Effect of hydrogen-peroxide-mediated oxidative stress on human dental pulp cells

To evaluate the effect of the oxidative stress on human dental pulp cells (HDPCs) promoted by toxic concentrations of hydrogen peroxide (H2O2) on its odontoblastic differentiation capability through time. Methods HDPCs were exposed to two different concentrations of H2O2 (0.1 and 0.3 μg/ml) for 30 min. Thereafter, cell viability (MTT assay) and oxidative stress generation (H2DCFDA fluorescence assay) were immediately evaluated. Data were compared with those for alkaline phosphatase (ALP) activity (thymolphthalein assay) and mineralized nodule deposition (alizarin red) by HDPCs cultured for 7 days in osteogenic medium. Results A significant reduction in cell viability and oxidative stress generation occurred in the H2O2-treated cells when compared with negative controls (no treatment), in a concentration-dependent fashion. Seven days after H2O2 treatment, the cells showed significant reduction in ALP activity compared with negative control and no mineralized nodule deposition. Conclusion Both concentrations of H2O2 were toxic to the cells, causing intense cellular oxidative stress, which interfered with the odontogenic differentiation capability of the HDPCs. Clinical significance The intense oxidative stress on HDPCs mediated by H2O2 at toxic concentrations promotes intense reduction on odontoblastic differentiation capability in a 7-day evaluation period, which may alter the initial pulp healing capability in the in vivo situation.

Atmospheric pressure gas plasma-induced colorectal cancer cell death is mediated by Nox2-ASK1 apoptosis pathways and oxidative stress is mitigated by Srx-Nrf2 anti-oxidant system

Atmospheric pressure gas plasma (AGP) generates reactive oxygen species (ROS) that induce apoptosis in cultured cancer cells. The majority of cancer cells develop a ROS-scavenging anti-oxidant system regulated by Nrf2, which confers resistance to ROS-mediated cancer cell death. Generation of ROS is involved in the AGP-induced cancer cell death of several colorectal cancer cells (Caco2, HCT116 and SW480) by activation of ASK1-mediated apoptosis signaling pathway without affecting control cells (human colonic sub-epithelial myofibroblasts; CO18, human fetal lung fibroblast; MRC5 and fetal human colon; FHC). However, the identity of an oxidase participating in AGP-induced cancer cell death is unknown. Here, we report that AGP up-regulates the expression of Nox2 (NADPH oxidase) to produce ROS. RNA interference designed to target Nox2 effectively inhibits the AGP-induced ROS production and cancer cell death. In some cases both colorectal cancer HT29 and control cells showed resistance to AGP treatment. Compared to AGP-sensitive Caco2 cells, HT29 cells show a higher basal level of the anti-oxidant system transcriptional regulator Nrf2 and its target protein sulfiredoxin (Srx) which are involved in cellular redox homeostasis. Silencing of both Nrf2 and Srx sensitized HT29 cells, leads to ROS overproduction and decreased cell viability. This indicates that in HT29 cells, Nrf2/Srx axis is a protective factor against AGP-induced oxidative stress. The inhibition of Nrf2/Srx signaling should be considered as a central target in drug-resistant colorectal cancer treatments.

Effects of temperature on complexes I and II mediated respiration, ROS generation and oxidative stress status in isolated gill mitochondria of the mud crab Scylla serrata

Effects of fluctuations in habitat temperature (18-30 degrees) on mitochondrial respiratory behavior and oxidative metabolic responses in the euryhaline ectotherm Scylla serrate are not fully understood. In the present study, effects of different temperatures ranging from 12 to 40 degrees C on glutamate and succinate mediated mitochondrial respiration, respiratory control ratio (RCR), ATP generation rate, ratio for the utilization of phosphate molecules per atomic oxygen consumption (P/O), levels of lipid peroxidation and H2O2 in isolated gill mitochondria of S. serrata are reported. The pattern of variation in the studied parameters was similar for the two substrates at different temperatures. The values recorded for RCR ( >= 3) and P/O ratio (1.4-2.7) at the temperature range of 15-25 degrees C were within the normal range reported for other animals (3-10 for RCR and 1.5-3 for P/O). Values for P/O ratio, ATP generation rate and RCR were highest at 18 degrees C when compared to the other assay temperatures. However, at low and high extreme temperatures, i.e. at 12 and 40 degrees C, states III and IV respiration rates were not clearly distinguishable from each other indicating that mitochondria were completely uncoupled. Positive correlations were noticed between temperature and the levels of both lipid peroxidation and H2O2. It is inferred that fluctuations on either side of ambient habitat temperature may adversely influence mitochondria respiration and oxidative metabolism in S. serrata. The results provide baseline data to understand the impacts of acute changes in temperature on ectotherms inhabiting estuarine or marine environments. (C) 2014 Elsevier Ltd. All rights reserved.

Magmas functions as a ROS regulator and provides cytoprotection against oxidative stress-mediated damages

Redox imbalance generates multiple cellular damages leading to oxidative stress-mediated pathological conditions such as neurodegenerative diseases and cancer progression. Therefore, maintenance of reactive oxygen species (ROS) homeostasis is most important that involves well-defined antioxidant machinery. In the present study, we have identified for the first time a component of mammalian protein translocation machinery Magmas to perform a critical ROS regulatory function. Magmas overexpression has been reported in highly metabolically active tissues and cancer cells that are prone to oxidative damage. We found that Magmas regulates cellular ROS levels by controlling its production as well as scavenging. Magmas promotes cellular tolerance toward oxidative stress by enhancing antioxidant enzyme activity, thus preventing induction of apoptosis and damage to cellular components. Magmas enhances the activity of electron transport chain (ETC) complexes, causing reduced ROS production. Our results suggest that J-like domain of Magmas is essential for maintenance of redox balance. The function of Magmas as a ROS sensor was found to be independent of its role in protein import. The unique ROS modulatory role of Magmas is highlighted by its ability to increase cell tolerance to oxidative stress even in yeast model organism. The cytoprotective capability of Magmas against oxidative damage makes it an important candidate for future investigation in therapeutics of oxidative stress-related diseases.

Serotonin receptor subtypes functional regulation and oxidative stress mediated apoptosis in 6-hydroxydopamine lesioned Parkinsonian rats

Parkinson’s disease is a chronic progressive neurodegenerative disorder characterized by the selective loss of dopaminergic neurons in the SNpc resulting in severe motor impairments. Serotonergic system plays an important regulatory role in the pathophysiology of PD in rats, the evaluation of which provides valuable insight on the underlying mechanisms of motor, cognitive and memory deficits in PD. We observed a decrease in 5-HT content in the brain regions of 6-OHDA infused rat compared to control. The decreased 5-HT content resulted in a decrease of total 5-HT, 5-HT2A receptors and 5-HTT function and an increase of 5-HT2C receptor function. 5-HT receptor subtypes - 5-HT2A and 5-HT2C receptors have differential regulatory role on the modulation of DA neurotransmission in different brain regions during PD. Our observation of impaired serotonergic neurotransmission in SNpc, corpus striatum, cerebral cortex, hippocampus, cerebellum and brain stem demonstrate that although PD primarily results from neurodegeneration in the SNpc, the associated neurochemical changes in other areas of the brain significantly contributes to the different motor and non motor symptoms of PD. The antioxidant enzymes – SOD, CAT and GPx showed significant down regulation which indicates increased oxidative damage resulting in neurodegeneration. We also observed an increase in the level of lipid peroxidation. Reduced expression of anti-apoptotic Akt and enhanced expression of NF-B resulting from oxidative stress caused an activation of caspase-8 thus leading the cells to neurodegeneration by apoptosis. BMC administration in combination with 5-HT and GABA to PD rats showed reversal of the impaired serotonergic neurotransmission and oxidative stress mediated apoptosis. The transplanted BMC expressed NeuN confirming that 5-HT and GABA induced the differentiation and proliferation of BMC to neurons in the SNpc along with an increase in DA content and an enhanced expression of TH. Neurotrophic factors – BDNF and GDNF rendered neuroprotective effects accompanied by improvement in behavioural deficits indicating a significant reversal of altered dopaminergic and serotonergic neurotransmission in PD. The restorative and neuroprotective effects of BMC in combination with 5-HT and GABA are of immense therapeutic significance in the clinical management of PD.

DNA hypomethylation and oxidative stress-mediated increase in genomic instability in equine sarcoid-derived fibroblasts

It is widely accepted that equine sarcoid disease, the most common skin associated neoplasm in equids, is induced by bovine papillomavirus (BPV-1). Although BPV-1 DNA has been found in almost all examined sarcoids so far, its detailed impact on the horse's host cell metabolism is largely unknown. We used equine fibroblast cell lines originating from sarcoid biopsies to study BPV-1-associated changes on DNA methylation status and oxidative stress parameters. Sarcoid-derived fibroblasts manifested increased proliferation in vitro, transcriptional rDNA activity (NORs expression) and DNA hypomethylation compared to control cells. Cells isolated from equine sarcoids suffered from oxidative stress: the expression of antioxidant enzymes was decreased and the superoxide production was increased. Moreover, increased ploidy, oxidative DNA damage and micronuclei formation was monitored in sarcoid cells. We postulate that both altered DNA methylation status and redox milieu may affect genomic stability in BPV-1-infected cells and in turn contribute to sarcoid pathology.

Cystine-mediated oxidative defence in Lactobacillus reuteri BR11

Lactobacillus reuteri BR11 possesses an abundant cystine uptake (Cyu) ABC-transporter that was previously found to be involved in a novel mechanism of oxidative defence mediated by cystine. The current study aimed to elucidate this mechanism with a focus on the role of the co-transcribed cystathionine ã-lyase (Cgl). Growth studies of wild-type L. reuteri BR11 and mutants inactivated in cgl and the cystine-binding protein encoding gene cyuC showed that in contrast to the Cyu transporter, whose inactivation led to growth arrest in aerated cultures, Cgl is not crucial for oxidative defence. However, the role of Cgl in oxidative defence became apparent in the presence of severe oxidative damage and cysteine deprivation. Cysteine was found to be protective against oxidative stress, and the action of Cgl in both cysteine biosynthesis and degradation poses a seemingly futile pathway that deprives the intracellular cysteine pool. To further characterise the relationship between Cgl activity and cysteine and their roles in oxidative defence, enzymatic assays were performed on purified Cgl, and intracellular concentrations of cysteine, cystathionine and methionine were determined. Cgl was highly active towards cystine and cystathionine and less active towards cysteine in vitro, suggesting the main function of Cgl to be cysteine biosynthesis. Cysteine was found at high concentrations in the cell, but the levels were not significantly affected by inactivation of cgl or growth under aerobic conditions. It was concluded that both anabolic and catabolic activities of Cgl towards cysteine contribute to oxidative defence, the former by maintaining an intracellular reservoir of thiol analogous to glutathione, and the latter by producing H2S which is readily secreted, thus creating a reducing extracellular environment. The significance of the Cyu transporter to the physiology of L. reuteri BR11 prompted a phylogenetic study to determine its presence in bacteria. Orthologs of the Cyu transporter that are closest matches to the Cyu transporter are only limited to several species of Lactobacillus and Leuconostoc. Outside the Lactobacillales order, the closest matching orthologs belong to Proteobacteria, and there are more orthologs in Proteobacteria than non-Lactobacillales Firmicutes, suggesting that the Cyu transporter locus was present in the ancestor of the Proteobacteria and Firmicutes, and over evolutionary time has been lost or diverged in many Firmicutes. The clustering of the Cyu transporter locus with a gene encoding a Cgl family protein is even rarer. It was only found in L. reuteri, Lactobacillus vaginalis, Weissella paramesenteroides, the Lactobacillus casei group, and several Campylobacter sp. An accompanying phylogenetic study of L. reuteri BR11 using multi-locus sequence analysis showed that L. reuteri BR11 had diverged from more than 100 strains of L. reuteri isolated from various hosts and geographical locations. However, comparison with other Lactobacillus species supported the current classification of BR11 as L. reuteri. The most closely related species to L. reuteri is L. vaginalis or Lactobacillus antri, depending on the housekeeping gene used for analysis. The close evolutionary relationship of L. vaginalis to L. reuteri and the high degree of sequence identity between the cgl-cyuABC loci in both species suggest that the Cyu system is highly likely to perform similar functions in L. vaginalis. In search of other genes that function in oxidative defence, a number of mutants which were inactivated in genes that confer increased resistance to oxidative stress in other bacteria were constructed. The genes targeted were ahpC (peroxidase component of the alkyl hydroperoxide reductase system), tpx (thiol peroxidase), osmC (osmotically induced protein C), mntH (Mn2+/Fe2+ transporter), gshA (ã-glutamylcysteine synthetase) and msrA (methionine sulfoxide reductase). The ahpC and mntH mutants had slightly lower minimum inhibitory concentrations of organic peroxides, suggesting these genes might be involved in resistance to organic peroxides in L. reuteri. However, none of the mutants exhibited growth defects in aerated cultures, in stark contrast to the cyuC mutant. This may be due to compensatory functions of other genes, a hypothesis which cannot be tested until a robust protocol for constructing markerless multiple gene deletion mutants in L. reuteri is developed. These results highlight the importance of the Cyu transporter in oxidative defence and provide a foundation for extending the research of this system in other bacteria.

SECIS-binding protein 2 promotes cell survival by protecting against oxidative stress

Reactive oxygen species (ROS) are a primary cause of cellular damage that leads to cell death. In cells, protection from ROS-induced damage and maintenance of the redox balance is mediated to a large extent by selenoproteins, a distinct family of proteins that contain selenium in form of selenocysteine (Sec) within their active site. Incorporation of Sec requires the Sec-insertion sequence element (SECIS) in the 3'-untranslated region of selenoproteins mRNAs and the SECIS-binding protein 2 (SBP2). Previous studies have shown that SBP2 is required for the Sec-incorporation mechanism; however, additional roles of SBP2 in the cell have remained undefined. We herein show that depletion of SBP2 by using antisense oligonucleotides (ASOs) causes oxidative stress and induction of caspase- and cytochrome c-dependent apoptosis. Cells depleted of SBP2 have increased levels of ROS, which lead to cellular stress manifested as 8-oxo-7,8-dihydroguanine (8-oxo-dG) DNA lesions, stress granules, and lipid peroxidation. Small-molecule antioxidants N-acetylcysteine, glutathione, and α-tocopherol only marginally reduced ROS and were unable to rescue cells fully from apoptosis, indicating that apoptosis might be directly mediated by selenoproteins. Our results demonstrate that SBP2 is required for protection against ROS-induced cellular damage and cell survival. Antioxid. Redox Signal. 12, 797–808.

Regulation of oxidative-stress responsive genes by arecoline in human keratinocytes

Background and Objective: Arecoline, an arecanut alkaloid present in the saliva of betel quid chewers, has been implicated in the pathogenesis of a variety of inflammatory oral diseases, including oral submucous fibrosis and periodontitis. To understand the molecular b asis of arecoline action in epithelial changes associated with these diseases, we investigated the effects of arecoline on human keratinocytes with respect to cell growth regulation and the expression of stress-responsive genes.Material and Methods:Human keratinocyte cells (of the HaCaT cell line) were treated with arecoline, following which cell viability was assessed using the Trypan Blue dye-exclusion assay, cell growth and proliferation were analyzed using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and 5-bromo-2-deoxyuridine incorporation assays, cell cycle arrest and generation of reactive oxygen species were examined using flow cytometry, and gene expression changes were investigated using the reverse transcription-polymerase chain reaction technique. The role of oxidative stress, muscarinic acetylcholine receptor and mitogen-activated protein kinase (MAPK) pathways were studied using specific inhibitors. Western blot analysis was performed to study p38 MAPK activation.Results:Arecoline induced the generation of reactive oxygen species and cell cycle arrest at the G1/G0 phase in HaCaT cells without affecting the expression of p21/Cip1. Arecoline-induced epithelial cell death at higher concentrations was caused by oxidative trauma without eliciting apoptosis. Sublethal concentrations of arecoline upregulated the expression of the following stress-responsive genes: heme oxygenase-1; ferritin light chain; glucose-6-phosphate dehydrogenase; glutamate-cysteine ligase catalytic subunit; and glutathione reductase.Additionally, there was a dose-dependent induction of interleukin-1alfa mRNA by arecoline via oxidative stress and p38 MAPK activation. Conclusion:our data highlight the role of oxidative stress in arecoline-mediated cell death, gene regulation and inflammatory processes in human keratinocytes.

A Mycobacterial Phosphoribosyltransferase Promotes Bacillary Survival by Inhibiting Oxidative Stress and Autophagy Pathways in Macrophages and Zebrafish

Mycobacterium tuberculosis employs various strategies to modulate host immune responses to facilitate its persistence in macrophages. The M. tuberculosis cell wall contains numerous glycoproteins with unknown roles in pathogenesis. Here, by using Concanavalin A and LC-MS analysis, we identified a novel mannosylated glycoprotein phosphoribosyltransferase, encoded by Rv3242c from M. tuberculosis cell walls. Homology modeling, bioinformatic analyses, and an assay of phosphoribosyltransferase activity in Mycobacterium smegmatis expressing recombinant Rv3242c (MsmRv3242c) confirmed the mass spectrometry data. Using Mycobacterium marinum-zebrafish and the surrogate MsmRv3242c infection models, we proved that phosphoribosyltransferase is involved in mycobacterial virulence. Histological and infection assays showed that the M. marinum mimG mutant, an Rv3242c orthologue in a pathogenic M. marinum strain, was strongly attenuated in adult zebrafish and also survived less in macrophages. In contrast, infection with wild type and the complemented Delta mimG: Rv3242c M. marinum strains showed prominent pathological features, such as severe emaciation, skin lesions, hemorrhaging, and more zebrafish death. Similarly, recombinant Msm Rv3242c bacteria showed increased invasion in non-phagocytic epithelial cells and longer intracellular survival in macrophages as compared with wild type and vector control M. smegmatis strains. Further mechanistic studies revealed that the Rv3242c- and mimG-mediated enhancement of intramacrophagic survival was due to inhibition of autophagy, reactive oxygen species, and reduced activities of superoxide dismutase and catalase enzymes. Infection with MsmRv3242c also activated the MAPK pathway, NF-kappa B, and inflammatory cytokines. In summary, we show that a novel mycobacterial mannosylated phosphoribosyltransferase acts as a virulence and immunomodulatory factor, suggesting that it may constitute a novel target for antimycobacterial drugs.

Ascorbate Protects Neurons against Oxidative Stress: A Raman Microspectroscopic Study

Oxidative stress due to excessive accumulation of reactive oxygen or nitrogen species in the brain as seen in certain neurodegenerative diseases can have deleterious effects on neurons. Hydrogen peroxide, endogenously generated in neurons under normal physiological conditions, can produce an excess of hydroxyl radical via a Fenton mediated mechanism. This may induce acute oxidative injury if not scavenged or removed effectively by antioxidants. There are several biochemical assay methods to estimate oxidative injury in cells; however, they do not provide information on the biochemical changes as the cells get damaged progressively under oxidative stress. Raman microspectroscopy offers the possibility of real time monitoring of the chemical composition of live cells undergoing oxidative stress under physiological conditions. In the present study, a hippocampal neuron coculture was used to observe the acute impact of hydroxyl radicals generated by hydrogen peroxide in the presence of Fe2+ (Fenton reaction). Raman peaks related to nucleic acids (725, 782, 1092, 1320, 1340, 1420, and 1576 cm(-1)) showed time-dependent changes over the experimental period (60 mm), indicating the breakdown of the phosphodiester backbone as well as nuclear bases. Interestingly, ascorbic acid (a potent antioxidant) when cotreated with Fenton reactants showed protection of cells as inferred from the Raman spectra, presumably by scavenging hydroxyl radicals. Little or no change in the Raman spectra was observed for untreated control cells and for cells exposed to Fe2+ only, H2O2 only, and ascorbate only. A live dead assay study also supported the current observations. Hence, Raman microspectroscopy has the potential to be an excellent noninvasive tool for early detection of oxidative stress that is seen in neurodegenerative diseases.

Robust Glyoxalase activity of Hsp31, a ThiJ/DJ-1/PfpI Family Member Protein, Is Critical for Oxidative Stress Resistance in Saccharomyces cerevisiae

Methylglyoxal (MG) is a reactive metabolic intermediate generated during various cellular biochemical reactions, including glycolysis. The accumulation of MG indiscriminately modifies proteins, including important cellular antioxidant machinery, leading to severe oxidative stress, which is implicated in multiple neurodegenerative disorders, aging, and cardiac disorders. Although cells possess efficient glyoxalase systems for detoxification, their functions are largely dependent on the glutathione cofactor, the availability of which is self-limiting under oxidative stress. Thus, higher organisms require alternate modes of reducing the MG-mediated toxicity and maintaining redox balance. In this report, we demonstrate that Hsp31 protein, a member of the ThiJ/DJ-1/PfpI family in Saccharomyces cerevisiae, plays an indispensable role in regulating redox homeostasis. Our results show that Hsp31 possesses robust glutathione-independent methylglyoxalase activity and suppresses MG-mediated toxicity and ROS levels as compared with another paralog, Hsp34. On the other hand, glyoxalase-defective mutants of Hsp31 were found highly compromised in regulating the ROS levels. Additionally, Hsp31 maintains cellular glutathione and NADPH levels, thus conferring protection against oxidative stress, and Hsp31 relocalizes to mitochondria to provide cytoprotection to the organelle under oxidative stress conditions. Importantly, human DJ-1, which is implicated in the familial form of Parkinson disease, complements the function of Hsp31 by suppressing methylglyoxal and oxidative stress, thus signifying the importance of these proteins in the maintenance of ROS homeostasis across phylogeny.

Exposure of spermatozoa to duroquinone may impair reproduction of the common carp (Cyprinus carpio) through oxidative stress

Toxicity of many waterborne organic contaminants to aquatic organisms is mediated through oxidative damages resulting from the production of reactive oxygen species (ROS). Using duroquinone as a model ROS inducer, we carried out in vitro and in vivo experiments to test the hypothesis that reproduction in common carp (Cyprinus carpio) can be impaired through oxidative damage of their spermatozoa. In vitro exposure of fish spermatozoa to 0, 12.5, 25, 50, 100 and 200 mu M duroquinone for 2 h showed a significant increase in the level of ROS in a dose-dependant manner. Sperm motility was significantly reduced in all exposure groups, but lipid peroxidation (LPO) and DNA strand break (measured by comet assay) were only enhanced at 50 mu M and above. A significant decrease in subsequent hatching rate was recorded in all the exposure groups, despite fertilization rate was not affected. In the in vivo experiment, spermatozoa were collected 24 and 72 h after fish received intra-peritoneal injections of 1.0 and 10 mg kg(-1) body weight duroquinone. DNA damage was clearly evident in spermatozoa of all treatment groups after 72 h exposure, and ROS was significantly enhanced in the high concentration group. LPO however, remained unchanged in both treatment groups. The overall results of both our in vitro and in vivo experiments demonstrated that duroquinone can induce ROS production in spermatozoa, which may impair sperm quality and subsequently reproductive success through oxidative stress. (c) 2006 Elsevier B.V. All rights reserved.