin and rofecoxib (inhibitors of cyclooxygenases), and dexamethasone (indirect inhibitor of PLA2) significantly attenuated venom-induced oedema, whereas methysergide, a serotonin 5-HT1/5-HT2 receptor antagonist, had no effect. The administration of antivenom 30 min before or immediately after venom injection also significantly inhibited venom-induced oedema. These results show that B. insularis venom causes oedema in the mouse hind paw and that this response is mediated by histamine, nitric oxide, and arachidonic acid metabolites formed by cyclooxygenases 1 and 2. The neutralization by commercial antivenom indicates that the venom components responsible for oedema are recognized by the antivenom and share immunological identity with their counterparts in the venoms of mainland Bothrops species. q 2003 Elsevier Ltd. All rights reserved. Keywords: Oedema; Hind paw; Histamine; Nitric oxide; Phospholipase A2; Prostaglandins; Serotonin

1. Introduction Envenomation by Central and South American pitvipers of the genera Bothrops and Lachesis produces marked local effects which include oedema, haemorrhage, and necrosis (Watt, 1989; Fan and Cardoso, 1995; Russell et al., 1997). Despite the widespread distribution of these two genera and * Corresponding author. Tel.: þ55-12-3947-1106; fax: þ 55-123947-1149. E-mail address: [email protected] (J.C. Cogo).

their clinical importance, in only a few species has the formation of oedema been studied in detail, i.e. B. asper (Gutie´rrez et al., 1980; Lomonte et al., 1993; Chaves et al., 1995), B. jararaca (Trebien and Calixto, 1989; Perales et al., 1992), B. lanceolatus (Loˆbo de Arau´jo et al., 2000; Faria et al., 2001) and Lachesis muta subspecies (Lomonte, 1985; Soares de Moura et al., 1998). The principal venom components responsible for the oedema induced by these venoms are metalloproteinases (Gutie´rrez and Rucavado, 2000) and (myotoxic) phospholipases A2 (Gutie´rrez and Lomonte, 1995).

0041-0101/03/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0041-0101(03)00230-7

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Bothrops insularis (jararaca ilhoa) is an endemic species restricted to the island of Queimada Grande, located 64 km off the coast of the State of Sa˜o Paulo, Brazil (Campbell and Lamar, 1989). Compared to other Brazilian species of Bothrops, the toxinology of B. insularis is still poorly understood. The components isolated so far from this venom include esterases (Selistre and Giglio, 1987; Selistre et al., 1990), a phospholipase A2 (PLA2) (Selistre et al., 1990; Cogo et al., 1998), a vascular endothelium growth factorrelated protein (Junqueira de Azevedo et al., 2001) and bradykinin-potentiating peptides (Cintra et al., 1990). More recently, a variety of peptidase activities was reported for this and other Bothrops venoms (Gasparello-Clemente and Silveira, 2002). B. insularis venom irreversibly inhibits neuromuscular transmission in mouse and chick neuromuscular preparations, with the latter being more sensitive to the venom (Cogo et al., 1993). This inhibition involves both a presynaptic action of the venom and a direct action on muscle fibers, as shown histologically by the damage to skeletal muscle. These effects are mediated principally by a PLA2-containing fraction of the venom (Cogo et al., 1998). The venom and PLA2 fraction also cause morphological alterations in avian kidney following intramuscular injection (Cruz-Ho¨fling et al., 2001). The finding that B. insularis venom causes muscle damage (Selistre et al., 1990; Cogo et al., 1998) suggests that this venom may produce local effects following injection. In this study, we examined the ability of B. insularis venom to cause oedema in the mouse hind paw and investigated some of the mediators involved in this response. The efficacy of commercial antivenom in neutralizing the oedema was also evaluated.

2. Material and methods 2.1. Reagents Cyproheptadine, dexamethasone, indomethacin, methysergide, N v-nitro-L -arginine methyl ester (L -NAME), D NAME and mepyramine were purchased from Sigma Chemical Co. (St Louis, MO, USA). Rofecoxib was obtained from Merck (Rio de Janeiro, RJ, Brazil). 2.2. Animals Adult male Swiss white mice (18– 22 g) were obtained from Animais de Laborato´rio (ANILAB, Paulı´nia, SP, Brazil). The mice were housed in conventional plastic cages at 22 8C on a 12 h light/dark cycle (lights on at 6 a.m.) with free access to water and rodent chow (Purinaw, Campinas, SP). The experiments described here were done within the guidelines established by the Brazilian College for Animal Experimentation (COBEA).

2.3. Venom and antivenom B. insularis venom was obtained by manual extraction from specimens captured on the island of Queimada Grande and was provided by the Instituto Butantan (Sa˜o Paulo, Brazil). The lyophilized venom was stored at 4 8C until used. Equine antivenom raised against a pool of Bothrops venoms (B. alternatus, B. jararaca, B. jararacussu, B. moojeni and B. neuwiedi) was obtained from the Instituto Butantan. 2.4. Paw oedema The ability of B. insularis venom to induce oedema was studied in mice. Fifty microliters of sterile 0.9% (w/v) saline solution with venom (0.01 – 2.5 mg/paw) were injected in the subplantar region of the right hind paw. The left hind paw received an equal volume of sterile saline alone and served as the control. Prior to injection, the venom solution was filtered through 0.22 mm Millipore filters. The volumes of both hind paws were measured by plethysmometry (model 7140 plethysmometer, Ugo Basile, Italy) at 0.25, 0.5, 1, 3, 6 and 24 h after venom administration according to Van Arman et al. (1965). Oedema was expressed as the percentage increase in the volume of the treated (right) paw relative to that of the control (left) paw at each time interval. 2.5. Drug treatments To determine the principal mediators involved in the venom-induced oedema, mice were pretreated with the following drugs for the indicated times prior to the injection of a fixed amount of venom (0.25 mg/paw): (1) dexamethasone, an indirect inhibitor of PLA2 activity, 0.2 mg/kg, i.m., 2 h before venom; (2) indomethacin, an inhibitor of cyclooxygenases 1 and 2 (COX-1 and COX-2), 4 mg/kg, i.v., 30 min before venom; (3) rofecoxib, an inhibitor of cyclooxygenase 2 (COX-2), 10 mg/kg, orally, 2 h before venom; (4) L -NAME, a non-specific inhibitor of nitric oxide (NO) synthase, 50 mmol/kg, i.v., 24 h and also 30 min before venom; (5) cyproheptadine, a dual antagonist of histamine H1 and serotonin 5-HT2 receptors, 6 mg/kg, i.p., 15 min before venom; (6) methysergide, a serotonin 5HT1/5-HT2 receptor antagonist, 6 mg/kg, i.p., 15 min before venom and (7) mepyramine, a histamine H1 receptor antagonist, 6 mg/kg, i.p., 15 min before venom. The drugs were generally dissolved and administered in sterile saline, except for indomethacin which was initially dissolved in 1 M Tris – HCl buffer, pH 8.0, and then diluted with saline solution to provide the desired concentration. The doses of the drugs used were chosen from relevant published reports of their activities. Following pretreatment and venom administration, the progress of oedema was monitored for up to 24 h as indicated above.

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2.6. Neutralization of oedema-inducing activity by antivenom The ability of commercial equine antivenom raised against Bothrops venoms to neutralize the oedema-inducing activity of the venom was examined by administering antivenom (0.1 ml, i.v.) either 30 min before or immediately after the injection of venom (0.25 mg/paw). The volume of antivenom used was sufficient to neutralize the amount of venom injected since 1 ml of antivenom neutralizes 5 mg of Bothrops venom (manufacturer’s specifications). The progress of oedema following injection of the venom/antivenom mixture was monitored as described above. 2.7. Statistical analysis The results were expressed as the mean ^ SEM of the percentage increase in right paw volume. Statistical comparisons among groups were done by ANOVA followed by the Tukey – Kramer test for multiple comparisons (Snedecor and Cochran, 1989).

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a maximum increase in paw volume of approximately 100%, whereas high doses (# 0.5 mg/paw) increased the paw volume by . 300%. For time intervals up to 180 min, there was little dose-dependence within the two broad groups indicated above, whereas at 360 min and, to a lesser extent, at 1440 min (only for high doses), there appeared to be some dose-dependence. The oedema caused by low doses was not accompanied by haemorrhage at the site of injection whereas with the higher doses, extensive haemorrhaging was observed in the injected paws after 3 h. Tissue necrosis was seen after 24 h in some mice given the highest doses. Haematuria was also observed in mice that received high doses of venom. Regardless of the dose administered, the oedema was maximal at 3 h and generally decreased thereafter, although with the highest dose tested (2.5 mg/paw) maximal oedema was still observed after 6 h. With the lower doses, the oedema

3. Results 3.1. Dose- and time-dependence of B. insularis-induced oedema Fig. 1 shows the mouse paw oedema induced by various doses of B. insularis venom. The responses to venom formed two well-defined groups: low doses (# 0.25 mg/ paw) caused a similar level of oedema which resulted in

Fig. 1. Mouse paw oedema induced by selected doses of B. insularis venom. The oedema, which was measured by plethysmometry as described in Section 2, was maximal after 3 h and decreased thereafter. Venom doses $0.5 mg/paw also caused haemorrhage. The points are the mean ^ SEM of five mice.

Fig. 2. Effect of biogenic amine receptor antagonists on oedema induced by B. insularis venom in mouse hind paw. Mice were pretreated with cyproheptadine or mepyramine (A) or methysergide (B) before the injection of venom (0.25 mg/paw), and oedema was evaluated by plethysmometry, as described in Section 2. The points are the mean ^ SEM of five mice. * p # 0:05 compared to the corresponding time points for venom alone.

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had disappeared after 24 h while with the higher doses paw volume was still elevated after this period. Based on these results, all subsequent experiments were done with a dose of 0.25 mg/paw in order to avoid possible complications in interpretation associated with the presence of haemorrhage. This dose of 0.25 mg/paw was the highest that could be used without visible haemorrhaging since a slightly greater dose (0.35 mg/paw; not shown in Fig. 1) produced haemorrhage in addition to oedema. 3.2. Pharmacological characterization of venom-induced oedema Fig. 2(A) shows that pretreating the mice with cyproheptadine or mepyramine significantly inhibited the venom-induced oedema from 60 min onwards. The nearly identical responses obtained with these two antagonists pointed to histamine as an important mediator in this effect.

Fig. 3. Effect of cyclooxygenase inhibitors (indomethacin and rofecoxib) or dexamethasone, an indirect inhibitor of PLA2, on oedema induced by B. insularis venom in mouse hind paw. Mice were pretreated with rofecoxib (A), indomethacin (B), or dexamethasone (A) before the injection of venom (0.25 mg/paw), and oedema was evaluated by plethysmometry, as described in Section 2. The points are the mean ^ SEM of five mice. * p # 0:05 compared to the corresponding time points for venom alone.

This conclusion was confirmed by the finding that methysergide, a 5-HT2 receptor antagonist, did not attenuate the oedema (Fig. 2(B)). Pretreating mice with inhibitors of the cyclooxygenase (COX) pathway of arachidonic acid metabolism (indomethacin and rofecoxib) or with an indirect inhibitor of endogenous PLA2 (dexamethasone) significantly attenuated the oedematogenic response to venom (Fig. 3). The COX-1 and COX-2 inhibitor indomethacin significantly reduced oedema from 1 h onwards whereas rofecoxib, a specific COX-2 inhibitor, markedly reduced oedema from 3 h onwards. Dexamethasone differed from these drugs in that it was more effective in preventing paw oedema. In this case, paw volume had returned to normal after 3 h while complete resolution with the COX inhibitors was seen only after 6 h. The involvement of nitric oxide (NO) in the venominduced oedema was assessed by pretreating the mice with

Fig. 4. Effect of NO synthase inhibition on oedema induced by B. insularis venom in mouse hind paw. Mice were pretreated with L NAME (A) or with the inactive enantiomer D -NAME (B) before the injection of venom (0.25 mg/paw), and oedema was evaluated by plethysmometry, as described in Section 2. The points are the mean ^ SEM of five mice. * p # 0:05 compared to the corresponding time points for venom alone.

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Fig. 5. Neutralization of the oedematogenic activity of B. insularis venom by commercial antivenom injected in vivo. Mice received antivenom (0.1 ml, i.v.) either 30 min before or immediately after the administration of venom (0.25 mg/paw). The points are the mean ^ SEM of five mice. * p # 0:05 compared to the corresponding time points for venom alone.

the NO synthase inhibitor L -NAME. As shown in Fig. 4(A), L -NAME markedly inhibited the venom-induced oedema. The specificity of this inhibition was confirmed by testing the biologically inactive enantiomer D -NAME which did not affect the oedema (Fig. 4(B)). 3.3. Neutralization of oedema formation by commercial bothropic antivenom The administration of commercial antivenom 30 min before or immediately after the injection of venom significantly inhibited venom-induced oedema (Fig. 5). This inhibition was observed from 60 min onwards; antivenom had no significant effect on the early phase (first 30 min) of the oedematogenic response.

4. Discussion B. insularis venom caused paw oedema in mice with a time course similar to that reported for other Bothrops venoms in mice and rats, i.e. a fairly rapid onset (generally # 3 h to peak) followed by a gradual decline over the following 24 h (Trebien and Calixto, 1989; Perales et al., 1992; Cury et al., 1994; Loˆbo de Arau´jo et al., 2000; Chacur et al., 2001; Faria et al., 2001; Carneiro et al., 2002; Kanashiro et al., 2002), although this resolution may extend to 72 h with high doses ($ 25 mg/paw) of Bothrops asper venom (Gutie´rrez et al., 1980; Lomonte et al., 1993; Chaves et al., 1995). This general time course differs from that reported for Lachesis muta rhombeata venom in mice in which maximal responses were obtained after 60 min but with resolution of the oedema after 4 h (Soares de Moura

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et al., 1998). The fact that some dose-dependence was observed within each of the two broad groups of responses only at 360 min and, to a lesser extent, at 1440 min (for high doses), suggests that various mechanisms contributed to oedema formation, but that in the early stages (up to 180 min) these may have been masked by one or more predominant mechanisms. The haemorrhage seen with high doses of B. insularis venom agreed with findings for B. asper (Lomonte et al., 1993; Chaves et al., 1995), B. jararaca (Trebien and Calixto, 1989; Perales et al., 1992), B. lanceolatus (Loˆbo de Arau´jo et al., 2000) and L. m. rhombeata (Soares de Moura et al., 1998) venoms. This haemorrhagic activity likely contributed to the markedly greater oedema seen with high doses of B. insularis venom compared to lower doses, and probably reflected the presence of haemorrhagic metalloproteinases able to induce oedema and inflammatory responses in laboratory animals (Rucavado et al., 1995; Moura-da-Silva et al., 1996; Escalante et al., 2000; Petretski et al., 2000; Rodrigues et al., 2001; Costa et al., 2002). This conclusion is supported by the recent demonstration of metalloproteinases in B. insularis venom glands based on gene analysis using expressed sequence tags which showed that metalloproteinases were the most abundant group of toxins expressed (Junqueira de Azevedo and Ho, 2002). The finding that inhibitors of cyclooxygenases (indomethacin, an inhibitor of COX-1 and COX-2, and rofecoxib, specific for COX-2) markedly attenuated the oedema indicated the involvement of arachidonic acid metabolites (prostaglandins) in the responses to B. insularis venom. The inhibitory effect of indomethacin on oedema was observed from 1 h onwards whereas a significant reduction in oedema by rofecoxib was seen only from 3 h onwards. This time difference for the effect of rofecoxib most likely reflects the lag period required for the induction of COX-2 isozyme expression and action (Kujubu et al., 1991; Ristimaki et al., 1994). Thus, our results suggest that B. insularis venominduced oedema depends on the initial activation of COX-1 with the quick generation of inflammatory prostanoids, the levels of which are maintained by the subsequent activation of COX-2. Paw oedema caused by the venom of B. asper (Chaves et al., 1995; but see Chacur et al., 2001), B. asper myotoxins I (Gutie´rrez et al., 1986a), II and III (Chaves et al., 1998), B. jararaca (Trebien and Calixto, 1989; Perales et al., 1992), B. lanceolatus in mice (Loˆbo de Arau´jo et al., 2000) and L. m. rhombeata (Soares de Moura et al., 1998) is also attenuated by cyclooxygenase inhibitors such as aspirin and indomethacin. In contrast, the oedema produced by B. lanceolatus in rats (Faria et al., 2001) was reported to involve lipoxygenase rather than cyclooxygenase products. Lipoxygenase products have also been implicated in B. jararaca venom-induced oedema in rats (Trebien and Calixto, 1989), but are apparently not involved in mice (at least not leukotrienes C4 and D4) (Perales et al., 1992). The participation of prostaglandins in B. insularis venom-induced oedema was further confirmed by

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the treatment with dexamethasone, which markedly reduced the oedema. However, these results with dexamethasone do not rule out the participation of leukotrienes. Dexamethasone has also been shown to inhibit the paw oedema caused by B. asper venom (Chaves et al., 1995; but see Chacur et al., 2001) and myotoxins II and III (Chaves et al., 1998), as well as the venoms of B. lanceolatus (Loˆbo de Arau´jo et al., 2000; Faria et al., 2001), B. jararaca (Perales et al., 1992) and L. m. rhombeata (Soares de Moura et al., 1998), but was ineffective when given after venom administration (Perales et al., 1992; Chaves et al., 1995). With the experimental design used here, it was not possible to determine whether the PLA2 involved in the oedema was from the venom, paws or both. Although dexamethasone can inhibit PLA2 activity indirectly through the formation of lipocortins (Flower, 1988; Cirino et al., 1989a), this glucocorticoid can also exert other antiinflammatory actions independent of the inhibition of PLA2 activity, including the inhibition of COX-2 gene expression and activity (O’Banion et al., 1991; Masferrer et al., 1992; Ferreira et al., 1997). Phospholipases A2 isolated from Bothrops venoms are frequently myotoxic (Gutie´rrez and Lomonte, 1995) and can cause oedema in rats and mice (Gutie´rrez et al., 1986a; Lloret and Moreno, 1993; Chaves et al., 1998; Landucci et al., 1998, 2000; Soares et al., 1998; Andria˜o-Escarso et al., 2000; Kanashiro et al., 2002). Although the catalytic activity of PLA2 contributes to oedema formation, it is not a prerequisite for this (Dı´az et al., 1992; Chaves et al., 1998; Landucci et al., 1998, 2000; Andria˜o-Escarso et al., 2002; Kanashiro et al., 2002). PLA2-induced oedema in rats and mice is mediated to varying extents by amines (histamine and serotonin) released from degranulating mast cells (Calhoun et al., 1989; Cirino et al., 1989b; Wang and Teng, 1990; Moreno et al., 1992; Landucci et al., 1998; Kanashiro et al., 2002). In contrast, in rabbits, in which the skin content of mast cells is very reduced, mast cell degranulation and, consequently, histamine and serotonin release, contributes little to the skin oedema induced by piratoxin-I, a Lys-49 PLA2 homologue isolated from the venom of Bothrops pirajai (Landucci et al., 2000). Considering that B. insularis venom contains PLA2 (Selistre et al., 1990; Cogo et al., 1998), we examined the involvement of amines, principally histamine and serotonin, in the venom-induced oedema by assessing the effects of cyproheptadine (dual histamine and serotonin receptor antagonist), mepyramine (histamine H1 receptor antagonist) and methysergide (serotonin 5-HT2 receptor antagonist) on venom-induced paw oedema. Cyproheptadine and mepyramine potently inhibited the oedema from 60 min onwards and showed very similar profiles, whereas methysergide had no effect on the oedema. These results indicate that histamine, but not serotonin, is involved in the venominduced oedema. Histamine is considered the major mediator of the early phase of increased vascular permeability, and causes venular gaps by activating endothelial cell H1 receptors. The activation of these receptors results in

increased production of pro-inflammatory prostaglandins by endothelial cells (Cole and Lewis, 1989). Thus, it is possible that histamine may further contribute to the amplification of venom-induced oedema by stimulating the synthesis of prostanoids. The observation that mepyramine prevented oedema formation more efficiently and earlier than the cyclooxygenase inhibitors is consistent with this hypothesis. Histamine and serotonin have been implicated in the oedema caused by B. lanceolatus venom in rats (Faria et al., 2001) but not in mice (Loˆbo de Arau´jo et al., 2000). These mediators also have little or no role in the oedema caused by B. asper venom in mice (Chaves et al., 1995) and by B. jararaca venom in rats (Trebien and Calixto, 1989) and mice (Perales et al., 1992). In constrast, the mouse paw oedema induced by L. m. rhombeata venom apparently involves a strong contribution by histamine and serotonin (Soares de Moura et al., 1998). The involvement of nitric oxide (NO) in venom-induced paw oedema has not been extensively investigated. The observation that kinins may be involved in B. lanceolatus venom-induced oedema in rats (Faria et al., 2001) suggested a role for NO in this species since NO is the principal mediator of kinin action in the microvasculature following the activation of bradykinin B2 receptors. In contrast, kinins are apparently not involved in the mouse and rat paw oedema caused by B. asper (Chacur et al., 2001) and B. jararaca (Trebien and Calixto, 1989) venom, respectively, or in the oedema caused by piratoxin-I in rabbits (Landucci et al., 2000). Soares de Moura et al. (1998) showed that L NAME, a non-specific inhibitor of NO synthase (NOS), markedly inhibited oedema formation by L. m. rhombeata venom in mice. Similarly, L -NAME attenuated the vascular component of the B. jararaca venom-induced inflammatory reaction, indicating the involvement of NO in this response (Guzzo et al., 2000). Our results also showed significant inhibition of B. insularis venom-induced oedema by L NAME, indicating that NO is an important mediator of this response. This was confirmed by the inability of D -NAME, the inactive enantiomer of L -NAME, to affect the oedema. The role of NO as a mediator of inflammatory reactions is controversial in the literature and the mechanism by which NO contributes to oedema formation after the injection of B. insularis venom remains to be determined. One possibility is that NO, through its potent vasodilating activity, may potentiate the increase in vascular permeability induced by mediators of this phenomenon such as histamine. Another possibility involves the increased formation of reactive nitrogen species such as peroxynitrite during the inflammatory response, as seen following the administration of B. jararaca venom in mice, where increased levels of NO derived from inducible NOS activity resulted in the formation of peroxynitrite in peritoneal leukocytes (Zamune´r et al., 2001). Since peroxynitrite is highly toxic to cells, these authors suggested a key role for this nitrogen product in the local injury caused by this

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venom. A similar role for peroxynitrite in B. insularis venom-induced oedema deserves investigation. The reduction in oedema observed with commercial antivenom indicated that although B. insularis venom was not included in the mixture of Bothrops venoms used in the immunization protocol for this antivenom, there was sufficient immunological identity between Bothrops venom components to allow for cross-neutralization. Cross-reactivity between commercial antivenom and venoms not included in the antigen pool has also been noted for other species such as B. asper and B. atrox (Bogarı´n et al., 1999) and Bothrops leucurus and Bothrops erythromelas (Camey et al., 2002). The neutralization of oedema-forming activity by antivenom is frequently assessed by using mixtures of venom preincubated with varying quantities of antivenom for up to 1 h at 37 8C before injection (Lomonte, 1985; Gutie´rrez et al., 1986b, 1998; Rojas et al., 1987; Lomonte et al., 1993; Rucavado and Lomonte, 1996; Bogarı´n et al., 1999; Leo´n et al., 2000). The ability of antivenom to prevent oedema when administered after venom is generally considerably less than in preincubation experiments and depends on the interval between venom and antivenom administration (Gutie´rrez et al., 1998). Thus, Leo´n et al. (1997, 2000) observed only , 30 – 50% inhibition of B. asper venom-induced oedema in mice when antivenom (IgG, F(ab0 )2 or Fab fragments) was given immediately after venom administration; the level of protection decreased progressively when antivenom was given 15 or 30 min after venom. Faria et al. (2001) reported that whereas preincubation of B. lanceolatus venom with antivenom partially reduced venom-induced oedema, there was no inhibition of oedema when antivenom was given immediately before venom. However, as shown here, antivenom given 30 min before or immediately after venom markedly inhibited oedema formation from 1 h onwards, but had little effect on the early response (,1 h). These findings agree with those of Rucavado and Lomonte (1996) who showed that the administration of antivenom 5 or 120 min before B. asper venom inhibited the late phase of oedema (.3 h), with little influence on the responses up to 3 h. For this same species, Lomonte et al. (1993) reported that preincubation with antivenom did not attenuate venom-induced mouse paw oedema. In conclusion, B. insularis venom induces paw oedema in mice through pathways involving histamine, NO and cyclooxygenase products. These mediators may interact with each other or be released sequentially. The neutralization of oedema by commercial antivenom indicates that the B. insularis venom components responsible for this phenomenon share immunological identity with their counterparts in the venoms of mainland Bothrops species. In view of the lack of effect of antivenom on the early oedematogenic response to the venom, further understanding of the venom components

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and of the pharmacological mechanisms involved in this phase is necessary.

Acknowledgements The authors thank Dr Rodrigo A.B. Lopes Martins for providing laboratory space and equipment for this study, Dr Alexandre P. Corrado for helpful discussions and Antonio C. Prianti and Luiz Prudeˆncio for technical assistance. This work was supported by Fundac¸a˜o Valeparaibana de Ensino (FVE).

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