Biochemical Characterization, Action on Macrophages, and Superoxide Anion Production of Four Basic Phospholipases A2 from Panamanian Bothrops asper Snake Venom

Bothrops asper (Squamata: Viperidae) is the most important venomous snake in Central America, being responsible for the majority of snakebite accidents. Four basic PLA2s (pMTX-I to -IV) were purified from crude venom by a single-step chromatography using a CM-Sepharose ion-exchange column (1.5 × 15 cm). Analysis of the N-terminal sequence demonstrated that pMTX-I and III belong to the catalytically active Asp49 phospholipase A2 subclass, whereas pMTX-II and IV belong to the enzymatically inactive Lys49 PLA2s-like subclass. The PLA2s isolated from Panama Bothrops asper venom (pMTX-I, II, III, and IV) are able to induce myotoxic activity, inflammatory reaction mainly leukocyte migration to the muscle, and induce J774A.1 macrophages activation to start phagocytic activity and superoxide production.


Introduction
Envenomation resulted from snakebites is the cause of considerable morbidity and mortality in many tropical and subtropical countries, being an important public health problem [1,2]. According to Kasturiratne et al. [2], the estimative number of deaths per year due to snakebites in 2007 for Central America ranged from 193 to 1,461. Since snakebites affect, in most cases, poor people living in rural parts of tropical countries [3], the World Health Organization (WHO), incorporated snakebite envenoming in its list of neglected diseases (http://www.who.int/neglected_diseases/ diseases/en/).
Lance-headed pit vipers belonging the Viperidae family, especially the Bothrops asper snake ( Figure 1) are responsible for the most severe cases of snakebite envenoming, being the main cause of the largest numbers of bites and fatalities in Central America [1]. Among the American countries, Panama has the highest incidence of snakebite cases, showing an average number of 40 to 65 cases per 100,000 population per year and a estimated total number of 1,300 to 1,800 cases per year [4,5], which B. asper is responsible for 90% of all snakebites cases of major clinical importance [4,6]. B. asper is able to inoculate a relative large amount of venom and is considered extreme aggressive, being able to cause severe accidents [7].
According to Gutiérrez et al. [5], the envenomation produced by B. asper induces prominent local tissue damage, characterized by swelling, blistering, prominent oedema, haemorrhage, dermonecrosis, and myonecrosis with clinical manifestations that include bleeding, effects on platelet aggregation, coagulopathy, hypotension, hemodynamic alterations, pulmonary oedema, and acute renal failure. Other less common effects include intravascular haemolysis, acute myocardial damage multiple organ failure, and death. e clinical features of the envenomation are affected by the venom components, which vary according to snake species, geographic region, age, sex, and environment [5,8,9]. Snake venoms are characterized as a complex mixture of bioactive molecules, which proteins compose more than 90% of the venom dry weight [10][11][12]. Many of these proteins are enzymes, in which the most abundant are phospholipases A 2 (PLA 2 s; E.C.3.1.1.4) [10]. PLA 2 s are members of a protein superfamily that comprise several groups of enzymes with different catalytic mechanisms, as well as different functional and structural features, that cleavage the sn-2 acyl ester bond of glycerolphospholipids producing free fatty acids and lysophospholipids [13,14]. Snake venom PLA 2 s (svPLA 2 s) have been grouped into four classi�cations according to minor structural differences as group I and II, both sub-classi�ed as type A or B. e group II is found in venoms from Viperidae family, while the group I is found in Elapidae and Hydrophiidae venoms [14]. svPLA 2 s from Viperidae family are placed into group IIB and are mainly subdivided in two types: Asp49 PLA 2 s, which have an Asp residue at position 49, and Lys49 PLA 2 s, showing a Lys residue at position 49. Different from Asp49 PLA 2 s, Lys49 PLA 2 s have low or any catalytic activity upon arti�cial substrates [13,15,16].
is present paper describes the biochemical and toxicological characterization of crude B. asper venom from Panama, and the isolation, puri�cation, and biochemical characterization of four basic cytotoxic PLA 2 from this venom and its effects on gastrocnemius muscle and in�ammation.

Materials and Methods
2.1. Materials. Bothrops asper snake venom was collected from adult specimens, captured in Caldera and Gomez (Provence of Chiriquí, Panama) and in Arraiján (Provence of Panama, Panama). e snakes were maintained in a serpentarium at the Gamboa Rainforest Resort, Panamá, where the crude venom was obtained by inducing the snake to bite a para�lm-wrapped jar. Venoms were centrifuged at 1,000 xg for 15 min, and supernatants were lyophilized and stored at −20 ∘ C in Microbiology Department at the Medicine Faculty of Panama University until used. Male albino Swiss mice, weighing 18-22 g, were used for the assays.
e murine macrophage cell lines (J774A.1) were obtained from Rio de Janeiro Cell Bank Collection (Brazil). RPMI-1640, penicillin, streptomycin, and L-glutamine were purchased from Sigma-Aldrich (MO, USA); fetal bovine serum (FBS) was from Cultilab (Brazil). All reagents were low endotoxin or endotoxin-free grades. Animal care was in accordance with the guidelines of the Brazilian College for Animal Experimentation (COBEA) and was approved by the Committee for Ethics in Animals Utilization of Universidade de São Paulo (CEUA no. 06.1.291.53.3). CM-Sepharose and Phenyl-Sepharose resins were purchased from Amersham Biosciences, Uppsala, Sweden. e Kit CK-UV-Kinetic was purchased from Bioclin, Brazil. e following reagents: ethylenediaminetetraacetic acid (EDTA), molecular weight protein standards, and acrylamide were obtained from Sigma Chemical Co. All other chemicals reagents were of analytical grade from Merck, Aldrich or Pharmacia Biotech.

P�ri�cation and Biochemical
Characterization o� PL� 2 s. B. asper crude venom (300 mg) was dissolved in 1.5 mL of 0.05 M ammonium bicarbonate buffer, pH 8.1 and applied on a CM-Sepharose column (1.5 × 15 cm) according to Soares et al. [17]. All fractions were analyzed by SDS-PAGE, and PLA 2 activity was evaluated in vitro by indirect erythrocyte lysis in agar containing human erythrocytes and egg yolk, as previously described [18], being the fraction with PLA 2 activity selected. Polyacrylamide gel electrophoresis was performed in the presence of sodium dodecyl sulfate (SDS-PAGE) [19]. Isoelectric focusing was performed according to previously described. Buffalyte, pH range 3.0-9.0 (Pierce, IL), was used to generate the pH gradient. To determinate the protein concentration, the microbiuret method was used. e mol. wt of PLA 2 s was estimated by mass spectrometry (Quattro II, Micromass). A Procise-491 (Applied Biosystems) automatic sequencer was used for the N-terminal sequencing [17]. e phenylthiohydantoin (PTH) amino acids were identi�ed by comparing their retention times with the 20 PTH-amino acid standard mixture. e peptides obtained were compared with the sequences of other related proteins in the SWISS-PROT/TrEMBL databases using the FASTA and BLAST tools.

Edema-Inducing Activity.
Groups of four male Swiss mice (18-22 g) were injected in the subplantar region with various amounts of crude venom (in a volume of 50 L) prepared with PBS, pH 7.2. en, the paw increase was measured at different time intervals (30, 60, 120, and 180 min), subtracting the initial paw measure (time 0 h). e paw edema was measured with the aid of a low-pressure pachymeter (Mitutoyo, Japan).

Hemorrhage.
Groups of four male Swiss mice (18-22 g) were injected by ID route in the dorsal region with various amounts of crude venom (in a volume of 50 L) prepared in PBS, pH 7.2. e hemorrhagic activity of the venom was determined as follows. Different doses of venom were injected intradermally, in a volume of 0.1 mL, into groups of four mice (18-22 g)� 2 h later, they were sacri�ced with CO 2 , their skin removed, and the area of the hemorrhagic spot was measured. Diameters were calculated, and the minimum hemorrhagic dose was de�ned as the dose of venom, which induced a lesion of 10 mm diameter [21].

Coagulant Effect.
Platelet-poor plasma was obtained from rabbit citrated blood by centrifuging the plasma twice at 2,500 xg for 15 min at 4 ∘ C. Aliquots of 0.5 mL of plateletpoor plasma were incubated with various amounts of crude venom (dissolved in 100 L of PBS, pH 7.2). Incubation was carried out for 5 min at 37 ∘ C. en, 0.1 mL of 0.25 M CaCL 2 was added to each tube, and they were checked for the formation of a clot every 30 seconds for a total period of 2 h. All experiments were carried out in triplicate.

Fibrinolytic
Activity. e �brinolytic activity was measured using 0.6% bovine plasminogen-free �brin plates [22]. For this purpose, 30 L of sample was placed on a �brin plate, and the lysis area was measured aer incubation at 37 ∘ C for 18 h. PBS was used as negative control. e speci�c activity was calculated from a standard curve for the lysis area obtained with plasmin on the plasminogen-free �brin plates. All experiments were carried out in triplicate.
2.3.6. PLA 2 Activity. It was determined by incubating 0.5 mL of crude venom solution (at various amounts) with 50 L of egg yolk diluted 1 : 5 with 0.1 M Tris, 10 mM CaCl 2 , and pH 8.5 buffer containing 1% Triton X-100. Incubations were carried out for 10 min at 37 ∘ C. e liberated free fatty acids were extracted and titrated according to the method of Dole [23]. Crude venom from Bothrops asper (10 g) and PBS were used as positive and negative controls, respectively. All experiments were carried out in triplicate.

Myotoxic Activity.
Groups of four male Swiss mice (18-22 g) were injected in the right gastrocnemius muscle with crude venom (50 g/50 L of PBS), MTXs (50 g/50 L of PBS), or PBS alone (50 L). Aer 3 h, blood was collected from the tail in heparinized capillary tubes and centrifuged for plasma separation. Activity of creatine kinase (CK) was then determined using 4 L of plasma, which was incubated for 3 min at 37 ∘ C with 1.0 mL of the reagent according to the kinetic CK-UV protocol from Bioclin, Brazil. e activity was expressed in U/L, where one unit corresponds to the production of 1 mmol of NADH per minute.

Histological Examination of Myonecrosis.
Myotoxic activity was assayed on the basis of the morphologic alterations induced by IM injections of crude venom or MTXs (50 g) and negative control PBS (50 L) in the right gastrocnemius muscle of Swiss mice (18-22 g, 4). Aer 24 h, the animals were euthanized with CO 2 , and a small section of the central region of the muscle was excised and soaked in �xing solution (10% formaldehyde in PBS, v/v). e material was then dehydrated by increasing concentrations of ethanol and processed for inclusion in paraffin. e resulting blocks were sliced in 2.5 m thick sections, stained with 0.25% (w/v) hematoxylin-eosin and examined under a light microscope [17].
2.3.9. Cytotoxic Assay. Cell viability was measured by Trypan blue exclusion. In brief, monolayers of J774A.1 cells grown in RPMI-1640 medium with 100 g/mL penicillin, 100 g/mL streptomycin, and 2 mM L-glutamine were withdrawn and aer counting 2 × 10 5 cells/80 L were added to plastic vials and incubated with 20 L of different concentrations of pMTX-I, II, and III (1.5, 3 and 6 g/mL) diluted in RPMI (control), for 1 h at 37 ∘ C in a humidi�ed atmosphere (5% CO 2 ). en, 20 L 0.1% Trypan blue was added to 100 L of J774A.1 macrophage suspension. Viable cell index was determined in a Neubauer's chamber by counting a total number of 100 cells. Results were expressed as percentage of viable cells.

2.3.10.
Colorimetric NBT Assay. e colorimetric NBT assay was conducted in J744A.1 cells. In this assay, the generation of superoxide was estimated by reducing nitroblue tetrazolium (NBT), a yellow liposoluble compound that becomes insoluble and blue in its reduced form [24]. For this test, the cells J774A.1 had their concentration adjusted to 2 × 10 5 /100 L and were incubated with 100 L of RPMI containing NBT 0.1% (control) or 100 L of 2 × 10 6 zymosan particles suspension diluted in RPMI containing NBT 0.1% (positive control) or 100 L of different concentrations of pMTX-I, II, and III (1.5, 3 and 6 g/mL) diluted in RPMI containing NBT 0.1%, and incubated for 1 h at 37 ∘ C in humid-i�ed atmosphere (5% CO 2 ). At the end of the incubation period, the vials were centrifugated for 30 seconds at 4,500 xg, and the cells were washed twice with warm PBS. e NBT reduced deposited inside the cells were then dissolved, �rst by adding 120 L of 2 M KOH to solubilize cell membranes and then by adding 140 L of DMSO to dissolve blue formazan with gentle shaking for 10 min at room temperature. e dissolved NBT solution was then transferred to a 96-well plate and absorbance was read on a microplate reader at 620 nm. Data were expressed as absorbance.

Phagocytic Activity of J774A.1 Cells of Nonopsonized
Zymosan. J774A.1 cells were plated on 13 mm diameter glass coverslips (Glass Tecnica, Brazil) in 24-well plates at a density of 2 × 10 5 cells per coverslip and allowed to attach for 2 h at 37 ∘ C under a 5% CO 2 atmosphere. Nonadherent cells were removed by washing with PBS. Cell monolayers were cultured for 1 h with RPMI supplemented with 100 g/mL penicillin, 100 g/mL streptomycin, and 2 mM L-glutamine at 37 ∘ C and 5% CO 2 , and then challenged with RPMI (control) or 6 g/mL of pMTX-I, II, and III diluted in RPMI. Aer washing in cold PBS, the monolayers were incubated for 1 h at 37 ∘ C and 5% CO 2 with nonopsonized zymosan, prepared as described below, and unbound particles were removed by washing with PBS. Cells were �xed with 2.5% glutaraldehyde for 15 min at room temperature, and the coverslips were mounted in microscope slides. e extent of phagocytosis was quanti�ed by contrast phase microscopic observation. At least 200 macrophages were counted in each determination, and those containing three or more internalized particles were considered positive for phagocytosis [25,26]. Results were presented as the percentage of cells positive for phagocytosis. e zymosan particles, obtained from yeast cell walls, were suspended in PBS providing a concentration of 3 mg/mL. Aer that, the zymosan suspension was sonicated for 15 min, and total zymosan particles were determined in a Neubauer's chamber. e ratio of zymosan per macrophage was 1 : 10.

Statistical Analysis.
Results are presented as mean ± S.D. obtained with the indicated number of tests. e statistical signi�cance of differences between groups was evaluated using student test. A 0.1 < < 0.05 value was considered to indicate signi�cance.

Results and Discussion
Panamanian B. asper snake venom induced hemorrhage, edema, myonecrosis, coagulation, and �brinolytic activities in vitro, and lethality, also presenting PLA 2 activity evidenced by titrimetric and indirect hemolytic assays (Table  1). e toxicological pro�le was qualitatively similar to that previously described for B. asper from Costa Rica [12] and Guatemala [27]. e nature and biological properties of snake venom components are peculiar to each species [8], whereas the presence and concentration of several venom components could vary intraspeci�cally as a function of geographic distribution, age, sex, feeding, size, season, and the time elapsed between venom extraction [9,11,28]. ese intraspeci�c variations have evident clinical and therapeutic implications and can affect the capacity of antivenoms to neutralize venoms from snakes of geographically separated populations [5,11,13,29,30].
Four basic svPLA 2 s were highly puri�ed in a single step puri�cation using an ion-exchange chromatography performed on a CM-Sepharose column. e elution of absorbed proteins with a linear gradient of concentrated buffer resulted in seven fractions (Figure 2(a)), which fractions Ba-4 to Ba-7 were related to PLA 2 s. Ba-4 and Ba-5 fraction were related to PLA 2 enzymatically active, whereas Ba-6 and Ba-7 were related to enzymatically inactive PLA 2 . e purity degree of the isolated proteins was further demonstrated by SDS-PAGE, mass spectrometry, and N-terminal sequence (Figures 2 and 3) and named as pMTX-I, pMTX-III, pMTX-IV, and pMTX-II, respectively. Different from our study, others research groups have exhaustively puri�ed PLA 2 s from Bothrops venoms using different combinations of chromatographic methods: gel �ltration, ion exchange, RP-HPLC, and affinity with antibodies and heparin [13,31].
e puri�ed proteins were characterized as single polypeptide chains, with an isoelectric point ranging from 8.1 to 8.3. e average molecular mass estimated by mass spectrometry was 14,156.5 for pMTX-I, 14,249.5 for pMTX-II, and 14,253.0 for pMTX-III ( Figure 3). e N-terminal sequence alignment of pMTX-I and pMTX-III with MTICR myotoxic III PLA 2 (Uniprot accession no.: P20474) from Costa Rican B. asper showed, respectively, 96% and 88% of identity, and the sequence alignment of pMTX-II and pMTX-IV showed, respectively, 96% and 94% of identity with B. asper MTIICR myotoxic IV PLA 2 (Uniprot accession no.: P24605). Additionally, multiple sequence alignment of pMTX-I, II, III, and IV with Costa Rica Bothrops PLA 2 s showed highly conserved amino acids, such as cysteine residues involved in disul�de bond formation. Several other conserved residues important to PLA 2 catalytic activity, such as the catalytic site (D 42 XCCXXHD 49 ) and the calciumbinding site (X 27 CGXGG 32 ) [13,31] were shown. e Nterminal sequences (Figure 2(b)) of the isolated MTXs demonstrated that pMTX-I and III are basic PLA 2 s with an aspartate residue at position 49 (Asp49), therefore catalytically active (Figure 4(a)), whereas MTX-II and IV are basic PLA 2 s displaying a lysine residue at the same position (Lys49) (Figure 4(a)), therefore, catalytically inactive.
Panamanian B. asper crude venom, pMTX-I, II, III, and IV showed a high myotoxic activity (Figure 4(b)). Histopathological analysis revealed a drastic myonecrosis, displaying contracted and clumped �bers in different stages of degeneration and leukocyte in�ltrate induced by myotoxins. Our results agree with Gutiérrez and Lomonte [32] and suggest that Lys49 myotoxins pMTX-II and pMTX-IV can affect the cell membrane of skeletal muscle �bers by a phospholipid hydrolysis independent mechanism. ese results suggest that, moreover the catalytic site, this toxin may possess another molecular region that can bind and disorganize skeletal muscle plasma membrane [31,32]. Some studies have suggested that myotoxic PLA 2 s may induce muscle cell damage by affecting the integrity of plasmatic membranes, thereby leading to hyper contraction and other intracellular effects [13,31,32].
In order to evaluate the activation of leukocytes, the toxicity of B. asper myotoxins on macrophage J774A.1 cell line were studied. e cells were incubated with different concentrations of pMTX-II, III, and IV during 1 hour. ese myotoxins did not affect the macrophage viability, which are in agreement with Zuliani et al. [25], showing their low toxicity on this cell type. Additionally, the effect of the same myotoxins on J774A.1 phagocytosis ability was evaluated via -glucan receptor, by the uptake of nonopsonized zymosan particles incubated with noncytotoxic concentrations of pMTX-II, III, and IV was investigated. Our data showed that J774A.1 macrophages incubated with RPMI showed an average of phagocytosis of 16.5 ± 0.5%. Incubation of macrophages with pMTX-II, III, and IV, at 6 g/mL, resulted in phagocytic indexes of 30.6 ± 0.6%, 29.3 ± 5.5%, and 36.5 ± 2.5%, respectively. ese results showed that the myotoxins studied were able to stimulate phagocytosis of non-opsonized zymosan particles by J774A.1 macrophages (Figure 5(a)), which are in agreement with Zuliani et al. [25]. Moreover, these results suggest that phospholipid hydrolysis catalytic activity is not essential for the activity observed and argue with the hypothesis that other molecular regions distinct from the active site may be involved in this effect.
One of the most immediate responses of macrophages during phagocytosis is the production of the potent oxygen free radical, superoxide anion. e enzyme complex primarily responsible for the production of this highly reactive oxygen species is the NADPH oxidase complex [33]. is reaction parallels the release of a variety of in�ammatory mediators that play crucial roles in the host defense by microbial killing, but may also cause injury to surrounding tissues [33][34][35]. In order to investigate the ability of pMTX-II, III, and IV to induce the production of superoxide by a macrophage cell line J774A.1, the cells were incubated with non-cytotoxic concentrations of myotoxins. As shown in Figure 5(b), J774A.1 macrophages incubated with RPMI (negative control) showed a superoxide production average of 0.316 ± 0.05 D.O., and J774A.1 incubated with RPMI plus non-opsonized zymosan (positive control) showed a superoxide production average of 0.455 ± 0.1 D.O. Incubation of macrophages with pMTX-II, III, and IV, at 3 and 6 g/mL, respectively, induced a signi�cant production of O 2 − in J774A.1 macrophages, showing that myotoxins are able to induce superoxide production by J774A.1 macrophages, indicating the ability of these toxins to activate these cells. Again, these results suggest that the PLA 2 catalytic activity is not important in macrophage activation. us, in accordance with our results, increments in hydrogen peroxide (H 2 O 2 ), another reactive oxygen specie generated by a multicomponent enzyme system, NADPH-oxidase, have been described in thioglycollate-elicited macrophages incubated with MTX-II and III from Costa Rica B. asper venom [25].
It is important to note that phagocytosis mediated by -glucan receptors and also by mannose and Fc receptors are coupled to the production of both proin�ammatory and microbicidal molecules, such ROS [36,37]. Release of ROS by phagocytic cells has been implicated in microbial killing [38] as well as in the damage to host surrounding tissue [39]. Considering that MT-II and MT-III isolated from B. asper venom from Costa Rica display a broad cytolytic activity and affect a variety of cell types in culture [40,41], our �ndings suggest the role of O 2 − in the cytotoxicity induced by these myotoxins, a hypothesis that can be addressed with the use of antioxidant agents.
In conclusion, the Panamanian B. asper venom has qualitatively a similar toxicological pro�le to those previously described for B. asper from Costa Rica and Guatemala despite the observation of quantitative variations in these activities. e PLA 2 s isolated from Panama Bothrops asper venom (pMTX-I, pMTX-II, pMTX-III, and pMTX-IV) induced myotoxic activity, in�ammatory reaction mainly leukocyte migration to the muscle, activation of macrophages to exert phagocytic activity, and production of superoxide. �on��ct o� �nte�ests e authors declare that there is no con�ict of interests.