[12] In Vivo visualization of oxygen radical-dependent photoemission

doi:10.1016/S0076-6879(94)33015-8

Methods in Enzymology

Volume 233, 1994, Pages 128-140

https://doi.org/10.1016/S0076-6879(94)33015-8 Get rights and content

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Oxygen radicals are mediators of tissue injury in a wide variety of pathophysiological conditions. These active oxygen metabolites can interact with cellular membrane structures, thereby inducing lipid peroxidation, which has been implicated as one of the major mechanisms of “oxidative stress” in tissue injury. This chapter presents evidence illustrating topographic correlation between oxidative changes and distribution of cell injury. It remains unclear whether in-site oxidant production may play a causal role in the same portion of cell injury, or whether malondialdehyde elevation or decrease in reduced glutathione contents in tissue may be merely a consequence of tissue injury that may result from oxidant-independent mechanisms. The chapter discusses current methodology to monitor visually spatial and temporal alterations of oxidative changes in addition to monitoring simultaneous cell function and viability in in vivo or ex vivo organ microcirculation. The chemiluminescence imaging method has several properties that prove useful for evaluation of oxidative stress in vivo: (1) the absence of an artificial photobleaching effect, (2) no need for excitation illumination, resulting in attenuation of cellular damages, and (3) the availability of different chemilumigenic probes, which makes it possible to pinpoint the active oxidants that are involved in different experimental models.

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Increase of reactive oxygen species generation in cerebral cortex slices after the transiently enhanced metabolic activity
2017, Neuroscience Research
Citation Excerpt :
This redox balance shift towards reduction during the high-potassium treatment was restored to its control-potassium condition (Fig. 7). In order to monitor ROS generation in living tissues and cells, optical imaging techniques have been investigated using fluorescence (Tsuchiya et al., 1994; Buxser et al., 1999) and chemiluminescent probes (Faulkner and Fridovich, 1993; Schreiber et al., 1995; Dirnagl et al., 1995; Daiber et al., 2004). However, only a few topographical investigations have been conducted on living animal skin and livers (Tsuchiya et al., 1994), isolated hearts (Näpänkangas et al., 2012), and brain tissue (Schreiber et al., 1995; Sasaki et al., 2006, 2008).
Under certain conditions such as hypoxia-reoxygenation, the generation of reactive oxygen species (ROS) increases following hypoxia caused by a decreased oxygen supply. As another hypoxic condition, an excess neural activity status including epileptic seizure induces a decrease in tissue oxygen partial pressure (pO₂) caused by enhanced oxygen utilization; however, whether ROS generation increases following the hypoxic status induced by transiently enhanced energy metabolism in brain tissue currently remains unknown. We herein investigated ROS-dependent chemiluminescence in cerebral cortex slices during the restoration of transiently enhanced energy metabolism induced by a high-potassium treatment with tissue pO₂ changes and redox balance. ROS generation in the tissue was enhanced after high-potassium-induced hypoxia, but not by the reversed order of the treatment: control-potassium then high-potassium treatment, high-potassium treatment alone, and control-potassium treatment alone. The high-potassium treatment induced a transient decrease in tissue pO₂ and a shift in the tissue redox balance towards reduction. The transient shift in the tissue redox balance towards reduction with enhanced metabolic activity and its recovery may correlate with ROS generation. This phenomenon may mimic ROS generation following the hypoxic status induced by transiently enhanced energy metabolism.
Heme deficiency sensitizes yeast cells to oxidative stress induced by hydroxyurea
2017, Journal of Biological Chemistry
Hydroxyurea (HU) has a long history of clinical and scientific use as an antiviral, antibacterial, and antitumor agent. It inhibits ribonucleotide reductase and reversibly arrests cells in S phase. However, high concentrations or prolonged treatment with low doses of HU can cause cell lethality. Although the cytotoxicity of HU may significantly contribute to its therapeutic effects, the underlying mechanisms remain poorly understood. We have previously shown that HU can induce cytokinesis arrest in the erg11-1 mutant of fission yeast, which has a partial defect in the biosynthesis of fungal membrane sterol ergosterol. Here, we report the identification of a new mutant in heme biosynthesis, hem13-1, that is hypersensitive to HU. We found that the HU hypersensitivity of the hem13-1 mutant is caused by oxidative stress and not by replication stress or a defect in cellular response to replication stress. The mutation is hypomorphic and causes heme deficiency, which likely sensitizes the cells to the HU-induced oxidative stress. Because the heme biosynthesis pathway is highly conserved in eukaryotes, this finding, as we show in our separate report, may help to expand the therapeutic spectrum of HU to additional pathological conditions.
Synchronism in mitochondrial ROS flashes, membrane depolarization and calcium sparks in human carcinoma cells
2017, Biochimica et Biophysica Acta - Bioenergetics
Mitochondria are major producers of reactive oxygen species (ROS) in many cells including cancer cells. However, complex interrelationships between mitochondrial ROS (mitoROS), mitochondrial membrane potential (ΔΨm) and Ca^2 + are not completely understood. Using human carcinoma cells, we further highlight biphasic ROS dynamics: - gradual mitoROS increase followed by mitoROS flash. Also, we demonstrate heterogeneity in rates of mitoROS generation and flash initiation time. Comparing mitochondrial and near-extra-mitochondrial signals, we show that mechanisms of mitoROS flashes in single mitochondria, linked to mitochondrial permeability transition pore opening (ΔΨm collapse) and calcium sparks, may involve flash triggering by certain levels of external ROS released from the same mitochondria. In addition, mitochondria-mitochondria interactions can produce wave propagations of mitoROS flashes and ΔΨm collapses in cancer cells similar to phenomena of ROS-induced ROS release (RIRR). Our data suggest that in cancer cells RIRR, activation of mitoROS flashes and mitochondrial depolarization may involve participation of extramitochondrial-ROS produced either by individual mitochondria and/or by neighboring mitochondria. This could represent general mechanisms in ROS-ROS signaling with suggested role in both mitochondrial and cellular physiology and signaling.
Enhanced anti-oxidative activity and lignocellulosic ethanol production by biotin addition to medium in Pichia guilliermondii fermentation
2015, Bioresource Technology
Commercialization of lignocellulosic ethanol fermentation requires its high titer, but the reactive oxygen species (ROS) accumulation during the bioprocess damaged the cells and compromised this goal. To improve the cellular anti-oxidative activity during non-detoxified corncob residue hydrolysate fermentation, seed cells were prepared to possess a higher level of intracellular biotin pool (IBP), which facilitated the biosyntheses of catalase and porphyrin. As a result, the catalase activity increased by 1.3-folds compared to control while the ROS level reduced by 50%. Cell viability in high-IBP cells was 1.7-folds of control and the final ethanol titer increased from 31.2 to 41.8 g L⁻¹ in batch fermentation. The high-IBP cells were further used for repeated-batch fermentation in the non-detoxified lignocellulosic hydrolysate, and the highest titer and average productivity of ethanol reached 63.7 g L⁻¹ and 1.2 g L⁻¹ h⁻¹. The results were favorable to future industrial application of this lignocellulosic bioethanol process.
Superoxide generation in different brain regions of rats during normoxia and hypoxia-reoxygenation
2012, Neuroscience Research
Citation Excerpt :
Topographic distribution of reactive oxygen species (ROS) has been investigated by photonic (Chen et al., 2003; Frank et al., 2000; Tsuchiya et al., 1994) and electron spin resonance (Berliner et al., 2001) computed tomography techniques; however, the ESR technique is still under development, and the photonic technique has been used for detecting the ROS produced by enzymatic and cellular systems, because ROS are highly reactive and have very short life in vivo (Sasaki, 2004).
The superoxide-dependent chemiluminescent intensity in different brain regions was examined in ex vivo tissue slices of rat brain during normoxia and hypoxia-reoxygenation with lucigenin. The chemiluminescent intensity increased during reoxygenation after hypoxic treatment. There was a higher level of chemiluminescent intensity in the hippocampus during normoxia, and a lower level in the white matter during normoxia and hypoxia-reoxygenation. A weak correlation was found between the chemiluminescent intensity and the glucose uptake rate during normoxia. Then we examined whether hypoxic strength correlates to superoxide generation. The chemiluminescent intensity increased in a hypoxic strength-dependent manner. The generation mechanism of superoxide was examined using carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, genipin, an inhibitor for uncoupling protein-2, alloprinol, a xanthine oxidase inhibitor, or apocynin, an NADPH oxidase inhibitor. The chemiluminescent signal was significantly inhibited by CCCP under normoxic condition and enhanced by genipin during normoxia and hypoxia-reoxygenation, but not by allopurinol or apocynin.
These results suggest that superoxide generation is high in the hippocampus during normoxia and low in the white matter during normoxia and hypoxia-reoxygenation, superoxide generation in the hypoxia-reoxygenation brain correlates with the strength of hypoxia influenced by oxygen delivery, and mitochondrion is the major sites of intracellular superoxide generation.
Studies on the photosensitized reduction of resorufin and implications for the detection of oxidative stress with Amplex Red
2011, Free Radical Biology and Medicine
The photosensitized reduction of resorufin (RSF), the fluorescent product of Amplex Red, was investigated using electron spin resonance (ESR), optical absorption/fluorescence, and oxygen consumption measurements. Anaerobic reaction of RSF in the presence of the electron donor reduced nicotinamide adenine dinucleotide (NADH) demonstrated that during visible light irradiation (λ > 300 nm), RSF underwent one-electron reduction to produce a semiquinoneimine-type anion radical (RSF^• ‾) as demonstrated by direct ESR. Spin-trapping studies of incubations containing RSF, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and NADH demonstrated, under irradiation with visible light, the production of the superoxide dismutase (SOD)-sensitive DMPO/^•OOH adduct. Both absorption and fluorescence spectra of RSF in the presence of NADH demonstrated that the RSF^• ‾ was further reduced during irradiation with formation of its colorless dihydroquinoneimine form, dihydroresorufin (RSFH₂). Both RSF^• ‾ and RSFH₂, when formed in an aerobic system, were immediately oxidized by oxygen, which regenerated the dye and formed superoxide. Oxygen consumption measurements with a Clark-type oxygen electrode showed that molecular oxygen was consumed in a light-dependent process. The suppression of oxygen consumption by addition of SOD or catalase further confirmed the production of superoxide and hydrogen peroxide.

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[12] In Vivo visualization of oxygen radical-dependent photoemission

Publisher Summary

Biochem. Biophys. Res. Commun.

FEBS Lett.

Anal. Biochem.

Cancer Lett.

Gastroenterology