Evaluation of fluorescent dyes for measuring intracellular glutathione content in primary cultures of human neurons and neuroblastoma SH‐SY5Y

increased ROS generation, depleted reduced glutathione, reduced GSH/GSSG ratio, and enhanced adaptive changes in the profile of the antioxidant defense system. These changes were also accompanied by mitochondrial dysfunctions, DNA damage and induction of apoptosis, accentuated activity of profibrogenic cytokine, and expression of fibronectin, the latter two being the major hallmarks of DN. These perturbations were largely blocked by various ROS inhibitors (Mito Q, diphenyleneiodonium chloride, and N-acetylcysteine) and MIOX/NOX4 siRNA. In conclusion, this study highlights a novel mechanism where MIOX under HG ambience exacerbates renal injury during the progression of diabetic nephropathy following the generation of excessive ROS via an unexplored G-X pathway.

Section: Methodsmentioning

confidence: 99%

myo-Inositol Oxygenase Overexpression Accentuates Generation of Reactive Oxygen Species and Exacerbates Cellular Injury following High Glucose Ambience

Sun

Dutta

Xie

et al. 2016

“…Detection of GSH and Apoptotic Cells-Total intracellular GSH was estimated using monochlorobimane (mBCL), which formed fluorescent adducts with GSH (42,43). Adherent cells were collected and preloaded with mBCl (40 M) in PBS for 10 min, followed by incubation with propyl iodide permeable only to dead cells.…”

Section: Methodsmentioning

confidence: 99%

Selenoprotein H Suppresses Cellular Senescence through Genome Maintenance and Redox Regulation

Cao

Chen

et al. 2014

Background: Selenoprotein H (SelH), a proposed redox-responsive DNA-binding protein, is little-studied. Results: SelH shRNA cells display severe proliferation defects and accelerated senescence with abnormal responses to DNA damage and oxidative stress. Conclusion: SelH suppresses senescence and may have important roles in gatekeeping genomic integrity. Significance: Learning how SelH keeps senescence in check is crucial for advancing our understanding linking selenium and aging intervention.

“…Changes in Intracellular Glutathione Content, GSH i -For intracellular GSH determinations, cells were preloaded for 15 min with 10 M mBCl, which forms blue fluorescent adducts with intracellular glutathione (30). Immediately prior to flow cytometry examination, PI was added.…”

Section: Methodsmentioning

confidence: 99%

Glutathione Depletion Is Necessary for Apoptosis in Lymphoid Cells Independent of Reactive Oxygen Species Formation

Franco

Panayiotidis²,

Cidlowski

2007

242

193

Changes in the intracellular redox environment of cells have been reported to be critical for the activation of apoptotic enzymes and the progression of programmed cell death. Glutathione (GSH) depletion is an early hallmark observed in apoptosis, and we have demonstrated that GSH efflux during death receptor-mediated apoptosis occurs via a GSH transporter. We now evaluate the relationship between GSH depletion, the generation of reactive oxygen species (ROS), and the progression of apoptosis. Simultaneous single cell analysis of changes in GSH content and ROS formation by multiparametric FACS revealed that loss of intracellular GSH was paralleled by the generation of different ROS including hydrogen peroxide, superoxide anion, hydroxyl radical, and lipid peroxides. However, inhibition of ROS formation by a variety of antioxidants showed that GSH loss was independent from the generation of ROS. Furthermore, GSH depletion was observed to be necessary for ROS generation. Interestingly, high extracellular thiol concentration (GSH and N-acetyl-cysteine) inhibited apoptosis, whereas, inhibition of ROS generation by other non-thiol antioxidants was ineffective in preventing cell death. Finally, GSH depletion was shown to be a necessary for the progression of apoptosis activated by both extrinsic and intrinsic signaling pathways. These results document a necessary and critical role for GSH loss in apoptosis and clearly uncouple for the first time GSH depletion from ROS formation.Apoptosis or programmed cell death is a ubiquitous, evolutionary conserved process. Under physiological conditions it is important not only in the turnover of cells in all tissues, but also during the normal development and senescence of the organism. Moreover, its deregulation has been observed to occur as either a cause or consequence of distinct pathologies (1, 2). For example, apoptosis mediated by Fas ligand (FasL) 3 receptor (Fas/CD95/Apo-1) activation is necessary for the immune system homeostasis because of its role in the rapid clearance of immunoreactive T cells maintaining the immune balance and reducing the risk of autoimmune diseases (3). Apoptosis is a highly organized process characterized by the progressive activation of precise pathways leading to specific biochemical and morphological alterations. Initial stages of apoptosis are characterized by reactive oxygen species (ROS) formation, changes in intracellular ionic homeostasis, cell shrinkage, loss of membrane lipid asymmetry, and chromatin condensation. Later stages associated with the execution phase of apoptosis are characterized by activation of execution caspases and endonucleases, apoptotic body formation, and cell fragmentation (1, 4, 5). Apoptosis has been widely reported to be modulated by changes in the redox status of the cell; however, the precise mechanisms involved are still unclear (6, 7). Reduced glutathione (GSH) is the most abundant low molecular weight thiol in animal cells and is involved in many cellular processes including antioxidant defense, drug det...