Oxidative Stress.

Oxidative stress is two sided: Whereas excessive oxidant challenge causes damage to biomolecules, maintenance of a physiological level of oxidant challenge, termed oxidative eustress, is essential for governing life processes through redox signaling. Recent interest has focused on the intricate ways by which redox signaling integrates these converse properties. Redox balance is maintained by prevention, interception, and repair, and concomitantly the regulatory potential of molecular thiol-driven master switches such as Nrf2/Keap1 or NF-κB/IκB is used for system-wide oxidative stress response. Nonradical species such as hydrogen peroxide (H2O2) or singlet molecular oxygen, rather than free-radical species, perform major second messenger functions. Chemokine-controlled NADPH oxidases and metabolically controlled mitochondrial sources of H2O2 as well as glutathione- and thioredoxin-related pathways, with powerful enzymatic back-up systems, are responsible for fine-tuning physiological redox signaling. This makes for a rich research field spanning from biochemistry and cell biology into nutritional sciences, environmental medicine, and molecular knowledge-based redox medicine.

Reactive Oxygen Species and Neutrophil Function.

Neutrophils are essential for killing bacteria and other microorganisms, and they also have a significant role in regulating the inflammatory response. Stimulated neutrophils activate their NADPH oxidase (NOX2) to generate large amounts of superoxide, which acts as a precursor of hydrogen peroxide and other reactive oxygen species that are generated by their heme enzyme myeloperoxidase. When neutrophils engulf bacteria they enclose them in small vesicles (phagosomes) into which superoxide is released by activated NOX2 on the internalized neutrophil membrane. The superoxide dismutates to hydrogen peroxide, which is used by myeloperoxidase to generate other oxidants, including the highly microbicidal species hypochlorous acid. NOX activation occurs at other sites in the cell, where it is considered to have a regulatory function. Neutrophils also release oxidants, which can modify extracellular targets and affect the function of neighboring cells. We discuss the identity and chemical properties of the specific oxidants produced by neutrophils in different situations, and what is known about oxidative mechanisms of microbial killing, inflammatory tissue damage, and signaling.

Redox metabolism: ROS as specific molecular regulators of cell signaling and function.

Redox reactions are intrinsically linked to energy metabolism. Therefore, redox processes are indispensable for organismal physiology and life itself. The term reactive oxygen species (ROS) describes a set of distinct molecular oxygen derivatives produced during normal aerobic metabolism. Multiple ROS-generating and ROS-eliminating systems actively maintain the intracellular redox state, which serves to mediate redox signaling and regulate cellular functions. ROS, in particular hydrogen peroxide (H2O2), are able to reversibly oxidize critical, redox-sensitive cysteine residues on target proteins. These oxidative post-translational modifications (PTMs) can control the biological activity of numerous enzymes and transcription factors (TFs), as well as their cellular localization or interactions with binding partners. In this review, we describe the diverse roles of redox regulation in the context of physiological cellular metabolism and provide insights into the pathophysiology of diseases when redox homeostasis is dysregulated.

Direct electrochemical evidence suggests that aqueous microdroplets spontaneously produce hydrogen peroxide.

Recent reports have detailed the striking observation that electroactive molecules, such as hydrogen peroxide (H2O2) and radical water species (H2O.+/H2O.-), are spontaneously produced in aqueous microdroplets. Stochastic electrochemistry allows one to study reactions in real-time occurring inside subfemtoliter droplets, one droplet at a time, when a microdroplet irreversibly adsorbs to an ultramicroelectrode surface (radius ~ 5 µm). Here, we use stochastic electrochemistry to probe the formation of hydrogen peroxide (H2O2) in single aqueous microdroplets suspended in 1,2-dichloroethane. The oxidation of H2O2 at alkaline pH (11.5) differs from near-neutral conditions (6.4), allowing us to create a digital, turn-off sensing modality for the presence of H2O2. Further, we show that the stochastic electrochemical signal is highest at the mass transfer limitation of the H2O2 couple and is dampened when the potential nears the formal potential. We validate these results by showing that the addition of a H2O2 selective probe, luminol, decreases the stochastic electrochemical response at alkaline pH (11.5). Our results support the observation that H2O2 is generated in water microdroplets at concentrations of ~100 s of µM.

Cysteine induces mitochondrial reductive stress in glioblastoma through hydrogen peroxide production.

Glucose and amino acid metabolism are critical for glioblastoma (GBM) growth, but little is known about the specific metabolic alterations in GBM that are targetable with FDA-approved compounds. To investigate tumor metabolism signatures unique to GBM, we interrogated The Cancer Genome Atlas for alterations in glucose and amino acid signatures in GBM relative to other human cancers and found that GBM exhibits the highest levels of cysteine and methionine pathway gene expression of 32 human cancers. Treatment of patient-derived GBM cells with the FDA-approved single cysteine compound N-acetylcysteine (NAC) reduced GBM cell growth and mitochondrial oxygen consumption, which was worsened by glucose starvation. Normal brain cells and other cancer cells showed no response to NAC. Mechanistic experiments revealed that cysteine compounds induce rapid mitochondrial H2O2 production and reductive stress in GBM cells, an effect blocked by oxidized glutathione, thioredoxin, and redox enzyme overexpression. From analysis of the clinical proteomic tumor analysis consortium (CPTAC) database, we found that GBM cells exhibit lower expression of mitochondrial redox enzymes than four other cancers whose proteomic data are available in CPTAC. Knockdown of mitochondrial thioredoxin-2 in lung cancer cells induced NAC susceptibility, indicating the importance of mitochondrial redox enzyme expression in mitigating reductive stress. Intraperitoneal treatment of mice bearing orthotopic GBM xenografts with a two-cysteine peptide induced H2O2 in brain tumors in vivo. These findings indicate that GBM is uniquely susceptible to NAC-driven reductive stress and could synergize with glucose-lowering treatments for GBM.

Molecular mechanism of the metal-independent production of hydroxyl radicals by thiourea dioxide and H2O2.

It is well-known that highly reactive hydroxyl radicals (HO•) can be produced by the classic Fenton system and our recently discovered haloquinone/H2O2 system, but rarely from thiol-derivatives. Here, we found, unexpectedly, that HO• can be generated from H2O2 and thiourea dioxide (TUO2), a widely used and environmentally friendly bleaching agent. A carbon-centered radical and sulfite were detected and identified as the transient intermediates, and urea and sulfate as the final products, with the complementary application of electron spin-trapping, oxygen-18 isotope labeling coupled with HPLC/MS analysis. Density functional theory calculations were conducted to further elucidate the detailed pathways for HO• production. Taken together, we proposed that the molecular mechanism for HO• generation by TUO2/H2O2: TUO2 tautomerizes from sulfinic acid into ketone isomer (TUO2-K) through proton transfer, then a nucleophilic addition of H2O2 on the S atom of TUO2-K, forming a S-hydroperoxide intermediate TUO2-OOH, which dissociates homolytically to produce HO•. Our findings represent the first experimental and computational study on an unprecedented new molecular mechanism of HO• production from simple thiol-derived sulfinic acids, which may have broad chemical, environmental, and biomedical significance for future research on the application of the well-known bleaching agent and its analogs.

Dependence on relative humidity in the formation of reactive oxygen species in water droplets.

Water microdroplets (7 to 11 µm average diameter, depending on flow rate) are sprayed in a closed chamber at ambient temperature, whose relative humidity (RH) is controlled. The resulting concentration of ROS (reactive oxygen species) formed in the microdroplets, measured by the amount of hydrogen peroxide (H2O2), is determined by nuclear magnetic resonance (NMR) and by spectrofluorimetric assays after the droplets are collected. The results are found to agree closely with one another. In addition, hydrated hydroxyl radical cations (•OH-H3O+) are recorded from the droplets using mass spectrometry and superoxide radical anions (•O2-) and hydroxyl radicals (•OH) by electron paramagnetic resonance spectroscopy. As the RH varies from 15 to 95%, the concentration of H2O2 shows a marked rise by a factor of about 3.5 in going from 15 to 50%, then levels off. By replacing the H2O of the sprayed water with deuterium oxide (D2O) but keeping the gas surrounding droplets with H2O, mass spectrometric analysis of the hydrated hydroxyl radical cations demonstrates that the water in the air plays a dominant role in producing H2O2 and other ROS, which accounts for the variation with RH. As RH increases, the droplet evaporation rate decreases. These two facts help us understand why viruses in droplets both survive better at low RH values, as found in indoor air in the wintertime, and are disinfected more effectively at higher RH values, as found in indoor air in the summertime, thus explaining the recognized seasonality of airborne viral infections.

Spatial and temporal control of mitochondrial H2 O2 release in intact human cells.

Hydrogen peroxide (H2 O2 ) has key signaling roles at physiological levels, while causing molecular damage at elevated concentrations. H2 O2 production by mitochondria is implicated in regulating processes inside and outside these organelles. However, it remains unclear whether and how mitochondria in intact cells release H2 O2 . Here, we employed a genetically encoded high-affinity H2 O2 sensor, HyPer7, in mammalian tissue culture cells to investigate different modes of mitochondrial H2 O2 release. We found substantial heterogeneity of HyPer7 dynamics between individual cells. We further observed mitochondria-released H2 O2 directly at the surface of the organelle and in the bulk cytosol, but not in the nucleus or at the plasma membrane, pointing to steep gradients emanating from mitochondria. Gradient formation is controlled by cytosolic peroxiredoxins, which act redundantly and with a substantial reserve capacity. Dynamic adaptation of cytosolic thioredoxin reductase levels during metabolic changes results in improved H2 O2 handling and explains previously observed differences between cell types. Our data suggest that H2 O2 -mediated signaling is initiated only in close proximity to mitochondria and under specific metabolic conditions.

Shear rate sensitizes bacterial pathogens to H2O2 stress.

Cells regularly experience fluid flow in natural systems. However, most experimental systems rely on batch cell culture and fail to consider the effect of flow-driven dynamics on cell physiology. Using microfluidics and single-cell imaging, we discover that the interplay of physical shear rate (a measure of fluid flow) and chemical stress trigger a transcriptional response in the human pathogen Pseudomonas aeruginosa. In batch cell culture, cells protect themselves by quickly scavenging the ubiquitous chemical stressor hydrogen peroxide (H2O2) from the media. In microfluidic conditions, we observe that cell scavenging generates spatial gradients of H2O2. High shear rates replenish H2O2, abolish gradients, and generate a stress response. Combining mathematical simulations and biophysical experiments, we find that flow triggers an effect like "wind-chill" that sensitizes cells to H2O2 concentrations 100 to 1,000 times lower than traditionally studied in batch cell culture. Surprisingly, the shear rate and H2O2 concentration required to generate a transcriptional response closely match their respective values in the human bloodstream. Thus, our results explain a long-standing discrepancy between H2O2 levels in experimental and host environments. Finally, we demonstrate that the shear rate and H2O2 concentration found in the human bloodstream trigger gene expression in the blood-relevant human pathogen Staphylococcus aureus, suggesting that flow sensitizes bacteria to chemical stress in natural environments.

Synergy of crystallinity modulation and intercalation engineering in carbon nitride for boosted H2O2 photosynthesis.

Photosynthesis of hydrogen peroxide (H2O2) by selective oxygen reduction is a green and cost-effective alternative to the energy-intensive anthraquinone process. Although inexpensive polymeric graphitic carbon nitride (g-C3N4) exhibits the ability to produce H2O2, its disordered and amorphous structure leads to a high recombination rate of photogenerated carriers and hinders charge transfer between layers. Herein, we predict that stacked polymeric g-C3N4 with ion intercalation (K+ and I-) can improve carrier separation and transfer by multiscale computational simulations. The electronic structures of g-C3N4 were tailored and modified by intercalating K+ and I- into the layer-by-layer structures. Guided by the computational predictions, we achieved efficient solar-driven H2O2 production by employing this facile and ion-intercalated crystalline g-C3N4. An H2O2 production rate of 13.1 mM g-1 h-1 and an apparent quantum yield of 23.6% at 400 nm were obtained. The synergistic effects of crystallinity regulation and dual interstitial doping engineering triggered the formation of new light absorption centers, the establishment of rapid charge diffusion channels, and the enhancement of two-electron oxygen reduction characteristics. This work sheds light on the dual tuning of crystallinity and electronic structure and broadens the design principles of organic-conjugated polymer photocatalysts for environmental remediation and energy conservation.

Proteome-wide tagging with an H2O2 biosensor reveals highly localized and dynamic redox microenvironments.

Hydrogen peroxide (H2O2) sensing and signaling involves the reversible oxidation of particular thiols on particular proteins to modulate protein function in a dynamic manner. H2O2 can be generated from various intracellular sources, but their identities and relative contributions are often unknown. To identify endogenous "hotspots" of H2O2 generation on the scale of individual proteins and protein complexes, we generated a yeast library in which the H2O2 sensor HyPer7 was fused to the C-terminus of all protein-coding open reading frames (ORFs). We also generated a control library in which a redox-insensitive mutant of HyPer7 (SypHer7) was fused to all ORFs. Both libraries were screened side-by-side to identify proteins located within H2O2-generating environments. Screening under a variety of different metabolic conditions revealed dynamic changes in H2O2 availability highly specific to individual proteins and protein complexes. These findings suggest that intracellular H2O2 generation is much more localized and functionally differentiated than previously recognized.

8-oxoguanine riboswitches in bacteria detect and respond to oxidative DNA damage.

Riboswitches rely on structured aptamer domains to selectively sense their target ligands and regulate gene expression. However, some riboswitch aptamers in bacteria carry mutations in their otherwise strictly conserved binding pockets that change ligand specificities. The aptamer domain of a riboswitch class originally found to selectively sense guanine forms a three-stem junction that has since been observed to exploit numerous alterations in its ligand-binding pocket. These rare variants have modified their ligand specificities to sense other purines or purine derivatives, including adenine, 2'-deoxyguanosine (three classes), and xanthine. Herein, we report the characteristics of a rare variant that is narrowly distributed in the Paenibacillaceae family of bacteria. Known representatives are always associated with genes encoding 8-oxoguanine deaminase. As predicted from this gene association, these variant riboswitches tightly bind 8-oxoguanine (8-oxoG), strongly discriminate against other purine derivatives, and function as genetic "ON" switches. Following exposure of cells to certain oxidative stresses, a representative 8-oxoG riboswitch activates gene expression, likely caused by the accumulation of 8-oxoG due to oxidative damage to G nucleobases in DNA, RNA, and the nucleotide pool. Furthermore, an engineered version of the variant aptamer was prepared that exhibits specificity for 8-oxoadenine, further demonstrating that RNA aptamers can acquire mutations that expand their ability to detect and respond to oxidative damage.

Contact between water vapor and silicate surface causes abiotic formation of reactive oxygen species in an anoxic atmosphere.

Spontaneous generation of reactive oxygen species (ROS) in aqueous microdroplets or at a water vapor-silicate interface is a new source of redox chemistry. However, such generation occurs with difficulty in liquid water having a large ionic strength. We report that ROS is spontaneously produced when water vapor contacts hydrogen-bonded hydroxyl groups on a silicate surface. The evolution of hydrogen-bonded species such as hydroxyl groups was investigated by using two-dimensional, time-resolved FT-IR spectroscopy. The participation of water vapor in ROS generation is confirmed by investigating the reaction of D2O vapor and hydroxyl groups on a silicate surface. We propose a reaction pathway for ROS generation based on the change of the hydrogen-bonding network and corresponding electron transfer onto the silicate surface in the water vapor-solid contact process. Our observations suggest that ROS production from water vapor-silicate contact electrification could have contributed to oxidation during the Archean Eon before the Great Oxidation Event.

ROS are evolutionary conserved cell-to-cell stress signals.

Cell-to-cell communication is fundamental to multicellular organisms and unicellular organisms living in a microbiome. It is thought to have evolved as a stress- or quorum-sensing mechanism in unicellular organisms. A unique cell-to-cell communication mechanism that uses reactive oxygen species (ROS) as a signal (termed the "ROS wave") was identified in flowering plants. This process is essential for systemic signaling and plant acclimation to stress and can spread from a small group of cells to the entire plant within minutes. Whether a similar signaling process is found in other organisms is however unknown. Here, we report that the ROS wave can be found in unicellular algae, amoeba, ferns, mosses, mammalian cells, and isolated hearts. We further show that this process can be triggered in unicellular and multicellular organisms by a local stress or H2O2 treatment and blocked by the application of catalase or NADPH oxidase inhibitors and that in unicellular algae it communicates important stress-response signals between cells. Taken together, our findings suggest that an active process of cell-to-cell ROS signaling, like the ROS wave, evolved before unicellular and multicellular organisms diverged. This mechanism could have communicated an environmental stress signal between cells and coordinated the acclimation response of many different cells living in a community. The finding of a signaling process, like the ROS wave, in mammalian cells further contributes to our understanding of different diseases and could impact the development of drugs that target for example cancer or heart disease.

Carbon dioxide/bicarbonate is required for sensitive inactivation of mammalian glyceraldehyde-3-phosphate dehydrogenase by hydrogen peroxide.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) contains an active site Cys and is one of the most sensitive cellular enzymes to oxidative inactivation and redox regulation. Here, we show that inactivation by hydrogen peroxide is strongly enhanced in the presence of carbon dioxide/bicarbonate. Inactivation of isolated mammalian GAPDH by H2O2 increased with increasing bicarbonate concentration and was sevenfold faster in 25 mM (physiological) bicarbonate compared with bicarbonate-free buffer of the same pH. H2O2 reacts reversibly with CO2 to form a more reactive oxidant, peroxymonocarbonate (HCO4-), which is most likely responsible for the enhanced inactivation. However, to account for the extent of enhancement, we propose that GAPDH must facilitate formation and/or targeting of HCO4- to promote its own inactivation. Inactivation of intracellular GAPDH was also strongly enhanced by bicarbonate: treatment of Jurkat cells with 20 µM H2O2 in 25 mM bicarbonate buffer for 5 min caused almost complete GAPDH inactivation, but no loss of activity when bicarbonate was not present. H2O2-dependent GAPDH inhibition in bicarbonate buffer was observed even in the presence of reduced peroxiredoxin 2 and there was a significant increase in cellular glyceraldehyde-3-phosphate/dihydroxyacetone phosphate. Our results identify an unrecognized role for bicarbonate in enabling H2O2 to influence inactivation of GAPDH and potentially reroute glucose metabolism from glycolysis to the pentose phosphate pathway and NAPDH production. They also demonstrate what could be wider interplay between CO2 and H2O2 in redox biology and the potential for variations in CO2 metabolism to influence oxidative responses and redox signaling.

Fatty acid oxidation drives mitochondrial hydrogen peroxide production by α-ketoglutarate dehydrogenase.

In the present study, we examined the mitochondrial hydrogen peroxide (mH2O2) generating capacity of α-ketoglutarate dehydrogenase (KGDH) and compared it to components of the electron transport chain using liver mitochondria isolated from male and female C57BL6N mice. We show for the first time there are some sex dimorphisms in the production of mH2O2 by electron transport chain complexes I and III when mitochondria are fueled with different substrates. However, in our investigations into these sex effects, we made the unexpected and compelling discovery that 1) KGDH serves as a major mH2O2 supplier in male and female liver mitochondria and 2) KGDH can form mH2O2 when liver mitochondria are energized with fatty acids but only when malate is used to prime the Krebs cycle. Surprisingly, 2-keto-3-methylvaleric acid (KMV), a site-specific inhibitor for KGDH, nearly abolished mH2O2 generation in both male and female liver mitochondria oxidizing palmitoyl-carnitine. KMV inhibited mH2O2 production in liver mitochondria from male and female mice oxidizing myristoyl-, octanoyl-, or butyryl-carnitine as well. S1QEL 1.1 (S1) and S3QEL 2 (S3), compounds that inhibit reactive oxygen species generation by complexes I and III, respectively, without interfering with OxPhos and respiration, had a negligible effect on the rate of mH2O2 production when pyruvate or acyl-carnitines were used as fuels. However, inclusion of KMV in reaction mixtures containing S1 and/or S3 almost abolished mH2O2 generation. Together, our findings suggest KGDH is the main mH2O2 generator in liver mitochondria, even when fatty acids are used as fuel.

Antioxidants are ineffective at quenching reactive oxygen species inside bacteria and should not be used to diagnose oxidative stress.

A wide variety of stresses have been proposed to exert killing effects upon bacteria by stimulating the intracellular formation of reactive oxygen species (ROS). A key part of the supporting evidence has often been the ability of antioxidant compounds to protect the cells. In this study, some of the most-used antioxidants-thiourea, glutathione, N-acetylcysteine, and ascorbate-have been examined. Their ability to quench superoxide and hydrogen peroxide was verified in vitro, but the rate constants were orders of magnitude too slow for them to have an impact upon superoxide and peroxide concentrations in vivo, where these species are already scavenged by highly active enzymes. Indeed, the antioxidants were unable to protect the growth and ROS-sensitive enzymes of E. coli strains experiencing authentic oxidative stress. Similar logic posits that antioxidants cannot substantially quench hydroxyl radicals inside cells, which contain abundant biomolecules that react with them at diffusion-limited rates. Indeed, antioxidants were able to protect cells from DNA damage only if they were applied at concentrations that slow metabolism and growth. This protective effect was apparent even under anoxic conditions, when ROS could not possibly be involved, and it was replicated when growth was similarly slowed by other means. Experimenters should discard the use of antioxidants as a way of detecting intracellular oxidative stress and should revisit conclusions that have been based upon such experiments. The notable exception is that these compounds can effectively degrade hydrogen peroxide from environmental sources before it enters cells.

Chemogenetic Approaches to Probe Redox Pathways: Implications for Cardiovascular Pharmacology and Toxicology.

Chemogenetics refers to experimental systems that dynamically regulate the activity of a recombinant protein by providing or withholding the protein's specific biochemical stimulus. Chemogenetic tools permit precise dynamic control of specific signaling molecules to delineate the roles of those molecules in physiology and disease. Yeast d-amino acid oxidase (DAAO) enables chemogenetic manipulation of intracellular redox balance by generating hydrogen peroxide only in the presence of d-amino acids. Advances in biosensors have allowed the precise quantitation of these signaling molecules. The combination of chemogenetic approaches with biosensor methodologies has opened up new lines of investigation, allowing the analysis of intracellular redox pathways that modulate physiological and pathological cell responses. We anticipate that newly developed transgenic chemogenetic models will permit dynamic modulation of cellularredox balance in diverse cells and tissues and will facilitate the identification and validation of novel therapeutic targets involved in both physiological redox pathways and pathological oxidative stress.

Hydrogen peroxide sensitivity connects the activity of COX5A and NPR3 to the regulation of YAP1 expression.

Reactive oxygen species (ROS) are among the most severe types of cellular stressors with the ability to damage essential cellular biomolecules. Excess levels of ROS are correlated with multiple pathophysiological conditions including neurodegeneration, diabetes, atherosclerosis, and cancer. Failure to regulate the severely imbalanced levels of ROS can ultimately lead to cell death, highlighting the importance of investigating the molecular mechanisms involved in the detoxification procedures that counteract the effects of these compounds in living organisms. One of the most abundant forms of ROS is H2 O2 , mainly produced by the electron transport chain in the mitochondria. Numerous genes have been identified as essential to the process of cellular detoxification. Yeast YAP1, which is homologous to mammalian AP-1 type transcriptional factors, has a key role in oxidative detoxification by upregulating the expression of antioxidant genes in yeast. The current study reveals novel functions for COX5A and NPR3 in H2 O2 -induced stress by demonstrating that their deletions result in a sensitive phenotype. Our follow-up investigations indicate that COX5A and NPR3 regulate the expression of YAP1 through an alternative mode of translation initiation. These novel gene functions expand our understanding of the regulation of gene expression and defense mechanism of yeast against oxidative stress.

Reducing oxidative protein folding alleviates senescence by minimizing ER-to-nucleus H2 O2 release.

Oxidative protein folding occurs in the endoplasmic reticulum (ER) to generate disulfide bonds, and the by-product is hydrogen peroxide (H2 O2 ). However, the relationship between oxidative protein folding and senescence remains uncharacterized. Here, we find that the protein disulfide isomerase (PDI), a key oxidoreductase that catalyzes oxidative protein folding, accumulated in aged human mesenchymal stem cells (hMSCs) and deletion of PDI alleviated hMSCs senescence. Mechanistically, knocking out PDI slows the rate of oxidative protein folding and decreases the leakage of ER-derived H2 O2 into the nucleus, thereby decreasing the expression of SERPINE1, which was identified as a key driver of cell senescence. Furthermore, we show that depletion of PDI alleviated senescence in various cell models of aging. Our findings reveal a previously unrecognized role of oxidative protein folding in promoting cell aging, providing a potential target for aging and aging-related disease intervention.