In vitro assessment of cytotoxic activities of Lachesis muta muta snake venom.

Envenomation by the bushmaster snake Lachesis muta muta is considered severe, characterized by local effects including necrosis, the main cause of permanent disability. However, cellular mechanisms related to cell death and tissue destruction, triggered by snake venoms, are poorly explored. The purpose of this study was to investigate the cytotoxic effect caused by L. m. muta venom in normal human keratinocytes and to identify the cellular processes involved in in cellulo envenomation. In order to investigate venom effect on different cell types, Alamar Blue assay was performed to quantify levels of cellular metabolism as a readout of cell viability. Apoptosis, necrosis and changes in mitochondrial membrane potential were evaluated by flow cytometry, while induction of autophagy was assessed by expression of GFP-LC3 and analyzed using fluorescence microscopy. The cytotoxic potential of the venom is shown by reduced cell viability in a concentration-dependent manner. It was also observed the sequential appearance of cells undergoing autophagy (by 6 hours), apoptosis and necrosis (12 and 24 hours). Morphologically, incubation with L. m. muta venom led to a significant cellular retraction and formation of cellular aggregates. These results indicate that L. m. muta venom is cytotoxic to normal human keratinocytes and other cell lines, and this toxicity involves the integration of distinct modes of cell death. Autophagy as a cell death mechanism, in addition to apoptosis and necrosis, can help to unravel cellular pathways and mechanisms triggered by the venom. Understanding the mechanisms that underlie cellular damage and tissue destruction will be useful in the development of alternative therapies against snakebites.

Preclinical assessment of a polyspecific antivenom against the venoms of Cerrophidion sasai, Porthidium nasutum and Porthidium ophryomegas: Insights from combined antivenomics and neutralization assays.

A polyspecific antivenom is used in Central America for the treatment of envenomings by viperid snakes. This antivenom is generated in horses hyperimmunized with a mixture of venoms from Bothrops asper, Crotalus simus and Lachesis stenophrys. The present study analyzed the ability of this antivenom to neutralize the venoms of three Central American viperid species of the 'Porthidium group', i.e. Porthidium nasutum, Porthidium ophryomegas and Cerrophidion sasai, formerly classified as Cerrophidion godmani. In addition, the immunorecognition of the components of these venoms was assessed by immunoaffinity antivenomics. The antivenom proved effective in neutralizing the lethal, hemorrhagic, myotoxic, phospholipase A(2) (PLA(2)) and proteinase activities of the three venoms, albeit exhibiting quantitative differences in the values of the Median Effective Doses (ED(50)). Excepting for certain low molecular mass bands corresponding to disintegrins, and some PLA(2)s and PI-metalloproteinases, Western blotting and immunoaffinity chromatography revealed immunorecognition of most Porthidium and Cerrophidion venom proteins. In agreement with in vivo neutralization assays, immobilized antivenom IgGs showed higher immunocapturing activity of toxins from both Porthidium taxa than from C. sasai. Overall our results demonstrate a significant paraspecific protection of the Costa Rican polyspecific antivenom against the three venoms sampled. They also stress the need to search for novel ways to enhance the immune response of horses against several weakly immunogenic venom components.

Gestation, thermoregulation, and metabolism in a viviparous snake, Vipera aspis: evidence for fecundity-independent costs.

Oxygen consumption of gestating Aspic vipers, Vipera aspis (L.), was strongly dependent on body temperature and mass. Temperature-controlled, mass-independent oxygen consumption did not differ between pregnant and nonpregnant females. Maternal metabolism was not influenced during early gestation by the number of embryos carried but was weakly influenced during late gestation. These results differ from previous investigations that show an increase in mass-independent oxygen consumption in reproductive females relative to nonreproductive females and a positive relationship between metabolism and litter size. These data also conflict with published field data on V. aspis that show a strong metabolic cost associated with reproduction. We propose that, under controlled conditions (i.e., females exposed to precise ambient temperatures), following the mobilisation of resources to create follicles (i.e., vitellogenesis), early gestation per se may not be an energetically expensive period in reproduction. However, under natural conditions, the metabolic rate of reproductive females is strongly increased by a shift in thermal ecology (higher body temperature and longer basking periods), enabling pregnant females to accelerate the process of gestation. Combining both laboratory and field investigation in a viviparous snake, we suggest that reproduction entails discrete changes in the thermal ecology of females to provide optimal temperatures to the embryos, whatever their number. This results in the counterintuitive notion that metabolism may well be largely independent of fecundity during gestation, at least in an ectothermic reptile.