Inflammatory events induced by brown spider venom and its recombinant dermonecrotic toxin: A pharmacological investigation

https://doi.org/10.1016/j.cbpc.2008.08.009Get rights and content

Abstract

Accidents involving Brown spider (Loxosceles sp.) venom produce a massive inflammatory response in injured region. This venom has a complex mixture of different toxins, and the dermonecrotic toxin is the major contributor to toxic effects. The ability of Loxosceles intermedia venom and a recombinant isoform of dermonecrotic toxin to induce edema and increase in vascular permeability was investigated. These toxins were injected into hind paws and caused a marked dose and time-dependent edema and increase in vascular permeability in mice. Furthermore, the enzymatic activity of venom toxins may be primal for these effects. A mutated recombinant isoform of dermonecrotic toxin, that has only residual enzymatic activity, was not able to induce these inflammatory events. Besides the previous heating of toxins markedly reduced the paw edema and vascular permeability showing that thermolabile constituents can trigger these effects. In addition, the ability of these venom toxins to evoke inflammatory events was partially reduced in compound 48/80-pretreated animals, suggesting that mast cells may be involved in these responses. Pretreating mice with histamine (prometazine and cetirizine) and serotonin (methysergide) receptor antagonists significantly attenuated toxins induced edema and vascular permeability. Moreover, HPLC analysis of whole venom showed the presence of histamine sufficient to induce inflammatory responses. In conclusion, these inflammatory events may result from the activation of mast cells, which in turn release bioamines and may be related to intrinsic histamine content of venom.

Introduction

Brown spiders are found worldwide and spider bites have been reported from all continents (Futrel, 1992, Hogan et al., 2004). Necrotic skin lesions with gravitational spreading and a massive inflammatory response are the hallmark of accidents with the Loxosceles genus (Futrel, 1992, da Silva et al., 2004). Whole venom is composed of a mixture of different proteic toxins, and the mechanisms that lead to these deleterious effects are under investigation.

The dermonecrotic toxin (sphingomyelinase-D, now recognized as phospholipase-D, Lee and Lynch, 2005) is the best known molecule found in different brown spider venoms, and studies have identified it as a contributor to the major toxic effect of spider bites (Binford and Wells, 2003). Previous studies cloned and expressed functional dermonecrotic toxins from Loxosceles intermedia venom glands that were able to reproduce the dermonecrosis and inflammatory response (Chaim et al., 2006, da Silveira et al., 2006, da Silveira et al., 2007).

Recent evidence has shown that the subcutaneous injections of whole L. intermedia venom and the recombinant isoform of the dermonecrotic toxin, LiRecDT1, cause a significant increase in edema and vascular permeability (da Silveira et al., 2006). However, there have been few scientific studies aimed at investigating the mechanisms underlying the brown spider toxin-induced inflammatory actions.

Accumulating data have demonstrated that mast cells are critical contributors to the development of peripheral inflammatory responses associated with tissue or nerve injury (Metcalfe et al., 1997, Mekori and Metcalfe, 2000, Zuo et al., 2003, Liu et al., 2007). Mast cells are intimately involved in the pathophysiology of inflammation (Mekori and Metcalfe, 2000), and their activation promotes the release of chemical mediators responsible for important tissue alterations such as vasodilatation, increases in vascular permeability and neutrophil chemotaxis among others (Metcalfe et al., 1997). Many of these effects are attributable to the activity of histamine and 5-hydroxitryptamine (5-HT, serotonin) of mast cell origin. Some venom phospholipases are known to lead directly to mast cell degranulation, like phospholipase-A (Cirino et al., 1989, Landucci et al., 1998). Additionally, mast cells have phospholipases-D localized in the plasma membrane that are associated to antigen mast cell degranulation (Rivera and Gilfillan, 2006).

The lack of an efficient protocol to alleviate the inflammatory response induced by brown spider envenomation may be related to a reduced number of studies carried out in experimental animals aimed at characterizing the mechanisms involved in inflammatory development. In the present study, we investigated the potential role of mast cells and the involvement of histamine and serotonin in the mechanisms by which the brown spider toxins cause inflammatory events.

Section snippets

Animals

Adult Swiss mice (Mus musculus, 20–30 g), with free access to food and water were used. All protocols employed were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the local ethics committee (process numbers 261/UFPR, 2007). All efforts were made to minimize the number of animals used and their suffering.

Reagents

Compound 48/80, cimetidine, cetirizine, histamine, serotonin and o-phthalaldehyde (OPA) were purchased from Sigma-Aldrich Chemical Co. (St.

Paw edema induced by subcutaneous injections of brown spider toxins

The subcutaneous (s.c.) injection of brown spider whole venom elicited a time (0 to 720 min, 10 µg/paw) (Fig. 1A) and dose-dependent (1 to 20 µg/paw) (Fig. 1B) edema formation that started at 5 min, peaked at 10 min and decreased thereafter, being absent 12 h later (Fig. 1A and B). The s.c. injection of LiRecDT1 also elicited a time (0 to 720 min, 10 µg/paw) (Fig. 1C) and dose-dependent (0.5 to 20 µg/paw) (Fig. 1D) edema formation that peaked at the 10 min time-point and was absent 12 h later (

Discussion

Inflammation is the basic mechanism available for the repair of tissue after an injury and consists of a cascade of cellular and microvascular reactions. This cascade includes edema, elevated permeability in microvessels and the migration of several cell types into damaged tissue (Schmid-Schönbein, 2006). The literature is well-supplied with studies relating the symptoms and signs evoked in accidents caused by brown spiders, but is deficient in studies about the mechanisms underlying the

Acknowledgments

This work was supported by grants from CNPq, CAPES, Fundação Araucária-PR, and Secretaria de Estado de Ciência, Tecnologia e Ensino Superior do Paraná, Brazil.

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