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

Abstract

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.

Pictolysin-III, a Hemorrhagic Type-III Metalloproteinase Isolated from Bothrops pictus (Serpentes: Viperidae) Venom, Reduces Mitochondrial Respiration and Induces Cytokine Secretion in Epithelial and Stromal Cell Lines

“…Notably, the inhibition of mitochondrial respiration stimulated by glutamate plus malate (complex I’s substrates), succinate (complex II’s substrate), and TMPD (an artificial complex IV’s substrate) plus ascorbate is a previous event for mtDAMPs production [ 90 ]; however, the toxin classes involved remain uncertain. Accordingly, the crude venom of viper Macrovipera lebetina obtusa affects respiration in the Vero monkey kidney epithelial cell line, with this effect sensitive to metalloproteinase and phospholipase inhibitors [ 21 ]. To our knowledge, there are no studies that identify the action of an isolated SVMP on mitochondrial respiration and its implication for the disruptive action of the cell–ECM interaction.…”

“…Some SVMPs also produce intravascular coagulation, edema, inflammation, necrosis, fibrin(ogen) degradation, induction of apoptosis, and inhibition of platelet aggregation [ 8 , 17 , 19 , 20 ]. Recently, a study using crude Macrovipera lebetina obtusa ( Levantine viper ) venom suggested that possibly SVMPs may disrupt the mitochondrial metabolism in the Vero monkey kidney epithelial cell line [ 21 ]. To our knowledge, there are no studies that identify the action of an isolated SVMP on mitochondrial respiration and its implication for the disruptive action of the cell–ECM interaction.…”

Pulmonary involvement from animal toxins: the cellular mechanisms

“…Numerous animal studies have provided supporting evidence that SVMPs, such as Jararhagin, the main SVMP found in the venom of Bothrops asper and Bothrops jararaca , primarily target the basal lamina of alveolar cells, leading to pulmonary hemorrhage [ 50 - 52 ]. The destructive effects on cellular structures can be further intensified by PLA 2 [ 53 , 54 ], another abundant enzyme in venom, as demonstrated by the increased detachment of endothelial cells when both enzymes are present [ 55 ]. Additionally, a C-type lectin (CTL) called aspercetin, which is another venom component, has been identified to potentiate the hemorrhagic effect [ 51 ].…”