Spatial, Temporal, and Quantitative Manipulation of Intracellular Hydrogen Peroxide in Cultured Cells

Abstract

Hydrogen peroxide (H2O2) is produced endogenously in a number of cellular compartments, including the mitochondria, the endoplasmic reticulum, peroxisomes, and at the plasma membrane, and can play divergent roles as a second messenger or a pathological toxin. It is assumed that the tuned production of H2O2 within neuronal and non-neuronal cells regulates a discreet balance between survival and death. However, a major challenge in understanding the physiological versus pathological role of H2O2 in cells has been the lack of validated methods that can spatially, temporally, and quantitatively modulate H2O2 production. A promising means of regulating endogenous H2O2 is through the expression of peroxide-producing enzyme D-amino acid oxidase (DAAO from Rhodotorula gracilis lacking a peroxisomal targeting sequence). Using viral vectors to express DAAO in distinct cell types and using targeting sequences to target DAAO to distinct subcellular sites, we can manipulate H2O2 production by applying the substrate D-alanine or permeable analogs of D-alanine. In this chapter, we describe the use of DAAO to produce H2O2 in culture models and the real-time visual validation of this technique using two-photon microscopy and chemoselective fluorescent probes.

Kinetic modeling of H2O2 dynamics in the mitochondria of HeLa cells

“…In order to experimentally investigate the trends predicted by the model, we used the genetically-encoded H 2 O 2 generator mito-DAAO, which localizes a H 2 O 2 perturbation to the mitochondrial matrix [ 10 , 70 ]. We varied the concentration of D-alanine (D-ala) substrate the cells were exposed to for up to 1 hr, then probed the Prx3 and Prx2 isoforms using redox Western blots.…”

“…It is important to note that the predicted steady states that result from this H 2 O 2 perturbation analysis are the net effect of the rate of H 2 O 2 generation by OxPhos and the additional k DAAO generation term; a higher OxPhos generation rate would result in a lower k DAAO needed to cause collapse of the Prx3 system. In order to experimentally investigate the trends predicted by the model, we used the genetically-encoded H 2 O 2 generator mito-DAAO, which localizes a H 2 O 2 perturbation to the mitochondrial matrix [10,70]. We varied the concentration of D-alanine (D-ala) substrate the cells were exposed to for up to 1 hr, then probed the Prx3 and Prx2 isoforms using redox Western blots.…”

Assessing the potential role of copper and cobalt in stimulating angiogenesis for tissue regeneration

“…Decrease of cell viability with high concentrations of ions might be due to cellular toxicity induced by reactive oxygen species (ROS). In the presence of superoxide (·O 2 -) (produced by the mitochondria during cellular respiration) or reducing agents, such as glutathione (GSH) or ascorbic acid (generally found in cells [ 22 ] and in cell culture media that we used for the experiments, respectively), Cu 2+ can be reduced to Cu + , which in turn, can catalyze the formation of hydroxyl radicals (OH · ) from hydrogen peroxide (H 2 O 2 ) (produced during normal cell metabolism [ 23 ]), through Haber-Weiss reaction [ 24 ]: …”

FLIM-Based Intracellular and Extracellular pH Measurements Using Genetically Encoded pH Sensor

“…In addition, long-wave excitation light has less scattering, lower phototoxicity, and reduced photobleaching of fluorophores due to its lower photon energy. Finally, since the required energy of excitation light is achieved in a very small focal volume, there is no fluorescent signal in the tissues located above and below the focal plane, which allows increased resolution compared to single-photon confocal microscopy [ 35 ].…”