scholarly journals Neurotoxic Effect of Flavonol Myricetin in the Presence of Excess Copper

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 845
Author(s):  
Anja Sadžak ◽  
Ignacija Vlašić ◽  
Zoran Kiralj ◽  
Marijana Batarelo ◽  
Nada Oršolić ◽  
...  
Keyword(s):  
Cell Death ◽  
Intracellular Signaling ◽  
Chromatin Condensation ◽  
Membrane Integrity ◽  
Toxic Effects ◽  
Trypan Blue Exclusion ◽  
Atp Production ◽  
Mtt Method ◽  
Biophysical Approach ◽  
Induced Changes

Oxidative stress (OS) induced by the disturbed homeostasis of metal ions is one of the pivotal factors contributing to neurodegeneration. The aim of the present study was to investigate the effects of flavonoid myricetin on copper-induced toxicity in neuroblastoma SH-SY5Y cells. As determined by the MTT method, trypan blue exclusion assay and measurement of ATP production, myricetin heightened the toxic effects of copper and exacerbated cell death. It also increased copper-induced generation of reactive oxygen species, indicating the prooxidative nature of its action. Furthermore, myricetin provoked chromatin condensation and loss of membrane integrity without caspase-3 activation, suggesting the activation of both caspase-independent programmed cell death and necrosis. At the protein level, myricetin-induced upregulation of PARP-1 and decreased expression of Bcl-2, whereas copper-induced changes in the expression of p53, p73, Bax and NME1 were not further affected by myricetin. Inhibitors of ERK1/2 and JNK kinases, protein kinase A and L-type calcium channels exacerbated the toxic effects of myricetin, indicating the involvement of intracellular signaling pathways in cell death. We also employed atomic force microscopy (AFM) to evaluate the morphological and mechanical properties of SH-SY5Y cells at the nanoscale. Consistent with the cellular and molecular methods, this biophysical approach also revealed a myricetin-induced increase in cell surface roughness and reduced elasticity. Taken together, we demonstrated the adverse effects of myricetin, pointing out that caution is required when considering powerful antioxidants for adjuvant therapy in copper-related neurodegeneration.

270 APOPTOSIS AND ONCOSIS ASSESSMENT IN CRYOPRESERVED MOUSE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 242
Author(s):  
M. E. O. A. Assumpção ◽  
A. R. S. Coutinho ◽  
W. B. Feitosa ◽  
C. M. Mendes ◽  
R. Simões ◽  
...  
Keyword(s):  
Cell Death ◽  
Liquid Nitrogen ◽  
Caspase 3 ◽  
Chromatin Condensation ◽  
Membrane Integrity ◽  
Water Bath ◽  
Slow Freezing ◽  
Control Group ◽  
Embryo Viability ◽  
Cryopreserved Embryos

Cryopreservation of mammalian embryos is an important tool for the application of reproductive biotechnology. Recent evidence indicates that apoptosis of cryopreserved embryos may be a negative factor for their viability. The aim of this study was to detect apoptosis and to characterize and quantify the embryonic cell death caused by cryopreservation. Mouse morulae were separated to be subjected to two cryopreservation protocols (slow freezing and vitrification) and a control group (fresh). In the slow-freezing procedure, embryos were exposed to 10% ethylene glycol (EG) for 10 min. Straws were placed in a methanol bath at -7�C until it reached -31�C and then plunged and stored in liquid nitrogen. The embryos were thawed in air for 10 s and in a 25�C water bath for 20 s. In the vitrification method, embryos were exposed to 10% and 20% EG for 5 min, followed by 40% EG + 18% Ficoll + 10% sucrose (EFS) for 30 s and then plunged and stored in liquid nitrogen. These embryos were thawed in a 25�C water bath for 20 s. For the cell death evaluation, cell membrane integrity from the fresh and cryopreserved embryos was assessed by Hoechst and propidium iodide (H/PI staining). Morphology and apoptosis were assessed by means of the haematoxylin-eosin staining (HE) and by electron microscopy (MET). To confirm apoptosis, 64 cryopreserved mouse morulae (34 submitted to slow freezing and 30 to vitrification) were used to evaluate Caspase-3 activity. The cryopreserved embryos were divided into experimental and control groups and incubated with Caspase-3 and buffer solution, respectively. Afterward, the embryos were incubated with rhodamine and the Caspase activity was determined under a fluorescence microscope. H/PI staining detected more membrane permeability in the vitrification (69.7%) than in the slow-freezing (48.4%) or fresh (13.8%) groups (P < 0.05; Wilcoxon's test). Nuclear evaluation by HE revealed that vitrification and slow freezing induced pyknosis and chromatin condensation. HE staining revealed weakly staining cytoplasm and degenerated cells in the vitrification group (indicating oncosis), whereas in the slow-freezing the presence of cytoplasmic condensation and eosinophilic structures indicating apoptosis were observed. MET examination of the ultrastructure confirmed the HE results. The Caspase-3 activity showed a fluorescence increase in both experimental groups compared with the control group. In conclusion, staining with HE allows detection of oncosis and apoptosis in cryopreserved embryos. Regarding the cryopreservation techniques, both slow freezing and vitrification showed oncosis and apoptosis injuries. However, in this experiment vitrification caused more cellular injuries, with less embryo viability, than slow freezing. This work was supported by FAPESP 04/01252-4 and CAPES.

scholarly journals Trypan Blue Exclusion Assay Verifies in Vitro Cytotoxicity of New Cis-Platinum (II) Complex in Human Cells

2019 ◽  
Vol 16 (3) ◽  
pp. 0555
Author(s):  
Hussein Et al.
Keyword(s):  
Cell Viability ◽  
Trypan Blue ◽  
Membrane Integrity ◽  
Platinum Complex ◽  
In Vitro Cytotoxicity ◽  
Toxic Effects ◽  
Polymorphonuclear Cells ◽  
Trypan Blue Exclusion ◽  
High Concentrations

          Various assays are used to determine the toxic effects of drugs at cellular levels in vitro.  One of these methods is the dye exclusion assay, which measures membrane integrity in the presence of Trypan blue. Trypan blue the dye which was used in this study to investigate cytotoxic effect of a new Cis –dichloroplatinum (II) complex [(Qu)2PtCl2] on the viability of polymorphonuclear cells (PMNs). Three concentrations of platinum complex were prepared (70, 35and 17.5 µg/ ml) and the results revealed that the percentage of cell viability decreased as the platinum complex concentration increased in comparison with control. The platinum complex exhibited low cytotoxic effects towards healthy cells at the concentrations of 17.5 µg/ ml and 35 µg/ ml, in which the percentage of cell viability was (77.01 ± 6.3) and (72.3± 0.50)respectively, with no significant differences as compared with the control(90.66 ±0.577). The viability was significantly decreased (67.59 ± 3.16) when the cells were treated with the concentration of 70 µg/ ml in comparison with control. These results indicated that the percentage of living cells decreased when treated with high concentrations of [(Qu)2PtCl2], which causes cells death, while low concentrations of the compound show low toxicity. This data indicates that this compound, at these concentrations may be suitable for use as a cancer treatment because it has low toxic effects on the healthy cells.        

scholarly journals Escherichia coli Shiga-Like Toxins Induce Apoptosis and Cleavage of Poly(ADP-Ribose) Polymerase via In Vitro Activation of Caspases

2002 ◽  
Vol 70 (8) ◽  
pp. 4669-4677 ◽  
Author(s):  
Joyce C. Y. Ching ◽  
Nicola L. Jones ◽  
Peter J. M. Ceponis ◽  
Mohamed A. Karmali ◽  
Philip M. Sherman
Keyword(s):  
Escherichia Coli ◽  
Cell Death ◽  
Intracellular Signaling ◽  
Chromatin Condensation ◽  
Specific Inhibitor ◽  
Caspase 8 ◽  
High Dose ◽  
Dependent Manner ◽  
Induced Apoptosis ◽  
Dose Dependent

ABSTRACT Shiga-like toxin-producing Escherichia coli causes hemorrhagic colitis and hemolytic-uremic syndrome in association with the production of Shiga-like toxins, which induce cell death via either necrosis or apoptosis. However, the abilities of different Shiga-like toxins to trigger apoptosis and the sequence of intracellular signaling events mediating the death of epithelial cells have not been completely defined. Fluorescent dye staining with acridine orange and ethidium bromide showed that Shiga-like toxin 1 (Stx1) induced apoptosis of HEp-2 cells in a dose- and time-dependent manner. Stx2 also induced apoptosis in a dose-dependent manner. Apoptosis induced by Stx1 (200 ng/ml) and apoptosis induced by Stx2 (200 ng/ml) were maximal following incubation with cells for 24 h (94.3% ± 1.8% and 81.7% ± 5.2% of the cells, respectively). Toxin-treated cells showed characteristic features of apoptosis, including membrane blebbing, DNA fragmentation, chromatin condensation, cell shrinkage, and the formation of apoptotic bodies, as assessed by transmission electron microscopy. Stx2c induced apoptosis weakly even at a high dose (1,000 ng/ml for 24 h; 26.7% ± 1.3% of the cells), whereas Stx2e did not induce apoptosis of HEp-2 cells. Thin-layer chromatography confirmed that HEp-2 cells express the Stx1-Stx2-Stx2c receptor, globotriaosylceramide (Gb3), but not the Stx2e receptor, globotetraosylceramide (Gb4). Western blot analysis of poly(ADP-ribose) polymerase (PARP), a DNA repair enzyme, demonstrated that incubation with Stx1 and Stx2 induced cleavage, whereas incubation with Stx2e did not result in cleavage of PARP. A pan-caspase inhibitor (Z-VAD-FMK) and a caspase-8-specific inhibitor (Z-IETD-FMK) eliminated, in a dose-dependent fashion, the cleavage of PARP induced by Shiga-like toxins. Caspase-8 activation was confirmed by detection of cleavage of this enzyme by immunoblotting. Cleavage of caspase-9 and the proapoptotic member of the Bcl-2 family BID was also induced by Stx1, as determined by immunoblot analyses. We conclude that different Shiga-like toxins induce different degrees of apoptosis that correlates with toxin binding to the glycolipid receptor Gb3 and that caspases play an integral role in the signal transduction cascade leading to toxin-mediated programmed cell death.

scholarly journals Racemoside A, an anti-leishmanial, water-soluble, natural steroidal saponin, induces programmed cell death in Leishmania donovani

2007 ◽  
Vol 56 (9) ◽  
pp. 1196-1204 ◽  
Author(s):  
Avijit Dutta ◽  
Angana Ghoshal ◽  
Debayan Mandal ◽  
Nirup B. Mondal ◽  
Sukdeb Banerjee ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Leishmania Donovani ◽  
Chromatin Condensation ◽  
Membrane Integrity ◽  
Water Soluble ◽  
Asparagus Racemosus ◽  
Steroidal Saponin ◽  
Significant Activity ◽  
Major Health Problem

Leishmaniasis remains a major health problem of the tropical and subtropical world. The visceral form causes the most fatalities if left untreated. Dramatic increases in the rates of infection and drug resistance and the non-availability of safe vaccines have highlighted the need for identification of novel and inexpensive anti-leishmanial agents. This study reports that racemoside A, a water-soluble steroidal saponin purified from the fruits of Asparagus racemosus, is a potent anti-leishmanial molecule effective against antimonial-sensitive (strain AG83) and -unresponsive (strain GE1F8R) Leishmania donovani promastigotes, with IC50 values of 1.15 and 1.31 μg ml−1, respectively. Incubation of promastigotes with racemoside A caused morphological alterations including cell shrinkage, an aflagellated ovoid shape and chromatin condensation. This compound exerts its leishmanicidal effect through the induction of programmed cell death mediated by the loss of plasma membrane integrity as detected by binding of annexin V and propidium iodide, loss of mitochondrial membrane potential culminating in cell-cycle arrest at the sub-G0/G1 phase, and DNA nicking shown by deoxynucleotidyltransferase-mediated dUTP end labelling (TUNEL). Racemoside A also showed significant activity against intracellular amastigotes of AG83 and GE1F8R at a 7–8-fold lower dose, with IC50 values of 0.17 and 0.16 μg ml−1, respectively, and was non-toxic to murine peritoneal macrophages up to a concentration of 10 μg ml−1. Hence, racemoside A is a potent anti-leishmanial agent that merits further pharmacological investigation.

scholarly journals The Calcineurin Pathway Inhibitor Tacrolimus Enhances the In Vitro Activity of Azoles against Mucorales via Apoptosis

2013 ◽  
Vol 12 (9) ◽  
pp. 1225-1234 ◽  
Author(s):  
F. Shirazi ◽  
D. P. Kontoyiannis
Keyword(s):  
Cell Death ◽  
Dna Fragmentation ◽  
Rhizopus Oryzae ◽  
Apoptotic Cell ◽  
Chromatin Condensation ◽  
Membrane Integrity ◽  
Ros Generation ◽  
Content Type ◽  
Intracellular Ca ◽  
Calcineurin Pathway

ABSTRACT The calcineurin pathway regulates antifungal drug resistance and the virulence of several major human-pathogenic fungi, including the recalcitrant Mucorales . We hypothesized that the fungistatic triazoles posaconazole (PCZ) and itraconazole (ICZ) become fungicidal in the setting of the calcineurin inhibitor tacrolimus (TCR) and that such an effect is mediated through apoptosis. Fungicidal activity and apoptosis were studied using standard microbiological techniques and hyphal metabolic and vital dye reduction assays at 37°C in RPMI 1640. Apoptosis was characterized by detecting intracellular Ca 2+ , phosphatidylserine (PS) externalization, DNA fragmentation, plasma membrane integrity, chromatin condensation, reactive oxygen species (ROS) generation, caspase-like activity, ATP, and cytochrome c release. MICs for PCZ and ICZ alone were significantly higher (8 to 128 μg/ml) than those of PCZ or ICZ plus TCR (0.25 to 4 μg/ml) for Rhizopus oryzae , Cunninghamella bertholletiae , and Mucor circinelloides . Both PCZ and ICZ in combination with TCR became fungicidal, and their activity was mediated through increased apoptotic cell death of R. oryzae (10 to 50%), C. bertholletiae (5 to 50%), and M. circinelloides (5 to 55%) germlings, with morphological apoptotic changes characterized by externalization of PS, nuclear condensation, and DNA fragmentation. Moreover, activation of the caspase-like activity was correlated with cell death induced by TCR plus PCZ or ICZ. These changes correlated with elevated intracellular Ca 2+ and ROS levels and disturbance of mitochondrial potential. We found that PCZ or ICZ in combination with TCR renders Mucorales sensitive to triazoles via apoptotic death. These observations could serve as a new paradigm for the development of new therapeutic strategies.

Protein Dynamics During Apoptosis

1999 ◽  
Vol 5 (S2) ◽  
pp. 1040-1041
Author(s):  
Brian Herman
Keyword(s):  
Cell Death ◽  
Clonal Selection ◽  
Chromatin Condensation ◽  
Critical Role ◽  
Membrane Integrity ◽  
Tissue Growth ◽  
Cell Shrinkage ◽  
Membrane Blebbing ◽  
Physiological Processes ◽  
Severe Tissue

Apoptosis is an actively regulated process of cell death necessary for proper control of tissue growth. The phenomenon is characterized by plasma membrane blebbing, cell shrinkage, chromatin condensation, and degradation of DNA. Apoptosis is distinctly different from necrosis. Unlike necrosis, which is a passive form of cell death, apoptosis appears to be an active physiological process using a controlled genetic “program” of gene expression. Furthermore, apoptosis does not involve severe tissue damage or inflammation. Membrane integrity is preserved, and there is no loss of cellular contents before phagocytosis. Apoptosis is also distinct from necrosis in that it can be triggered or suppressed by tissue-specific hormones and growth factors.Apoptosis plays a critical role in tissue homeostasis by counterbalancing mitosis. It is an essential component of many physiological processes, including embryonic development, clonal selection in thymocytes, and in protection against disease.

scholarly journals 82 EVALUATION OF CELL DEATH IN CRYOPRESERVED MOUSE EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 191
Author(s):  
A.R.S. Coutinho ◽  
A.B. Nascimeto ◽  
C.M. Mendes ◽  
R. Simoes ◽  
C.F. Lucio ◽  
...  
Keyword(s):  
Cell Death ◽  
Liquid Nitrogen ◽  
Chromatin Condensation ◽  
Membrane Integrity ◽  
Embryonic Cell ◽  
Slow Freezing ◽  
Cell Membrane Integrity ◽  
Frozen Embryos ◽  
Mammalian Embryos ◽  
Cryopreserved Embryos

Cryopreservation of mammalian embryos is an important tool for the application of reproductive biotechnology. Recent evidence indicates that apoptosis may be important in determining the viability of cryopreserved embryos. Our goal was to detect apoptosis and characterize and quantify the embryonic cell death caused by cryopreservation. Mouse morulae were collected, selected, and separated into three groups: fresh, slow-freezing, and vitrification. In the slow-freezing procedure, embryos were exposed to 10% ethylene glycol (EG) for 10 min. After loading, the straws were placed into methanol at −7°C for 5 min, seeded and after 5 min cooled at 0.5°C/minute. After 10 minutes at −31°C, straws were plunged into and stored in liquid nitrogen. Slow-frozen straws were thawed in air for 10 s, and then immersed in a 25°C water bath for 20 s. Embryos were vitrified by exposing them to 10% and 20% EG for 5 min followed by 40% EG + 18% Ficoll + 10% sucrose (EFS) for 30 s and the 0.25-mL straws then plunged into and stored in liquid nitrogen. The vitrified straws were warmed by immersing them in 25°C water for 20 s. Cell membrane integrity was assessed by Hoechst and propidium iodide double staining (H/PI). Fresh and thawed embryos were scored (following IETS recommendations) and then fixed after 30 min in PBS + 10% FCS. Morphology and apoptosis were assessed with Haematoxylin-Eosin staining (HE) and by electron microscopy (MET). The number of Grade I embryos recovered after thawing was higher for slow-frozen embryos (61.5%) than vitrified embryos (29.5%). H/PI detected more membrane permeability in the vitrified embryos (69.7%), than in the slow-frozen (48.4%) or non-frozen (13.8%) groups (P < 0,05, Wilcoxon's test). Nuclear evaluation by HE revealed that vitrification and slow-freezing induced pyknosis and chromatin condensation. Mitotic pattern was observed in the fresh and slow-frozen group, but not in vitrification group suggesting that the embryos were either not randomly allocated to the groups or not-treated and fixed at the same age, or that vitrification changed the nuclear status of the embryos. HE staining revealed weakly staining cytoplasm and degenerated cells in the vitrification group (indicating oncosis), while in the slow-frozen group the presence of cytoplasmic condensation and eosinophilic structures indicated apoptosis. The ultrastructure examination confirmed the HE observations. In conclusion, the results demonstrated that staining with HE allows detection of oncosis and apoptosis in cryopreserved embryos. According to these data, vitrification caused more cellular injuries than slow-freezing, and oncosis was the predominant injury. It is important to point that specific molecular apoptosis tests must be performed to confirm these results. This work was supported by FAPESP 04/01252-4.

Involvement of ICE family proteases in apoptosis induced by reoxygenation of hypoxic hepatocytes

1996 ◽  
Vol 271 (6) ◽  
pp. G949-G958 ◽  
Author(s):  
S. Shimizu ◽  
Y. Eguchi ◽  
W. Kamiike ◽  
Y. Akao ◽  
H. Kosaka ◽  
...  
Keyword(s):  
Cell Death ◽  
Chromatin Condensation ◽  
Interleukin 1 ◽  
Membrane Integrity ◽  
Morphological Characteristics ◽  
Electrophoretic Analysis ◽  
Dna Fragments ◽  
Electron Microscopic ◽  
Triggering Factor ◽  
Induced Apoptosis

Cell death due to reoxygenation after hypoxia was characterized in primary cultured hepatocytes. Fluorescence and electron microscopic analyses of reoxygenated hepatocytes revealed morphological characteristics of apoptosis, including chromatin condensation, nuclear fragmentation, and formation of apoptotic bodies. Few necrotic hepatocytes, defined by loss of plasma membrane integrity, mitochondrial swelling, and formation of large vacuoles, were observed. Activation of interleukin-1 beta-converting enzyme (ICE)-like and CPP32/Yama-like proteases, which are known to drive apoptosis, was observed during reoxygenation, and addition of their respective inhibitors inhibited the induction of apoptosis, indicating the involvement of ICE family proteases in apoptosis by reoxygenation. Production of oxygen radicals was enhanced by reoxygenation of hypoxic cells, and reoxygenation-induced apoptosis was inhibited by oxygen radical scavengers, suggesting a role for reactive oxygen species as a triggering factor in cell death. Electrophoretic analysis revealed the presence of 50-kb DNA fragments but not oligonucleosomal DNA fragments in reoxygenation-induced apoptotic hepatocytes.

Absence of temporal ordering of apoptotic features in heat-shock treated leukemia and lymphoma cell lines

1996 ◽  
Vol 54 ◽  
pp. 758-759
Author(s):  
D. W. Fairbain ◽  
M.D. Standing ◽  
K.L. O'Neill
Keyword(s):  
Heat Shock ◽  
Cell Lines ◽  
Chromatin Condensation ◽  
Membrane Integrity ◽  
Resistant Cell ◽  
Membrane Blebbing ◽  
Late Loss ◽  
Induced Apoptosis ◽  
Dying Cells ◽  
In Cells

Apoptosis is a genetically defined response to physiological stimuli that results in cellular suicide. Features common to apoptotic cells include chromatin condensation, oligonucleosomal DNA fragmentation, membrane blebbing, nuclear destruction, and late loss of ability to exclude vital dyes. These characteristics contrast markedly from pathological necrosis, in which membrane integrity loss is demonstrated early, and other features of apoptosis, which allow a non-inflammatory removal of dead and dying cells, are absent. Using heat shock-induced apoptosis as a model for examining stress response in cells, we undertook to categorize a variety of human leukemias and lymphomas with regard to their response to heat shock. We were also interested in determining whether a common temporal order was followed in cells dying by apoptosis. In addition, based on our previous results, we investigated whether increasing heat load resulted in increased apoptosis, with particular interest in relatively resistant cell lines, or whether the mode of death changed from apoptosis to necrosis.

Quercetin in attenuation of ischemic/reperfusion injury: A review

2020 ◽  
Vol 13 ◽  
Author(s):  
Milad Ashrafizadeh ◽  
Saeed Samarghandian ◽  
Kiavash Hushmandi ◽  
Amirhossein Zabolian ◽  
Md Shahinozzaman ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Membrane ◽  
Blood Supply ◽  
Apoptotic Cell ◽  
Ischemia Reperfusion ◽  
Membrane Integrity ◽  
Therapeutic Effects ◽  
Molecular Pathways ◽  
Cell Membrane Integrity ◽  
Cell Membrane Disruption

Background: Ischemia/reperfusion (I/R) injury is a serious pathologic event that occurs due to restriction in blood supply to an organ, followed by hypoxia. This condition leads to enhanced levels of pro-inflammatory cytokines such as IL-6 and TNF-, and stimulation of oxidative stress via enhancing reactive oxygen species (ROS) levels. Upon reperfusion, blood supply increases, but it deteriorates condition, and leads to generation of ROS, cell membrane disruption and finally, cell death. Plant derived-natural compounds are well-known due to their excellent antioxidant and anti-inflammatory activities. Quercetin is a flavonoid exclusively found in different vegetables, herbs, and fruits. This naturally occurring compound possesses different pharmacological activities making it appropriate option in disease therapy. Quercetin can also demonstrate therapeutic effects via affecting molecular pathways such as NF-B, PI3K/Akt and so on. Methods: In the present review, we demonstrate that quercetin administration is beneficial in ameliorating I/R injury via reducing ROS levels, inhibition of inflammation, and affecting molecular pathways such as TLR4/NF-B, MAPK and so on. Results and conclusion: Quercetin can improve cell membrane integrity via decreasing lipid peroxidation. Apoptotic cell death is inhibited by quercetin via down-regulation of Bax, and caspases, and upregulation of Bcl-2. Quercetin is able to modulate autophagy (inhibition/induction) in decreasing I/R injury. Nanoparticles have been applied for delivery of quercetin, enhancing its bioavailability and efficacy in alleviation of I/R injury. Noteworthy, clinical trials have also confirmed the capability of quercetin in reducing I/R injury.

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