Biochemical, Pharmacological, and Structural Characterization of New Basic P L A 2 Bbil-TX from Bothriopsis bilineata Snake Venom

Bbil-TX, a PLA2, was purified from Bothriopsis bilineata snake venom after only one chromatographic step using RP-HPLC on μ-Bondapak C-18 column. A molecular mass of 14243.8 Da was confirmed by Q-Tof Ultima API ESI/MS (TOF MS mode) mass spectrometry. The partial protein sequence obtained was then submitted to BLASTp, with the search restricted to PLA2 from snakes and shows high identity values when compared to other PLA2s. PLA2 activity was presented in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 25–37°C. Maximum PLA2 activity required Ca2+ and in the presence of Cd2+, Zn2+, Mn2+, and Mg2+ it was reduced in the presence or absence of Ca2+. Crotapotin from Crotalus durissus cascavella rattlesnake venom and antihemorrhagic factor DA2-II from Didelphis albiventris opossum sera under optimal conditions significantly inhibit the enzymatic activity. Bbil-TX induces myonecrosis in mice. The fraction does not show a significant cytotoxic activity in myotubes and myoblasts (C2C12). The inflammatory events induced in the serum of mice by Bbil-TX isolated from Bothriopsis bilineata snake venom were investigated. An increase in vascular permeability and in the levels of TNF-a, IL-6, and IL-1 was was induced. Since Bbil-TX exerts a stronger proinflammatory effect, the phospholipid hydrolysis may be relevant for these phenomena.


Introduction
Viperidae snakes are represented in South America by Crotalus, Bothrops, Bothriopsis and Lachesis. Bothriopsis bilineata is the endemic and rare bothropic snake species [1]. e envenomation is characterized by a generalized in�ammatory state. e normal reaction to envenomation involves a series of complex immunologic cascades that ensures a prompt protective response to venom in humans [2]. Although activation of the immune system during envenomation is generally protective, septic shock develops in a number of patients as a consequence of excessive or poorly regulated immune response to the injured organism [3]. is imbalanced reaction may harm the host through a maladaptitive release of endogenous mediators that include cytokines and nitric oxide.
PLA 2 s are abundant in snake venoms and have been widely employed as pharmacological tools to investigate their role in diverse pathophysiological processes. Viperid and crotalid venoms contain PLA 2 s with the ability to cause rapid necrosis of skeletal muscle �bers, thus being referred to as myotoxic PLA 2 s [4]. Local in�ammation is a prominent characteristic of snakebite envenomations by viperid and crotalid species [5].
Furthermore, PLA 2 myotoxins are relevant tools for the study of key general in�ammatory mechanisms. High levels of secretory PLA 2 (sPLA 2 ) are detected in a number of in�ammatory disorders in humans, such as bronchial asthma [6], allergic rhinitis [7], septic shock [8], acute pancreatitis [9], extensive burning [10], and autoimmune diseases [11]. In addition, increased expression and release of sPLA 2 have been found in rheumatoid arthritis, in�ammatory bowel diseases, and atherosclerosis [12,13]. Mechanisms involved in the proin�ammatory action of sPLA 2 are being actively investigated, and most of this knowledge is based on studies using puri�ed venom PLA 2 s. is paper describes the isolation and biochemical and pharmacological characterization of new PLA 2 s from Bothriopsis bilineata venom, Bbil-TX, and also the study of its various toxic activities, including myotoxicity, cytotoxicity, and in�ammation.

Venom and Reagents.
Bothriopsis bilineata venom was donated by Dr. Corina Vera Gonzáles. All chemicals and reagents used in this work were of analytical or sequencing grade and purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).

Reversed-Phase HPLC (RP-HPLC).
Five milligrams of the whole venom from Bothriopsis bilineata was dissolved in 200 L ammonium bicarbonate 0.2 M pH 8.0. 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. 2 Activity. PLA 2 activity was measured using the assay described by Holzer and Mackessy, [14] modi�ed for 96-well plates. e standard assay mixture contained 200 L of buffer (10 mM tris-HCl, 10 mM CaCl 2 and 100 mM NaCl, pH 8.0), 20 L of substrate (4-nitro-3-octanoyloxy-benzoic acid), 20 L of water, and 20 L of Bbil-TX in a �nal volume of 260 L. Aer adding Bbil-TX (20 g), the mixture was incubated for up to 40 min at 37 ∘ C, with the reading of absorbance at intervals of 10 min. e enzyme activity, expressed as the initial velocity of the reaction ( ), was calculated based on the increase of absorbance aer 20 min. e optimum pH and temperature of the PLA 2 were determined by incubating the enzyme in four buffers of different pH values (4)(5)(6)(7)(8)(9)(10) and at different temperatures, respectively. e effect of substrate concentration (0.1, 0.2, 0.3, 0.5, 1, 2, 5, 10, 20, and 30 mM) on enzyme activity was determined by measuring the increase of absorbance aer 20 min. e inhibition of PLA 2 activity by crotapotins from Crotalus durissus cascavella and DAII-2 from Didephis albiventris serum was determined by preincubating the protein (Bbil-TX) and each inhibitor for 30 min at 37 ∘ C prior to assaying the residual enzyme activity under optimal conditions. All assays were done in triplicate and the absorbances at 425 nm were measured with a VersaMax 190 multiwell plate reader (Molecular Devices, Sunnyvale, CA, USA).

Electrophoresis.
Tricine SDS-PAGE in a discontinuous gel and buffer system was used to estimate the molecular mass of the proteins, under reducing and nonreducing conditions [15].

Amino Acid Analysis.
Amino acid analysis was performed on a Pico-Tag Analyzer (Waters Systems) as described by [16]. e puri�ed Bbil-TX sample (30 g) was hydrolyzed at 105 ∘ C for 24 h in 6 M HCl (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 which the PTC-amino acids were identi�ed and quanti�ed by HPLC, by comparing their retention times and peak areas with those from a standard amino acid mixture.

2.�. Determination of the Molecular Mass of the Puri�ed Protein by Mass
Spectrometry. An aliquot (4.5 L) of the protein was inject by C18 (100 m × 100 mm) RP-UPLC (nanoAcquity UPLC, Waters) coupled with nanoelectrospray tandem mass spectrometry on a Q-T of Ultima API mass spectrometer (MicroMass/Waters) at a �ow rate of 600 nl/min. e gradient was 0-50% acetonitrile in 0.1% formic acid over 45 min. e instrument was operated in MS continuum mode and the data acquisition was from 100-3,000 at a scan rate of 1 s and an interscan delay of 0.1 s. e spectra were accumulated over about 300 scans and the multiple charged data produced by the mass spectrometer on the scale were converted to the mass (molecular weight) scale using maximum-entropy-based soware (1) supplied with Masslynx 4.1 soware package. e processing parameters were: output mass range 6,000-20,000 Da at a "resolution" of 0.1 Da/channel; the simulated isotope pattern model was used with the spectrum blur width parameter set to 0.2 Da and the minimum intensity ratios between successive peaks were 20% (le and right). e deconvoluted spectrum was then smoothed (2 × 3 channels, Savitzky Golay smooth) and the mass centroid values obtained using 80% of the peak top and a minimum peak width at half height of 4 channels.

Analysis of Tryptic
Digests. e protein was reduced (DTT 5 mM for 25 min to 56 ∘ C) and alkylated (Iodoacetamide 14 mM for 30 min) prior to the addition of trypsin (Promega�s sequencing grade modi�ed). Aer trypsin addition (20 ng/ L in ambic 0.05 M), the sample was incubated for 16 hr at 37 ∘ C. To stop the reaction, formic acid 0.4% was added and the sample centrifuged at 2500 rpm for 10 min. e pellet was discarded and the supernatant dried in a speed vac. e resulting peptides were separated by C18 (100 m × BioMed Research International 3 100 mm) RP-UPLC (nanoAcquity UPLC, Waters) coupled with nanoelectrospray tandem mass spectrometry on a Q-Tof Ultima API mass spectrometer (MicroMass/Waters) at a �ow rate of 600 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  , in order to select the ion of interest; subsequently, these ions were fragmented in the collision cell (TOF MS/MS mode).
Raw data �les from LC-MS/MS runs were processed using Masslynx 4.1 soware package (Waters) and analyzed using the MASCOT search engine version 2.3 (Matrix Science Ltd.) against the snakes database, using the following parameters: peptide mass tolerance of ±0.1 Da, fragment mass tolerance of ±0.1 Da, and oxidation as variable modi-�cations in methionine and trypsin as enzyme.

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 the Bbil-TX. Samples (50 L) containing 0.1, 1, and 5 g of the PLA 2 Bbil-TX were injected in the right gastrocnemius. A control group received 50 L of PBS. At different intervals, blood was collected from the tail into heparinized capillary tubes aer 2, 4, 6, 9, 12 and 24 hours, and the plasma creatine kinase (CK; EC 2.7.3.2) activity was determined by a kinetic assay Ck-Nac, (creatine kinase, Beacon, Diagnostics, Germany). To the reaction mixture 10 L of the plasma obtained by centrifugation from mice blood was added. e solution is incubated for 2 minutes and reads at 430 nm. e results were expressed as U/L according to the manufacturer.
2.9. Cytotoxicity Assays. Cytotoxic activity was assayed on murine skeletal muscle C2C12 myoblasts and myotubes (ATCC CRL-1772). Variable amounts of Bbil-TX were diluted in assay medium (Dulbecco's Modi�ed Eagle's Medium supplemented with 1% fetal-calf serum) and added to cells in 96-well plates, in 150 L. Controls for 0 and 100% toxicity consisted of assay medium, and 0.1% Triton X-100, respectively. Aer 3 h at 37 ∘ C, a supernatant aliquot was collected for determination of lactic dehydrogenase (LDH; EC 1.1.1.27) activity released from damaged cells, using a kinetic assay (Wiener LDH-P UV). Experiments were carried out in triplicate.

Edema-Forming
Activity. e ability of Bbil-TX to induce edema was studied in groups of �ve Swiss mice (18-20 g). Fiy microliters of phosphate-buffered saline (PBS; 0.12 M NaCl, 0.04 M sodium phosphate, pH 7.2) with Bbil-TX (0.1; 1 and 5 g/paw) were injected in the subplantar region of the right footpad. e control group received an equal volume of PBS alone. e paw swelling was measured with an Electronic Caliper Series 1101 (INSIZE LTDA, SP, Brazil) at 0.5, 1, 3, 6, 9, and 24 h aer administration. Edema was expressed as the percentage increase in the size of the treated group to that of the control group at each time equal to 24 hrs.
2.11. Cytokines. e levels of cytokines IL-6 and IL-1 in the serum from BALB/c mice were assayed by a twosite sandwich enzyme-like immunosorbent assay (ELISA) as described by [17]. In brief, ELISA plates were coated with 100 L (1 g/mL) of the monoclonal antibodies anti-IL-6 and anti-IL-1 placed 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% fetalcalf 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 antibody anti-IL-6 and anti-IL-1 as a second antibody 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. 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-6 and IL-1.
To measure the cytotoxicity of TNF-present in the serum from BALB/c mice, a standard assay with L-929 cells, a �broblast continuous cell line, was used as described previously by [18]. e percentage cytotoxicity was calculated as follows: (A control − A sample A control ) × 100. Titres were calculated as the reciprocal of the dilution of the sample in which 50% of the cells in the monolayer were lysed. TNFactivity is expressed as units/mL, estimated from the ratio of a 50% cytotoxic dose of the test to that of the standard mouse recombinant TNF-.

Statistical Analysis.
Results are reported as means ± SEM. e signi�cance of differences between the means was assessed by ANOVA followed by Dunnett's test when various experimental groups were compared with the control group. A value of indicated signi�cance.

Results
Fractionation of Bothriopsis bilineata venom by RP-HPLC on a -Bondapak C18 column resulted in eleven peaks (1-11) ( Figure 1). e 11 peaks were screened for myotoxic and PLA 2 activities. Peak 7 caused local myotoxicity at concentrations ranging from 0.1 to 5 g/mL in mouse gastrocnemius muscle. In addition, peak 7, named Bbil-TX-I (Bothriopsis bilineata toxin) showed high PLA 2 activity and was selected for biochemical and pharmacological characterization. e purity of this peak was con�rmed by rechromatography on an analytical RP-HPLC -Bondapack C18 column, showing the presence of only one peak and by Tricine SDS-PAGE, which revealed the presence of one electrophoretic band with Mr  around 15 kDa, in the absence and presence of DTT (1 M) (data not shown). Q-Tof Ultima API ESI/MS (TOF MS mode) mass spectrometry analysis con�rmed the homogeneity of the peak Bbil-TX and determined the exact molecular mass of 14243.8 Da (Figure 2). is value of molecular mass was used in calculating the molar concentrations of toxin used in the experiments described below.
e alkylated and reduced protein was digested with trypsin and the resulting tryptic peptides (10) were fractionated by RP-UPLC (nanoAcquity UPLC, Waters) (data not shown). 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 100-2000 , in order to select the ion of interest; subsequently, these ions were fragmented in the collision cell (TOF MS/MS mode). e data �les obtained from LC-MS/MS runs were processed using the Masslynx 4.1 soware package (Waters) and analyzed using the MASCOT search engine version 2.3 (http://www.matrixscience.com/). Table 1 shows the deduced sequence and measured masses of alkylated peptides obtained for Bbil-TX PLA 2 . Isoleucine and leucine residues were not discriminated in any of the sequences reported (3) y(6) + + b(6) + + y0 (7)  since they were indistinguishable in low energy CID spectra. Because of the external calibration applied to all spectra, it was also not possible to resolve the 0.041 Da difference between glutamine and lysine residues, except for the lysine that was deduced based on the cleavage and missed cleavage of the enzyme. e ten peptides obtained in Q-Tof Ultima API ESI/MS (TOF MS mode) mass spectrometry of the Bbil-TX PLA 2 were submitted to the NCBI database, using the protein search program BLASTp with the search being restricted to the sequenced proteins from the basic protein with phospholipase A 2 activity family. Based on the positional matches of the de novo sequenced peptides with other homologous proteins, it was possible to deduce the original positions of these peptides in the native protein ( Figure 4).
e results of the primary structures show that Bbil-TX PLA 2 is composed of 122 amino acid residues and shares the conserved sequence domains common to PLA 2 group, including the 14 cysteines, the calcium-binding site located on (Y)27, (G)28, (C)31, and (G)32, and the catalytic network commonly formed by (H)48, (D)49, (Y)52, and (D)90. A comparative analysis of the sequence of Bbil-TX PLA 2 with other neurotoxins "ex vivo" and myotoxic PLA 2 s belonging to Viperidae family, LmTX-I and LmTX-II (Lachesis muta muta) [19] and Crotalus scutulatus scutulatus (Mojave rattlesnake) (accession number P62023), showed similarity of 81.1-91.0%. (SwissProt database http://br.expasy.org/). e tandem mass spectra shown in Figure 3, relative to the peptide eluted in fraction 3 having the sequence C C F V H D C C Y G K, allows to classify both enzymes as PLA 2 .
e local myotoxic effect (i.m.) in vivo was observed with PLA 2 Bbil-TX studied. It was observed that the PLA 2 induced a conspicuous effect evidenced by the rapid elevation of plasma CK activity through a time course, reaching its maximum effect 2 h aer injection and returning to normal levels aer 24 h (Figure 6(a)). Our results showed that the PLA 2 Bbil-TX did not show systemic myotoxic effect (i.v.) (Figure 6(b)).
Compared to PBS-injected animals, those which received subplantar injections of the Bbil-TX (0.1, 1, and 5 g/paw) presented marked paw edema (Figure 7(a)). Maximal activity was attained 1 h to the Bbil-TX aer injection and receded to normal levels aer 24 h. e level of edema induction by 5 g of PLA 2 1 hour aer administration was 61.57%, showing a dose-dependent activity. To further analyze the mechanisms of the in�ammatory events induced by Bbil-TX (0.1 g), TNF-, IL-6, and IL-1 concentrations were measured in the serum. TNF-levels were increased 1 h aer injection of Bil-TX and no detectable production was observed at the later time intervals studied (Figures 7(b), 7(c), and 7(d)). Bbil-TX caused a signi�cant increase in IL-6 release between 1 and 3 h, respectively, in serum collected aer injection of venom compared with the control (Figure 7(c)). However, increased levels of IL-1 were detected between 1, 3, 6, and 12 h, respectively (Figure 7(d)).

Discussion
e puri�cation procedure for basic PLA 2 s developed by [20][21][22] showed to be also efficient for the obtainment of neurtoxin "ex vivo" and myotoxin from Bothriopsis bilineata snake venom. Fractionation of this crude venom by single-step chromatography in a column -Bondapack C-18 coupled to a system of reversed-phase HPLC was carried out and as a result of the proposed method, several toxins have been efficiently puri�ed. Fraction 7 was named Bbil-TX (PLA 2 ). SDS-PAGE showed evidence that Bbil-TX isolated PLA 2 s have an Mr of ∼14 kDa for the monomers, similar to basic PLA 2 s isolated from other venoms (data not shown) [23]. e conserved residues Y28, G30, G32, D49, H48, and Y52 are directly or indirectly linked in the catalyses of the Bbil-TX. e molecular masses obtained by mass spectrometry showed to be similar to that of other snake venom PLA 2 s [22,24,25]. e amino acid composition of the Bbil-TX PLA 2 toxin suggests the presence of 14 half-Cys residues, providing the basis for a common structural feature of PLA 2 in the formation of its seven disul�de bridges [20,21,26] and a high content of basic and hydrophobic residues, that provides a explication important in the interaction of the PLA 2 with negatively charged phospholipids of cells membranes [27]. Such an interaction is important to explain the effect of these enzymes on different cells types, both prokariotes and eukariotes [28,29].
Comparison of the amino acid sequence of Bbil-TX PLA 2 showed high homology with other neurotoxic and myotoxic PLA 2 s from Lachesis and Crotalus genera (Figure 4). Sequence homology studies had shown that there are extremely conserved positions in the PLA 2 s. In positions 1 and 2, there is a predominance of the amino acid sequence (HL), in position 4 (Q), and in positions 5 to 7 (FNK). 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. e calcium ion is coordinated by three main chain oxygen atoms from residues (Y)28, (G)30, (G)32, and two carboxylate oxygen atoms of (D)49. Two generally conserved solvent water molecules complete the coordination sphere of the calcium ion forming a pentagonal bipyramidal geometry. It is believed that two disul�de bridges (C)27-(C)119 and (C)29-(C)45 ensure the correct relative orientation of the calcium-binding loop in relation to the amino acids of the catalytic network [30]. e residues (H)48, (Y)52, and (D)99 which are responsible for catalytic activity have an ideal stereochemistry with the presence of the so-called "catalytic network", a system of hydrogen bonds which involves the catalytic triad [30,31]. Residues forming the Ca 2+ -binding loop and the catalytic network of Bbil-TX PLA 2 show a high conservation grade, re�ecting the nondecreased catalytic activity. e PLA 2 activity was shown to be higher in Bbil-TX PLA 2 (24.75 ± 2.28 nmoles/min/mg) when compared with the whole venom (8.15 ± 1.24 nmoles/min/mg). PLA 2 enzyme from snake venom shows classic Michaelis-Menten behavior against micellar substrates [32]. With a synthetic substrate, Bbil-TX PLA 2 behaved allosterically, especially at low concentrations, which is in agreement with the results obtained by [23] for the PLA 2 of Bothrops jararacussu venom and Damico et al. [19] for the PLA 2 isoform puri�ed from Lachesis muta muta venom. Using the same synthetic nonmicellar substrate, it was also possible to observe that the dependence of activity on substrate concentration was markedly sigmoidal for the PrTX-III from Bothrops pirajai [33]. PLA 2 s from crotalic venoms have showed a similar behavior to the one presented by bothropic PLA 2 s with the same substrate used in the kinetic studies to Bbil-TX PLA 2 [14,34]. Despite the structural and functional differences among bothropic and crotalic PLA 2 s, both show allosteric behavior in the presence of the same substrate.
e PLA 2 activity could be veri�ed with different pH levels; the optimum pH of basic PLA 2 s is around 7.0 and 8.5 [32,35]. Bbil-TX PLA 2 can be considered basic since its highest activity is evidenced at pH 8.0. Temperature is another kinetic parameter utilized to characterize the PLA 2 (Asp49). It has been shown that PLA 2 from Naja naja naja is very stable in extreme temperatures such as 100 ∘ C [35]. e optimum temperature of Bbil-TX PLA 2 was around 37 ∘ C, but at 40-45 ∘ C, the Bbil-TX PLA 2 activity did not present a huge decrease.
Bbil-TX PLA 2 increases the plasmatic CK levels aer i.m. injection (Figure 6(a)), revealing drastic local myotoxicity. is myotoxicity induced by snake venoms, including Botrhiopsis bilineata, may result from the direct action of myotoxins on the plasma membranes of muscle cells, or indirectly, as a consequence of vessel degenerations and ischemia caused by hemorrhagins or metaloproteases. Bbil-X PLA 2 contributes signi�cantly to local myotoxic action in vivo. It was already demonstrated that the snake venom PLA 2 s are the principal cause of local damage [40]. Myotoxic PLA 2 s affect directly the plasma membrane integrity of muscle cells, originating an in�ux of Ca 2+ ions to the citosol that starts several degenerative events with irreversible cell injures [41]. e binding sites of myotoxins on the plasma membranes are not clearly established, although two types have been proposed: (a) negatively charged phospholipids [42], present on membranes of several cell types, explaining the high in vitro cytotoxic action of these enzymes [28,43,44], and (b) protein receptors, which make muscle cells more susceptible to myotoxin action [28].
All these biological effects induced by the toxin occur in the presence of a measurable PLA 2 activity. Although the catalytic activity of PLA 2 s contributes to pharmacological effects, it is not a prerequisite [21,26,29]. However, further studies are necessary to identify the structural determinants involved in these pharmacological activities.
Some authors, [21,41,45,46], 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 catalytic activity and the other for biological activity expression. According to them, the pharmacological place would be located on the surface of PLA 2 molecules. According to the model proposed by [47], the anti-coagulant place would be located in a region between the 53 and 76 residues, considering this region charged positively in the PLA 2 with high anticoagulant activity. In PLA 2 with moderate or low anticoagulant activity, there is a predominancy of negative charges.
Further research in identifying target proteins will help determine details of the mechanisms of the pharmacological effects at the cellular and molecular levels [48]. Studies in these areas will result in new, exciting, and innovative opportunities and avenues in the future, both in �nding answers to the toxicity of PLA 2 enzymes and in developing proteins with novel functions. PLA 2 s from snake venoms exert a large number of pharmacological activities due to a process of accelerated 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 [29]. e integral analysis of the in�ammation elicited by Bbil-TX in the mouse serum performed in the present study allowed a parallel evaluation of the increase in microvascular permeability and the production of various in�ammatory mediators. Bbil-TX induced an increase in vascular permeability in the paw of mice. is is in agreement with previous observations on the edema-forming activity of similar molecules in the rodent footpad model [49,50].
e increase of vascular permeability was detected early aer Bbil-TX injection and developed rapidly, indicating that the observed plasma extravasation is primarily due to formation of endothelial gaps in vessels of microcirculation. e main edema formation occurred 1 h aer the injection of Bbil-TX with constant decrease. Bbil-TX caused paw edema in mice with a time course similar to that reported for other PLA 2 s from Bothrops venoms in mice and rats, that is, a fairly rapid onset (generally ≤ 3 h to peak) followed by a gradual decline over the following 24 h [51][52][53][54].
e mediators involved in this effect of Bbil-TX myotoxin were not addressed in this study. However, the immediate plasma extravasation in response to Bbil-TX strongly suggests the involvement of vasoactive mediators derived from mast-cell granules. is strongly suggests that enzymatic phospholipid hydrolysis plays a signi�cant role in this event.
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 Bbil-TX induced an increase in TNF-, IL-6, and IL-1 in the serum [55]. us, our results suggest that IL-1 may contribute to the leukocyte in�ux induced by Bbil-TX. In addition, the similarity observed in the time course of IL-6 and IL-1 increase in the serum may indicate a positive regulatory role for IL-1 on the release of IL-6 induced by Bbil-TX. IL-6, an important mediator of in�ammation, causes leukocytosis characterized by a rapid neutrophilia by releasing of PMN leukocytes from the bone marrow [56,57]. In addition, IL-6 upregulates intercellular-adhesion-molecule-1 (ICAM-1) expression by endothelial cells but decreases the levels of Lselectin on circulating PMN leukocytes contributing to �rm adhesion, the next step of cell migration [58].
TNF-is also likely to be involved in leukocyte in�ltration induced by Bbil-TX, since the PLA 2 caused a signi�cant increase of TNF-levels in the serum. TNF-is likely to induce the expression of E-selectin, CD11b/CD18, and intercellular adhesion molecule-1 (ICAM-1) and triggers the release of several cytokines such as IL-1 and IL-6 and eicosanoids. us, our results suggest that TNF-may have a role in the expression of CD18 and the release of other cytokines following Bbil-TX injection, thereby being relevant for neutrophil in�ux and for increase of vascular permeability. It is interesting that TNF-and IL-6, as well as IL-1, may induce or potentiate the expression and release of group IIA PLA 2 s [59,60].
In conclusion, Bbil-TX induces a marked in�ammatory reaction in the mouse serum. Since basic myotoxic PLA 2 s are abundant in snake venoms, these toxins must play a relevant role in the proin�ammatory activity that characterizes this venom. e fact that Bbil-TX elicited a stronger in�ammatory reaction argues in favor of a role of enzymatic phospholipid hydrolysis in this phenomenon, either through the direct release of arachidonic acid from plasma membranes or through activation of intracellular processes in target cells.
Accumulating evidences have strongly shown that venom PLA 2 s are among the major mediators of myonecrosis [40], hemolysis, mast cell degranulation, and edema formation [3].
PLA 2 s isolated from Bothrops venoms are frequently myotoxic [26] and can cause edema in rats and mice [39,45,49,54]. ese results suggest that, for some PLA 2 s, catalytic activity plays a role in the edematogenic effect.
Ethical Approval e animals and research protocols used in this study followed the guidelines of the Ethical Committee for use of animals of ECAE-IB-UNICAMP SP, Brazil (protocol number 1931-1) and international laws and policies. All efforts were made to minimize the number of animals used and their suffering.