Abstract
Viperidae venom has several local and systemic effects, such as pain, edema, inflammation, kidney failure and coagulopathy. Additionally, bothropic venom and its isolated components directly interfere on cellular metabolism, causing alterations such as cell death and proliferation. Inflammatory cells are particularly involved in pathological envenomation mechanisms due to their capacity of releasing many mediators, such as nitric oxide (NO). NO has many effects on cell viability and it is associated to the development of inflammation and tissue damage caused by Bothrops and Bothropoides venom. Bothropoides insularis is a snake found only in Queimada Grande Island, which has markedly toxic venom. Thus, the aim of this work was to evaluate the biological effects of Bothropoides insularis venom (BiV) on RAW 264.7 cells and assess NO involvement. The venom was submitted to colorimetric assays to identify the presence of some enzymatic components. We observed that BiV induced H2O2 production and showed proteolytic and phospholipasic activities. RAW 264.7 murine macrophages were incubated with different concentrations of BiV and then cell viability was assessed by MTT reduction assay after 2, 6, 12 and 24 hours of incubation. A time- and concentration-dependent effect was observed, with a tendency to cell proliferation at lower BiV concentrations and cell death at higher concentrations. The cytotoxic effect was confirmed after lactate dehydrogenase (LDH) measurement in the supernatant from the experimental groups. Flow cytometry analyses revealed that necrosis is the main cell death pathway caused by BiV. Also, BiV induced NO release. The inhibition of both proliferative and cytotoxic effects with L-NAME were demonstrated, indicating that NO is important for these effects. Finally, BiV induced an increase in iNOS expression. Altogether, these results demonstrate that B. insularis venom have proliferative and cytotoxic effects on macrophages, with necrosis participation. We also suggest that BiV acts by inducing iNOS expression and causing NO release.
Highlights
Snake venoms consist of 95% proteins, with or without enzymatic activities, such as neurotoxins, cardiotoxins, lecithins, disintegrins, L-amino acid oxidases natriuretic peptides, metalloproteases, phospholipases A2 and phosphodiesterases [1,2,3]
The presence of important components of Bothropoides insularis venom (BiV) was verified by enzymatic assays, which demonstrated that B. insularis venom has proteolytic and phospholipasic doi:10.1371/journal.pone.0151029.g001
Activities, and results in H2O2 production. These findings suggest the presence of metalloproteases, PLA2 and LAAO in BiV, respectively (Fig 1)
Summary
Snake venoms consist of 95% proteins, with or without enzymatic activities, such as neurotoxins, cardiotoxins, lecithins, disintegrins, L-amino acid oxidases (svLAAOs) natriuretic peptides, metalloproteases (svMPs), phospholipases A2 (svPLA2) and phosphodiesterases [1,2,3]. These proteins are the main responsible agents for the toxic effects observed in animal and human envenomations. The severity of these effects is associated with many factors, their qualitative and quantitative composition is essential to determine the type and intensity of their toxicity [6] These venoms, as well as their proteins, have shown marked cytotoxic effects on many cell types [7,8]. Events such as cell death, proliferation and expression of mediators can interfere with both their function and tissue response to the venom-induced lesion [8,10]
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