Characterization of cell death caused by diplodiatoxin and dipmatol, toxic metabolites of Stenocarpella maydis

Masango, Mxolisi Goodwill; Ellis, Charlotte; Botha, Charl P.

doi:10.1016/j.toxicon.2015.05.013

Cited by 8 publications

(7 citation statements)

References 37 publications

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“…Phosphatidylserine, an amino-phospholipid located on the inner surface of the plasma membrane, has been used as an early marker of cells undergoing apoptosis (Krysko, D’Herde & Vandenabeele 2006). During apoptotic cell death, phosphatidylserine is actively translocated to the outer surface of the plasma membrane, where it can be detected by using annexin V (Masango, Ellis & Botha 2015). In the current study, apoptosis was induced in the C2C12 myoblasts only at the 24 h period following exposure to geigerin (Figure 3).…”

Section: Discussionmentioning

confidence: 99%

Geigerin-induced cytotoxicity in a murine myoblast cell line (C2C12)

Botha

Clift

Ferreira

et al. 2017

Onderstepoort j. vet. res.

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Geigeria poisoning in sheep, locally known as ‘vermeersiekte’, is an economically important plant poisoning in southern Africa. The toxic principles contained by the toxic plants are believed to be several sesquiterpene lactones, such as geigerin, vermeeric acid and vermeerin, which cause striated muscle lesions in small stock. Because of ethical issues surrounding the use of live animals in toxicity studies, there is currently a dire need to establish an in vitro model that can be used to replace traditional animal experimentation. The objective of this study was to determine the cytotoxicity of geigerin in a murine myoblast cell line (C2C12) using methyl-thiazol-tetrazolium (MTT) and lactate dehydrogenase (LDH) assays, annexin V and propidium iodide (PI) flow cytometry and transmission electron microscopy (TEM). Mouse myoblasts were exposed to 2.0 mM, 2.5 mM and 5.0 mM geigerin for 24, 48 and 72 h. A concentration-dependent cytotoxic response was observed. Apoptosis was detected by means of annexin V flow cytometry during the first 24 h and apoptotic bodies were also visible on TEM. According to the LDH and PI flow cytometry results, myoblast cell membranes were not injured. We concluded that the murine myoblast cell line (C2C12) is a suitable model for future studies planned to evaluate the cytotoxicity of other and combinations of sesquiterpene lactones, with and without metabolic activation, implicated in ‘vermeersiekte’ and to elucidate the subcellular effects of these myotoxins on cultured myoblasts.

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Section: Discussionmentioning

confidence: 99%

Geigerin-induced cytotoxicity in a murine myoblast cell line (C2C12)

Botha

Clift

Ferreira

et al. 2017

Onderstepoort j. vet. res.

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“…The evaluation of plasma membrane permeability was realized using propidium iodide (PI) staining (Sigma-Aldrich). This probe is a fluorescent substance that labels the DNA of cells with damaged plasma membrane, since under these conditions it has the ability to penetrate the cell (Masango et al, 2015). After treatment with PF-429242 (3 and 6 µM), the parasite concentration was adjusted to 1 × 10 7 cells/mL in 200 µL PBS.…”

Section: Evaluation Of Plasma Membrane Permeabilitymentioning

confidence: 99%

PF-429242, a Subtilisin Inhibitor, Is Effective in vitro Against Leishmania infantum

et al. 2021

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PF-429242 is an inhibitor of subtilisin, an important protease found in Leishmania. However, studies regarding the effect of PF-429242 on Leishmania are scarce. In this work we evaluated the antileishmanial effect of PF-429242 against Leishmania infantum and the mechanism involved in the death of the parasite. PF-429242 had low toxicity against mammalian cells (peritoneal macrophages) (CC50 = 189.07 μM) and presented activity against L. infantum promastigotes (IC50 = 2.78 μM) and intracellular amastigotes (IC50 = 14.07 μM), indicating selectivity toward the parasite. Transmission electron microscopy (TEM), as well as staining of L. infantum promastigotes with MitoTracker® Red, rhodamine 123 and MitoSOX, revealed that the mitochondria was a potential target of PF-429242. In addition, PF-429242 caused an accumulation of neutral lipids in promastigotes, which was demonstrated by Nile Red staining and TEM, and induced oxidative stress (H2DCFDA staining). Furthermore the formation of autophagic vacuoles in L. infantum promastigotes was observed by MDC staining and TEM. However, the killing induced by PF-429242 in L. infantum promastigotes appeared to be unrelated to apoptosis and/or necrosis as there was no phosphatidylserine externalization, DNA fragmentation or alterations in the permeability of the parasite plasma membrane, as assessed by annexin V-FITC, TUNEL and propidium iodide staining, respectively. The morphological and ultrastructural evaluation of the promastigotes by optical microscopy, scanning electron microscopy (SEM) and TEM, revealed the presence of parasites with flagellar defects. TEM analysis of the intracellular amastigotes indicated that mitochondrial damage and autophagy could also be involved in the death of these forms after treatment with PF-429242. In addition, PF-429242 treatment stimulated NO production from infected macrophage, but only at a high concentration (100 μM), as well as an increase of TNF levels after treatment with 10 μM of PF-429242. The compound did not stimulate ROS or IL-10 production. Together, these data highlight the antileishmanial potential of PF-429242, inducing several cellular alterations in the parasite, such as mitochondrial damage, neutral lipids accumulation, oxidative stress and autophagy which culminate in the death of L. infantum, as well as modulating host cellular responses that favor the development of an immune response against the parasite.

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“…About apoptosis/necrosis: after exposure to melittin, 2μL of fluorescein isothiocyanateconjugated Annexin V antibody (FITC) was added and the cells were incubated for 1h. After, propidium iodide (PI, 50μg/mL) was added with further incubation for 10min (Masango et al, 2015). The cells were removed from the plate and placed under refrigeration for readings in the flow cytometer.…”

Section: Methodsmentioning

confidence: 99%

Melittin-induced metabolic changes on the Madin-Darby Bovine Kidney cell line

Picoli

Peter

Lopes

et al. 2021

Arq. Bras. Med. Vet. Zootec.

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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.

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Characterization of cell death caused by diplodiatoxin and dipmatol, toxic metabolites of Stenocarpella maydis

Cited by 8 publications

References 37 publications

Geigerin-induced cytotoxicity in a murine myoblast cell line (C2C12)

Geigerin-induced cytotoxicity in a murine myoblast cell line (C2C12)

PF-429242, a Subtilisin Inhibitor, Is Effective in vitro Against Leishmania infantum

Melittin-induced metabolic changes on the Madin-Darby Bovine Kidney cell line

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