Cytotoxic potency of H2O2 in cell cultures: Impact of cell concentration and exposure time

Gülden, Michael; Jess, Anne; Kammann, Julia; Maser, Edmund; Seibert, Hasso

doi:10.1016/j.freeradbiomed.2010.07.015

Cited by 186 publications

(180 citation statements)

References 34 publications

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“…Because the extent of oxidative stress depends on cellular antioxidant capacity, we first determined the variance resulting from differences in plated cell numbers and antioxidant enzyme distribution between astrocytes and microglia. In line with previous reports (43,44), the total antioxidant capacities of the two cell types did not differ significantly (data not shown). Thus, difference in the cellular distribution of enzymes involved in phosphorylation/dephosphorylation and redox signaling between the two cell types might be responsible for this differential sensitivity.…”

Section: Discussionsupporting

confidence: 93%

“…The decrease in the extent of phosphorylation evident at later times was attributable to destruction of H 2 O 2 by astroglial cells. Exogenously added H 2 O 2 is taken up by such cells via a process characterized by first-order kinetics, with the time taken to achieve 50% of the ultimate uptake depending on both cell type and cell numbers (43,44). To minimize the impact of astroglial uptake and destruction of H 2 O 2 on peroxide levels, as well as to exclude indirectly propagating signals, we chose to confine the H 2 O 2 (0.5 mM) treatment time to only 10 min; the peroxide was then washed away.…”

Section: Discussionmentioning

confidence: 99%

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Astrocytes, but Not Microglia, Rapidly Sense H2O2 via STAT6 Phosphorylation, Resulting in Cyclooxygenase-2 Expression and Prostaglandin Release

Park

Lee

Kim

et al. 2012

The Journal of Immunology

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Emerging evidence has established that astrocytes, once considered passive supporting cells that maintained extracellular ion levels and served as a component of the blood–brain barrier, play active regulatory roles during neurogenesis and in brain pathology. In the current study, we demonstrated that astrocytes sense H2O2 by rapidly phosphorylating the transcription factor STAT6, a response not observed in microglia. STAT6 phosphorylation was induced by generators of other reactive oxygen species (ROS) and reactive nitrogen species, as well as in the reoxygenation phase of hypoxia/reoxygenation, during which ROS are generated. Src–JAK pathways mediated STAT6 phosphorylation upstream. Experiments using lipid raft disruptors and analyses of detergent-fractionated cells demonstrated that H2O2-induced STAT6 phosphorylation occurred in lipid rafts. Under experimental conditions in which H2O2 did not affect astrocyte viability, H2O2-induced STAT6 phosphorylation resulted in STAT6-dependent cyclooxygenase-2 expression and subsequent release of PGE2 and prostacyclin, an effect also observed in hypoxia/reoxygenation. Finally, PGs released from H2O2-stimulated astrocytes inhibited microglial TNF-α expression. Accordingly, our results indicate that ROS-induced STAT6 phosphorylation in astrocytes can modulate the functions of neighboring cells, including microglia, through cyclooxygenase-2 induction and subsequent release of PGs. Differences in the sensitivity of STAT6 in astrocytes (highly sensitive) and microglia (insensitive) to phosphorylation following brief exposure to H2O2 suggest that astrocytes can act as sentinels for certain stimuli, including H2O2 and ROS, refining the canonical notion that microglia are the first line of defense against external stimuli.

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Section: Discussionsupporting

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Astrocytes, but Not Microglia, Rapidly Sense H2O2 via STAT6 Phosphorylation, Resulting in Cyclooxygenase-2 Expression and Prostaglandin Release

Park

Lee

Kim

et al. 2012

The Journal of Immunology

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“…H 2 O 2 has been extensively used to study the effects of antioxidant and cytoprotective role of phytochemicals (Gulden et al 2010;Brenner et al 2010). Hence, the cytoprotective propensity of BME has been evaluated against H 2 O 2 induced oxidative stress in PC12 and L132 cells.…”

Section: Discussionmentioning

confidence: 99%

“…H 2 O 2 is a well known potent inducer of ROS capable of inducing cell injury both in vitro and in vivo (Kim et al 2012;Terashvili et al 2012). Studies have shown that H 2 O 2 is known to induce oxidative damage, leading to lipid peroxidation, ROS generation, GSH depletion and reduction in mitochondrial membrane potential, antioxidant enzymes (SOD, catalase and GPx) activity preceding cell death (Chan et al 2009;Brenner et al 2010;Gulden et al 2010).…”

Section: Discussionmentioning

confidence: 99%

Cytoprotective propensity of Bacopa monniera against hydrogen peroxide induced oxidative damage in neuronal and lung epithelial cells

2014

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Hydrogen peroxide (H 2 O 2 ), a major reactive oxygen species (ROS) produced during oxidative stress, is toxic to the cells. Hence, H 2 O 2 has been extensively used to study the effects of antioxidant and cytoprotective role of phytochemicals. In the present investigation H 2 O 2 was used to induce oxidative stress via ROS production within PC12 and L132 cells. Cytoprotective propensity of Bacopa monniera extract (BME) was confirmed by cell viability assays, ROS estimation, lipid peroxidation, mitochondria membrane potential assay, comet assay followed by gene expression studies of antioxidant enzymes in PC12 and L132 cells treated with H 2 O 2 for 24 h with or without BME pre-treatment. Our results elucidate that BME possesses radical scavenging activity by scavenging 2,2-diphenyl-1-picrylhydrazyl, 2,2 0 -azinobis(3-ethylbenzothiazoline-6-sulphonic acid), superoxide radical, and nitric oxide radicals. The IC 50 value of BME against these radicals was found to be 226.19, 15.17, 30.07, and 34.55 lg/ml, respectively). The IC 50 of BME against ROS, lipid peroxidation and protein carbonylation was found to be 1296.53, 753.22, and 589.04 lg/ml in brain and 1137.08, 1079.65, and 11101.25 lg/ml in lung tissues, respectively. Further cytoprotective potency of the BME ameliorated the mitochondrial and plasma membrane damage induced by H 2 O 2 as evidenced by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase leakage assays in both PC12 and L132 cells. H 2 O 2 induced cellular, nuclear and mitochondrial membrane damage was restored by BME pre-treatment. H 2 O 2 induced depleted antioxidant status was also replenished by BME pre-treatment. This was confirmed by spectrophotometric analysis, semi-quantitative RT-PCR and western blot studies. These results justify the traditional usage of BME based on its promising antioxidant and cytoprotective property.

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“…In addition to the experimental setup itself, the compound's dynamics also can influence the system's kinetics: compounds with a high reactivity can react with a cellular component, causing an immediate effect on or in the cells and thereby leading to a decrease in the compound's concentration (Gülden et al, 2010).…”

Section: Adversity Versus Adaptationmentioning

confidence: 99%

The use of biomarkers of toxicity for integrating in vitro hazard estimates into risk assessment for humans

Blaauboer

Boekelheide

Clewell

et al. 2012

ALTEX

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SummaryThe role that in vitro systems can play in toxicological risk assessment is determined by the appropriateness of the chosen methods, with respect to the way in which in vitro data can be extrapolated to the in vivo situation. This report presents the results of a workshop aimed at better defining the use of in vitro-derived biomarkers of toxicity (BoT) and determining the place these data can have in human risk assessment. As a result, a conceptual framework is presented for the incorporation of in vitro-derived toxicity data into the risk assessment process. The selection of BoT takes into account that they need to distinguish adverse and adaptive changes in cells. The framework defines the place of in vitro systems in the context of data on exposure, structural and physico-chemical properties, and toxicodynamic and biokinetic modeling. It outlines the determination of a proper point-of-departure (PoD) for in vitro-in vivo extrapolation, allowing implementation in risk assessment procedures. A BoT will need to take into account both the dynamics and the kinetics of the compound in the in vitro systems. For the implementation of the proposed framework it will be necessary to collect and collate data from existing literature and new in vitro test systems, as well as to categorize biomarkers of toxicity and their relation to pathways-of-toxicity. Moreover, data selection and integration need to be driven by their usefulness in a quantitative in vitro-in vivo extrapolation (QIVIVE). Keywords: biomarker of toxicity, integrated testing strategies, quantitative in vitro-in vivo extrapolations

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Cytotoxic potency of H2O2 in cell cultures: Impact of cell concentration and exposure time

Cited by 186 publications

References 34 publications

Astrocytes, but Not Microglia, Rapidly Sense H2O2 via STAT6 Phosphorylation, Resulting in Cyclooxygenase-2 Expression and Prostaglandin Release

Astrocytes, but Not Microglia, Rapidly Sense H2O2 via STAT6 Phosphorylation, Resulting in Cyclooxygenase-2 Expression and Prostaglandin Release

Cytoprotective propensity of Bacopa monniera against hydrogen peroxide induced oxidative damage in neuronal and lung epithelial cells

The use of biomarkers of toxicity for integrating in vitro hazard estimates into risk assessment for humans

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