Exposing cells to H2O2: A quantitative comparison between continuous low-dose and one-time high-dose treatments

Sobotta, Mirko C.; Barata, Ana G.; Schmidt, Ulrich; Mueller, Sebastian; Millonig, Gunda; Dick, Tobias P.

doi:10.1016/j.freeradbiomed.2013.02.017

Cited by 96 publications

(78 citation statements)

References 27 publications

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“…We have used this approach because we believe that such delivery of H 2 O 2 is more relevant from a pathophysiological standpoint, than sudden 'bursts' of authentic extracellular H 2 O 2 [44,45]. According to prior studies, low concentrations of glucose oxidase induce secondary alterations in intracellular pathways, including depletion of intracellular antioxidants, deterioration of mitochondrial membrane potential, and the impairment of mitochondrial electron transport, culminating in a secondary production of ROS from endogenous sources [46–49].…”

Section: Discussionmentioning

confidence: 99%

AP39, a novel mitochondria-targeted hydrogen sulfide donor, stimulates cellular bioenergetics, exerts cytoprotective effects and protects against the loss of mitochondrial DNA integrity in oxidatively stressed endothelial cells in vitro

et al. 2014

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The purpose of the current study was to investigate the effect of the recently synthesized mitochondrially-targeted H2S donor, AP39 [10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol-5yl)phenoxy)decyl) triphenylphosphonium bromide], on bioenergetics, viability, and mitochondrial DNA integrity in bEnd.3 murine microvascular endothelial cells in vitro, under normal conditions, and during oxidative stress. Intracellular H2S was assessed by the fluorescent dye 7-azido-4-methylcoumarin. For the measurement of bioenergetic function, the XF24 Extracellular Flux Analyzer was used. Cell viability was estimated by the combination of the MTT and LDH methods. Oxidative protein modifications were measured by the Oxyblot method. Reactive oxygen species production was monitored by the MitoSOX method. Mitochondrial and nuclear DNA integrity were assayed by the Long Amplicon PCR method. Oxidative stress was induced by addition of glucose oxidase. AP39 (30 – 300 nM) to bEnd.3 cells increased intracellular H2S levels, with a preferential response in the mitochondrial regions. AP39 exerted a concentration-dependent effect on mitochondrial activity, which consisted of a stimulation of mitochondrial electron transport and cellular bioenergetic function at lower concentrations (30–100 nM) and an inhibitory effect at the higher concentration of 300 nM. Under oxidative stress conditions induced by glucose oxidase, an increase in oxidative protein modification and an enhancement in MitoSOX oxidation was noted, coupled with an inhibition of cellular bioenergetic function and a reduction in cell viability. AP39 pretreatment attenuated these responses. Glucose oxidase induced a preferential damage to the mitochondrial DNA; AP39 (100 nM) pretreatment protected against it. In conclusion, the current paper documents antioxidant and cytoprotective effects of AP39 under oxidative stress conditions, including a protection against oxidative mitochondrial DNA damage.

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Section: Discussionmentioning

confidence: 99%

AP39, a novel mitochondria-targeted hydrogen sulfide donor, stimulates cellular bioenergetics, exerts cytoprotective effects and protects against the loss of mitochondrial DNA integrity in oxidatively stressed endothelial cells in vitro

et al. 2014

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“…The wells were treated with 2.5 ml of 10 lM PL, PEITC, or 100 lM of BSO for a period of 10 h. After treatment, the cells were trypsinized, washed once with phosphate-buffered saline (PBS), followed by resuspension in 2 ml of 100 mM MMTS to convert sulfhydryl groups to -S-CH 3 . to prevent oxidation artifacts (44). The MMTS cell suspension was placed on ice for 20 min, followed by two more PBS washes.…”

Section: Immunoblotting For Prx-2 Oxidationmentioning

confidence: 99%

Using Sensors and Generators of H₂O₂to Elucidate the Toxicity Mechanism of Piperlongumine and Phenethyl Isothiocyanate

Huang

Langford

Sikes

2016

Antioxidants & Redox Signaling

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Aims: Chemotherapeutics target vital functions that ensure survival of cancer cells, including their increased reliance on defense mechanisms against oxidative stress compared to normal cells. Many chemotherapeutics exploit this vulnerability to oxidative stress by elevating the levels of intracellular reactive oxygen species (ROS). A quantitative understanding of the oxidants generated and how they induce toxicity will be important for effective implementation and design of future chemotherapeutics. Molecular tools that facilitate measurement and manipulation of individual chemical species within the context of the larger intracellular redox network present a means to develop this understanding. In this work, we demonstrate the use of such tools to elucidate the roles of H 2 O 2 and glutathione (GSH) in the toxicity mechanism of two ROS-based chemotherapeutics, piperlongumine and phenethyl isothiocyanate. Results: Depletion of GSH as a result of treatment with these compounds is not an important part of the toxicity mechanisms of these drugs and does not lead to an increase in the intracellular H 2 O 2 level. Measuring peroxiredoxin-2 (Prx-2) oxidation as evidence of increased H 2 O 2 , only piperlongumine treatment shows elevation and it is GSH independent. Using a combination of a sensor (HyPer) along with a generator (D-amino acid oxidase) to monitor and mimic the drug-induced H 2 O 2 production, it is determined that H 2 O 2 produced during piperlongumine treatment acts synergistically with the compound to cause enhanced cysteine oxidation and subsequent toxicity. The importance of H 2 O 2 elevation in the mechanism of piperlongumine promotes a hypothesis of why certain cells, such as A549, are more resistant to the drug than others. Innovation and Conclusion: The approach described herein sheds new light on the previously proposed mechanism of these two ROS-based chemotherapeutics and advocates for the use of both sensors and generators of specific oxidants to isolate their effects. Antioxid. Redox Signal. 24, 924-938.

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“…This system has been used to induce hypoxia in solutions and in microfluidic devices (Askoxylakis et al, 2011;Baumann et al, 2008;Huang et al, 2013;Li et al, 2016;Millonig et al, 2009;Mueller et al, 2009;Rajan et al, 2013;Sobotta et al, 2013;Zitta et al, 2012). The use of GOX/CAT is beneficial in that the system provides a rapid onset of hypoxia (usually within a few minutes) (Askoxylakis et al, 2011;Baumann et al, 2008;Huang et al, 2013;Li et al, 2016;Millonig et al, 2009;Mueller et al, 2009;Rajan et al, 2013;Sobotta et al, 2013;Zitta et al, 2012). One drawback to any GOX system, however, is the production of hydrogen peroxide, a reactive oxygen species (ROS) (Fruehauf and Meyskens, 2007) whose accumulation would not only cause undesired cellular response but also inactivate both GOX and CAT (Hielscher and Gerecht, 2015;Pal et al, 2000;Trachootham et al, 2009;Tse and Gough, 1987).…”

Section: Introductionmentioning

confidence: 99%

“…Some recent work has started to explore the ability of GOX/CAT reactions to induce hypoxia for in vitro cell culture (Askoxylakis et al, 2011;Baumann et al, 2008;Huang et al, 2013;Li et al, 2016;Millonig et al, 2009;Mueller et al, 2009;Rajan et al, 2013;Sobotta et al, 2013). The GOX/CAT system has also been adapted to 3D printed inserts (Li et al, 2016) where GOX and CAT were coated on printed disks and the degrees of solution hypoxia were controlled by the distance between the enzyme-immobilized disks and the solution in the culture plate.…”

Section: Introductionmentioning

confidence: 99%

Enzyme-immobilized hydrogels to create hypoxia for in vitro cancer cell culture

Dawes

König

Lin

2017

Journal of Biotechnology

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Hypoxia is a critical condition governing many aspects of cellular fate processes. The most common practice in hypoxic cell culture is to maintain cells in an incubator with controlled gas inlet (i.e., hypoxic chamber). Here, we describe the design and characterization of enzymeimmobilized hydrogels to create solution hypoxia under ambient conditions for in vitro cancer cell culture. Specifically, glucose oxidase (GOX) was acrylated and co-polymerized with poly(ethylene glycol)-diacrylate (PEGDA) through photopolymerization to form GOXimmobilized PEG-based hydrogels. We first evaluated the effect of soluble GOX on inducing solution hypoxia (O 2 < 5%) and found that both unmodified and acrylated GOX could sustain hypoxia for at least 24 hours even under ambient air condition with constant oxygen diffusion from the air-liquid interface. However, soluble GOX gradually lost its ability to sustain hypoxia after 24 hours due to the loss of enzyme activity over time. On the other hand, GOXimmobilized hydrogels were able to create hypoxia within the hydrogel for at least 120 hours, potentially due to enhanced protein stabilization by enzyme 'PEGylation' and immobilization. As a proof-of-concept, this GOX-immobilized hydrogel system was used to create hypoxia for in vitro culture of Molm14 (acute myeloid leukemia (AML) cell line) and Huh7 (hepatocellular carcinoma (HCC) cell line). Cells cultured in the presence of GOX-immobilized hydrogels remained viable for at least 24 hours. The expression of hypoxia associated genes, including carbonic anhydrase 9 (CA9) and lysyl oxidase (LOX), were significantly upregulated in cells cultured with GOX-immobilized hydrogels. These results have demonstrated the potential of using enzyme-immobilized hydrogels to create hypoxic environment for in vitro cancer cell culture.

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Exposing cells to H2O2: A quantitative comparison between continuous low-dose and one-time high-dose treatments

Cited by 96 publications

References 27 publications

AP39, a novel mitochondria-targeted hydrogen sulfide donor, stimulates cellular bioenergetics, exerts cytoprotective effects and protects against the loss of mitochondrial DNA integrity in oxidatively stressed endothelial cells in vitro

AP39, a novel mitochondria-targeted hydrogen sulfide donor, stimulates cellular bioenergetics, exerts cytoprotective effects and protects against the loss of mitochondrial DNA integrity in oxidatively stressed endothelial cells in vitro

Using Sensors and Generators of H₂O₂to Elucidate the Toxicity Mechanism of Piperlongumine and Phenethyl Isothiocyanate

Enzyme-immobilized hydrogels to create hypoxia for in vitro cancer cell culture

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Exposing cells to H2O2: A quantitative comparison between continuous low-dose and one-time high-dose treatments

Cited by 96 publications

References 27 publications

AP39, a novel mitochondria-targeted hydrogen sulfide donor, stimulates cellular bioenergetics, exerts cytoprotective effects and protects against the loss of mitochondrial DNA integrity in oxidatively stressed endothelial cells in vitro

AP39, a novel mitochondria-targeted hydrogen sulfide donor, stimulates cellular bioenergetics, exerts cytoprotective effects and protects against the loss of mitochondrial DNA integrity in oxidatively stressed endothelial cells in vitro

Using Sensors and Generators of H2O2to Elucidate the Toxicity Mechanism of Piperlongumine and Phenethyl Isothiocyanate

Enzyme-immobilized hydrogels to create hypoxia for in vitro cancer cell culture

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Using Sensors and Generators of H₂O₂to Elucidate the Toxicity Mechanism of Piperlongumine and Phenethyl Isothiocyanate