Concentrations Of Tert-butyl Hydroperoxide Research Articles

Redox profiles of amyotrophic lateral sclerosis lymphoblasts with or without known SOD1 mutations.

Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressing neurodegenerative disease that affects motor neurons. This disease is associated with oxidative stress especially in mutant superoxide dismutase 1 (mutSOD1) patients. However, less is known for the most prevalent sporadic ALS form, due to a lack of disease models. Here, we studied oxidative stress profiles in lymphoblasts from ALS patients with mutSOD1 or unknown (undSOD1) mutations. mutSOD1 and undSOD1 lymphoblasts, as well as sex/age-matched controls (3/group) were obtained from Coriell and divided into 46years-old-men (C1), 46years-old-women (C2) or 26/27years-old-men (C3) cohorts. Growth curves were performed, and several parameters associated with redox homeostasis were evaluated, including SOD activity and expression, general oxidative stress levels, lipid peroxidation, response to oxidative stimulus, glutathione redox cycle, catalase expression, and activity, and Nrf2 transcripts. Pooled (all cohorts) and paired (intra-cohort) statistical analyses were performed, followed by clustering and principal component analyses (PCA). Although a high heterogeneity among lymphoblast redox profiles was found between cohorts, clustering analysis based on 7 parameters with high chi-square ranking (total SOD activity, oxidative stress levels, catalase transcripts, SOD1 protein levels, metabolic response to mM concentrations of tert-butyl hydroperoxide, glutathione reductase activity, and Nrf2 transcript levels) provided a perfect cluster segregation between samples from healthy controls and ALS (undSOD1 and mutSOD1), also visualized in the PCA. Our results show distinct redox signatures in lymphoblasts from mutSOD1, undSOD1 and healthy controls that can be used as therapeutic targets for ALS drug development.

Modulating glutathione thiol status alters pancreatic β-cell morphogenesis in the developing zebrafish (Danio rerio) embryo

Emerging evidence suggests that redox-active chemicals perturb pancreatic islet development. To better understand potential mechanisms for this, we used zebrafish (Danio rerio) embryos to investigate roles of glutathione (GSH; predominant cellular redox buffer) and the transcription factor Nrf2a (Nfe2l2a; zebrafish Nrf2 co-ortholog) in islet morphogenesis. We delineated critical windows of susceptibility to redox disruption of β-cell morphogenesis, interrogating embryos at 24, 48 and 72 h post fertilization (hpf) and visualized Nrf2a expression in the pancreas using whole-mount immunohistochemistry at 96 hpf. Chemical GSH modulation at 48 hpf induced significant islet morphology changes at 96 hpf. Pro-oxidant exposures to tert-butylhydroperoxide (77.6 μM; 10-min at 48 hpf) or tert-butylhydroquinone (1 μM; 48-56 hpf) decreased β-cell cluster area at 96 hpf. Conversely, exposures to antioxidant N-acetylcysteine (bolsters GSH pools; 100 μM; 48-72 hpf) or sulforaphane (activates Nrf2a; 20 μM; 48-72 hpf) significantly increased islet areas. Nrf2a was also stabilized in β-cells: 10-min exposures to 77.6 μM tert-butylhydroperoxide significantly increased Nrf2a protein compared to control islet cells that largely lack stabilized Nrf2a; 10-min exposures to higher (776 μM) tert-butylhydroperoxide concentration stabilized Nrf2a throughout the pancreas. Using biotinylated-GSH to visualize in situ protein glutathionylation, islet cells displayed high protein glutathionylation, indicating oxidized GSH pools. The 10-min high (776 μM) tert-butylhydroperoxide exposure (induced Nrf2a globally) decreased global protein glutathionylation at 96 hpf. Mutant fish expressing inactive Nrf2a were protected against tert-butylhydroperoxide-induced abnormal islet morphology. Our data indicate that disrupted redox homeostasis and Nrf2a stabilization during pancreatic β-cell development impact morphogenesis, with implications for disease states at later life stages. Our work identifies a potential molecular target (Nrf2) that mediates abnormal β-cell morphology in response to redox disruptions. Moreover, our findings imply that developmental exposure to exogenous stressors at distinct windows of susceptibility could diminish the reserve redox capacity of β-cells, rendering them vulnerable to later-life stresses and disease.

Antioxidant Properties of Tonsil-Derived Mesenchymal Stem Cells on Human Vocal Fold Fibroblast Exposed to Oxidative Stress

The therapeutic potential of tonsil-derived mesenchymal stem cells (TMSCs) has been proved in several in vitro and in vivo models based on their antioxidative capacity. Oxidative stress is involved in the formation of vocal fold scars and the aging of vocal folds. However, few studies have examined the direct correlation between oxidative damage and reconstitution of extracellular matrix (ECM) in the vocal fold fibrosis. We, therefore, sought to investigate the impact of oxidative stress on cell survival and ECM production of human vocal fibroblasts (hVFFs) and the protective effects elicited by TMSCs against oxidative damages in hVFFs. hVFFs were exposed to different concentrations of tert-butyl hydroperoxide in the presence or absence of TMSCs. Cell viability and reactive oxygen species (ROS) production were assessed to examine the progression of oxidative stress in vitro. In addition, expression patterns of ECM-associated factors including various collagens were examined by real-time PCR and immunocytochemical analysis. We found that both cell viability and proliferation capacity of hVFFs were decreased following the exposure to tBHP in a dose-dependent manner. Furthermore, tBHP treatment induced the generation of ROS and reactive aldehydes, while it decreased endogenous activity of antioxidant enzymes in hVFF. Importantly, TMSCs could rescue these oxidative stress-associated damages of hVFFs. TMSCs also downregulated tBHP-mediated production of proinflammatory cytokines in hVFFs. In addition, coculture with TMSC could restore the endogenous matrix metalloproteinase (MMP) activity of hVFFs upon tBHP treatment and, in turn, reduce the oxidative stress-induced ECM accumulation in hVFFs. We have, therefore, shown that the changes in hVFF proliferative capacity and ECM gene expression induced by oxidative stress are consistent with in vivo phenotypes observed in aging vocal folds and vocal fold scarring and that TMSCs may function to reduce oxidative stress in aging vocal folds.

Deletion of the fungus specific protein phosphatase Z1 exaggerates the oxidative stress response in Candida albicans

BackgroundCandida albicans is an opportunistic pathogen which is responsible for widespread nosocomial infections. It encompasses a fungus specific serine/threonine protein phosphatase gene, CaPPZ1 that is involved in cation transport, cell wall integrity, oxidative stress response, morphological transition, and virulence according to the phenotypes of the cappz1 deletion mutant.ResultsWe demonstrated that a short-term treatment with a sublethal concentration of tert-butyl hydroperoxide suppressed the growth of the fungal cells without affecting their viability, both in the cappz1 mutant and in the genetically matching QMY23 control strains. To reveal the gene expression changes behind the above observations we carried out a global transcriptome analysis. We used a pilot DNA microarray hybridization together with extensive RNA sequencing, and confirmed our results by quantitative RT-PCR. Novel functions of the CaPpz1 enzyme and oxidative stress mechanisms have been unraveled. The numbers of genes affected as well as the amplitudes of the transcript level changes indicated that the deletion of the phosphatase sensitized the response of C. albicans to oxidative stress conditions in important physiological functions like membrane transport, cell surface interactions, oxidation-reduction processes, translation and RNA metabolism.ConclusionsWe conclude that in the wild type C. albicans CaPPZ1 has a protective role against oxidative damage. We suggest that the specific inhibition of this phosphatase combined with mild oxidative treatment could be a feasible approach to topical antifungal therapy.

Characterization of Bulk Rigidity of Rigid Red Blood Cell Populations in Sickle Cell Patients Using a Parameterization Model of Ektacytometry

Introduction: One of the critical characteristics of sickle-cell disease (SCD) is the increased rigidity of the red blood cell (RBC) membrane. This decreased flexibility of hemoglobin S (HbS)-rich RBCs contributes to reduced lifespan of the cell and is known to be a primary cause of vaso-occlusion, leading to painful crisis episodes and other complications in SCD. Although often overlooked as a factor in disease severity, increased RBC rigidity has been recently shown to reduce the vascular wall adhesion of white blood cells (WBCs) drastically - hinting at a larger role for RBC rigidity in patient well-being beyond instigating vasocclusion. The rigidity and composition of RBCs in SCD patients varies highly, i.e., there can be a large fraction of HbS cells with moderate stiffness or a small fraction of extremely rigid HbS cells. Ektacytometry is a primary technique used for measuring RBC deformability in SCD via laser diffraction viscometry, but is limited in that it renders an average measurement of deformability for the entire RBC population, i.e., across both stiff and healthy RBCs. This average measurement of deformability may underestimate the rigidity of HbS-rich (%S fraction) or sickle RBC population in the blood. Here, we present an approach to estimate rigidity of HbS cells in SCD via a method that implements artificially rigidified RBCs mixed into whole blood at different ratios. Methods: We develop an approach to estimate rigidity of HbS cells in SCD via a method that implements artificially rigidified RBCs. RBCs from healthy (non-SCD) are incubated in solutions with varying concentrations of tert-butyl hydroperoxide to stiffen the cell membrane. These artificially rigidified RBCs are then mixed into whole blood at different ratios/fractions. Four parent concentrations of TBHP are chosen as base RBC rigidities: 1.0, 0.9, 0.75, and 0.5 mM TBHP. After adequate washing, stiffened RBCs are mixed with healthy RBCs in whole blood in increments of 10% rigid RBC fractions up to a total of 100% while holding the hematocrit constant at ~40%. All samples are then measured independently using a LoRRca MaxSis Ektacytometer (Mechatronics Instruments BV, Zwaag, The Netherlands). We then parameterize these ektacytometry deformability measurements to develop a numerical model that predicts the bulk rigidity of the rigid RBC population in SCD blood samples. We measured the EI for fifteen different SCD patient blood samples, details including patient SCD genotype, age, gender, current medical intervention/therapies, %S fraction, and measured maximum bulk EI. Results: With the predicted maximum EI of the rigid RBC population in patient blood samples, we plotted deformability curves using techniques described by Baskurt et al. We utilized a simplified Skreestra-Bronkhorst model for plotting predicted deformability curves implementing critical parameters such as maximum EI, shear where half the maximum EI is achieved, and the range of shear stress in the standard ektacytometry deformability measurement. The predicted bulk SCD whole blood deformability curves are overlaid with the healthy blood curve, experimental bulk SCD whole blood standard measurement, and with the predicted curve of the sickle RBC population for patients 2, 6, and 7 in Figure 1a, 1b, and 1c. These plots show that the precision of the parameterized deformability curves compared to a standard healthy curve and actual measurement of the patient sample. Specifically, in Figure 1a and 1b shows there is large differences in the elongation curve of the sickle population in comparison to the bulk measurements, yielding a maximum EIs that are ~2 times smaller than the bulk measurement. Conversely, in Figure 1c we see there is not much difference in the elongation curve of the sickle population compared to the actual raw standard measurement. Conclusions: Overall, this work presents an innovative method for a more thorough examination of ektacytometry deformability measurements of SCD patients. An understanding of bulk stiffness of the HbS population is crucial as it would allow for more accurate characterization of the impact of RBC rigidity on SCD patient crisis and general wellbeing. More detailed information on the rigidity of the HbS RBC population in SCD has the potential to be utilized to understand phenomena in hemodynamics and connection between RBC rigidity and occurrences of painful crisis in SCD patients. Disclosures No relevant conflicts of interest to declare.

The effect of AP-2\u03b4 on transcription of the Prestin gene in HEI-OC1 cells upon oxidative stress

BackgroundThe study aimed to investigate the effect of oxidative stress on Prestin expression, and explore the transcription factors (TFs) that are involved in regulating the expression of Prestin in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells upon oxidative stress.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to detect the expression level of Prestin. Reverse chromatin immunoprecipitation (reverse ChIP) assay was performed to identify proteins that could bind to the Prestin gene. Small interfering RNA (siRNA) and chromatin immunoprecipitation (ChIP) experiments were used to further verify the results. HEI-OC1 cells were incubated with four different concentrations of tert-butyl hydroperoxide (t-BHP) for 24 h or 48 h to construct the oxidative stress model.ResultsOxidative stress induced Prestin increase at the mRNA level but with a concomitant decrease at the protein level. TF activating enhancer binding protein-2δ (AP-2δ) screened by reverse ChIP assay was demonstrated to bind to transcriptional start site 1441 of the Prestin promoter region and negatively regulate the expression of Prestin by siRNA and ChIP experiments. Furthermore, AP-2δ was down-regulated under oxidative stress.ConclusionsIn conclusion, oxidative stress inhibits the expression of Prestin protein, and the transcription mechanism is triggered to compensate for the loss of Prestin protein. AP-2δ is one of the important TFs that suppresses transcription of the Prestin gene, and AP-2δ suppression further boosted Prestin mRNA activation under oxidative stress.

On the Effects of Reactive Oxygen Species and Nitric Oxide on Red Blood Cell Deformability.

The main function of red blood cells (RBCs) is the transport of respiratory gases along the vascular tree. To fulfill their task, RBCs are able to elastically deform in response to mechanical forces and, pass through the narrow vessels of the microcirculation. Decreased RBC deformability was observed in pathological conditions linked to increased oxidative stress or decreased nitric oxide (NO) bioavailability, like hypertension. Treatments with oxidants and with NO were shown to affect RBC deformability ex vivo, but the mechanisms underpinning these effects are unknown. In this study we investigate whether changes in intracellular redox status/oxidative stress or nitrosation reactions induced by reactive oxygen species (ROS) or NO may affect RBC deformability. In a case-control study comparing RBCs from healthy and hypertensive participants, we found that RBC deformability was decreased, and levels of ROS were increased in RBCs from hypertensive patients as compared to RBCs from aged-matched healthy controls, while NO levels in RBCs were not significantly different. To study the effects of oxidants on RBC redox state and deformability, RBCs from healthy volunteers were treated with increasing concentrations of tert-butylhydroperoxide (t-BuOOH). We found that high concentrations of t-BuOOH (≥ 1 mM) significantly decreased the GSH/GSSG ratio in RBCs, decreased RBC deformability and increased blood bulk viscosity. Moreover, RBCs from Nrf2 knockout (KO) mice, a strain genetically deficient in a number of antioxidant/reducing enzymes, were more susceptible to t-BuOOH-induced impairment in RBC deformability as compared to wild type (WT) mice. To study the role of NO in RBC deformability we treated RBC suspensions from human volunteers with NO donors and nitrosothiols and analyzed deformability of RBCs from mice lacking the endothelial NO synthase (eNOS). We found that NO donors induced S-nitrosation of the cytoskeletal protein spectrin, but did not affect human RBC deformability or blood bulk viscosity; moreover, under unstressed conditions RBCs from eNOS KO mice showed fully preserved RBC deformability as compared to WT mice. Pre-treatment of human RBCs with nitrosothiols rescued t-BuOOH-mediated loss of RBC deformability. Taken together, these findings suggest that NO does not affect RBC deformability per se, but preserves RBC deformability in conditions of oxidative stress.

Partial Isobutane Oxidation to tert‐Butyl Hydroperoxide in a Micro Reactor – Comparison of DTBP and Aqueous TBHP as Initiator

AbstractThe oxidation of isobutane has been investigated for the first time in a micro reactor for a wide range of process conditions comparing the product ratios obtained with di‐tert‐butyl peroxide (DTBP) and tert‐butyl hydroperoxide (TBHP) as initiator. DTBP led to nearly the double conversion compared to the same concentration of TBHP, but the selectivity of TBHP using TBHP as initiator was higher. Using aqueous TBHP, the conversions reached up to 6 % in case of supercritical conditions but stayed below 2 % in case of two‐phase flow experiments. With TBHP in decane as initiator a high conversion was reached but nearly no TBHP was formed. It seems that the silicon layer of the micro reactor was not stable under such experimental conditions. In the present work, for the first time a conversion level near to that of the industrial batch process has been reached in a micro reactor for the isobutane oxidation.

Development of graft copolymer of carboxymethylcellulose and N-vinylcaprolactam towards strong antioxidant and antibacterial polymeric materials

The present paper reports graft copolymerization of N-vinylcaprolactam (NVCL) onto carboxymethylcellulose (CMC) using tert-butyl hydroperoxide (TBHP) initiator in N2 atmosphere. The grafting was found to be highest when concentrations of CMC, NVCL and TBHP were 6.25×10−2gdm−3, 10×10−3moldm−3 and 20×10−5moldm−3 respectively. The graft copolymer (CMC-g-PNVCL-1) was characterized by FTIR, 1HNMR, SEM, AFM, XRD and TGA analysis. The antioxidant activity of the graft copolymer was found to be higher (3.81%) than CMC and slightly less (5.47%) than the standard one (butylated hydroxytoluene, BHT). The hydroxyl radical scavenging activity of graft copolymer was found to be slightly less (13.69%) than CMC and more (44.20%) than the BHT. The hydrogen peroxide scavenging activity of graft copolymer was observed to be less (20.42%) than CMC and 53.34% less than BHT. The superoxide scavenging activity of graft copolymer was greater than the both. Compared to CMC the graft copolymer has shown greater antibacterial activity against S. aureus, Proteus vulgaris and S. typhi bacteria and less against Klebsiellapneumonia and Salmonella typhimurium.

Synthesis, characterisation and catalytic activity of dithiocarbazate Schiff base complexes in oxidation of cyclohexane

Schiff base complexes of transition metals can be used for catalytic oxidation of hydrocarbons in the presence of tert-butyl hydroperoxide (TBHP). In this work, metal complexes of dithiocarbazate Schiff bases (abbreviated as NiSBdiAP, CuSBdiAP, CoSBdiAP, FeSBdiAP, MnSBdiAP and ZnSBdiAP, in which SBdiAP represents the Schiff base) were synthesized and characterized using several techniques; Fourier transforms infrared spectroscopy, nuclear magnetic resonance, 1H NMR and 13C NMR, magnetic susceptibility measurements, molar conductivity, ultraviolet-visible spectroscopy, and inductively coupled plasma spectrometry. The oxidation of cyclohexane by the prepared metal complexes as catalysts was investigated in the presence of TBHP as an oxidative source under mild conditions. The product was analyzed using gas chromatography. It was found that cyclohexanol and cyclohexanone were the main products of the oxidation reaction. The iron complex of Schiff base derived from 2,6-diacetyl pyridine and S-benzyldithiocarbazate showed the highest activity during the screening studies towards cyclohexane oxidation. The results showed that the time of the reaction, temperature, and the concentration of TBHP and the catalyst type influenced the selectivity and conversion of cyclohexane oxidation. The selectivity of the reaction was 96% indicating the significance of the work toward industrial oxidation of cyclohexane.

326 - The Role of Cytoskeletal S-Nitrosation in Red Blood Cell Deformability

The deformability of red blood cells (RBC) is an important characteristic of RBC functionality, and contributes to define blood rheological properties and blood viscosity. The ability of RBCs to change their shape mainly depends on the flexibility of the interactions between cytoskeletal proteins, in particular of the structure of the main cysteine rich protein spectrin. In this study we aimed to analyze the effects of S-nitrosation of spectrin on changes in RBC deformability. We found that treatment with S-nitrosocystein (SNOC) leads to S-nitrosation of purified spectrin as assessed by chemiluminescence detection (CLD). Similarly, SNOC-induced nitrosation of cytoskeletal proteins of RBC ghosts and RBC were measured by CLD and the biotin switch assay. Western blotting following the biotin switch assay confirmed that spectrin was one of the nitrosated cytoskeletal proteins in RBC ghosts and RBCs. However, treatment with SNOC or DEA/NO did not affect deformability of RBCs as assessed by ektacytometry measured in a LORRCA system, nor blood viscosity measured in a low shear rotational viscometer under the same shear rate conditions. Instead, we found that changes in redox state of RBC by treatment with high concentrations of tert-Butyl hydroperoxide decreased deformability and increased blood viscosity. Our results demonstrate that incubation of RBC, RBC ghosts and purified spectrin with a nitrosothiol result in significantly increased S-nitrosation of RBC cytoskeletal spectrin. However, nitrosation did not improve RBC deformability or change blood viscosity. Hence, in our setting change in redox state/oxidative modification rather than nitrosation reactions of cytoskeletal proteins like spectrin have an impact on RBC deformability and blood viscosity.

Solvent-controlled synthesis of tetranuclear cage-like copper(ii) silsesquioxanes. Remarkable features of the cage structures and their high catalytic activity in oxidation with peroxides

Two principally different in their molecular architecture isomeric tetranuclear copper(ii) silsesquioxanes, "Globule"-like compound [(PhSiO1.5)12(CuO)4(NaO0.5)4] (1) and "Sandwich"-like derivative [(PhSiO1.5)6(CuO)4(NaO0.5)4(PhSiO1.5)6] (2), were synthesized by the partial cleavage of polymeric copper(ii) silsesquioxane [(PhSiO1.5)2(CuO)]n by tetraphenylcyclotetrasiloxanolate. The route leading to the formation of either 1 or 2 entirely depends on the nature and composition of the solvent used for this reaction. Thus, the process in an ethanol-1-butanol solution gives compound 1. When a 1,4-dioxane-methanol mixture was used, compound 2 was prepared. The structures and unusual crystal packing of the cages were confirmed by the X-ray studies. It has been found that the reaction of benzene with H2O2 in acetonitrile solution at 50 °C catalyzed by 1 requires addition of trifluoroacetic acid (TFA) in low concentration and gives phenol with a turnover number (TON) of 250 after 3 h. The initial reaction rate W0 linearly depends on the concentration of catalyst 2. The oxidation of 1-phenylethanol to acetophenone with hydrogen peroxide catalyzed by complex 1 in the presence of TFA is not efficient. In contrast, 1 exhibited excellent activity in the oxidation with tert-butyl hydroperoxide (TBHP) in the absence of any acid (the yield of acetophenone was close to the quantitative, TON attained 475 after 2 h). A kinetic study of this reaction led to the conclusion that the process occurs with the participation of radicals tert-BuO˙ produced in the Cu-promoted decomposition of TBHP. The mode of dependence of W0 on the initial concentration of TBHP indicates the formation of an intermediate adduct between the catalyst 1 and TBHP (characterized by the equilibrium constant K1≈ 2 M(-1) for the conditions of conducted experiments) followed by subsequent decomposition of the adduct (k2≈ 0.2 s(-1)) to generate an intermediate species tert-BuO˙ which induces the alcohol oxidation.

Mechanism of induction of oxidative stress in liver mitochondria by low concentrations of tert-butyl hydroperoxide

The mechanism of the effect of tert-butyl hydroperoxide (tBHP) on the kinetics of decrease in liver mitochondrial ΔΨ (transmembrane electric potential) in response to successive additions of tBHP in low concentrations has been studied. FeSO(4) was found to increase significantly the damaging effect of tBHP; this effect was shown to increase in the presence of low concentrations of Ca2+ starting from 2 µM CaCl(2). Cyclosporin A prevents these effects. The data show that the damaging effect of low concentrations of tBHP in the course of pyruvate oxidation in isolated liver mitochondria is caused by the opening of the nonspecific Ca2+-dependent cyclosporin A-sensitive pore in the inner mitochondrial membrane. Application of a method of studying oxidative stress regulators, developed in this work, is illustrated by an example of the prooxidant action of ascorbate. This method is proposed for studying mitochondria in hemochromatosis, a pathology caused by excessive accumulation of iron.

Pronounced catalytic activity of oxo-vanadium(IV) Schiff base complexes in the oxidation of cyclooctene and styrene by tert-butyl hydroperoxide

Abstract A practical catalytic method for efficient oxidation of olefins using tert-butyl hydroperoxide catalyzed by N2O2 tetradentate Schiff base–VO(IV) complexes has been developed. Using these catalytic systems for the oxidation of cyclooctene gives the epoxide as the sole product with yields up to 98% while oxidation of styrene produce styrene oxide and benzaldehyde as major products in excellent yields (100%). The efficiency of the catalysts is strongly influenced by the nature of solvent, reaction time, temperature, tert-butyl hydroperoxide concentration and also catalyst concentration.

Profiles of oxidative stress-related microRNA and mRNA expression in auditory cells

Oxidative stress and high levels of reactive oxygen species (ROS) are risk factors of auditory cell injury and hearing impairment. MicroRNAs (miRNAs) are critical for the post-transcriptional regulation of gene expression and cell proliferation and survival. However, little is known about the impact of oxidative stress on the expression of miRNAs and their targeted mRNAs in auditory cells. We employed a cell model of oxidative stress by treatment of House Ear Institute-Organ of Corti 1 (HEI-OC1) cells with different concentrations of tert-butyl hydroperoxide (t-BHP) to examine the t-BHP-induced production of ROS and to determine the impact of t-BHP treatment on the relative levels of miRNA and mRNA transcripts in HEI-OC1 cells. We found that treatment with different concentrations of t-BHP promoted the production of ROS, but inhibited the proliferation of HEI-OC1 cells in a dose- and time-dependent manner. Furthermore, treatment with t-BHP induced HEI-OC1 cell apoptosis. Further microarray analyses revealed that treatment with t-BHP increased the transcription of 35 miRNAs, but decreased the expression of 40 miRNAs. In addition, treatment with t-BHP up-regulated the transcription of 2076 mRNAs, but down-regulated the levels of 580 mRNA transcripts. Notably, the up-regulated (or down-regulated) miRNAs were associated with the decreased (or increased) expression of predicted targeted mRNAs. Importantly, these differentially expressed mRNAs belonged to different functional categories, forming a network participating in the oxidative stress-related process in HEI-OC1 cells. Therefore, our findings may provide new insights into understanding the regulation of miRNAs on the oxidative stress-related gene expression and function in auditory cells.

Sialic acid attenuates the cytotoxicity of the lipid hydroperoxides HpODE and HpETE

Reduction of peroxide molecular species is an essential function in living organisms. In previous studies, we proposed a new function for the sialic acid N-acetylneuraminic acid (Neu5Ac)—that of antioxidant/hydrogen peroxide scavenging agent. On the basis of the reaction scheme, Neu5Ac is thought to act as a general antioxidant of all hydroperoxide-type species (R–OOHs). The concentration of tert-butyl hydroperoxide ( t-BuOOH) decreased after co-incubation with N-acetylneuraminic acid. Neu5Ac also decreased the R–OOH concentration in solutions of peroxylinolenic acid (13( S)-hydroperoxy-(9 Z,11 E)-octadecadienoic acid, HpODE) and peroxyarachidonic acid (15( S)-hydroperoxy-(5 Z,8 Z,11 Z,13 E)-eicosatetraenoic acid, HpETE)—two lipid hydroperoxides that participate in many physiological events. Moreover, the cytotoxicity of both these lipid hydroperoxides was attenuated by reaction with Neu5Ac acid. Our results suggest that N-acetylneuraminic acid is a potential antioxidant of most hydroperoxides that accumulate in organisms.

Decreased iPLA2γ expression induces lipid peroxidation and cell death and sensitizes cells to oxidant-induced apoptosis

Our previous studies showed that renal proximal tubular cells (RPTC) express Ca(2+)-independent phospholipase A(2)gamma (iPLA(2)gamma) in endoplasmic reticulum (ER) and mitochondria and that iPLA(2)gamma prevents and/or repairs lipid peroxidation induced by oxidative stress. Our present studies determined the importance of iPLA(2)gamma in mitochondrial and cell function using an iPLA(2)gamma-specific small hairpin ribonucleic acid (shRNA) adenovirus. iPLA(2)gamma expression and activity were decreased in the ER by 24 h and in the mitochondria by 48 h compared with scrambled shRNA adenovirus-treated cells. Lipid peroxidation was elevated by 2-fold at 24 h and remained elevated through 72 h in cells with decreased iPLA(2)gamma. Using electrospray ionization-mass spectrometry, primarily phosphatidylcholines and phosphatidylethanolamines were increased in iPLA(2)gamma-shRNA-treated cells. At 48 h after exposure to the iPLA(2)gamma shRNA, uncoupled oxygen consumption was inhibited by 25% and apoptosis was observed at 72 and 96 h. RPTC with decreased iPLA(2)gamma expression underwent apoptosis when exposed to a nonlethal concentration of the oxidant tert-butyl hydroperoxide (TBHP). Exposure of control cells to a nonlethal concentration of TBHP induced iPLA(2)gamma expression in RPTC. These results suggest that iPLA(2)gamma is required for the prevention and repair of basal lipid peroxidation and the maintenance of mitochondrial function and viability, providing further evidence for a cytoprotective role for iPLA(2)gamma from oxidative stress.

Screening of senescence-associated genes with specific DNA array reveals the role of IGFBP-3 in premature senescence of human diploid fibroblasts

Repeated exposures to sublethal concentrations of tert-butylhydroperoxide and ethanol trigger premature senescence of WI-38 human diploid fibroblasts. We found 16 replicative senescence-related genes with similar alterations in expression level in replicative senescence and two models of stress-induced premature senescence. Among these genes was IGFBP-3. Using a siRNA approach, we showed that IGFBP-3 regulates the appearance of several biomarkers of senescence after repeated exposures of WI-38 fibroblasts to tert-butylhydroperoxide and ethanol.

Spectrophotometric Determination of Free Radicals and Reactive Oxygen Species wih N,N-Dimethyl-p-phenylendiamine

A novel spectrophotometry for the determination of total free radicals and peroxides was developed using N,N,-dimethyl-p-phenylenediamine (DMPD) as a detection reagent. The stability of a DMPD aqueous solution was improved by dissolving DMPD in dimethyl sulfoxide (DMSO). The optimal conditions for the detection of free radicals in human serum were examined with DMPD. DMPD, the sample and 100 μmol L−1 ammonium iron(II) sulfate were mixed in a 0.1 mol L−1 acetate buffer solution (pH 5.0) containing 5% DMSO and left standing at 40°C for 10 minutes, followed by a measurement of the absorbance at 550 nm. The calibration curves of free radicals (alkoxyl radical, hydroxyl radical and nitrogen monoxide) showed good linearity over the range of 2.9 nmol L−1∼40 μmol L−1 (r2 = 0.995), 75.2∼500 nmol L−1 (r2 = 0.992) and 44.5∼300 μmol L−1 (r2 = 0.991), respectively. The detection limits of alkoxyl radical, hydroxyl radical and nitrogen monoxide were 0.9 nmol L−1, 22.6 nmol L−1 and 13.3 μmol L−1, respectively. The recovery of 10 μmol L−1 alkoxyl radical in human serum was 119%. The average of the total concentration for free radicals and peroxides as the concentration of tert-butyl hydroperoxide in human serum was 7.0 μmol L−1 (n = 6).

Heme oxygenase-1 and interleukin-11 are overexpressed in stress-induced premature senescence of human WI-38 fibroblasts induced by tert-butylhydroperoxide and ethanol

Acute repeated exposures to subcytotoxic concentrations of tert-butylhydroperoxide and ethanol trigger premature senescence of human diploid fibroblasts. In the present work we found an increased mRNA and protein level of interleukin-11 and heme oxygenase-1 in premature senescence of WI-38 human diploid foetal lung fibroblasts induced by both tert-butylhydroperoxide and ethanol. We tested whether interleukin-11 and heme oxygenase-1 could protect against tert-butylhydroperoxide- or ethanol-induced premature senescence when stable overexpression was established using a retroviral vector-based transduction. No protective effect was found against the decrease of the proliferative potential, the increase of the proportion of senescence-associated ss-galactosidase positive cells and the increase of the mRNA levels of six senescence-associated genes.