Abstract
ABSTRACT In this study, the toxic effects of melittin on Madin-Darby Bovine Kidney cells (MDBK) were analyzed with respect to mitochondrial functionality by reduction of MTT and flow cytometry, apoptosis potential, necrosis, oxygen reactive species (ROS) production, lipid peroxidation, and DNA fragmentation using flow cytometry and cell membrane destabilization by confocal microscopy. The toxicity presented dose-dependent characteristics and mitochondrial activity was inhibited by up to 78.24 ±3.59% (P<0.01, n = 6) in MDBK cells exposed to melittin (10μg/mL). Flow cytometry analysis revealed that melittin at 2μg/mL had the highest necrosis rate (P<0.05) for the cells. The lipoperoxidation of the membranes was also higher at 2μg/mL of melittin (P<0.05), which was further confirmed by the microphotographs obtained by confocal microscopy. The highest ROS production occurred when the cells were exposed to 2.5μg/mL melittin (P<0.05), and this concentration also increased DNA fragmentation (P<0.05). There was a significative and positive correlation between the lipoperoxidation of membranes with ROS (R=0.4158), mitochondrial functionality (R=0.4149), and apoptosis (R=0.4978). Thus, the oxidative stress generated by melittin culminates in the elevation of intracellular ROS that initiates a cascade of toxic events in MDBK cells.
Highlights
Apitoxin or bee venom is secreted by a specialized gland present in the worker bees and confined in a vesicle until the moment of stinging (Benton et al, 1963)
The objective of this study was to examine the effects of melittin in Madin-Darby Bovine Kidney cells (MDBK) (Madin-Darby Bovine Kidney) cells by analysis of mitochondrial functionality, apoptosis potential, necrosis, reactive oxygen species (ROS) production, lipid peroxidation, DNA fragmentation and cell membrane destabilization
Dimethyl sulfoxide (DMSO), MTT (3- (4,5-dimethylthiazol-2yl)-2-5-diphenyl2H-tetrazolate reagent), as well as other reagents used in flow cytometry and confocal microscopy, were commercially purchased from SigmaAldrich Corp
Summary
Apitoxin or bee venom is secreted by a specialized gland present in the worker bees and confined in a vesicle until the moment of stinging (Benton et al, 1963). It is a complex mixture of nitrogenous compounds, containing several biologically active components, including enzymes, peptides, and biogenic amines, conferring a wide variety of allergic and pharmacological properties, such as anti-inflammatory, antimicrobial, and antitumor activities (Cardoso et al, 2003; Abreu et al, 2010). Phospholipase A2 is present in the venom, which further amplifies the catalytic actions of melittin (Cardoso et al, 2003; Koumanov et al, 2003)
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