Biochemical Characterization and Pharmacological Properties of New Basic PLA2 BrTX-I Isolated from Bothrops roedingeri (Roedinger's Lancehead) Mertens, 1942, Snake Venom

BrTX-I, a PLA2, was purified from Bothrops roedingeri venom after only one chromatographic step using reverse-phase HPLC on μ-Bondapak C-18 column. A molecular mass of 14358.69 Da was determined by MALDI-TOF mass spectrometry. Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The total amino acid sequence was obtained using SwissProt database and showed high amino acid sequence identity with other PLA2 from snake venom. The amino acid composition showed that BrTX-I has a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA2. BrTX-I presented PLA2 activity and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0, 35–45°C, and required Ca2+. In vitro, the whole venom and BrTX-I caused a neuromuscular blockade in biventer cervicis preparations in a similar way to other Bothrops species. BrTX-I induced myonecrosis and oedema-forming activity analyzed through injection of the purified BrTX-I in mice. Since BrTX-I exerts a strong proinflammatory effect, the enzymatic phospholipid hydrolysis might be relevant for these phenomena; incrementing levels of IL-1, IL-6, and TNFα were observed at 15 min, 30 min, one, two, and six hours postinjection, respectively.


Introduction
PLA 2 s (phosphatide 2-acylhydrolase, EC 3.1.14) represent a superfamily of lipolytic enzymes which speci�cally catalyze the hydrolysis of the ester bond at the sn-2 position of glycerophospholipids resulting in the generation of fatty acid (arachidonate) and lysophospholipids. e PLA 2 superfamily consists of about 15 groups which are further subdivided into several subgroups, all of which display differences in terms of their structural and functional speci�cities. However, the four main types or classes of PLA 2 s are the secreted, the cytosolic, the Ca 2+ -independent and the lipoproteinassociated PLA 2 [1], PLA 2 structure/function, mechanism, and signaling [2]. Snake venom PLA 2 s displays a variety of activities, such as neurotoxicity, myotoxicity, cardiotoxicity, and hemolysis that may be modulated by speci�c receptors located on target cells [3][4][5][6]. Indeed, PLA 2 receptors classi�ed as kinds M and N [7] have been identi�ed in various kinds of cells, including vascular smooth muscle cells, platelets, neutrophils, chondrocytes, �broblasts, hepatocytes, and mesangial cells, as well as in brain, lung, and skeletal muscle [8,9]. Snake venom PLA 2 can bind to M receptors, which are the most common kind found in human macrophages and muscle cells, and these may mediate some of the deleterious actions of venom PLA 2 s, although that was not conclusively demonstrated [5,6].
Peru has a rich and diverse herpetofauna that includes venomous snake species of the families Elapidae (16 species of Micrurus and the pelagic sea snake Pelamis platurus) and Viperidae (15 species) [10]. Snakebite envenomations represent a public health problem in this country. e vast majority of snakebites in Peru are in�icted by species of the genus Bothrops (familyViperidae) [11]. Bothrops atrox, Bothrops brazili, and Bothrops bilineatus are distributed in the tropical rainforests located in the eastern part of the country, whereas Bothrops barnetti and Bothrops roendingeri are found in the western dry coastal regions [10][11][12].
is variety of pharmacological roles derives from an accelerated microevolutionary process through which a high rate of amino acid substitutions have occurred in molecular regions located mainly at the surface of these molecules [13][14][15]. e purpose of this paper is to isolate, biochemically and pharmacologically characterize a basic PLA 2 from Bothrops roedingeri venom, BrTX-I.

Venom and Reagents
. e venom was obtained from the adult specimens of Bothrops roedingeri captured in the vicinity of Arequipa-Perú. Swiss mice (18-20 g) were supplied by the Animal Services Unit of the State University of Campinas (UNICAMP). All experiments were conduced in accordance with guidelines of the Committee for Ethics in Animal Research, UNICAMP No. 2006-1 (Campinas- Brazil). e reagents used in this work were of analytical or sequencing grade. 2 Activity. PLA 2 activity was measured using the assay described in [16,17], modi�ed for 96-well plates [18]. e standard assay mixture contained 200 L of buffer (10 mM Tris-HCl, 10 mM CaCl 2 , 100 mM NaCl, pH 8.0), 20 L of substrate (4-nitro-3-octanoyloxy-benzoic acid), 20 L of water, and 20 L of PLA 2 in a �nal volume of 260 L. Aer the addition of PLA 2 (20 g), the mixture was incubated for up to 40 min at 37 ∘ C, with the absorbance being read at 10 min intervals. e enzyme activity, expressed as the initial velocity of the reaction (Vo), was calculated based on the increase in absorbance aer 20 min.

PLA
All assays were done three times and the absorbances at 425 nm were measured using a VersaMax 190 multiwell plate reader (Molecular Devices, Sunnyvale, CA, USA).

Reversed-Phase HPLC (RP-HPLC).
Five milligrams of the venom was dissolved in 200 L solvent A (TFA 0.1%, pH 3.5). e resulting solution was clari�ed by centrifugation and the supernatant was applied to a -Bondapak C18 column (0.78 × 30 cm; Waters 991-PDA system). Fractions were eluted using a linear gradient (0-100%, v/v) of acetonitrile (solvent B) at a constant �ow rate of 1.0 mL/min over 40 min. e elution pro�le was monitored at 280 nm, and the collected fractions were lyophilized and conserved at −20 ∘ C.

Amino Acid Analysis.
Amino acid analysis was done on a Pico-Tag amino acid analyzer (Waters Corporation, Massachusetts, USA) as described by [20]. e puri�ed protein (30 g) was hydrolyzed at 105 ∘ C for 24 h in 6 M HCl acid (Pierce sequencing grade) containing 1% phenol (w/v). e hydrolyzates were reacted with 20 L of derivatization solution (ethanol : triethylamine : water : phenylisothiocyanate, 7 : 1 : 1 : 1, v/v) for 1 h at room temperature aer the phenylthiohydantoin (PTC)-amino acids were identi�ed and quanti�ed by HPLC by the comparison of their retention times and peak areas with those of a standard amino acid mixture.
2.6. Reduction and Alkylation. Puri�ed lyophilized protein from RP-HPLC was resuspended in 8 M urea containing 10 mM DTT at pH 8.0 and the disul�de bridges were then reduced by incubation at 37 ∘ C for 2 h. Since the number of cysteine residues in the protein was initially unknown, the optimum concentration of iodoacetamide for alkylating the free thiols was derived empirically, based on results obtained from incubations using various concentrations of iodoacetamide and different amounts of protein, with each mixture being analyzed by mass spectrometry [21]. Based on these preliminary experiments, a 30% molar excess of iodoacetamide relative to the total number of thiols was eventually chosen and the mixture was incubated for 1.5 h at 37 ∘ C in the dark. e reaction was ceased by injecting the mixture onto a RP-HPLC column followed by lyophilization of the collected peak.

Enzymatic
Hydrolysis. e puri�ed proteins were hydrolyzed with sequencing grade bovine pancreatic trypsin in 0.4% ammonium bicarbonate, pH 8.5, for 4 h at 37 ∘ C, at an enzyme : substrate ratio of 1 : 100 (w/w). e reaction was ceased by lyophilization.

Mass Spectrometry.
All mass spectra were acquired using a quadrupole-time of �ight (Q-TOF) hybrid mass spectrometer Q-TOF Ultima from Micromass (Manchester, UK) equipped with a nano Zspray source operating in a positive ion mode. e ionization conditions of usage included a capillary voltage of 2.3 kV, a cone voltage and RF1 lens of 30 V and 100 V, respectively, and a collision energy of 10 V. e source temperature was 70 ∘ C and the cone gas was N 2 at a �ow of 80 L/h; nebulizing gas was not used to obtain the sprays. Argon was used for collisional cooling and for fragmentation of ions in the collision cell. External calibration with sodium iodide was made over a mass range from 50 to 3000 m/z. All spectra were acquired with the TOF analyzer in "Vmode" (TOF kV = 9.1) and the MCP voltage set at 2150 V.

Analysis of Native and Alkylated
Protein. Lyophilised RP-HPLC fractions of intact native and alkylated protein were dissolved in 10% acetonitrile in 0.1% TFA and was introduced into the mass spectrometer source with a syringe pump at a �ow rate of 500 nL/min. Mass spectra were acquired over the mass range of 1000-2800 m/z for the native protein and over the range of 800-2000 m/z for the alkylated protein, both at a scan speed of 1 s/scan. e masses were analyzed by the MassLynx-MaxEnt 1 deconvolution algorithm. e data obtained were processed using the Mascot MS/MS Ion Search soware http://www.matrixscience.com/.

De Novo
Sequencing of Tryptic Peptides. Alkylated tryptic peptides fractionated by RP-HPLC were lyophilized and re-suspended in 20% acetonitrile in 0.1% TFA prior to injection into the mass spectrometer source at a �ow rate of 500 nL/min. Before performing a tandem mass spectrum, an ESI/MS mass spectrum (TOF MS mode) was acquired for each HPLC fraction over the mass range of 400-2000 m/z, in order to select the ion of interest, subsequently, these ions were fragmented in the collision cell (TOF MS/MS mode). Different collision energies were used, depending on the mass and charge state of the ions. e resulting ion spectra was acquired in the TOF analyser and deconvoluted using the MassLynx-MaxEnt 3 algorithm. Singly charged spectra were processed manually using the PepSeq application included in MassLynx.

Myotoxic Activity.
Groups of four Swiss mice (18-20 g) received an intramuscular (i.m.) or an intravenous (i.v.) injection of variable amounts of BrTX-I, in 100 L of PBS, in the gastrocnemius. A control group received 100 L of PBS. At different intervals of time (2, 4, 6, 9, and 24 h) blood was collected from the tail into heparinized capillary tubes, and the plasma creatine kinase (CK; EC 2.7.3.2) activity was determined by a kinetic assay (Sigma 47-UV). Activity was expressed in U/L, one unit de�ned as the phosphorylation of 1 mol of creatine/min at 25 ∘ C.

Edema-Forming
Activity. e ability of BrTX-I to induce edema was studied in groups of �ve Swiss mice (18-20 g) according Ponce-Soto et al. [6,23,24]. Twenty microliters of phosphate-buffered saline (PBS; 0.12 M NaCl, 0.04 M sodium phosphate, pH 7.2) with BrTX-I (1, 5, 10 and 20 g/paw) were injected in the subplantar region of the right footpad. e control group received an equal volume of PBS alone. e swelling of the paw was measured at 0.5; 1; 3; 6, and 24 h aer administration. Edema was expressed as the percentage increased in the volume of the treated group to that of the control group at each time interval.
2.11.4. Cytokines. e percentage of cytotoxicity was of IL-1, IL-6, and TNF-in the plasma were collected and measured at 30, 60, 180, and 360 min aer i.p. injection of the BrTX-I PLA 2 (1.0 mg/kg) (20 g/100 L) or sterile saline. Aer centrifugation, the supernatants were used for determination of IL-1 and IL-6 levels by a speci�c EIA. e levels of cytokines IL-1, IL-6, and TNF-in the serum from BALB/c mice were assayed by a two-site sandwich enzyme-like immunosorbent assay (ELISA). In brief, ELISA plates were coated with 100 L (1 g/mL) of the monoclonal antibodies anti-IL-1, in 0.1 M sodium carbonate buffer (pH 8.2) and incubated for 6 hours at room temperature. e wells were then washed with 0.1% phosphate-buffered saline (PBS/Tween-20) and blocked with 100 L of 10% fetal calf serum (FCS) in PBS for 2 hours at room temperature. Aer washing, duplicate sera samples of 50 L were added to each well. Aer 18 hours of incubation at 4 ∘ C, the wells were washed and incubated with 100 L (2 g/mL) of the biotinylated monoclonal antibodies anti-IL-1, anti-IL-6,as second antibodies for 45 minutes at room temperature. Aer a �nal wash, the reaction was developed by the addition of orthophenyldiamine (OPD) to each well. Optical densities were measured at 405 nm in a microplate reader were measured using a VersaMax 190 multiwell plate reader (Molecular Devices, Sunnyvale, CA, USA).
e cytokine content of each sample was read from a standard curve established with the appropriate recombinant cytokines (expressed in picograms per millilitre). e minimum levels of each cytokine detectable in the conditions of the assays were 10 pg/mL for IL-1, IL-6.
2.12. Statistical Analysis. e results are reported as the means ± SEM. e signi�cance of differences among the means was assessed by ANOVA followed by Dunnett's test when various experimental groups were compared to the control group. A value of indicated signi�cance.

Results
e elution pro�le of Bothrops roendigeri venom following RP-HPLC performed on a C18 column showed �een fractions (1-15) ( Figure 1). e �een eluted peaks were screened for PLA 2 activity. Only the fraction labeled in �gure  Table 1 shows the masses of the tryptic peptides obtained for from the BrTX-I. It is possible to see that these proteins presented �ve common peptides to the other Bothrops snake venoms. e data obtained were processed using the Mascot MS/MS Ion Search soware (http://www.matrixscience.com/).
To obtain detailed structural information, the native protein was alkylated and then digested to be analyzed through ESI-MS/MS. e alkylated protein digest was fractionated by RP-HPLC and each chromatographic peak marked in the chromatogram was manually collected and lyophilized. De novo sequencing by ESI-MS/MS was carried out for each peptide peak. e sequences were deduced using ESI-MS/MS and 5 peptides were obtained from the alkylated BrTX-I (Table 1).
Ile and Leu residues were not discriminated in any of the sequences, since they were indistinguishable in lowenergy collision-induced dissociation spectra. Due to the external calibration applied to all the spectra, it was also not possible to distinguish between Gln and Lys residues based on the 0.035 Da that separates these amino acids, except for Lys, marked in bold in Table 1 analysis of the cleavage and missed cleavage sites of the enzyme.
Each de novo sequenced peptide of the BrTX-I was submitted separately to the NCBI database, using the protein search program BLAST-p with the search being restricted to the sequenced proteins from the PLA 2 from snake venom family. In order to determine the presence and number of cysteine residues, BrTX-I was reduced and alkylated as described in Section 2.6. e protein mass registered in peak 1-4 aer alkylation was 15170.    (peak 3), 1,120.28 Da (peak 4), and 616.79 Da (Peak 5). Aer the determination of these molecular masses and with the utilization of iodoacetamide, the cysteines presented in the peptides were alkylated (Table 1).
e peptide eluted in fraction 4 of BrTX-I, having the sequence Y G C Y C G W G G R (tandem MS spectra shown in Figure 3) and the sequence of the BrTX-I protein was deduced and returns high homology with the others PLA 2 s from snake from Bothrops snake genus present of the venoms snake registered in the date base Blast-p and showed high sequence homology with other PLA 2 in the region associated with the catalytic site ( Figure 4).
e PLA 2 activity was examined in the Bothrops roedingeri venom and in BrTX-I using the synthetic substrate 4nitro-3(octanoyloxy) benzoic acid [25]. e PLA 2 activity was higher in BrTX-I c ( Figure 5(a)). Under the conditions used, BrTX-I showed a discrete sigmoidal behavior ( Figure  5(b) insert), mainly at low substrate concentrations. Maximum enzyme activity occurred at 35-40 ∘ C ( Figure 5(c)) and the pH optimum was 8.0 ( Figure 5(d)). PLA 2 s require Ca +2 for full activity, being only 1 mM of Ca +2 needed for BrTX-I to present phospholipase A 2 activity. e addition of Zn 2+ , Mg 2+ , Mn 2+ , and Cd 2+ (10 mM) in the presence of low Ca 2+ concentration (1 mM) decreases the enzyme activity. e substitution of Ca 2+ by Mg 2+ , Cd 2+ and Mn 2+ also reduced the activity to levels similar to those in the absence of Ca 2+ ( Figure 5(e)).
In the neuromuscular activity in chick nerve-muscle preparation, the whole venom concentrations of the 50 g/mL were tested as well as the concentrations of 5, 20, 50, and 100 g/mL of BrTX-I. e tested concentration, in both venom and BrTX-I, caused an irreversible dose-dependent blockade of the neuromuscular transmission ( ). e time required for the venom to achieve 50% twitch tension blockade, through an indirect stimulation, was: 22.60 ± 0.61 min (50 g/mL) ( Figure 6). e time required for BrTX-I to achieve 50% twitch tension blockade, also through indirect stimulation only doses of 50 (31 1 ± 2 min) and 100 g/mL (2 29 ± 28 min) (Figure 6(a)). e twitch tension records of the control preparation remain stable at 98% to the venom and 97% to the BrTX-I (5 g) along the 120 min of incubation with Krebs solution.
Regarding the venom, the concentration of 50 g/mL altered signi�cantly the ACh (110 M) and KCl (20 mM) induced contractures when compared to the control values. In the concentration of the 50 g/mL, the complete blockade was not accompanied by signi�cantly inhibition of the response to ACh and KCl (Figure 6(b)). In the control preparations, the contracture to ACh and KCl was kept stable aer a 120 min indirect stimulation.
In vivo, BrTX-I induced a conspicuous local myotoxic effect when injected by the i.m. route, only doses 10 and 20 g (Figure 6(c)), but no increase in plasma CK levels occurred aer their i.v. injection even in the same dose of 20 g. Timecourse analysis showed a maximum increase in plasma CK 1 h aer i.m. injection, returning to normal by 24 h ( Figure  6(d)).
To further analyze and compare the mechanisms of the in�ammatory events induced by BrTX-I PLA 2 , the concentrations of the IL-1, IL-6, and TNF-in the serum were measured. BrTX-I caused a marked increase in the TNFconcentrations only at 1 h (Figure 7(b)). In both the case of IL-1, the maximum peak was recorded at 6 h, on the other hand for IL-6 level the peak was at 3 h (Figures 7(c) and 7(d)).

Discussion
e puri�cation procedure for basic PLA 2 s developed by Ponce-Soto et al. [6,15,23,24] showed to be also efficient for the obtainment of the Bothropsroedingeritoxin I PLA 2 (BrTX-I) from Bothrops roedingeri snake venom. Fractionation protocol of this crude venom using a single pass chromatographic in a column -Bondapack C-18 coupled to a system of reverse phase HPLC (0.78 cm-30 cm; Waters 991-PDA system) gave rise to 15 fractions at 280 nm, the eight last being the basic PLA 2 named BrTX-I (Figure 1).
SDS-PAGE showed (Figure 1 insert) the isolated toxin, BrTX-I have Mr of ∼14 kDa similarly to basic PLA 2 isolated from other myotoxins from Bothrops snake venoms. e molecular masses obtained by MALDI TOF mass spectrometry showed to be similar to that of other snake venom PLA 2 s (14358.69 Da) (Figure 2). Sequence homology studies had showed that there are extremely conserved positions in the PLA 2 s. In positions 1 and 2, there is a predominance of the amino acids sequence (DL), in position 4 (Q). One of the highly conserved regions in the amino acid sequences of PLA 2 is the Ca 2+ -binding loop, segment from…YGCYCGXGG… and HD(49)CC (Figure 3). Residues forming the Ca 2+ -binding loop and the catalytic network of BrTX-I PLA 2 show a high conservation grade, re�ecting the nondecreased catalytic activity.
e primary structure of BrTX-I determined by deduced sequencing (SwissProt database http://br.expasy.org/) method is aligned with the sequences of some other homologous snake venom PLA 2 from snake of the crotalidae family. It was very similar to that of other PLA 2 (Figure 4). e PLA 2 activity showed to be higher in BrTX-I (16.87 ± 0.643 nmoles/min/mg) when compared with the whole venom (2.59 ± 0.617 nmoles/min/mg) ( Figure 5(a)). e PLA 2 from �rotalus durissus terri�cus venom is a typical PLA 2 , since it hydrolyzes synthetic substrates at position 2 and preferentially attacks substrates in their micellar state [28]. ey can hydrolyze phospholipids in monomeric, micellar or lipid bilayer phases. PLA 2 enzymes exhibit a large and abrupt increase (up to 10,000 times) in their catalytic activity when monomeric phospholipids aggregate forms micelles at their critical micellar concentration [29]. is is due to the higher efficiency of interfacial catalysis, which depends on the absorption of the enzyme onto the lipid-water interface, strongly promoted by the presence of anionic amphipatic molecules within the membrane [30]. With synthetic substrate, BrTX-I behaved allosterically, especially at low substrate concentrations, which is in agreement with the results obtained by Beghini et al. [31], Bon�m et al. [32,33], Ponce-Soto et al. [18], Calgarotto et al. [26], Huancahuire-Vega et al. [27] and for other PLA 2 using the same nonmicellar substrate also observed that the dependence of activity on substrate concentration was markedly sigmoidal (Figure 5(b)).
e amino acid composition of the BrTX-I PLA 2 toxin revealed a high content of basic and hydrophobic residues, with 14 half-Cys, in agreement with the reported compositions and primary structures of PLA 2 toxins isolated from Bothrops venoms ( Figure 5(f)), [6,15,38,39]. e pharmacological activities investigated for BrTX-I PLA 2 includes neurotoxicity ex vivo in preparation BCP, in vivo inducing rapid damaging action to skeletal muscle tissue, paw oedema and increase of IL-1, IL-6 and TNF-in the mice serum.
Some authors [3,4,6,15,[40][41][42][43][44] have proposed several models to explain PLA 2 catalytic and pharmacological activities. In these models PLA 2 has two separated places; one is responsible for catalitic activity and other for biological activity expression. In according to them, the pharmacological place would be located in the surface of PLA 2 molecules.
e BrTX-I caused an irreversible concentrationdependent blockade of the indirectly elicited twitch responses of the chick biventer cervicis muscle preparation (BCP). Only doses 20, 50, and 100 g/mL caused an irreversible dose-dependent blockade of the neuromuscular transmission ( Figure 6(a)). e complete blockade of the muscle contraction all of the doses, was not accompanied by any signi�cant inhibition of the responses to ACh. Inhibition response to KCl was progressive in terms of increasing the dose, suggesting a myotoxic effects due to destabilization of the membrane (Figure 6(b)).
us, the neuromuscular blockade produced by BrTX-I may be attributed to presynaptic activity, either by blocking axonal conduction or by affecting transmitter release at the motor nerve-terminal. e fact that the BrtX-I from Bothrops moojeni did not signi�cantly affect the response to ACh and KCl, except when high doses were used, suggests that the venom presents a primordial presynaptic nature. Such neuromuscular blockade characteristics have been attributed to presynaptic-acting PLA 2 from snake [45,46] as those of Crotalus durissus terri�cus [47], Micrurus species [48,49], and other Bothrops, Bothrops insularis [50], Bothrops pauloensis [47,51], and Bothriopsis bilineata smargadina [52], which did not show any detectable effect on the nicotinic receptor and, in some cases, showed only a mild muscle alteration.
In according to the model proposed by [42], the anticoagulant place would be located in a region between the 53 and 76 residues, considering this region charged positively in the PLA 2 with high anti-coagulant activity. In PLA 2 with moderate or low anti-coagulant activity, there is a predominancy of negative chargings. is region is placed in a distinct local and separated of foreseen regions by neurotoxicity and myotoxicity.
Local and systemic skeletal muscle degeneration is a common consequence of envenomations due to snakebites and mass bee attacks. PLA 2 is an important myotoxic component in these venoms, inducing a similar pattern of degenerative events in muscle cells. e bothropics PLA 2 myotoxins generally present low systemic toxicity, in contrast to myotoxic PLA 2 that are also strongly neurotoxic [5,53].
Our studies on local and systemic myotoxicity in vivo reveal the BrTX-I is nonsystemic myotoxin with local action due to decrease of the plasmatic CK levels (Figures 6(a) and 6(b)). is fact reinforces the hypothesis of differentiated action of local and systemic myotoxicity proposed by Gutiérrez and Ownby [5] and also the unspeci�city and speci�city proposed by Kini [3], Ponce-Soto et al. [6] and Gutiérrez et al. [54]. PLA 2 s from snake venoms exert a large number of pharmacological activities [35,54] due to a process of accelerated micro-evolution through which a high mutational rate in the coding regions of their genes has allowed the development of new functions, mainly associated with the exposed regions of the molecules [13]. e integral analysis of the in�ammation elicited by BrTX-I from Bothrops roedingeri venom in the mouse serum performed in the present study allowed a parallel evaluation of the increase in microvascular permeability, by paw oedema and the production of various in�ammatory mediators.
e PLA 2 s from snake induced an increase in vascular permeability in peritoneal cavity of mice. is is in agreement with previous observations on the edema forming activity of similar molecules in the rodent footpad model [55,56]. e increase of vascular permeability was detected aer BrTX-I injection and developed rapidly, indicating that the observed plasma extravasation is primarily due to formation of endothelial gaps in vessels of microcirculation ( Figure  7(a)). Previous studies have documented polymorphonuclear and mononuclear cellular in�ltrate aer injection of myotoxic PLA 2 s from the venoms of Bothrops asper [57], Bothrops nummifer [58], and Bothrops jararacussu [59] in mouse skeletal muscle, and aer intrapleural administration of similar myotoxins from Bothrops jararacussu and Bothrops pirajai venoms [60]. e mediators involved in this effect of BrTX-I was not addressed in this study. However, the immediate plasma extravasation in response to BrTX-I, strongly suggests the involvement of vasoactive mediators derived from mast cell granules. Previously, the ability of venom PLA 2 to degranulate mast cells has been shown [55].
TNF-is also likely to be involved in in�ammation induced by BrTX-I, since the PLA 2 caused a signi�cant increase of TNF-levels in the serum. TNF-is also likely to be involved in leukocyte in�ltration induced by BrTX-I, since the PLA 2 caused a signi�cant increase of TNFlevels in the serum. TNF-induces the expression of Eselectin, CD11b/CD18 and ICAM-1 and triggers the release of several cytokines such as IL-1 and IL-6 ( Figure 7(b)). us, our results suggest that TNF-may have a role in the expression of CD18 and the release of other cytokines following BrTX-I injection, thereby being relevant for neutrophil in�ux and for increase of vascular permeability on the paw edema.
Cytokines, such as IL-1, IL-6, and TNF-, are also relevant mediators for leukocyte migration and participate in several in�ammatory conditions. �ur results showed that BrTX-I induce increase in IL-1 and IL-6 in the serum, exerting a stronger effect (Figures 7(c) and 7(d)). IL-1 induced the expression of adhesion molecules by endothelial cells and stimulates the release of both IL-6 and TNF- [61]. us, our results suggest that IL-1 may contribute for the leukocyte migration.
All these biological effects induced by the BrTX-I occur in the presence of a measurable PLA 2 activity. Although the catalytic activity of PLA 2 contributes to pharmacological effects, it is not a prerequisite [55,56,[62][63][64]. However, further studies are necessary to identify the structural determinants involved in these pharmacological activities.