Mitochondrial uncoupling proteins protect human airway epithelial ciliated cells from oxidative damage.

Apical cilia on epithelial cells defend the lung by propelling pathogens and particulates out of the respiratory airways. Ciliated cells produce ATP that powers cilia beating by densely grouping mitochondria just beneath the apical membrane. However, this efficient localization comes at a cost because electrons leaked during oxidative phosphorylation react with molecular oxygen to form superoxide, and thus, the cluster of mitochondria creates a hotspot for oxidant production. The relatively high oxygen concentration overlying airway epithelia further intensifies the risk of generating superoxide. Thus, airway ciliated cells face a unique challenge of producing harmful levels of oxidants. However, surprisingly, highly ciliated epithelia produce less reactive oxygen species (ROS) than epithelia with few ciliated cells. Compared to other airway cell types, ciliated cells express high levels of mitochondrial uncoupling proteins, UCP2 and UCP5. These proteins decrease mitochondrial protonmotive force and thereby reduce production of ROS. As a result, lipid peroxidation, a marker of oxidant injury, decreases. However, mitochondrial uncoupling proteins exact a price for decreasing oxidant production; they decrease the fraction of mitochondrial respiration that generates ATP. These findings indicate that ciliated cells sacrifice mitochondrial efficiency in exchange for safety from damaging oxidation. Employing uncoupling proteins to prevent oxidant production, instead of relying solely on antioxidants to decrease postproduction oxidant levels, may offer an advantage for targeting a local area of intense ROS generation.

Sequestrase chaperones protect against oxidative stress-induced protein aggregation and [PSI+] prion formation.

Misfolded proteins are usually refolded to their functional conformations or degraded by quality control mechanisms. When misfolded proteins evade quality control, they can be sequestered to specific sites within cells to prevent the potential dysfunction and toxicity that arises from protein aggregation. Btn2 and Hsp42 are compartment-specific sequestrases that play key roles in the assembly of these deposition sites. Their exact intracellular functions and substrates are not well defined, particularly since heat stress sensitivity is not observed in deletion mutants. We show here that Btn2 and Hsp42 are required for tolerance to oxidative stress conditions induced by exposure to hydrogen peroxide. Btn2 and Hsp42 act to sequester oxidized proteins into defined PQC sites following ROS exposure and their absence leads to an accumulation of protein aggregates. The toxicity of protein aggregate accumulation causes oxidant sensitivity in btn2 hsp42 sequestrase mutants since overexpression of the Hsp104 disaggregase rescues oxidant tolerance. We have identified the Sup35 translation termination factor as an in vivo sequestrase substrate and show that Btn2 and Hsp42 act to suppress oxidant-induced formation of the yeast [PSI+] prion, which is the amyloid form of Sup35. [PSI+] prion formation in sequestrase mutants does not require IPOD (insoluble protein deposit) localization which is the site where amyloids are thought to undergo fragmentation and seeding to propagate their heritable prion form. Instead, both amorphous and amyloid Sup35 aggregates are increased in btn2 hsp42 mutants consistent with the idea that prion formation occurs at multiple intracellular sites during oxidative stress conditions in the absence of sequestrase activity. Taken together, our data identify protein sequestration as a key antioxidant defence mechanism that functions to mitigate the damaging consequences of protein oxidation-induced aggregation.

Superoxide: The enigmatic chemical chameleon in neutrophil biology.

The burst of superoxide produced when neutrophils phagocytose bacteria is the defining biochemical feature of these abundant immune cells. But 50 years since this discovery, the vital role superoxide plays in host defense has yet to be defined. Superoxide is neither bactericidal nor is it just a source of hydrogen peroxide. This simple free radical does, however, have remarkable chemical dexterity. Depending on its environment and reaction partners, superoxide can act as an oxidant, a reductant, a nucleophile, or an enzyme substrate. We outline the evidence that inside phagosomes where neutrophils trap, kill, and digest bacteria, superoxide will react preferentially with the enzyme myeloperoxidase, not the bacterium. By acting as a cofactor, superoxide will sustain hypochlorous acid production by myeloperoxidase. As a substrate, superoxide may give rise to other forms of reactive oxygen. We contend that these interactions hold the key to understanding the precise role superoxide plays in neutrophil biology. State-of-the-art techniques in mass spectrometry, oxidant-specific fluorescent probes, and microscopy focused on individual phagosomes are needed to identify bactericidal mechanisms driven by superoxide. This work will undoubtably lead to fascinating discoveries in host defense and give a richer understanding of superoxide's varied biology.

How a natural antibiotic uses oxidative stress to kill oxidant-resistant bacteria.

Natural products that possess antibiotic and antitumor qualities are often suspected of working through oxidative mechanisms. In this study, two quinone-based small molecules were compared. Menadione, a classic redox-cycling compound, was confirmed to generate high levels of reactive oxygen species inside Escherichia coli. It inactivated iron-cofactored enzymes and blocked growth. However, despite the substantial levels of oxidants that it produced, it was unable to generate significant DNA damage and was not lethal. Streptonigrin, in contrast, was poorer at redox cycling and did not inactivate enzymes or block growth; however, even in low doses, it damaged DNA and killed cells. Its activity required iron and oxygen, and in vitro experiments indicated that its quinone moiety transferred electrons through the adjacent iron atom to oxygen. Additionally, in vitro experiments revealed that streptonigrin was able to damage DNA without inhibition by catalase, indicating that hydrogen peroxide was not involved. We infer that streptonigrin can reduce bound oxygen directly to a ferryl species, which then oxidizes the adjacent DNA, without release of superoxide or hydrogen peroxide intermediates. This scheme allows streptonigrin to kill a bacterial cell without interference by scavenging enzymes. Moreover, its minimal redox-cycling behavior avoids alerting either the OxyR or the SoxRS systems, which otherwise would block killing. This example highlights qualities that may be important in the design of oxidative drugs. These results also cast doubt on proposals that bacteria can be killed by stressors that merely stimulate intracellular O2- and H2O2 formation.

Oxidation of caspase-8 by hypothiocyanous acid enables TNF-mediated necroptosis.

Necroptosis is a form of regulated cell death triggered by various host and pathogen-derived molecules during infection and inflammation. The essential step leading to necroptosis is phosphorylation of the mixed lineage kinase domain-like protein by receptor-interacting protein kinase 3. Caspase-8 cleaves receptor-interacting protein kinases to block necroptosis, so synthetic caspase inhibitors are required to study this process in experimental models. However, it is unclear how caspase-8 activity is regulated in a physiological setting. The active site cysteine of caspases is sensitive to oxidative inactivation, so we hypothesized that oxidants generated at sites of inflammation can inhibit caspase-8 and promote necroptosis. Here, we discovered that hypothiocyanous acid (HOSCN), an oxidant generated in vivo by heme peroxidases including myeloperoxidase and lactoperoxidase, is a potent caspase-8 inhibitor. We found HOSCN was able to promote necroptosis in mouse fibroblasts treated with tumor necrosis factor. We also demonstrate purified caspase-8 was inactivated by low concentrations of HOSCN, with the predominant product being a disulfide-linked dimer between Cys360 and Cys409 of the large and small catalytic subunits. We show oxidation still occurred in the presence of reducing agents, and reduction of the dimer was slow, consistent with HOSCN being a powerful physiological caspase inhibitor. While the initial oxidation product is a dimer, further modification also occurred in cells treated with HOSCN, leading to higher molecular weight caspase-8 species. Taken together, these findings indicate major disruption of caspase-8 function and suggest a novel mechanism for the promotion of necroptosis at sites of inflammation.

Oxidant-Dependent Sensitizing, Protective, and Mitigative Effects in X-Ray-Irradiated Pulmonary Endothelial Cells.

The primary response of proliferating bovine pulmonary artery endothelial cells (BPAECs) after X-ray irradiation [≤10 gray (Gy)] is shown to be transient cell-cycle arrest. Accompanying oxidant-linked functional changes within the mitochondria are readily measured, but increased autophagy is not. Radiation-induced apoptosis is negligible in this line-important because cells undergoing apoptosis release oxygen-derived species that can overwhelm/mask the radiation-associated species and their effects that we wish to investigate. Cells irradiated and cultured at 3% oxygen exhibited delayed cell-cycle arrest (6-8 hours after 10 Gy irradiation) compared with those maintained at 20% oxygen (2-4 hours after 10 Gy irradiation). At 3% oxygen, either only during or only after irradiation, results intermediate between 20% and 3% oxygen throughout were obtained. No variability in cell-cycle distribution was observed for unirradiated cells cultured under different prevailing oxygen levels. Mitochondrially localized manganese superoxide dismutase delayed the X-ray-induced cell-cycle changes when over-expressed in BPAEC, indicating superoxide to be one of the key oxygen-derived cytotoxic species involved in the radiobiological response. Also, the peroxynitrite biomarker 3-nitrotyrosine was elevated, whereas hydrogen peroxide levels were not. Lastly, the utility of the BPAEC for screening potential countermeasures to ionizing radiation is demonstrated with some quinoline derivatives. Three of the five compounds appeared mitigative, and all were protective. It is suggested that the oxidation-reduction chemistry of these compounds probably offers a reasonable explanation for their observed ameliorative properties. Furthermore, the results suggest a promising new direction in the search for lead compounds as countermeasures to the effects of ionizing radiation. SIGNIFICANCE STATEMENT: The primary radiological response of proliferating bovine pulmonary artery endothelial cells is cell-cycle arrest, starting soon after X-ray irradiation (1-10 Gy) at 20% O2 but delayed by 4 hours at systemic (3%) O2. Oxygen/superoxide is found to be radio-sensitizing in at least two distinct time windows, during and after the irradiation, with both responses antagonized by various hydroxyquinoline derivatives. Similar responses in many other cell lines are likely to be masked by elevated oxidants associated with apoptosis.

Effect of hydrogen peroxide on lens transparency, intracellular pH, gap junction coupling, hydrostatic pressure and membrane water permeability.

Clouding of the eye lens or cataract is an age-related anomaly that affects middle-aged humans. Exploration of the etiology points to a great extent to oxidative stress due to different forms of reactive oxygen species/metabolites such as Hydrogen peroxide (H2O2) that are generated due to intracellular metabolism and environmental factors like radiation. If accumulated and left unchecked, the imbalance between the production and degradation of H2O2 in the lens could lead to cataracts. Our objective was to explore ex vivo the effects of H2O2 on lens physiology. We investigated transparency, intracellular pH (pHi), intercellular gap junction coupling (GJC), hydrostatic pressure (HP) and membrane water permeability after subjecting two-month-old C57 wild-type (WT) mouse lenses for 3 h or 8 h in lens saline containing 50 µM H2O2; the results were compared with control lenses incubated in the saline without H2O2. There was a significant decrease in lens transparency in H2O2-treated lenses. In control lenses, pHi decreases from ∼7.34 in the surface fiber cells to 6.64 in the center. Experimental lenses exposed to H2O2 for 8 h showed a significant decrease in surface pH (from 7.34 to 6.86) and central pH (from 6.64 to 6.56), compared to the controls. There was a significant increase in GJC resistance in the differentiating (12-fold) and mature (1.4-fold) fiber cells compared to the control. Experimental lenses also showed a significant increase in HP which was ∼2-fold higher at the junction between the differentiating and mature fiber cells and ∼1.5-fold higher at the center compared to these locations in control lenses; HP at the surface was 0 mm Hg in either type lens. Fiber cell membrane water permeability significantly increased in H2O2-exposed lenses compared to controls. Our data demonstrate that elevated levels of lens intracellular H2O2 caused a decrease in intracellular pH and led to acidosis which most likely uncoupled GJs, and increased AQP0-dependent membrane water permeability causing a consequent rise in HP. We infer that an abnormal increase in intracellular H2O2 could induce acidosis, cause oxidative stress, alter lens microcirculation, and lead to the development of accelerated lens opacity and age-related cataracts.

Silver nanoparticles stimulate 5-Fluorouracil-induced colorectal cancer cells to kill through the upregulation TRPV1-mediated calcium signaling pathways.

The involvement of the TRP vanilloid 1 (TRPV1) cation channel on the 5-Fluorouracil (5-FU)-caused Ca2+ signals through the activation of the apoptotic signaling pathway and stimulating the mitochondrial Ca2+ and Zn2+ accumulation-induced reactive oxygen species (ROS) productions in several cancer cells, except the colorectal cancer (HT-29) cell line, was recently reported. I aimed to investigate the action of silver nanoparticles (SiNPs) and 5-FU incubations through the activation of TRPV1 on ROS, apoptosis, and cell death in the HT-29 cell line. The cells were divided into four groups: control, SiNP (100 µM for 48 h), 5-FU (25 µM for 24 h), and 5-FU + SiNP. SiNP treatment through TRPV1 activation (via capsaicin) stimulated the oxidant and apoptotic actions of 5-FU in the cells, whereas they were diminished in the cells by the TRPV1 antagonist (capsazepine) treatment. The apoptotic and cell death actions of 5-FU were determined by increasing the propidium iodide/Hoechst rate, caspase-3, -8, and -9 activations, mitochondrial membrane depolarization, lipid peroxidation, and ROS, but decreasing the glutathione and glutathione peroxidase. The increase of cytosolic free Ca2+ and Zn2+ into mitochondria via the stimulation of TRPV1 current density increased oxidant and apoptotic properties of 5-FU in the cells. For the therapy of HT-29 tumor cells, I found that the combination of SiNPs and 5-FU was synergistic via TRPV1 activation.

Oxidative Stress: The Role of Antioxidant Phytochemicals in the Prevention and Treatment of Diseases.

Oxidative stress, characterized by an imbalance favouring oxidants over antioxidants, is a key contributor to the development of various common diseases. Counteracting these oxidants is considered an effective strategy to mitigate the levels of oxidative stress in organisms. Numerous studies have indicated an inverse correlation between the consumption of vegetables and fruits and the risk of chronic diseases, attributing these health benefits to the presence of antioxidant phytochemicals in these foods. Phytochemicals, present in a wide range of foods and medicinal plants, play a pivotal role in preventing and treating chronic diseases induced by oxidative stress by working as antioxidants. These compounds exhibit potent antioxidant, anti-inflammatory, anti-aging, anticancer, and protective properties against cardiovascular diseases, diabetes mellitus, obesity, and neurodegenerative conditions. This comprehensive review delves into the significance of these compounds in averting and managing chronic diseases, elucidating the key sources of these invaluable elements. Additionally, it provides a summary of recent advancements in understanding the health benefits associated with antioxidant phytochemicals.

Proteomics and Beyond: Cell Decision-Making Shaped by Reactive Electrophiles.

Revolutionary proteomic strategies have enabled rapid profiling of the cellular targets of electrophilic small molecules. However, precise means to directly interrogate how these individual electrophilic modifications at low occupancy functionally reshape signaling networks have until recently been largely limited. We highlight here new methods that transcend proteomic platforms to forge a quantitative link between protein target-selective engagement and downstream signaling. We focus on recent progress in the study of non-enzyme-assisted signaling mechanisms and crosstalk choreographed by native reactive electrophilic species (RES). Using this as a model, we offer a long-term vision of how these toolsets together with fundamental biochemical knowledge of precision electrophile signaling may be harnessed to assist covalent ligand-target matching and ultimately amend disease-specific signaling dysfunction.

Expression of RcrB confers resistance to hypochlorous acid in uropathogenic Escherichia coli.

To eradicate bacterial pathogens, neutrophils are recruited to the sites of infection, where they engulf and kill microbes through the production of reactive oxygen and chlorine species (ROS/RCS). The most prominent RCS is the antimicrobial oxidant hypochlorous acid (HOCl), which rapidly reacts with various amino acid side chains, including those containing sulfur and primary/tertiary amines, causing significant macromolecular damage. Pathogens like uropathogenic Escherichia coli (UPEC), the primary causative agent of urinary tract infections, have developed sophisticated defense systems to protect themselves from HOCl. We recently identified the RcrR regulon as a novel HOCl defense strategy in UPEC. Expression of the rcrARB operon is controlled by the HOCl-sensing transcriptional repressor RcrR, which is oxidatively inactivated by HOCl resulting in the expression of its target genes, including rcrB. The rcrB gene encodes a hypothetical membrane protein, deletion of which substantially increases UPEC's susceptibility to HOCl. However, the mechanism behind protection by RcrB is unclear. In this study, we investigated whether (i) its mode of action requires additional help, (ii) rcrARB expression is induced by physiologically relevant oxidants other than HOCl, and (iii) expression of this defense system is limited to specific media and/or cultivation conditions. We provide evidence that RcrB expression is sufficient to protect E. coli from HOCl. Furthermore, RcrB expression is induced by and protects from several RCS but not from ROS. RcrB plays a protective role for RCS-stressed planktonic cells under various growth and cultivation conditions but appears to be irrelevant for UPEC's biofilm formation. IMPORTANCE Bacterial infections pose an increasing threat to human health, exacerbating the demand for alternative treatments. Uropathogenic Escherichia coli (UPEC), the most common etiological agent of urinary tract infections (UTIs), are confronted by neutrophilic attacks in the bladder, and must therefore be equipped with powerful defense systems to fend off the toxic effects of reactive chlorine species. How UPEC deal with the negative consequences of the oxidative burst in the neutrophil phagosome remains unclear. Our study sheds light on the requirements for the expression and protective effects of RcrB, which we recently identified as UPEC's most potent defense system toward hypochlorous acid (HOCl) stress and phagocytosis. Thus, this novel HOCl stress defense system could potentially serve as an attractive drug target to increase the body's own capacity to fight UTIs.

Alveolar Mitochondrial Quality Control During Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) is a leading cause of respiratory failure and death in patients in the intensive care unit. Experimentally, acute lung injury resolution depends on the repair of mitochondrial oxidant damage by the mitochondrial quality control (MQC) pathways, mitochondrial biogenesis, and mitophagy, but nothing is known about this in the human lung. In a case-control autopsy study, we compared the lungs of subjects dying of ARDS (n = 8; cases) and age-/gender-matched subjects dying of nonpulmonary causes (n = 7; controls). Slides were examined by light microscopy and immunofluorescence confocal microscopy, randomly probing for co-localization of citrate synthase with markers of oxidant stress, mitochondrial DNA damage, mitophagy, and mitochondrial biogenesis. ARDS lungs showed diffuse alveolar damage with edema, hyaline membranes, and neutrophils. Compared with controls, a high degree of mitochondrial oxidant damage was seen in type 2 epithelial (AT2) cells and alveolar macrophages by 8-hydroxydeoxyguanosine and malondialdehyde co-staining with citrate synthase. In ARDS, antioxidant protein heme oxygenase-1 and DNA repair enzyme N-glycosylase/DNA lyase (Ogg1) were found in alveolar macrophages but not in AT2 cells. Moreover, MAP1 light chain-3 (LC3) and serine/threonine-protein kinase (Pink1) staining were absent in AT2 cells, suggesting a mitophagy failure. Nuclear respiratory factor-1 staining was missing in the alveolar region, suggesting impaired mitochondrial biogenesis. Widespread hyperproliferation of AT2 cells in ARDS could suggest defective differentiation into type 1 cells. ARDS lungs show profuse mitochondrial oxidant DNA damage but little evidence of MQC activity in AT2 epithelium. Because these pathways are important for acute lung injury resolution, our findings support MQC as a novel pharmacologic target for ARDS resolution.

Unanchored tri-NEDD8 inhibits PARP-1 to protect from oxidative stress-induced cell death.

NEDD8 is a ubiquitin-like protein that activates cullin-RING E3 ubiquitin ligases (CRLs). Here, we identify a novel role for NEDD8 in regulating the activity of poly(ADP-ribose) polymerase 1 (PARP-1) in response to oxidative stress. We show that treatment of cells with H2O2 results in the accumulation of NEDD8 chains, likely by directly inhibiting the deneddylase NEDP1. One chain type, an unanchored NEDD8 trimer, specifically bound to the second zinc finger domain of PARP-1 and attenuated its activation. In cells in which Nedp1 is deleted, large amounts of tri-NEDD8 constitutively form, resulting in inhibition of PARP-1 and protection from PARP-1-dependent cell death. Surprisingly, these NEDD8 trimers are additionally acetylated, as shown by mass spectrometry analysis, and their binding to PARP-1 is reduced by the overexpression of histone de-acetylases, which rescues PARP-1 activation. Our data suggest that trimeric, acetylated NEDD8 attenuates PARP-1 activation after oxidative stress, likely to delay the initiation of PARP-1-dependent cell death.

Protective effects of Japanese sake yeast on depressive-like behaviors, oxidative stress and inflammatory parameters in a rat model of global cerebral ischemia/reperfusion.

Stroke is a serious cerebrovascular disease that causes post-stress depression and death. Stress and inflammation have pivotal roles in the induction of the disease. Several drugs and agents have been used for the treatment of disease, but their uses are faced with limitations owing to their side effects. Natural agents are more efficient for the treatment of stroke due to lower toxicity and their pharmaceutical properties. Sake yeast or Japanese rice wine is an antioxidant compound that could be used to treat stroke and post-stress depression. This study evaluates the effects of sake yeast on depressive-like behaviors, oxidative stress and inflammatory parameters in a rat model of global cerebral ischemia/reperfusion. Rats were divided into four groups, including 1) control: without bilateral common carotid artery occlusion (BCCAO) and sake supplement, 2) Ischemia group: rats induced with BCCAO and lack of therapeutic supplement, and 3 and 4) Ischemia + sake groups: rats induced with BCCAO and treated with 25 and 50 mg/kg sake yeast, respectively. Depressive-like behaviors antioxidant enzymes activities were assessed. The induction of stroke increased oxidant status, inflammatory parameters, and depressive-like behaviors, while the administration of sake could decrease inflammation, depressive-like behaviors, and oxidant status and increase antioxidant enzymes. The yeast could be used as a supplement in combination with other drugs to treat stroke.

Transcranial direct current stimulation alleviates nociceptive behavior in male rats with neuropathic pain by regulating oxidative stress and reducing neuroinflammation.

Transcranial direct curent stimulation (tDCS) and trans-spinal direct current stimulation (tsDCS) are promising therapies for pain that can alter the excitability of neuronal activity in cerebral cortex. The aim of the study is to investigate the therapeutic effects of direct current stimulation (DCS) over the spinal cord and cerebral cortex on oxidative stress and neuroinflammation in rats with chronic constriction injury (CCI). Male Wistar rats were randomly divided into four experimental groups: Sham, CCI, CCI + tDCS and CCI + tsDCS. The neuropathic pain model was induced by using the CCI model. Rats with neuropathy were treated with cathodal tDCS and tsDCS stimulations consisting of 0.5 mA for 30 min a day for 7 days from day 8 onwards. Locomotor activity was measured by open-field test and nociceptive behavior was assessed by hot-plate, tail-flick and Randall-Selitto tests. Following the behavioral experiments, total oxidant capacity (TOC), total antioxidant capacity (TAC) and proinflammatory cytokine levels were evaluated in spinal cord and cerebral cortex tissues. The CCI model induced significant mechanical and thermal hyperalgesia. Nociceptive behaviors in rats with CCI were reversed by DCS treatment. Higher TOC and lower TAC levels were detected in the spinal cord and cerebral cortex tissues of the CCI rats compared to the control. tsDCS treatment amended oxidant/antioxidant status. Moreover, tsDCS modulated the central levels of Tumor necrosis factor-α (TNF-α), interleukin 1-beta (IL-1ß), IL-6 and IL-18. tsDCS stimulation showed better therapeutic effect on neuropathic pain by regulating oxidant/antioxidant levels and reducing neuroinflammation. DCS, especially at spinal level, may be a promising therapeutic strategy that can be used alone or in combination with other effective treatments for alleviating neuropathic pain.

Characterization of the Trr/Trx system in the fungal pathogen Candida glabrata.

C. glabrata, an opportunistic fungal pathogen, can adapt and resist to different stress conditions. It is highly resistant to oxidant stress compared to other Candida spp and to the phylogenetically related but non-pathogen Saccharomyces cerevisiae. In this work, we describe the Trx/Trr system of C. glabrata composed of Trr1 and Trr2 (thioredoxin reductases) and Trx2 (thioredoxin) that are localized in the cytoplasm and Trx3 present in the mitochondrion. The transcriptional induction of TRR2 and TRX2 by oxidants depends on Yap1 and Skn7 and TRR1 and TRX3 have a low expression level. Both TRR2 and TRX2 play an important role in the oxidative stress response. The absence of TRX2 causes auxotrophy of methionine and cysteine. Trr1 and Trr2 are necessary for survival at high temperatures and for the chronological life span of C. glabrata. Furthermore, the Trx/Trr system is needed for survival in the presence of neutrophils. The role of TRR1 and TRX3 is not clear, but in the presence of neutrophils, they have non-overlapping functions with their TRR2 and TRX2 paralogues.

Boronate-Based Oxidant-Responsive Derivatives of Acetaminophen as Proinhibitors of Myeloperoxidase.

Myeloperoxidase (MPO) is an important component of the human innate immune system and the main source of a strong oxidizing and chlorinating species, hypochlorous acid (HOCl). Inadvertent, misplaced, or excessive generation of HOCl by MPO is associated with multiple human inflammatory diseases. Therefore, there is a considerable interest in the development of MPO inhibitors. Here, we report the synthesis and characterization of a boronobenzyl derivative of acetaminophen (AMBB), which can function as a proinhibitor of MPO and release acetaminophen, the inhibitor of chlorination cycle of MPO, in the presence of inflammatory oxidants, i.e., hydrogen peroxide, hypochlorous acid, or peroxynitrite. We demonstrate that the AMBB proinhibitor undergoes conversion to acetaminophen by all three oxidants, with the involvement of the primary phenolic product intermediate, with relatively long half-life at pH 7.4. The determined rate constants of the reaction of the AMBB proinhibitor with hydrogen peroxide, hypochlorous acid, or peroxynitrite are equal to 1.67, 1.6 × 104, and 1.0 × 106 M-1 s-1, respectively. AMBB showed lower MPO inhibitory activity (IC50 > 0.3 mM) than acetaminophen (IC50 = 0.14 mM) toward MPO-dependent HOCl generation. Finally, based on the determined reaction kinetics and the observed inhibitory effects of two plasma components, uric acid and albumin, on the extent of AMBB oxidation by ONOO- and HOCl, we conclude that ONOO- is the most likely potential activator of AMBB in human plasma.

Molsidomine decreases hyperoxia-induced lung injury in neonatal rats.

BACKGROUND: The study's objective is to evaluate if Molsidomine (MOL), an anti-oxidant, anti-inflammatory, and anti-apoptotic drug, is effective in treating hyperoxic lung injury (HLI). METHODS: The study consisted of four groups of neonatal rats characterized as the Control, Control+MOL, HLI, HLI + MOL groups. Near the end of the study, the lung tissue of the rats were evaluated with respect to apoptosis, histopathological damage, anti-oxidant and oxidant capacity as well as degree of inflammation. RESULTS: Compared to the HLI group, malondialdehyde and total oxidant status levels in lung tissue were notably reduced in the HLI + MOL group. Furthermore, mean superoxide dismutase, glutathione peroxidase, and glutathione activities/levels in lung tissue were significantly higher in the HLI + MOL group as compared to the HLI group. Tumor necrosis factor-α and interleukin-1ß elevations associated with hyperoxia were significantly reduced following MOL treatment. Median histopathological damage and mean alveolar macrophage numbers were found to be higher in the HLI and HLI + MOL groups when compared to the Control and Control+MOL groups. Both values were increased in the HLI group when compared to the HLI + MOL group. CONCLUSIONS: Our research is the first to demonstrate that bronchopulmonary dysplasia may be prevented through the protective characteristics of MOL, an anti-inflammatory, anti-oxidant, and anti-apoptotic drug. IMPACT: Molsidomine prophylaxis significantly decreased the level of oxidative stress markers. Molsidomine administration restored the activities of antioxidant enzymes. Molsidomine prophylaxis significantly reduced the levels of inflammatory cytokines. Molsidomine may provide a new and promising therapy for BPD in the future. Molsidomine prophylaxis decreased lung damage and macrophage infiltration in the tissue.

Quercetin handles cellular oxidant/antioxidant systems and mitigates immunosenescence hallmarks in human PBMCs: An in vitro study.

Immunosenescence, oxidative stress, and low vaccine efficacy are important symptoms of aging. The goal of our study was to test if quercetin had antiaging and stimulating effects on peripheral blood mononuclear cell (PBMC) immune cells in vitro in the presence of concanavalin a, PBMCs were isolated from healthy elderly and young people and cultured in a complete Roswell Park Memorial Institute 1640 medium supplemented with quercetin. Cell proliferation was assessed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide colorimetric assay after a 48-h incubation period. Spectrophotometric assays were used to assess oxidative biomarkers (proteins carbonyl [PC], malondialdehydes [MDA], and reduced glutathione [GSH]). The enzyme-linked immunosorbent assay method was used to determine the amount of interleukin (IL)-2 released. A Griess reagent was used to investigate inducible nitrite oxide synthase (iNOS) activity. When compared to young control cells, aged PBMCs had lower proliferation potency, lower IL-2 and NO release, and higher MDA and PC levels. Importantly, quercetin-treated aged PBMCs have a high proliferative response comparable to young cells, restored iNOS activity, and increased levels of GSH antioxidant defences. In comparison to untreated aged PBMCs, treated PBMCs have lower lipo-oxidative damage but higher PC levels. Quercetin may be used as a promising dietary vaccinal adjuvant in the elderly, it has significant effects in reducing immunosenescence hallmarks, as well as mitigating the lipo-oxidative stress in PBMCs cells.

Molecular mechanisms of oxidative stress-related neonatal jaundice.

Oxidative stress is a pathological condition characterized by an overload of oxidant products, named free radicals, which are not well counteracted by antioxidant systems. Free radicals induce oxidative damage to many body organs and systems. In neonatal red blood cells, free-radical mediated-oxidative stress leads to eryptosis, a suicidal death process of erythrocytes consequent to alteration of cell integrity. Neonatal red blood cells are targets and at the same time generators of free radicals through the Fenton and Haber-Weiss reactions. Enhanced eryptosis in case of oxidative stress damage may cause anemia if the increased loss of erythrocytes is not enough compensated by enhanced new erythrocytes synthesis. The oxidative disruption of the red cells may cause unconjugated idiopathic hyperbilirubinemia in neonates. High levels of bilirubin are recognized to be dangerous for the central nervous system in newborns, however, many studies have highlighted the antioxidant function of bilirubin. Recently, it has been suggested that physiologic concentration of bilirubin correlates with higher antioxidant status while high pathological bilirubin levels are associated with pro-oxidants effects. The aim of this educational review is to provide an updated understanding of the molecular mechanisms underlying erythrocyte oxidant injury and its reversal in neonatal idiopathic hyperbilirubinemia.