Changes in attachment and metabolic activity of rat neonatal cardiomyocytes and nonmyocytes caused by Macrovipera lebetina obtusa venom.

The Caucasian viper Macrovipera lebetina obtusa (MLO) is one of the most prevalent and venomous snakes in the Caucasus and the surrounding regions, yet the effects of MLO venom on cardiac function remain largely unknown. We examined the influence of MLO venom (crude and with inhibited metalloproteinases and phospholipase A2) on attachment and metabolic activity of rat neonatal cardiomyocytes (CM) and nonmyocytes (nCM), assessed at 1 and 24 h. After exposing both CM and nCM to varying concentrations of MLO venom, we observed immediate cytotoxic effects at a concentration of 100 µg/ml, causing detachment from the culture substrate. At lower MLO venom concentrations both cell types detached in a dose-dependent manner. Inhibition of MLO venom metalloproteinases significantly improved CM and nCM attachment after 1-hour exposure. At 24-hour exposure to metalloproteinases inhibited venom statistically significant enhancement was observed only in nCM attachment. However, metabolic activity of CM and nCM did not decrease upon exposure to the lower dose of the venom. Moreover, we demonstrated that metalloproteinases and phospholipases A2 are not the components of the MLO venom that change metabolic activity of both CM and nCM. These results provide a valuable platform to study the impact of MLO venom on prey cardiac function. They also call for further exploration of individual venom components for pharmaceutical purposes.

The Contribution of Phospholipase A2 and Metalloproteinases to the Synergistic Action of Viper Venom on the Bioenergetic Profile of Vero Cells.

Increasing concern about the use of animal models has stimulated the development of in vitro cell culture models for analysis of the biological effects of snake venoms. However, the complexity of animal venoms and the extreme synergy of the venom components during envenomation calls for critical review and analysis. The epithelium is a primary target for injected viper venom's toxic substances, and therefore, is a focus in modern toxinology. We used the Vero epithelial cell line as a model to compare the actions of a crude Macrovipera lebetina obtusa (Levantine viper) venom with the actions of the same venom with two key enzymatic components inhibited (specifically, phospholipase A2 (PLA2) and metalloproteinases) in the bioenergetic cellular response, i.e., oxygen uptake and reactive oxygen species generation. In addition to the rate of free-radical oxidation and lipid peroxidation, we measured real-time mitochondrial respiration (based on the oxygen consumption rate) and glycolysis (based on the extracellular acidification rate) using a Seahorse analyzer. Our data show that viper venom drives an increase in both glycolysis and respiration in Vero cells, while the blockage of PLA2 or/and metalloproteinases affects only the rates of the oxidative phosphorylation. PLA2-blocking in venom also increases cytotoxic activity and the overproduction of reactive oxygen species. These data show that certain components of the venom may have a different effect within the venom cocktail other than the purified enzymes due to the synergy of the venom components.

Hemorrhagic changes and microglia activation induced by Macrovipera lebetina obtusa venom with the inhibited enzymatic activity in rat brain.

The metalloproteinases and phospholipase A2 are the main enzymes in the venom of Macrovipera lebetina obtusa that play a decisive role in the destructive and toxic effects on the organism of the prey. Metalloproteinases cause hemorrhagic damage, destroy the basement membrane of the blood vessel and disrupt the connections between endothelial cells. Phospholipase A2 causes hemolysis of erythrocytes, destroy the cell membranes, and inhibits the adhesion of platelets and so on. The state of the capillaries of the rat brain and microglia under the action of the venom with separately inhibited enzymes was investigated and compared to the action of the crude venom. Also, the toxicity LD50 of the venom of Macrovipera lebetina obtusa with the inhibited enzymatic activity was determined. The histochemical study showed that the inhibition of phospholipase A2 enzymatic activity did not significantly change the vasodestructive effect of the venoms. In case of action of a venom with inhibited enzymatic activity of metalloproteinases, low activity of microglia and less damaged capillaries were observed. The toxicity of the venom with inhibited phospholipase A2 and with inhibited metalloproteinases was respectively 1.8 and 3.7 times weaker than that of the crude venom. We can claim that both the toxicity of the venom of Macrovipera lebetina obtusa, the damaged brain vessels and the increased activity of CNS microglia are determined mainly by the action of metalloproteinases.

Antinociceptive, anti-inflammatory, and cytotoxic properties of Origanum vulgare essential oil, rich with ß-caryophyllene and ß-caryophyllene oxide.

Background: Essential oils are of great interest for their analgesic and anti-inflammatory properties. We aimed to study the content of the essential oil of the Origanum vulgare of the Armenian highlands (OVA) in different periods of vegetation and to investigate its antinociceptive and anti-inflammatory effects in mice (in vivo) and cytotoxic action in cultured cells (in vitro). OVA essential oil was extracted from fresh plant material by hydro-distillation. Methods: For OVA essential oil contents determination the gas chromatography-mass spectrometry method was used. Formalin and hot plate tests and analysis of cell viability using the methyl-thiazolyl-tetrazolium (MTT) assay were used. Results: The maximal content of ß-caryophyllene and ß-caryophyllene oxide in OVA essential oil was revealed in the period of blossoming (8.18% and 13.36%, correspondently). In the formalin test, 4% OVA essential oil solution (3.5 mg/mouse) exerts significant antinociceptive and anti-inflammatory effects (P = 0.003). MTT assay shows approximately 60% cytotoxicity in HeLa and Vero cells for 2.0 µL/mL OVA essential oil in media. Conclusions: The wild oregano herb of Armenian highlands, harvested in the blossoming period, may be considered as a valuable source for developing pain-relieving preparations.

The development and evaluation of the efficacy of ovine-derived experimental antivenom immunoserum against Macrovipera lebetina obtusa (MLO) venom.

Here we describe the processing and development of animal-derived monovalent antibody serum against Macrovipera lebetina obtusa venom by purification and concentration of the immunoglobulins using caprylic acid. We demonstrate that this new viper venom antiserum is pre-clinically effective in neutralizing lethal toxicity and hemorrhagicity of the venom of the Armenian Levantine viper - a significant public health problem in Armenia and a wide region from south-east parts of Europe to south-west Asia. The developed product shows a high capacity to inhibit metalloproteinases and phospholipase activity of venom included in the study in comparison to current specific antivenoms, and following additional experimental approvals, it will be possible to derive the monovalent antivenom satisfying international standards, which will be much cheaper and accessible compared with the current market rivals.