toxins Article
Enzymatic and Pro-Inflammatory Activities of Bothrops lanceolatus Venom: Relevance for Envenomation Marie Delafontaine 1 , Isadora Maria Villas-Boas 2 , Laurence Mathieu 1 , Patrice Josset 3 , Joël Blomet 1 and Denise V. Tambourgi 2, * ID 1 2 3
*
Prevor Laboratory, Moulin de Verville, Valmondois 95760, France; [email protected] (M.D.); [email protected] (L.M.); [email protected] (J.B.) Immunochemistry Laboratory, Butantan Institute, São Paulo 05503-900, Brazil; [email protected] Trousseau Hospital, Paris 75012, France; [email protected] Correspondence: [email protected]; Tel.: +55-11-2627-9722
Academic Editor: Syed A. Ali Received: 2 July 2017; Accepted: 31 July 2017; Published: 7 August 2017
Abstract: Bothrops lanceolatus, commonly named ‘Fer-de-Lance’, is an endemic snake of the French Caribbean Island of Martinique. Envenomations by B. lanceolatus present clinical aspects characterized by systemic thrombotic syndrome and important local inflammation, involving edema and pain but limited hemorrhage. To investigate mechanisms of venom-induced inflammation, B. lanceolatus venom was characterized, its cross-reactivity with bothropic antivenom explored, its cytotoxicity on human keratinocytes and vascular cells, and the production of cytokines and chemokines were analyzed. We used electrophoretic separation, zymography, colorimetric or fluorimetric enzymatic assays, and immunochemical assays. Therapeutic South American bothropic antivenom cross-reacted with B. lanceolatus venom and completely or partially abolished its PLA2, hyaluronidase, and proteolytic activities, as well as its cytotoxicity for keratinocytes. The substrate specificity of B. lanceolatus venom proteases was emphasized. B. lanceolatus venom cytotoxicity was compared to the B. jararaca venom. Both venoms were highly cytotoxic for keratinocytes (HaCaT), whereas B. lanceolatus venom showed particularly low toxicity for endothelial cells (EAhy926). Patterns of cytokine and chemokine production by cells exposed to the venoms were highly pro-inflammatory. Thus, the results presented here show that B. lanceolatus venom toxins share important antigenic similarities with South American Bothrops species toxins, although their proteases have acquired particular substrate specificity. Moreover, the venom displays important cytotoxic and pro-inflammatory action on human cell types such as keratinocytes and endothelial cells, which are important players in the local and systemic compartments affected by the envenomation. Keywords: snake venom; Bothrops lanceolatus; toxic activities; antivenom
1. Introduction In South and Central America, Bothrops species account for the majority of venomous ophidian accidents. Envenomation by these snakes has a complex pathophysiology, characterized by prominent local effects (edema, pain, hemorrhage, and necrosis) and systemic effects such as coagulation disturbances, hemorrhage, and renal failure [1,2]. Bothrops lanceolatus, commonly known as ‘Fer-de-lance’, is an endemic species confined to the island of Martinique in the Caribbean, where it is responsible for an average of 30 cases of human envenomation per year [3]. B. lanceolatus venom induces local and systemic effects comparable to bothropic syndrome, but the envenomation is characterized by a predominant prothrombotic profile. Systemic thrombosis development can lead to cerebral,
Toxins 2017, 9, 244; doi:10.3390/toxins9080244
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myocardial, or pulmonary infarctions that only rapid treatment with the monospecific commercial antivenom (Bothrofav® , Sanofi-Pasteur, France) can prevent [2–5]. B. lanceolatus venom contains five major types of enzymes; zinc-dependent snake venom metalloproteases (SVMPs), snake venom serine proteinases (SVSPs), phospholipases A2 (PLA2), L-amino acid oxidases, and a specific C-type lectin-like molecule [6,7]. SVMPs are particularly abundant in Bothrops venoms [8,9]. They all share a highly conserved zinc-dependent active site but vary in their non-catalytic domains composition, an important factor in the classification of these toxins [10]. Class I SVMPs (PI-SVMPs) are only composed of the catalytic protease domain. Class II proteins also present a disintegrin domain, whereas class III SVMPs have both a disintegrin-like and a cysteine-rich domain. In addition to the class III structure, some PIII-SVMPs present two C-type lectin-like domains [11,12]. SVMPs induce hemorrhage, myonecrosis, cutaneous lesions and inflammation, and degradation of coagulation factors and extracellular matrix components. Through their non-catalityc domains, they interfere with platelet functions and cell adhesion molecules [13]. SVSPs have a serine residue in their active site. They were shown to disturb haemostasis by affecting platelet function and degrading coagulation cascade components [14]. PLA2s hydrolyse glycerophospholipids in the sn-2 position. They are present in enzymatically active or inactive forms in Bothrops venoms. They display a wide range of biological effects such as neurotoxicity, cardiotoxicity, myotoxicity, hemolysis, and, anti-coagulating, anti-platelet, and edema-forming activities [15]. Three enzymes have already been purified and characterized from B. lanceolatus venom; a hemorrhagic PI-SVMP, an acidic phospholipase, and a potent fibrinogenolytic enzyme [16,17]. Inflammation induced by B. lanceolatus venom has been described in rat and mouse models [18–21]. B. lanceolatus venom-induced edema is accompanied by local hemorrhage, involves neutrophil infiltration, mast cell degranulation, and the release of arachidonic acid metabolites, bradykinin, histamine, and serotonin in the first hours after inoculation [18,19,21]. Intraperitoneal injection of the venom was followed by intense neutrophil migration, a chemotactic process in which macrophages and lipoxygenase metabolites were involved [20]. Intravenous administration of the specific commercial antivenom shows low efficacy at inhibiting venom-induced rat hind paw edema [19]. The mechanism of thrombosis observed in envenomation by B. lanceolatus has not yet been elucidated. The venom cleaves purified human fibrinogen but is unable to clot citrated human plasma, and almost normal coagulation profile can be observed in patients developing thrombosis, suggesting that the thrombosis mechanism could involve vascular endothelial cells or platelet activation [2]. It is deprived of defibrinating activity in mice [22,23]. The venom thrombotic syndrome observed in cases of human envenomations is not reproducible in mice [6]. Like the monospecific antivenom raised against B. lanceolatus venom, the therapeutic Costa Rican polyvalent antivenom (Clodomiro Picado Institute, Costa Rica) was shown to recognize venom toxic compounds in vitro and to be fully effective in mice in neutralizing the lethal, hemorrhagic, edema-forming, myotoxic, and indirect hemolytic activities of the venom [6,22]. The commercial Brazilian bothropic antivenom is obtained from the hyperimmune sera of horses immunized with a pool of five Brazilian bothropic venoms: Bothrops jararaca (50%), Bothrops alternatus (12.5%), Bothrops jararacussu (12.5%), Bothrops moojeni (12.5%), and Bothrops neuwiedi (12.5%). This antivenom cross reacts with B. lanceolatus venom and its hemorrhagic PI-SVMP, BlaH1, and neutralizes the hemorrhagic activity of this toxin [17]. The main B. lanceolatus venom activities have been studied [7,15–18,22,24]. Here, we extended the characterization of B. lanceolatus venom enzymatic and cytotoxic activities to improve the understanding of the venom-induced local inflammation mechanism. The venom enzymatic activities were confirmed and further investigated. The antivenom cross-reaction and the inhibition of in vitro B. lanceolatus venom enzymatic activities were studied to highlight antigenic similarities between B. lanceolatus and South American Bothrops species venoms. To approach human inflammation mechanisms, we studied B. lanceolatus venom cytotoxicity on human cell lines of keratinocytes and vascular endothelial cells and cytokines/chemokines production upon venom-exposure.
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2. Results 2.1. Immunochemical Characterization and Cross-Reactivity of B. lanceolatus Venom The electrophoretic profile analysis of B. lanceolatus venom in non-reducing conditions reveals the presence of proteins with molecular masses between 10 and 180 kDa (Figure 1(Aa)). In reducing conditions, the venom’s profile consists of three major bands, the molecular masses of which are 70 and 37 kDa (Figure 1(Ab)). The bidimensional electrophoresis of the venom (Figure 1B) revealed that the majority of its components are acidic, with an isoelectric point (pI) between 4 and 7 and with only one isoform. Some basic proteins were revealed with Mr between 15 and 19 kDa. A protein with a molecular mass of 30 kDa presented three isoforms, with an pI between 6.6 and 6.9. The lectin western blot analysis shows that several proteins contain, N-acetyl-D-glucosamine and/or sialic acid and terminal α-D-mannosyl and/or α-D-glucosyl groups, as determined using WGA and Con A, respectively (Figure 1C,D). The cross-reactivity of the venom with bothropic antivenom was validated by two methods; western blotting and ELISA, using B. jararaca venom as the positive control, with botulinum toxin antiserum as the negative control. Figure 1E shows a strong recognition of several components of B. lanceolatus venom by the bothropic antivenom, mainly proteins with Mr between 26 and 70 kDa. By ELISA, the ability of the bothropic antivenom to recognize B. lanceolatus venom was confirmed with a 1:320,000 antibody titer (Figure 1F). The control antiserum did not recognize any of the venom components, neither by western blot (data not shown) nor by ELISA (Figure 1F). 2.2. Enzymatic Activities of B. lanceolatus Venom The hyaluronidase activity of the venom was determined using hyaluronic acid as the substrate and B. jararaca venom as the positive control. The specific hyaluronidase activity of B. lanceolatus venom was 20.5 ± 2.4 TRU/mg, which was about five times lower than the B. jararaca venom activity in the same conditions (Table 1). A fluorimetric assay was used to study the presence of PLA2 activity in the venom, using B. jararaca and C. durissus terrificus venoms as positive controls. The specific PLA2 activity of B. lanceolatus venom reached 231.3 ± 6.4 UF/min/µg, which was significantly higher than that of B. jararaca venom but lower than Crotalus venom (Table 1). B. lanceolatus venom (25 µg) was submitted to gelatin and fibrinogen zymography. Figure 2A exhibits the presence of high molecular mass gelatinases (Mr > 115 kDa), as well as smaller ones, between 64 and 85 kDa. Several proteins are able to cleave fibrinogen with molecular masses between 20 and 180 kDa (Figure 2B). However, a band of about 30 kDa presents higher activity, compared with the other proteins. The cleavage of fibrinogen was also investigated by incubating samples of fibrinogen with the venom and submitting them to SDS-PAGE analysis in reducing conditions. α and β chains of fibrinogen were completely cleaved in the presence of 0.5 µg of venom (Figure 2C). EDTA and PMSF completely prevented β chain degradation and showed partial inhibition of α chain cleavage, whereas 1,10-phenanthroline abolished both α and β chain cleavage. Table 1. Hyaluronidase and phospholipase activities of B. lanceolatus venom; specific activities and neutralization by bothropic antivenom. Enzymatic Activities Hyaluronidase Specific activity (TRU/mg) Amount of bothropic antivenom (mL/mg of venom) required to inhibit activity by 95% Phospholipase Specific activity (UF/min/µg) Amount of bothropic antivenom (mL/mg of venom) required to inhibit activity by 95%
C. durissus terrificus
B. jararaca
B. lanceolatus
NT
116.6 ± 8.6
20.5 ± 2.4 *
NT
0.056
≤0.056
461.4 ± 11.7
121.9 ± 6.9 #
231.3 ± 6.4 *
NT
0.32
2.29
#
NT: not tested; * p < 0.05 compared to B. jararaca venom; p < 0.05 compared to C. d. terrificus venom.
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Figure 1. Uni‐ and bi‐dimensional electrophoretic analysis of B. lanceolatus venom, its cross‐reaction Figure 1. Uniand bi-dimensional electrophoretic analysis of B. lanceolatus venom, its cross-reaction with bothropic antivenom, and the presence of glycosylated proteins in the venom. (A,C,D) Samples with bothropic antivenom, and the presence of glycosylated proteins in the venom. (A,C,D) Samples of of B. lanceolatus venom were submitted to electrophoretic SDS‐PAGE separation (12% of acrylamide) B. lanceolatus venom were submitted to electrophoretic SDS-PAGE separation (12% of acrylamide) in in non‐reducing (a) and reducing (b) conditions. (A) The gel on which 30 μg of venom was separated was stained with Coomassie Blue R‐250. (B) Strips with pH gradient (3 to 10) were rehydrated with non-reducing (a) and reducing (b) conditions. (A) The gel on which 30 µg of venom was separated was stainedbuffer containing B. lanceolatus venom (100 μg) before isofocalization. After washing with reducing with Coomassie Blue R-250. (B) Strips with pH gradient (3 to 10) were rehydrated and chelating buffers, the focalized strips were submitted to SDS‐PAGE electrophoresis (12% of with bufferacrylamide). The resulting gels were silver stained. (C,D) Venom samples (15 μg) were separated by containing B. lanceolatus venom (100 µg) before isofocalization. After washing with reducing and chelating buffers, the focalized strips were submitted to SDS-PAGE electrophoresis electrophoresis and electrotransferred to nitrocellulose membranes. The membranes were incubated with the peroxidase‐conjugated lectins, WGA and Con A. Recognized bands were visualized with (12% of acrylamide). The resulting gels were silver stained. (C,D) Venom samples (15 µg) were separated by electrophoresis and electrotransferred to nitrocellulose membranes. The membranes were incubated with the peroxidase-conjugated lectins, WGA and Con A. Recognized bands were visualized with DAB. (E) After electrophoresis in non-reducing conditions, samples (5 µg) of B. jararaca (Bj) and B. lanceolatus (Bl) venoms were electrotransferred to nitrocellulose membranes and incubated with bothropic antivenom diluted 1:10,000 followed by GAH/IgG-AP (1:7500). Cross-reacting bands were visualized with NBT-BCIP. (F) ELISA plates were coated with B. lanceolatus venom (1 µg/well). They were incubated with serial dilutions of bothropic antivenom or botulinum toxin antiserum, as negative control, followed by GAH/IgG-AP (1:3000). The results showed are representative of two experiments, realized in duplicates. The titer was determined as the highest antivenom dilution, which produced an absorbance eight times greater than the absorbance determined for the control serum. This absorbance value is represented by a dotted line.
were incubated with serial dilutions of bothropic antivenom or botulinum toxin antiserum, as negative control, followed by GAH/IgG‐AP (1:3000). The results showed are representative of two experiments, realized in duplicates. The titer was determined as the highest antivenom dilution, which produced an absorbance eight times greater than the absorbance determined for the control Toxinsserum. This absorbance value is represented by a dotted line. 2017, 9, 244 5 of 19
Figure 2. 2. Gelatinolytic Gelatinolyticand andfibrinogenolytic fibrinogenolyticactivities activitiesof ofB. B. lanceolatus venom. (A,B) Samples of Figure lanceolatus venom. (A,B) Samples of B. B. lanceolatus venom (25 µg) were separated by SDS-PAGE in 12% acrylamide gels containing (A) 10% lanceolatus venom (25 μg) were separated by SDS‐PAGE in 12% acrylamide gels containing (A) 10% gelatin or (B) 10% fibrinogen under non-reducing conditions at 4 ◦ C. The gels were then incubated gelatin or (B) 10% fibrinogen under non‐reducing conditions at 4 °C. The gels were then incubated overnight at 37 ◦ C in substrate buffer (pH 8.3) and stained with Coomassie Blue R250. (C) Samples of overnight at 37 °C in substrate buffer (pH 8.3) and stained with Coomassie Blue R250. (C) Samples of fibrinogen (30 µg) were incubated with B. lanceolatus venom (0.5 µg) for 1 h at 37 ◦ C in the absence fibrinogen (30 μg) were incubated with B. lanceolatus venom (0.5 μg) for 1 h at 37 °C in the absence or or presence of proteases inhibitors (20 mM), EDTA, 1,10-phenanthroline (Phen), or PMSF. Samples presence of proteases inhibitors (20 mM), EDTA, 1,10‐phenanthroline (Phen), or PMSF. Samples were were then separated by SDS-PAGE electrophoresis in reducing conditions Coomassie Blue then separated by SDS‐PAGE electrophoresis in reducing conditions before before Coomassie Blue R250 R250 staining. staining.
Finally, Finally, the the proteolytic proteolytic activity activity of of B. B. lanceolatus lanceolatus venom venom upon upon FRET FRET peptidic peptidic substrates substrates was was investigated. In linear kinetics conditions (excess of substrate), the specific activities of B. lanceolatus investigated. In linear kinetics conditions (excess of substrate), the specific activities of B. lanceolatus venom on the peptides Abz‐FRSSRQ and Abz‐RPPGFSPFRQ were of the values 111.0 ± 4.7 and 78.3 venom on the peptides Abz-FRSSRQ and Abz-RPPGFSPFRQ were of the values 111.0 ± 4.7 and ± 9.6 UF/min/μg, respectively (Table 2). Upon pre‐incubation with inhibitors, those activities could 78.3 ± 9.6 UF/min/µg, respectively (Table 2). Upon pre-incubation with inhibitors, those activities be partially or completely abolished. Cleavage of the peptide substrate Abz‐FRSSRQ could be fully could be partially or completely abolished. Cleavage of the peptide substrate Abz-FRSSRQ could be inhibited by the whereas full inhibition of ofAbz‐RPPGFSPFRQ fully inhibited byuse the of usePMSF, of PMSF, whereas full inhibition Abz-RPPGFSPFRQcleavage cleavagecould could be be achieved with EDTA and 1,10‐phenanthroline. achieved with EDTA and 1,10-phenanthroline. We tested the ability of Brazilian bothropic antivenom to neutralize the hyaluronidase, PLA2, Table 2. Proteolytic of The B. lanceolatus venom on FRET peptidic substrates. and proteolytic activities of the activity venom. IC95 for B. jararaca venom was first determined for hyaluronidase and PLA2 activities and then tested on B. lanceolatus venom. This concentration was Fluorescent Peptides sufficient to completely inhibit B. lanceolatus hyaluronidase activity. On the contrary, IC95 for B. Abz-FRSSRQ Abz-RPPGFSPFRQ lanceolatus PLA2 activity was found to be about seven times higher than IC95 for B. jararaca venom (Table 1). No complete inhibition of B. lanceolatus venom proteolytic activity could be reached in the Venom activity (UF/min/µg) 111.0 ± 4.7 78.3 ± 9.6 fluorimetric assay (Table 2). EDTA (100 mM) 0.8 ± 1.7 94.1 ± 6.8 ** 1,10-phenanthroline (5 mM) 4.9 ± 4.6 100 ± 0 * Inhibition (%) PMSF (5 mM)
93.5 ± 4.5 *
79.5 ± 13.8 *
Bothropic antivenom
33.5 ± 33.6 *
60.8 ± 21.2 *
Venom samples (50 µg/mL for Abz-RPPGFSPFRQ substrate; 25 µg/mL for Abz-FRSSRQ substrate) were incubated with inhibitors or bothropic antivenom (200 µL/mL) for 30 min at room temperature. Then substrate (5 µM) was added to the wells and the venom activity was measured at 37 ◦ C for 15 min by spectrophotometry. The data represent the mean ± SD (n = three assays, each done in duplicate for the inhibitors and n = six assays for the neutralization experiments). * p < 0.05 compared to the activity of venom alone.
We tested the ability of Brazilian bothropic antivenom to neutralize the hyaluronidase, PLA2, and proteolytic activities of the venom. The IC95 for B. jararaca venom was first determined for hyaluronidase and PLA2 activities and then tested on B. lanceolatus venom. This concentration was sufficient to completely inhibit B. lanceolatus hyaluronidase activity. On the contrary, IC95 for
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B. lanceolatus PLA2 activity was found to be about seven times higher than IC95 for B. jararaca venom (Table 1). No complete inhibition of B. lanceolatus venom proteolytic activity could be reached in the fluorimetric assay (Table 2). 2.3. Cytotoxicity of the Venom for Human Cells Incubation of human keratinocytes HaCaT with B. lanceolatus venom induced a loss of more than 95% of cell viability for concentrations above 2.7 µg/mL during 24 h (Figure 3A). 50% of viability was reached with the concentration of 2.5 µg/mL (CI 95: 2.3–2.7 µg/mL; Figure 3C). After 48 and 72 h of incubation, a 50% loss of viability was reached for approximately 1.7 µg/mL (CI 95: 1.6–1.9 µg/mL) and 1.5 µg/mL (CI 95: 0.9–2.0 µg/mL), respectively. After 24 h of incubation, B. jararaca venom presented an IC50 of 2.9 µg/mL (CI 95: 1.7–4.1 µg/mL; Figure 3A,C), which was not significantly different from B. lanceolatus venom IC50. The two Bothrops venoms presented a significative difference of cytotoxic potential for endothelial vascular cells EAhy926 (Figure 3B,D). After 24 h of exposure, concentrations of B. jararaca venom above 4 µg/mL induced at least 90% of viability loss, whereas the same effect was observed with a minimum of 125 µg/mL of B. lanceolatus venom (Figure 3B). The calculated IC50 were 0.8 µg/mL (CI 95: 0.5–1.0 µg/mL) and 51.7 µg/mL (CI 95: 0–107.2 µg/mL), for B. jararaca and B. lanceolatus venom, respectively (Figure 3D). After 48 h of exposure to B. lanceolatus venom; the IC50 was 42.4 µg/mL (CI 95: 0–97.5 µg/mL), and, after 72 h, it was 17.6 µg/mL (CI 95: 0–51.2 µg/mL). Neutralization of the cytotoxicity of B. lanceolatus venom for the two cell lineages by bothropic antivenom was tested by preincubating venom with antivenom. B. jararaca venom was used as the positive control, and botulinum toxin antiserum was used as the negative control. The concentrations of venom were set so as to obtain about 100% loss of viability within 24 h. Figure 3E shows the complete inhibition of both Bothrops venoms’ cytotoxicity in keratinocytes by the bothropic antivenom, using 1:20 antivenom dilution. However, no inhibition of the venoms’ cytotoxicity was observed for endothelial vascular cells, using antivenom dilution up to 1:5 (Figure 3F). The negative control showed no interference of non-specific antibodies with the assay. 2.4. Inflammatory Potential of the Venom for Human Keratinocytes and Vascular Endothelial Cells The effect of venom on human keratinocytes and endothelial vascular cells was further investigated by measuring the production of cytokines and chemokines by venom-treated cells in conditions of partial loss of viability. In the supernatant of venom-exposed keratinocytes and endothelial vascular cells, four cytokines and chemokines were detected: IL-8, MCP-1, RANTES, and IL-6, the latter observed only in EAhy926 supernatants (Figure 4). IP-10, MIG, IL-12p70, TNF-α, IL-10, IL-1β, IL-17a, IFN-γ, IL-4, and IL-2 were not detected in any of the culture supernatants (data not shown). Concentrations of IL-8, MCP-1, and RANTES were stable along the time in the supernatants of DMEM-treated keratinocytes (Figure 4A,C,E). From 48 h of incubation, concentrations of IL-8, MCP-1, and RANTES increased significantly in the supernatants of venom-treated keratinocytes when compared to DMEM-treated cells. Concentrations of the three proteins increased between 24 to 48 h of incubation with the venom. IL-8 and MCP-1 concentrations stayed stable between 48 and 72 h of incubation, whereas RANTES concentrations decreased in this period of time (Figure 4A,C,E).
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to 48 h of incubation with the venom. IL‐8 and MCP‐1 concentrations stayed stable between 48 and 7 of 19 72 h of incubation, whereas RANTES concentrations decreased in this period of time (Figure 4A,C,E).
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Figure 3.3. Cytotoxicity ofof B. lanceolatus venom on human keratinocytes HaCaTHaCaT and endothelial vascular Figure Cytotoxicity B. lanceolatus venom on human keratinocytes and endothelial cells EAhy926 and its inhibition by bothropic antivenom. (A–D) After being subcultured in 96-well vascular cells EAhy926 and its inhibition by bothropic antivenom. (A–D) After being subcultured in plates in DMEM medium, HaCaT and EAhy926 cells were exposed to different concentrations of 96‐well plates in DMEM medium, HaCaT and EAhy926 cells were exposed to different concentrations Bothrops venoms or only with medium as control. Cellular viability was assessed after 24, 48, and of Bothrops venoms or only with medium as control. Cellular viability was assessed after 24, 48, and 72 h of incubation by MTT assay. The percentage of viability and IC50 were calculated using software 72 h of incubation by MTT assay. The percentage of viability and IC50 were calculated using software GraphPad Prism. The experiments were done three times, in triplicate. (A,B) are representatives of one GraphPad Prism. The experiments were done three times, in triplicate. (A,B) are representatives of experiment. (C,D) represent the IC50 values, calculated as the means of the three assays for each time one experiment. (C,D) represent the IC50 values, calculated as the means of the three assays for each interval, using venom concentrations represented in (A,B). (E,F) Venom samples were pre-incubated time interval, using venom concentrations represented in (A,B). (E,F) Venom samples were pre‐ with bothropic antivenom for 30 min at room temperature. Cells were exposed to these samples for incubated with bothropic antivenom for 30 min at room temperature. Cells were exposed to these 24 h before cell viability was assessed by MTT assay. Data are represented as average ± standard samples for 24 h before cell viability was assessed by MTT assay. Data are represented as average ± error. The columns represent the mean ± SD (n = 3 each). NS: not significant. * p < 0.05 compared to standard error. The columns represent the mean ± SD (n = 3 each). NS: not significant. * p
Enzymatic and Pro-Inflammatory Activities of Bothrops lanceolatus Venom: Relevance for Envenomation Marie Delafontaine 1 , Isadora Maria Villas-Boas 2 , Laurence Mathieu 1 , Patrice Josset 3 , Joël Blomet 1 and Denise V. Tambourgi 2, * ID 1 2 3
*
Prevor Laboratory, Moulin de Verville, Valmondois 95760, France; [email protected] (M.D.); [email protected] (L.M.); [email protected] (J.B.) Immunochemistry Laboratory, Butantan Institute, São Paulo 05503-900, Brazil; [email protected] Trousseau Hospital, Paris 75012, France; [email protected] Correspondence: [email protected]; Tel.: +55-11-2627-9722
Academic Editor: Syed A. Ali Received: 2 July 2017; Accepted: 31 July 2017; Published: 7 August 2017
Abstract: Bothrops lanceolatus, commonly named ‘Fer-de-Lance’, is an endemic snake of the French Caribbean Island of Martinique. Envenomations by B. lanceolatus present clinical aspects characterized by systemic thrombotic syndrome and important local inflammation, involving edema and pain but limited hemorrhage. To investigate mechanisms of venom-induced inflammation, B. lanceolatus venom was characterized, its cross-reactivity with bothropic antivenom explored, its cytotoxicity on human keratinocytes and vascular cells, and the production of cytokines and chemokines were analyzed. We used electrophoretic separation, zymography, colorimetric or fluorimetric enzymatic assays, and immunochemical assays. Therapeutic South American bothropic antivenom cross-reacted with B. lanceolatus venom and completely or partially abolished its PLA2, hyaluronidase, and proteolytic activities, as well as its cytotoxicity for keratinocytes. The substrate specificity of B. lanceolatus venom proteases was emphasized. B. lanceolatus venom cytotoxicity was compared to the B. jararaca venom. Both venoms were highly cytotoxic for keratinocytes (HaCaT), whereas B. lanceolatus venom showed particularly low toxicity for endothelial cells (EAhy926). Patterns of cytokine and chemokine production by cells exposed to the venoms were highly pro-inflammatory. Thus, the results presented here show that B. lanceolatus venom toxins share important antigenic similarities with South American Bothrops species toxins, although their proteases have acquired particular substrate specificity. Moreover, the venom displays important cytotoxic and pro-inflammatory action on human cell types such as keratinocytes and endothelial cells, which are important players in the local and systemic compartments affected by the envenomation. Keywords: snake venom; Bothrops lanceolatus; toxic activities; antivenom
1. Introduction In South and Central America, Bothrops species account for the majority of venomous ophidian accidents. Envenomation by these snakes has a complex pathophysiology, characterized by prominent local effects (edema, pain, hemorrhage, and necrosis) and systemic effects such as coagulation disturbances, hemorrhage, and renal failure [1,2]. Bothrops lanceolatus, commonly known as ‘Fer-de-lance’, is an endemic species confined to the island of Martinique in the Caribbean, where it is responsible for an average of 30 cases of human envenomation per year [3]. B. lanceolatus venom induces local and systemic effects comparable to bothropic syndrome, but the envenomation is characterized by a predominant prothrombotic profile. Systemic thrombosis development can lead to cerebral,
Toxins 2017, 9, 244; doi:10.3390/toxins9080244
www.mdpi.com/journal/toxins
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myocardial, or pulmonary infarctions that only rapid treatment with the monospecific commercial antivenom (Bothrofav® , Sanofi-Pasteur, France) can prevent [2–5]. B. lanceolatus venom contains five major types of enzymes; zinc-dependent snake venom metalloproteases (SVMPs), snake venom serine proteinases (SVSPs), phospholipases A2 (PLA2), L-amino acid oxidases, and a specific C-type lectin-like molecule [6,7]. SVMPs are particularly abundant in Bothrops venoms [8,9]. They all share a highly conserved zinc-dependent active site but vary in their non-catalytic domains composition, an important factor in the classification of these toxins [10]. Class I SVMPs (PI-SVMPs) are only composed of the catalytic protease domain. Class II proteins also present a disintegrin domain, whereas class III SVMPs have both a disintegrin-like and a cysteine-rich domain. In addition to the class III structure, some PIII-SVMPs present two C-type lectin-like domains [11,12]. SVMPs induce hemorrhage, myonecrosis, cutaneous lesions and inflammation, and degradation of coagulation factors and extracellular matrix components. Through their non-catalityc domains, they interfere with platelet functions and cell adhesion molecules [13]. SVSPs have a serine residue in their active site. They were shown to disturb haemostasis by affecting platelet function and degrading coagulation cascade components [14]. PLA2s hydrolyse glycerophospholipids in the sn-2 position. They are present in enzymatically active or inactive forms in Bothrops venoms. They display a wide range of biological effects such as neurotoxicity, cardiotoxicity, myotoxicity, hemolysis, and, anti-coagulating, anti-platelet, and edema-forming activities [15]. Three enzymes have already been purified and characterized from B. lanceolatus venom; a hemorrhagic PI-SVMP, an acidic phospholipase, and a potent fibrinogenolytic enzyme [16,17]. Inflammation induced by B. lanceolatus venom has been described in rat and mouse models [18–21]. B. lanceolatus venom-induced edema is accompanied by local hemorrhage, involves neutrophil infiltration, mast cell degranulation, and the release of arachidonic acid metabolites, bradykinin, histamine, and serotonin in the first hours after inoculation [18,19,21]. Intraperitoneal injection of the venom was followed by intense neutrophil migration, a chemotactic process in which macrophages and lipoxygenase metabolites were involved [20]. Intravenous administration of the specific commercial antivenom shows low efficacy at inhibiting venom-induced rat hind paw edema [19]. The mechanism of thrombosis observed in envenomation by B. lanceolatus has not yet been elucidated. The venom cleaves purified human fibrinogen but is unable to clot citrated human plasma, and almost normal coagulation profile can be observed in patients developing thrombosis, suggesting that the thrombosis mechanism could involve vascular endothelial cells or platelet activation [2]. It is deprived of defibrinating activity in mice [22,23]. The venom thrombotic syndrome observed in cases of human envenomations is not reproducible in mice [6]. Like the monospecific antivenom raised against B. lanceolatus venom, the therapeutic Costa Rican polyvalent antivenom (Clodomiro Picado Institute, Costa Rica) was shown to recognize venom toxic compounds in vitro and to be fully effective in mice in neutralizing the lethal, hemorrhagic, edema-forming, myotoxic, and indirect hemolytic activities of the venom [6,22]. The commercial Brazilian bothropic antivenom is obtained from the hyperimmune sera of horses immunized with a pool of five Brazilian bothropic venoms: Bothrops jararaca (50%), Bothrops alternatus (12.5%), Bothrops jararacussu (12.5%), Bothrops moojeni (12.5%), and Bothrops neuwiedi (12.5%). This antivenom cross reacts with B. lanceolatus venom and its hemorrhagic PI-SVMP, BlaH1, and neutralizes the hemorrhagic activity of this toxin [17]. The main B. lanceolatus venom activities have been studied [7,15–18,22,24]. Here, we extended the characterization of B. lanceolatus venom enzymatic and cytotoxic activities to improve the understanding of the venom-induced local inflammation mechanism. The venom enzymatic activities were confirmed and further investigated. The antivenom cross-reaction and the inhibition of in vitro B. lanceolatus venom enzymatic activities were studied to highlight antigenic similarities between B. lanceolatus and South American Bothrops species venoms. To approach human inflammation mechanisms, we studied B. lanceolatus venom cytotoxicity on human cell lines of keratinocytes and vascular endothelial cells and cytokines/chemokines production upon venom-exposure.
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2. Results 2.1. Immunochemical Characterization and Cross-Reactivity of B. lanceolatus Venom The electrophoretic profile analysis of B. lanceolatus venom in non-reducing conditions reveals the presence of proteins with molecular masses between 10 and 180 kDa (Figure 1(Aa)). In reducing conditions, the venom’s profile consists of three major bands, the molecular masses of which are 70 and 37 kDa (Figure 1(Ab)). The bidimensional electrophoresis of the venom (Figure 1B) revealed that the majority of its components are acidic, with an isoelectric point (pI) between 4 and 7 and with only one isoform. Some basic proteins were revealed with Mr between 15 and 19 kDa. A protein with a molecular mass of 30 kDa presented three isoforms, with an pI between 6.6 and 6.9. The lectin western blot analysis shows that several proteins contain, N-acetyl-D-glucosamine and/or sialic acid and terminal α-D-mannosyl and/or α-D-glucosyl groups, as determined using WGA and Con A, respectively (Figure 1C,D). The cross-reactivity of the venom with bothropic antivenom was validated by two methods; western blotting and ELISA, using B. jararaca venom as the positive control, with botulinum toxin antiserum as the negative control. Figure 1E shows a strong recognition of several components of B. lanceolatus venom by the bothropic antivenom, mainly proteins with Mr between 26 and 70 kDa. By ELISA, the ability of the bothropic antivenom to recognize B. lanceolatus venom was confirmed with a 1:320,000 antibody titer (Figure 1F). The control antiserum did not recognize any of the venom components, neither by western blot (data not shown) nor by ELISA (Figure 1F). 2.2. Enzymatic Activities of B. lanceolatus Venom The hyaluronidase activity of the venom was determined using hyaluronic acid as the substrate and B. jararaca venom as the positive control. The specific hyaluronidase activity of B. lanceolatus venom was 20.5 ± 2.4 TRU/mg, which was about five times lower than the B. jararaca venom activity in the same conditions (Table 1). A fluorimetric assay was used to study the presence of PLA2 activity in the venom, using B. jararaca and C. durissus terrificus venoms as positive controls. The specific PLA2 activity of B. lanceolatus venom reached 231.3 ± 6.4 UF/min/µg, which was significantly higher than that of B. jararaca venom but lower than Crotalus venom (Table 1). B. lanceolatus venom (25 µg) was submitted to gelatin and fibrinogen zymography. Figure 2A exhibits the presence of high molecular mass gelatinases (Mr > 115 kDa), as well as smaller ones, between 64 and 85 kDa. Several proteins are able to cleave fibrinogen with molecular masses between 20 and 180 kDa (Figure 2B). However, a band of about 30 kDa presents higher activity, compared with the other proteins. The cleavage of fibrinogen was also investigated by incubating samples of fibrinogen with the venom and submitting them to SDS-PAGE analysis in reducing conditions. α and β chains of fibrinogen were completely cleaved in the presence of 0.5 µg of venom (Figure 2C). EDTA and PMSF completely prevented β chain degradation and showed partial inhibition of α chain cleavage, whereas 1,10-phenanthroline abolished both α and β chain cleavage. Table 1. Hyaluronidase and phospholipase activities of B. lanceolatus venom; specific activities and neutralization by bothropic antivenom. Enzymatic Activities Hyaluronidase Specific activity (TRU/mg) Amount of bothropic antivenom (mL/mg of venom) required to inhibit activity by 95% Phospholipase Specific activity (UF/min/µg) Amount of bothropic antivenom (mL/mg of venom) required to inhibit activity by 95%
C. durissus terrificus
B. jararaca
B. lanceolatus
NT
116.6 ± 8.6
20.5 ± 2.4 *
NT
0.056
≤0.056
461.4 ± 11.7
121.9 ± 6.9 #
231.3 ± 6.4 *
NT
0.32
2.29
#
NT: not tested; * p < 0.05 compared to B. jararaca venom; p < 0.05 compared to C. d. terrificus venom.
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Figure 1. Uni‐ and bi‐dimensional electrophoretic analysis of B. lanceolatus venom, its cross‐reaction Figure 1. Uniand bi-dimensional electrophoretic analysis of B. lanceolatus venom, its cross-reaction with bothropic antivenom, and the presence of glycosylated proteins in the venom. (A,C,D) Samples with bothropic antivenom, and the presence of glycosylated proteins in the venom. (A,C,D) Samples of of B. lanceolatus venom were submitted to electrophoretic SDS‐PAGE separation (12% of acrylamide) B. lanceolatus venom were submitted to electrophoretic SDS-PAGE separation (12% of acrylamide) in in non‐reducing (a) and reducing (b) conditions. (A) The gel on which 30 μg of venom was separated was stained with Coomassie Blue R‐250. (B) Strips with pH gradient (3 to 10) were rehydrated with non-reducing (a) and reducing (b) conditions. (A) The gel on which 30 µg of venom was separated was stainedbuffer containing B. lanceolatus venom (100 μg) before isofocalization. After washing with reducing with Coomassie Blue R-250. (B) Strips with pH gradient (3 to 10) were rehydrated and chelating buffers, the focalized strips were submitted to SDS‐PAGE electrophoresis (12% of with bufferacrylamide). The resulting gels were silver stained. (C,D) Venom samples (15 μg) were separated by containing B. lanceolatus venom (100 µg) before isofocalization. After washing with reducing and chelating buffers, the focalized strips were submitted to SDS-PAGE electrophoresis electrophoresis and electrotransferred to nitrocellulose membranes. The membranes were incubated with the peroxidase‐conjugated lectins, WGA and Con A. Recognized bands were visualized with (12% of acrylamide). The resulting gels were silver stained. (C,D) Venom samples (15 µg) were separated by electrophoresis and electrotransferred to nitrocellulose membranes. The membranes were incubated with the peroxidase-conjugated lectins, WGA and Con A. Recognized bands were visualized with DAB. (E) After electrophoresis in non-reducing conditions, samples (5 µg) of B. jararaca (Bj) and B. lanceolatus (Bl) venoms were electrotransferred to nitrocellulose membranes and incubated with bothropic antivenom diluted 1:10,000 followed by GAH/IgG-AP (1:7500). Cross-reacting bands were visualized with NBT-BCIP. (F) ELISA plates were coated with B. lanceolatus venom (1 µg/well). They were incubated with serial dilutions of bothropic antivenom or botulinum toxin antiserum, as negative control, followed by GAH/IgG-AP (1:3000). The results showed are representative of two experiments, realized in duplicates. The titer was determined as the highest antivenom dilution, which produced an absorbance eight times greater than the absorbance determined for the control serum. This absorbance value is represented by a dotted line.
were incubated with serial dilutions of bothropic antivenom or botulinum toxin antiserum, as negative control, followed by GAH/IgG‐AP (1:3000). The results showed are representative of two experiments, realized in duplicates. The titer was determined as the highest antivenom dilution, which produced an absorbance eight times greater than the absorbance determined for the control Toxinsserum. This absorbance value is represented by a dotted line. 2017, 9, 244 5 of 19
Figure 2. 2. Gelatinolytic Gelatinolyticand andfibrinogenolytic fibrinogenolyticactivities activitiesof ofB. B. lanceolatus venom. (A,B) Samples of Figure lanceolatus venom. (A,B) Samples of B. B. lanceolatus venom (25 µg) were separated by SDS-PAGE in 12% acrylamide gels containing (A) 10% lanceolatus venom (25 μg) were separated by SDS‐PAGE in 12% acrylamide gels containing (A) 10% gelatin or (B) 10% fibrinogen under non-reducing conditions at 4 ◦ C. The gels were then incubated gelatin or (B) 10% fibrinogen under non‐reducing conditions at 4 °C. The gels were then incubated overnight at 37 ◦ C in substrate buffer (pH 8.3) and stained with Coomassie Blue R250. (C) Samples of overnight at 37 °C in substrate buffer (pH 8.3) and stained with Coomassie Blue R250. (C) Samples of fibrinogen (30 µg) were incubated with B. lanceolatus venom (0.5 µg) for 1 h at 37 ◦ C in the absence fibrinogen (30 μg) were incubated with B. lanceolatus venom (0.5 μg) for 1 h at 37 °C in the absence or or presence of proteases inhibitors (20 mM), EDTA, 1,10-phenanthroline (Phen), or PMSF. Samples presence of proteases inhibitors (20 mM), EDTA, 1,10‐phenanthroline (Phen), or PMSF. Samples were were then separated by SDS-PAGE electrophoresis in reducing conditions Coomassie Blue then separated by SDS‐PAGE electrophoresis in reducing conditions before before Coomassie Blue R250 R250 staining. staining.
Finally, Finally, the the proteolytic proteolytic activity activity of of B. B. lanceolatus lanceolatus venom venom upon upon FRET FRET peptidic peptidic substrates substrates was was investigated. In linear kinetics conditions (excess of substrate), the specific activities of B. lanceolatus investigated. In linear kinetics conditions (excess of substrate), the specific activities of B. lanceolatus venom on the peptides Abz‐FRSSRQ and Abz‐RPPGFSPFRQ were of the values 111.0 ± 4.7 and 78.3 venom on the peptides Abz-FRSSRQ and Abz-RPPGFSPFRQ were of the values 111.0 ± 4.7 and ± 9.6 UF/min/μg, respectively (Table 2). Upon pre‐incubation with inhibitors, those activities could 78.3 ± 9.6 UF/min/µg, respectively (Table 2). Upon pre-incubation with inhibitors, those activities be partially or completely abolished. Cleavage of the peptide substrate Abz‐FRSSRQ could be fully could be partially or completely abolished. Cleavage of the peptide substrate Abz-FRSSRQ could be inhibited by the whereas full inhibition of ofAbz‐RPPGFSPFRQ fully inhibited byuse the of usePMSF, of PMSF, whereas full inhibition Abz-RPPGFSPFRQcleavage cleavagecould could be be achieved with EDTA and 1,10‐phenanthroline. achieved with EDTA and 1,10-phenanthroline. We tested the ability of Brazilian bothropic antivenom to neutralize the hyaluronidase, PLA2, Table 2. Proteolytic of The B. lanceolatus venom on FRET peptidic substrates. and proteolytic activities of the activity venom. IC95 for B. jararaca venom was first determined for hyaluronidase and PLA2 activities and then tested on B. lanceolatus venom. This concentration was Fluorescent Peptides sufficient to completely inhibit B. lanceolatus hyaluronidase activity. On the contrary, IC95 for B. Abz-FRSSRQ Abz-RPPGFSPFRQ lanceolatus PLA2 activity was found to be about seven times higher than IC95 for B. jararaca venom (Table 1). No complete inhibition of B. lanceolatus venom proteolytic activity could be reached in the Venom activity (UF/min/µg) 111.0 ± 4.7 78.3 ± 9.6 fluorimetric assay (Table 2). EDTA (100 mM) 0.8 ± 1.7 94.1 ± 6.8 ** 1,10-phenanthroline (5 mM) 4.9 ± 4.6 100 ± 0 * Inhibition (%) PMSF (5 mM)
93.5 ± 4.5 *
79.5 ± 13.8 *
Bothropic antivenom
33.5 ± 33.6 *
60.8 ± 21.2 *
Venom samples (50 µg/mL for Abz-RPPGFSPFRQ substrate; 25 µg/mL for Abz-FRSSRQ substrate) were incubated with inhibitors or bothropic antivenom (200 µL/mL) for 30 min at room temperature. Then substrate (5 µM) was added to the wells and the venom activity was measured at 37 ◦ C for 15 min by spectrophotometry. The data represent the mean ± SD (n = three assays, each done in duplicate for the inhibitors and n = six assays for the neutralization experiments). * p < 0.05 compared to the activity of venom alone.
We tested the ability of Brazilian bothropic antivenom to neutralize the hyaluronidase, PLA2, and proteolytic activities of the venom. The IC95 for B. jararaca venom was first determined for hyaluronidase and PLA2 activities and then tested on B. lanceolatus venom. This concentration was sufficient to completely inhibit B. lanceolatus hyaluronidase activity. On the contrary, IC95 for
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B. lanceolatus PLA2 activity was found to be about seven times higher than IC95 for B. jararaca venom (Table 1). No complete inhibition of B. lanceolatus venom proteolytic activity could be reached in the fluorimetric assay (Table 2). 2.3. Cytotoxicity of the Venom for Human Cells Incubation of human keratinocytes HaCaT with B. lanceolatus venom induced a loss of more than 95% of cell viability for concentrations above 2.7 µg/mL during 24 h (Figure 3A). 50% of viability was reached with the concentration of 2.5 µg/mL (CI 95: 2.3–2.7 µg/mL; Figure 3C). After 48 and 72 h of incubation, a 50% loss of viability was reached for approximately 1.7 µg/mL (CI 95: 1.6–1.9 µg/mL) and 1.5 µg/mL (CI 95: 0.9–2.0 µg/mL), respectively. After 24 h of incubation, B. jararaca venom presented an IC50 of 2.9 µg/mL (CI 95: 1.7–4.1 µg/mL; Figure 3A,C), which was not significantly different from B. lanceolatus venom IC50. The two Bothrops venoms presented a significative difference of cytotoxic potential for endothelial vascular cells EAhy926 (Figure 3B,D). After 24 h of exposure, concentrations of B. jararaca venom above 4 µg/mL induced at least 90% of viability loss, whereas the same effect was observed with a minimum of 125 µg/mL of B. lanceolatus venom (Figure 3B). The calculated IC50 were 0.8 µg/mL (CI 95: 0.5–1.0 µg/mL) and 51.7 µg/mL (CI 95: 0–107.2 µg/mL), for B. jararaca and B. lanceolatus venom, respectively (Figure 3D). After 48 h of exposure to B. lanceolatus venom; the IC50 was 42.4 µg/mL (CI 95: 0–97.5 µg/mL), and, after 72 h, it was 17.6 µg/mL (CI 95: 0–51.2 µg/mL). Neutralization of the cytotoxicity of B. lanceolatus venom for the two cell lineages by bothropic antivenom was tested by preincubating venom with antivenom. B. jararaca venom was used as the positive control, and botulinum toxin antiserum was used as the negative control. The concentrations of venom were set so as to obtain about 100% loss of viability within 24 h. Figure 3E shows the complete inhibition of both Bothrops venoms’ cytotoxicity in keratinocytes by the bothropic antivenom, using 1:20 antivenom dilution. However, no inhibition of the venoms’ cytotoxicity was observed for endothelial vascular cells, using antivenom dilution up to 1:5 (Figure 3F). The negative control showed no interference of non-specific antibodies with the assay. 2.4. Inflammatory Potential of the Venom for Human Keratinocytes and Vascular Endothelial Cells The effect of venom on human keratinocytes and endothelial vascular cells was further investigated by measuring the production of cytokines and chemokines by venom-treated cells in conditions of partial loss of viability. In the supernatant of venom-exposed keratinocytes and endothelial vascular cells, four cytokines and chemokines were detected: IL-8, MCP-1, RANTES, and IL-6, the latter observed only in EAhy926 supernatants (Figure 4). IP-10, MIG, IL-12p70, TNF-α, IL-10, IL-1β, IL-17a, IFN-γ, IL-4, and IL-2 were not detected in any of the culture supernatants (data not shown). Concentrations of IL-8, MCP-1, and RANTES were stable along the time in the supernatants of DMEM-treated keratinocytes (Figure 4A,C,E). From 48 h of incubation, concentrations of IL-8, MCP-1, and RANTES increased significantly in the supernatants of venom-treated keratinocytes when compared to DMEM-treated cells. Concentrations of the three proteins increased between 24 to 48 h of incubation with the venom. IL-8 and MCP-1 concentrations stayed stable between 48 and 72 h of incubation, whereas RANTES concentrations decreased in this period of time (Figure 4A,C,E).
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to 48 h of incubation with the venom. IL‐8 and MCP‐1 concentrations stayed stable between 48 and 7 of 19 72 h of incubation, whereas RANTES concentrations decreased in this period of time (Figure 4A,C,E).
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Figure 3.3. Cytotoxicity ofof B. lanceolatus venom on human keratinocytes HaCaTHaCaT and endothelial vascular Figure Cytotoxicity B. lanceolatus venom on human keratinocytes and endothelial cells EAhy926 and its inhibition by bothropic antivenom. (A–D) After being subcultured in 96-well vascular cells EAhy926 and its inhibition by bothropic antivenom. (A–D) After being subcultured in plates in DMEM medium, HaCaT and EAhy926 cells were exposed to different concentrations of 96‐well plates in DMEM medium, HaCaT and EAhy926 cells were exposed to different concentrations Bothrops venoms or only with medium as control. Cellular viability was assessed after 24, 48, and of Bothrops venoms or only with medium as control. Cellular viability was assessed after 24, 48, and 72 h of incubation by MTT assay. The percentage of viability and IC50 were calculated using software 72 h of incubation by MTT assay. The percentage of viability and IC50 were calculated using software GraphPad Prism. The experiments were done three times, in triplicate. (A,B) are representatives of one GraphPad Prism. The experiments were done three times, in triplicate. (A,B) are representatives of experiment. (C,D) represent the IC50 values, calculated as the means of the three assays for each time one experiment. (C,D) represent the IC50 values, calculated as the means of the three assays for each interval, using venom concentrations represented in (A,B). (E,F) Venom samples were pre-incubated time interval, using venom concentrations represented in (A,B). (E,F) Venom samples were pre‐ with bothropic antivenom for 30 min at room temperature. Cells were exposed to these samples for incubated with bothropic antivenom for 30 min at room temperature. Cells were exposed to these 24 h before cell viability was assessed by MTT assay. Data are represented as average ± standard samples for 24 h before cell viability was assessed by MTT assay. Data are represented as average ± error. The columns represent the mean ± SD (n = 3 each). NS: not significant. * p < 0.05 compared to standard error. The columns represent the mean ± SD (n = 3 each). NS: not significant. * p
